Time Course of Enrofloxacin and Its Active Metabolite in Peripheral Leukocytes of Dogs

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1 Time Course of Enrofloxacin and Its Active Metabolite in Peripheral Leukocytes of Dogs Albert Boeckh, DVM, DACVCP Dawn Boothe, DVM, PhD, DACVIM, DACVCP Scott Wilkie, BS Sarah Jones, DVM Department of Veterinary Physiology and Pharmacology College of Veterinary Medicine Texas A&M University College Station, TX ABSTRACT The accumulation of fluoroquinolones in peripheral leukocytes is well established for several members of this family of drugs. This study documented accumulation of enrofloxacin and its active metabolite, ciprofloxacin, in canine peripheral leukocytes after oral administration at 7.5, 10, or 20 mg/kg, doses that are within the currently approved label of Baytril (Bayer Corporation Animal Health, Shawnee Mission, KS) in the United States. Accumulation index, as defined by C max in white blood cells (WBCs) divided by C max in plasma at the same time points, ranged from 47-fold to 63-fold for enrofloxacin and 28- to 35-fold for ciprofloxacin. Time course of both enrofloxacin and ciprofloxacin in WBCs paralleled the time course in plasma. Disappearance half-life of ciprofloxacin from WBCs was significantly (P =.001) longer than was enrofloxacin (P =.015) for all doses studied. The accumulation of enrofloxacin in peripheral leukocytes may contribute to the antimicrobial efficacy of this compound. INTRODUCTION The fluorinated quinolone (FQ) antimicrobials are widely used in veterinary medicine due to their marked efficacy, safety, and convenient dosing regimen. 1 Enrofloxacin, the first FQ approved globally for veterinary medicine, has been marketed for 10 years (Baytril, Bayer Corporation Animal Health, Shawnee Mission, KS) and new therapeutically beneficial features of this quinolone continue to be identified. Among the attributes of the FQs is their accumulation by phagocytic leukocytes. 2 Although accumulation is well documented for FQs in human phagocytes, the only report of accumulation of enrofloxacin involved canine alveolar macrophages. 1 The accumulation of drug in phagocytes (e.g., macrophages and neutrophils) makes FQs an appealing choice for treatment of microorganisms that can survive intracellular phagocytosis. Organisms that cause infections in dogs include Staphylococcus, Salmonella, Nocardia, Chlamydia, Mycoplasma, Brucella, and atypical Mycobacterium. Another potential advantage of accumulation of enrofloxacin by white blood cells (WBCs) is the ability of WBCs to serve as carriers of enrofloxacin to the sites of inflammation. Subsequent release at the site of infection may increase concentrations of enrofloxacin to which infecting microbes are exposed, 3 thus increasing the potential of therapeutic success

2 Veterinary Therapeutics Vol. 2, No. 4, Fall 2001 Ciprofloxacin, a commercially available antimicrobial approved for use in humans (Cipro, Bayer Corporation Pharmaceutical Division, West Haven, CT), accumulates in leukocytes. 4 The antibacterial action of ciprofloxacin is additive with that of enrofloxacin. 5 In dogs, enrofloxacin is metabolized to ciprofloxacin, which can achieve therapeutic concentrations against some organisms. 5 Thus, in vivo studies focusing on enrofloxacin should also examine the presence of the active metabolite ciprofloxacin. The objective of this study was to characterize the accumulation of enrofloxacin and ciprofloxacin by canine peripheral leukocytes after single oral administration of enrofloxacin. MATERIALS AND METHODS Animals Six spayed female bloodhounds, 2 to 3 years of age, weighing between 23 and 26 kg each, were studied. Animals were housed in a facility approved by the US Department of Agriculture, and the study was approved by the Texas A&M University Laboratory Animal Care Committee. The dogs were fed a high-quality commercially available diet once daily and had access to fresh water ad libitum. The dogs were not receiving any medication except for monthly heartworm preventative (Heartgard, Merial Limited, Duluth, GA) and an oral supplement of iron sulfate due to the volume of blood collected (280 ml per treatment three treatments over 6 weeks). All dogs were clinically healthy as determined by results of physical examinations and complete blood counts performed before each trial day. Each dog was also observed at least once daily throughout the 6 weeks of the study. Treatments All six dogs received three different single oral doses of enrofloxacin (7.5, 10, and 20 mg/kg) using a randomized, crossover design with washout periods of at least 2 weeks between each treatment. The dosages of enrofloxacin were selected as the middle to the maximum in the range approved for dogs by the Food and Drug Administration. Dogs were fasted for 12 hours prior to administration of the drug, weighed immediately prior to administration, and were dosed orally with commercially available enrofloxacin (Baytril ) nonflavored 22.7-mg and 68-mg tablets to the nearest 11.3 mg (half of a mg tablet). Blood Sampling and Preparation Before administration of the drug, a catheter was placed aseptically into the left jugular vein of each dog to facilitate collection of blood samples. Whole-blood samples (5 ml) were collected in tubes containing sodium citrate as an anticoagulant prior to administration of enrofloxacin and 15, 30, 45, 60, 75, and 90 minutes and 2, 3, 4, 6, 8, 10, 12, 18, and 24 hours after dosing. Samples intended for measurement of drug concentrations in plasma were centrifuged at 1150 g for 15 minutes within 2 hours of collection. Plasma was harvested and frozen at 20 C until analysis. A wholeblood sample (20 ml) from each dog was collected in a tube with sodium citrate for isolation of peripheral WBCs prior to dosing and 1, 2, 3, 6, 8, 10, 12, 18, and 24 hours after administration of the drug. For quantification of enrofloxacin in WBCs, blood was centrifuged at 1125 g for 15 minutes within 2 hours of collection. The leukocyte-rich layer (buffy coat) was then transferred to a tube of smaller volume using a Pasteur pipette. Red blood cells (RBCs) in the WBC samples were lysed by adding 2 ml of hypotonic saline (0.2% NaCl, 64.8 mosm) and gently inverting the sample for 30 seconds. Isotonicity was then restored by adding 1 ml of 335

3 hypertonic saline (1.6% NaCl, mosm). 6 Each sample was visually inspected for a red pellet beneath the WBC pellet after centrifugation to verify there were no intact RBCs in the WBC samples. The resulting fluid (after hypotonic lysis) was centrifuged at 700 g for 10 minutes. The supernatant was transferred to another vial and frozen at 20 C. All sample manipulation, including initial whole-blood centrifugation, was performed at 5 C to minimize any drug movement from the WBC into the drug-free supernatant. The WBC pellet was resuspended in modified (calcium-free) Hanks balanced salt solution 6 (HBSS) to a volume of 1 ml by weighing the sample and adding HBSS to a final weight of 1 gram after subtracting the weight of the empty tube. A 200-µl aliquot of this sample was submitted for WBC quantification and differential counting using an automated cell-counting system. No RBCs were detected in the samples during the automated counting. Supernatant was harvested for confirmation of the magnitude of diffusion of either enrofloxacin or ciprofloxacin from the WBCs into the surrounding fluid. The remaining 800 µl of suspended WBCs were lysed by freezing, defrosting, and sonication. Samples were frozen overnight at 20 C, rapidly defrosted in a hot waterbath at 56 C the following morning, and immediately sonicated for 15 minutes in an ultrasonic waterbath. This procedure was repeated two more times to ensure there were no remaining intact cells in the samples. To further verify that there were no intact cells in the WBC samples after the lysing procedure, random samples were evaluated under low-power optical microscopy. The lysed WBC suspensions were frozen at 20 C until analysis. All samples were analyzed within 2 weeks of sample collection. At the time of WBC collection, a 0.5-ml aliquot of RBCs was simultaneously collected from the midcore of the centrifuged sample in 500 µl of HBSS. Cell counting was not performed because the viscous consistency precluded counting by either manual or automated methods. For quantification of enrofloxacin in RBCs, 500 µl of HBSS was added to 500 µl of RBCs. The suspension was whirled, frozen, defrosted, and sonicated three times, using the same procedure as for WBCs. Samples were frozen at 20 C until analysis at the same time as for other samples. Standards of enrofloxacin prepared in HBSS were frozen with each set of the samples collected from study animals. These standards were assayed simultaneously with those samples with which they were frozen to verify stability of drug during storage. Similarly, to determine any negative impacts of freezing, defrosting, and sonication procedures on drug stability, standards were also prepared in HBSS and subsequently subjected to the lysing protocol. Sample Analysis On the day of analysis, samples (WBC, RBC, and supernatant) were defrosted and centrifuged through a 10-kD membrane at 1750 g for 2 hours to remove cellular debris and proteins. The prepared samples were analyzed by high-performance liquid chromatography (HPLC) using fluorescence detection with excitation at 280 nm and emission at 450 nm in a method developed in the authors laboratory, based on previously described methods. 7,8 Briefly, using a Waters HPLC system (Waters Corporation, Milford, MA), samples were injected through a reverse-phase C-18 column and measured by fluorescence detection. Samples were compared to a standard curve prepared with canine plasma, with controls prepared with canine serum and HBSS. The lower and upper limits of quantification (LOQ) for this study were 25 ng/ml and 5400 ng/ml, respectively, for enrofloxacin and

4 Veterinary Therapeutics Vol. 2, No. 4, Fall 2001 ng/ml and 6100 ng/ml for ciprofloxacin. Precision was 94.6% for enrofloxacin and 93.3% for ciprofloxacin. Accuracy was 100.0% for enrofloxacin and 102.1% for ciprofloxacin. Samples with concentrations below the LOQ were not included. Data Analysis Concentration of enrofloxacin and ciprofloxacin versus time curves for each animal were subjected to noncompartmental linear regression analysis to determine the following pharmacokinetic parameters: maximum concentration (C max ); time for maximum concentration (T max ); time for drug appearance in plasma; area under the curve (AUC) to infinity using the trapezoidal method; mean residence time (MRT); disappearance constants (k d ); and disappearance half-life (t 1/2d ). Enrofloxacin and ciprofloxacin were not administered intravenously; therefore, neither clearance nor apparent volume of distribution was determined for either compound. Likewise, the terminal slope of the plasma drug concentration-versustime curve could not be confirmed to represent elimination. To determine the intracellular concentration of enrofloxacin or ciprofloxacin in WBCs, it was assumed that any drug detected in the WBC samples originated from inside the WBCs. The intracellular concentration of enrofloxacin and ciprofloxacin was determined by dividing the concentration measured in the cellular filtrate by the volume of the WBCs originally present in the sample (i.e., drug concentration in WBCs = drug concentration in filtrate/volume of WBC pellet). White blood cells were reconstituted to a volume of 1 ml. The volume of the WBC pellet was determined by multiplying the number of WBCs in the sample by 523 µm 3, which is the average volume of canine WBCs. 9 Statistical Analysis Descriptive statistics reported for enrofloxacin and ciprofloxacin in plasma, 10 WBCs, and RBCs were means and standard deviations for all variables except for t 1/2d which was reported as the harmonic mean and pseudo-standard deviation. Comparisons were made among different doses within each drug for C max, T max, MRT, k d, and t 1/2d, using a generalized linear model that allows designation of a random variable (PROC MIXED; SAS Institute, Cary, NC). In this test, the individual dog was designated as the random effect variable. Least square means were computed for dose and time effects. Significant differences among means were detected using Tukey s test of multiple comparisons. Differences were considered to be significant at P <.05. RESULTS Concentrations of enrofloxacin in standards frozen with the experimental samples were unchanged, indicating lack of degradation from freezing or lysing. Administration of higher doses of enrofloxacin to the dogs resulted in proportional increases in drug concentrations of both enrofloxacin and ciprofloxacin in plasma and in WBCs (Figure 1). White and Red Blood Cells The number of WBCs harvested was variable, with a mean of 84,000 WBC/µl ± 37,000. Differential counts revealed a mean of 65% granulocytes and 35% lymphocytes. White blood cell differential counts were statistically similar among the three doses administered. In general, the time course of enrofloxacin and ciprofloxacin in WBCs tended to parallel those in plasma (Figure 1). However, compared to plasma, concentrations in WBCs and AUC for both enrofloxacin and ciprofloxacin were markedly increased. Concentrations of enrofloxacin in WBCs peaked (T max ) at ap- 337

5 Enrofloxacin plasma Ciprofloxacin plasma Enrofloxacin WBC Ciprofloxacin WBC Enrofloxacin RBC Ciprofloxacin RBC Concentration (µg/ml) Figure 1A Hours Enrofloxacin plasma Ciprofloxacin plasma Enrofloxacin WBC Ciprofloxacin WBC Enrofloxacin RBC Ciprofloxacin RBC Concentration (µg/ml) Figure 1B Hours Figure 1. Concentrations (mean ± standard deviation) of enrofloxacin and ciprofloxacin in white blood cells (WBC), plasma, and red blood cells (RBC) from six dogs after oral administration of enrofloxacin at 7.5 (A), 10 (B), or 20 (C) mg/kg. 338

6 Veterinary Therapeutics Vol. 2, No. 4, Fall Enrofloxacin plasma Ciprofloxacin plasma Enrofloxacin WBC Ciprofloxacin WBC Enrofloxacin RBC Ciprofloxacin RBC Concentration (µg/ml) Figure 1C Hours proximately the same time as they did in plasma at all dosages (P >.05) (Figure 1). Ciprofloxacin peaked later than enrofloxacin in plasma and WBCs (P =.04). No significant difference was seen (P >.05) for T max among the three different dosages of enrofloxacin or ciprofloxacin (Table 1). Maximum concentration of enrofloxacin and ciprofloxacin in WBCs increased as the dosage increased, but not significantly among the three dosages (Table 1). For enrofloxacin, the differences in AUC approached significance at 7.5 mg/kg (P =.053) and 10 mg/kg (P =.058) when compared with 20 mg/kg. Area under the curve for ciprofloxacin was significantly greater in WBCs at 20 mg/kg compared with 7.5 mg/kg (P =.009) and 10 mg/kg (P =.025). Mean residence time, t 1/2d, and k d did not differ significantly among dosages for either enrofloxacin or ciprofloxacin. Accumulation index, defined as C max in WBC divided by C max in plasma at the same time points, ranged from 47-fold to 63-fold for enrofloxacin and 28- to 35-fold for ciprofloxacin (Figure 2A). This C max accumulation index was significantly different (P =.03) between enrofloxacin and ciprofloxacin. Similarly, the ratio of AUC in WBC:plasma was also calculated for each of the three dosages, and significant differences (P =.019) were observed between enrofloxacin and ciprofloxacin (Figure 2B). The AUC ratio was between 33- and 46- fold for enrofloxacin and 20- and 22-fold for ciprofloxacin (P =.034). Disappearance half-life of ciprofloxacin from WBCs was significantly longer than for enrofloxacin (P =.039) for all doses studied (Table 1). The harmonic mean t 1/2d of enrofloxacin in WBCs was 4.92 ± 1.17 hours for 7.5 mg/kg, 3.48 ± 1.09 hours for 10 mg/kg, and 5.38 ± 2.48 hours for 20 mg/kg. For ciprofloxacin, the values were 6.00 ± 1.13 hours, 8.63 ± 4.85 hours, and 8.75 ± 6.69 hours for 7.5, 10, and 20 mg/kg, respectively. 339

7 TABLE 1. Pharmacokinetic Parameters for Enrofloxacin and Ciprofloxacin in White Blood Cells After Oral Administration of Enrofloxacin to Dogs at Different Dosages Enrofloxacin 7.5 mg/kg Enrofloxacin 10 mg/kg Enrofloxacin 20 mg/kg Variables Mean SD Mean SD Mean SD Enrofloxacin C max (µg/ml) T max (hr) AUC (µg/ml/hr) MRT (hr) t 1/2d (hr) k d (hr 1 ) Ciprofloxacin C max (µg/ml) T max (hr) AUC (µg/ml/hr) MRT (hr) t 1/2d (hr) k d (hr 1 ) C max = maximum concentration; T max = time to maximum concentration; AUC = area under time curve; MRT = mean residence time; t 1/2d = disappearance half-life; k d = disappearance constant. No significant differences were detected among the dosages for t 1/2d of either enrofloxacin or ciprofloxacin. Red blood cell concentrations for both the parent compound and the metabolite paralleled those in plasma, but were on average ten times lower than in plasma (Figure 1). All samples of supernatant collected from the WBC samples before the lysing procedure were drug free. DISCUSSION Results of differential WBC counts were similar to those of normal canine blood in that there were 65% granulocytes and 35% lymphocytes. It is not known if the kinetics of FQ accumulation vary among different types of WBCs, and the contribution of each individual type of WBC in the accumulation of enrofloxacin and ciprofloxacin cannot be estimated in this study. The lack of significant differences in C max for enrofloxacin among the dosages tested most likely reflects the small number of animals studied, biological variability among animals, and techniques for WBC isolation. The power to detect a significant difference in C max between 7.5 and 20 mg/kg using a sample size of six was only 35% in this study. However, AUC (which takes into account both C max and elimination half-life) did differ between 7.5 and 20 mg/kg, supporting a dose-dependent effect on drug accumulation in WBCs. The accumulation of enrofloxacin and ciprofloxacin in WBCs in this study was greater 340

8 Veterinary Therapeutics Vol. 2, No. 4, Fall 2001 Accumulation Index Enrofloxacin Ciprofloxacin Enrofloxacin dosage (mg/kg) Accumulation Index Enrofloxacin Ciprofloxacin Enrofloxacin dosage (mg/kg) Figure 2A Figure 2B Figure 2. Maximum concentrations (A) and area under the curve (B) for enrofloxacin and its metabolite ciprofloxacin (ratios of white blood cells:plasma) after oral administration of enrofloxacin to dogs at 7.5, 10, or 20 mg/kg. than that previously reported for ciprofloxacin in human neutrophils 4 and for enrofloxacin in canine alveolar macrophages. 1 In the latter report, Hawkins and Boothe presented concentrations in macrophages that were 10- to 30- fold lower than those found in the study reported here and ratios between macrophages and plasma that were 4- to 9-fold lower than those found in this study. The difference is due, in part, to a miscalculation in the first study. The macrophage volume in that study was incorrect in that a volume of ml was used rather than ml. As a consequence, the calculated pellet volume was 10- fold higher than it should have been, resulting in intracellular concentrations of drug that were less than actual by the same factor. Another study that documented the accumulation of ciprofloxacin in human neutrophils showed C max of 10 µg/ml. 11 An additional reason for the disparity in results may be differences in techniques for cell isolation used in this study compared with those used in previous studies. The technique developed in the authors laboratory avoids sequential cell washes required for separation of WBCs when using density gradient separation. 4,11 Those washes may lead to loss of drug by diffusion from the cell into the drug-free supernatant. However, the amount of drug potentially lost through efflux into the wash was not likely to be sufficient to negatively impact the conclusions of the study. Based on the LOQ of the assay in the study reported here (25 ng/ml), the maximum amount of enrofloxacin lost through efflux into the hypotonic and hypertonic wash (total = 3 ml) would have been 75 ng. This amount was much smaller (less than 1% at initial time points) than the intracellular concentrations in WBCs. If this amount of enrofloxacin efflux did occur, intracellular concentrations would have been even higher than reported here. Processing the samples at 5 C may have reduced drug loss by decreasing membrane permeability. One disadvantage of the technique used in this study is possible contamination of WBC samples with RBCs. Hypotonic lysis of the RBCs, followed by a single wash, minimized contamination of the WBC samples with RBC. Both visual inspection and automated cell counts confirmed the absence of intact RBCs in these samples. Because intracellular concentrations of both enrofloxacin and ciprofloxacin in RBCs were so much lower than in the WBCs, their contribution to levels of parent compound and metabolite in the samples should be 341

9 considered negligible. This study intended to measure only intracellular concentrations of the compounds, not drug that is bound to membranes. Most of the intracellular fluoroquinolones do not accumulate in subcellular organelles but stay free in the cytosol. 2,12 An advantage of this assay was the use of an ultrafiltration technique which removed cellular debris and any drug bound to cellular components while maintaining drug present in the cytosol. The contribution of ciprofloxacin to antimicrobial efficacy following administration of enrofloxacin to dogs is often not considered, 13 yet results of this study revealed that ciprofloxacin was present in substantial concentrations following oral administration of the parent compound. The two compounds appear to act additively. 5 In this study, the contribution of ciprofloxacin to total C max was 22% for the 7.5 mg/kg dosage, resulting in a combined C max of 175 µg/ml. Similarly, for 10 and 20 mg/kg, the increase was 29% and 20%, respectively, resulting in combined C max levels of 171 µg/ml and 280 µg/ml. For combined AUC, the contribution of ciprofloxacin at the various dosages of enrofloxacin administered was 36% (7.5 mg/kg), 40% (10 mg/kg), and 37% (20 mg/kg). The contribution of ciprofloxacin to the AUC is higher than to C max because it has a longer half-life than does enrofloxacin, decreasing the slope of the concentration-versustime line and thus prolonging elimination. The clinical relevance of a larger AUC for drugs described as concentration-dependent is not clear, although some investigators have suggested that the total AUC divided by the minimum inhibitory concentration (MIC) of the organism (referred to as area under the inhibitory concentration curve) is a more relevant ratio upon which antimicrobial efficacy might be predicted for the FQs. 14 Ratios of drug concentration between plasma and WBCs show greater accumulation of enrofloxacin than ciprofloxacin inside WBCs. This may reflect the greater lipophilicity of the parent compound compared with the metabolite. 14 The source of ciprofloxacin, either accumulated from plasma or metabolized from enrofloxacin by WBCs, cannot be established by the results of this study. Accumulation of FQs in WBCs has been attributed to passive diffusion into the cells followed by intracellular trapping due to binding or ionization (ph mediated). 2,15 Although this may be true, the magnitude of the accumulation suggests that an active transport process also is involved. Studies that are more recent suggest that, although not clearly understood, the process is active and energy dependent. 16 This was demonstrated in vitro with the use of compounds (sodium fluoride) that prevented mitochondrial oxidative phosphorylation and therefore production of adenosine triphosphate necessary for active transport. 17 Similarly, inhibition of glycolysis by sodium cyanide prevented intracellular quinolone accumulation. 16,17 In another study, pefloxacin uptake was demonstrated to be carrier-mediated, activated by a Ca 2+ -dependent system. Uptake was inhibited by verapamil, a calcium channel blocker. 17 However, this transport mechanism may be unique to monocytes, differing from that of polymorphonucleated leukocytes. 18,19 Further evidence suggests that once accumulated in macrophages fluoroquinolones may enhance phagocytic and antimicrobial activities of macrophages, 20 possibly leading to improved efficacy, especially in the treatment of infections caused by intracellular organisms. Regardless of the mechanism of accumulation, an increase in antimicrobial efficacy can be anticipated to occur. As a concentration-dependent antimicrobial drug, the efficacy of enrofloxacin is predicted by the inhibitory quotient (IQ), or the ratio of C max to the MIC of 342

10 Veterinary Therapeutics Vol. 2, No. 4, Fall 2001 the microbe. 13 Staphylococcus intermedius and Salmonella spp organisms that can survive intracellular phagocytosis can serve as examples. The reported MIC 90 for enrofloxacin for each organism is 0.25 µg/ml. 21 With accumulation of enrofloxacin in WBCs, the resulting IQ is 508 at 7.5 mg/kg and greater than 890 times that at 20 mg/kg. Enrofloxacin is also reported to be highly effective against Mycoplasma, 13 another organism that can survive phagocytosis. Efficacy may be due to the high intracellular concentrations of enrofloxacin to which this microorganism is exposed. The reported MIC 90 for Mycoplasma spp is 0.5 µg/ml, 22 and the IQ is 254 at 7.5 mg enrofloxacin/kg and 445 at 20 mg/kg. Inhibitory quotients, if ciprofloxacin is included in total antimicrobial activity, become 25% to 28% higher. Accumulation may also facilitate antimicrobial efficacy by increased drug delivery. White blood cells likely can serve as carriers of drug to sites of infection, leading to enhanced efficacy. 22 This may provide increased concentrations of enrofloxacin and ciprofloxacin at the site of infection, compared with the same tissue when not infected. The release of enrofloxacin and ciprofloxacin from the leukocyte at the site of infection may occur as phagocytic cells die at the site or as a result of active drug transport processes out of the cell. High intracellular concentrations of FQ offer an advantage specific for intracellular organisms when compared to other drugs not accumulated by phagocytes. The quinolone antimicrobials located inside WBCs remain active, 22 maintaining the same killing potency inside the cell as they do outside. 4,23 This is a distinct and somewhat unique advantage of FQs. Other selected antimicrobials also accumulate inside phagocytes, but the accumulation is in specific subcellular compartments. Examples include macrolides such as erythromycin and lincosamides, which accumulate in lysosomes. 15 The function of WBCs does not appear to be inhibited by the presence of high intracellular concentrations of enrofloxacin and ciprofloxacin, another advantage of FQs compared with most other accumulated drugs. 23 In summary, this study demonstrates that enrofloxacin and ciprofloxacin accumulate inside canine peripheral leukocytes. This accumulation may be one of the reasons for the remarkable efficacy of enrofloxacin in treating bacterial infections in veterinary medicine. REFERENCES 1. Hawkins E, Boothe D: Concentration of enrofloxacin and its active metabolite in alveolar macrophages and pulmonary epithelial lining fluid of dogs. J Vet Pharmacol Ther 21:1823, Tulkens PM: Accumulation and subcellular distribution of antibiotics in macrophages in relation to activity against intracellular bacteria. In: Ciprofloxacin in Pulmonology. Zucksschwerdt Verlag: Munchen; 1990: Walters J, Zhang F, Nakkula R: Mechanisms of fluoroquinolone transport by human neutrophils. Antimicrob Agents Chemother 43(11): , Schuler P, Zemper K, Borner K, Koeppe P, Schaberg T, Lode H: Penetration of sparfloxacin and ciprofloxacin into alveolar macrophages, epithelial lining fluid and polymorphonuclear leucocytes. Eur Resp J 10: , Pirro R, Scheer M, de Jong A: Additive in vitro activity of enrofloxacin and its main metabolite ciprofloxacin. Proc 14th Annu Congr Eur Soc Vet Dermatol, Pasloske, K: Ketorolac Pharmacokinetics and Effects on Neutrophil Function and Prostaglandin E2 Concentration in Dogs [master s thesis]. Guelph, Ontario, Canada: Ontario Veterinary College, University of Guelph; Kung K, Riond JL: Comparison of an HPLC and bioassay method to determine antimicrobial concentrations after intravenous and oral administration of enrofloxacin in 4 dogs. Res Vet Sci 54:47248, Bayer Corporation Agricultural Division: Method for the analysis of enrofloxacin and ciprofloxacin in canine plasma. Shawnee Mission KS, Snell HS: In: Clinical and Functional Histology for Medical Students. Little, Brown and Co: London; 1984: Boothe DM, Boeckh A, Boothe HW, Wilkie S: Tissue concentrations of enrofloxacin and ciprofloxacin in anesthetized dogs following single intravenous administration. Vet Ther 2:120128,

11 11. Garrafo R, Jambou D, Chichmanian RM, Ravoire S, Lapalus P: In vitro and in vivo ciprofloxacin pharmacokinetics in human neutrophils. Antimicrob Agents Chemother 35(11): , Vazifeh D, Bryskier A, Labro M: Mechanism underlying levofloxacin uptake by human polymorphonuclear neutrophils. Antimicrob Agents Chemother 43(2): , Kung K, Riond J, Wanner M: Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther 16:462468, Papich M: Comparison of fluoroquinolone antibacterials. Proc ACVIM 5355, Tulkens PM: Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis 10(2): , Loo K, Cario A, Zhang F, Walters J: Regulation of ciprofloxacin uptake in human promyelocytic leukemia cells and polymorphonuclear leukocytes. J Leukoc Biol 61:619623, Knoller J, Brom J, Schonfeld W, Konig W: Influence of ciprofloxacin on leukotriene generation from various cells in vitro. J Antimicrob Chemother 25: , Memin E, Panteix G, Revol A: Carrier-mediated system for pefloxacin uptake in human monocytes. J Antimicrob Chemother 40:263268, Bounds S, Nakkula R, Walters D: Fluoroquinolone transport by human monocytes: Characterization and comparison to other cells of myeloid lineage. Antimicrob Agents Chemother 44(10): , Tullio V, Cuffini AM, Bonino A, et al: Influence of a new fluoroquinolone, AF3013 (the active metabolite of plurifloxacin), on macrophage functions against Klebsiella pneumoniae: An in vitro comparison with pefloxacin. J Antimicrob Chemother 46:241247, National Committee of Clinical Laboratory Standards: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standards. NCCLS. June 1999; Document M31-A:19(11). 22. Carlier MB, Scorneaux B, Zenebergh A, Desnottes JF, Tulkens PM: Cellular uptake, localization and activity of fluoroquionolones in uninfected and infected macrophages. J Antimicrob Chemother 25(Suppl B): 2739, Bailly S, Fay M, Roche Y, Gougerot-Pocidalo MA: Effects of quinolones on tumor necrosis factor production by human monocytes. Int J Immunopharmacol. 12(1):3136,