Tissue Concentrations of Enrofloxacin and Ciprofloxacin in Anesthetized Dogs Following Single Intravenous Administration*

Transcription

1 Tissue Concentrations of Enrofloxacin and Ciprofloxacin in Anesthetized Dogs Following Single Intravenous Administration* Dawn M. Boothe, DVM, PhD a Albert Boeckh, DVM a Harry W. Boothe, DVM, MS b Scott Wilkie, BS b a Department of Veterinary Physiology and Pharmacology b Department of Small Animal Medicine and Surgery College of Veterinary Medicine Texas A&M University College Station, TX ABSTRACT Concentrations of enrofloxacin and its active metabolite, ciprofloxacin, were detected in tissues following single intravenous administration of 20 mg/kg to four anesthetized dogs. Two hours after treatment, serum samples were collected, animals were euthanized, and tissues were collected from each dog. Solid tissues were homogenized, and both enrofloxacin and ciprofloxacin were detected using high-performance liquid chromatography. Enrofloxacin was detected in all tissues examined; the highest concentrations were found in the gall bladder, urine, bile, stomach, and liver. Concentrations in the cerebral cortex, cerebrospinal fluid, skin, and prostate were 5.7, 5.3, 9.2, and 23.5 µg/ml, respectively. Tissues for which the concentration of enrofloxacin was 4 µg/ml or lower included the trachea, articular cartilage, aqueous humor, fat, and tendon. An inhibitory quotient of 8 or more was achieved for enrofloxacin in the majority of tissues studied. Ciprofloxacin was detected in 29 of 40 tissues examined. *This study was sponsored by Bayer Corporation Animal Health, Shawnee Mission, KS. INTRODUCTION Enrofloxacin has been approved in the United States for several years for treatment of susceptible bacterial infections in small animals. 1 Most recently, a flexible label has been approved for this drug to be given once daily at 5 to 20 mg/kg. Serum drug concentration data are available either on the current label (2.5 mg/kg/day) 2 or in the literature for single 3 or multiple 4,5 (5-day) dosing at 2.5 to 11 mg/kg/day. A recent report 6 addresses plasma drug concentrations at the maximum label dosage rate of 20 mg/kg. However, for tissues other than serum, drug concentration data are available only for 2.5 mg/kg. Additional information is available for a limited number of tissues (e.g., bone, 7 skin, 8 urine 5 ). Achieving effective concentrations of a drug at the site of infection is a requirement for therapeutic success with antimicrobials This will facilitate eradication of the infection while decreasing the risk of antimicrobial resistance. 14 Administration of enrofloxacin at the maximum dosage should meet these objectives, particularly for organisms such as Pseudomonas, with high 120

2 D. M. Boothe, A. Boeckh, H. W. Boothe, and S. Wilkie (greater than 1 but less than 4 µg/ml) 15 minimum inhibitory concentrations (MICs). However, this expectation would be better met if data were available that supported evidence of tissue concentrations that would equal or exceed the MIC. Enrofloxacin is deethylated to its active metabolite, ciprofloxacin. 3,6 Previous studies have documented that the maximum serum concentration (C max ) achieved for ciprofloxacin is at least 40% of that achieved for enrofloxacin. 6 Therefore, administration of enrofloxacin at 20 mg/kg could also provide therapeutic concentrations of ciprofloxacin with activity against susceptible organisms in certain tissues. Additionally, enrofloxacin and ciprofloxacin act in an additive fashion, 16 and the metabolite increases the concentration and duration of antimicrobial drug at the site of infection. 6 Thus, in vivo studies that focus on the activity or kinetics of enrofloxacin should also include data for ciprofloxacin. The purpose of this study was to describe tissue concentrations of enrofloxacin and its active metabolite following intravenous administration of a single dose of enrofloxacin at 20 mg/kg. MATERIALS AND METHODS Four hounds (two male and two female), 2 to 3 years of age and weighing 25 to 28 kg each, were used in the study. The dogs were apparently healthy at the start of the trial as determined by clinical laboratory tests (complete blood count and serum chemistry panel) and physical examination. Animals were fed a standard diet and provided free access to water. All experimental protocols were approved by the University Laboratory Animal Care Committee. Tissue Sampling Anesthesia was induced with pentobarbital. Immediately after induction, a baseline blood sample was collected, and enrofloxacin (Baytril Antibacterial Injectable Solution 22.7%, Bayer Corporation Animal Health, Agriculture Division, Shawnee Mission, KS) was given as a rapid intravenous infusion (20 mg/kg) via the cephalic vein. Blood samples were collected from the contralateral cephalic vein at 30, 60, and 120 minutes following administration, and serum was harvested from the samples. Dogs were euthanized with an overdose of pentobarbital immediately following collection of the final blood sample, and tissues were collected for drug analysis. Animals were not exsanguinated prior to tissue collection. Approximately 3 g of each tissue was excised, blotted gently on a tissue to remove excess blood and other fluids (e.g., gastrointestinal luminal contents, bile, urine), and placed in a plastic storage tube. Tissue collection took place over a 30-minute period. Although the general sequence of tissue collection was similar for each animal, the exact timing of collection compared with time of euthanasia varied by up to 10 minutes. Additionally, the same tissues were not necessarily collected from all dogs, even those of the same sex. All samples were frozen in microcentrifuge tubes within 3 hours of collection and frozen at 20 o C until analysis. Sample Analysis On the day of analysis, serum, urine, and cerebrospinal fluid (CSF) samples were defrosted and centrifuged through a 10-kD membrane at 3000 rpm for 2 hours to remove cellular debris and proteins. Solid tissues were homogenized in 2 ml of physiologic saline and prepared in the same manner as fluid tissue samples. Filtrate was analyzed by high-performance liquid chromatography (HPLC) using reverse-phase chromatography with fluorescence detection, with excitation at 280 nm 121

3 and emission at 450 nm. The upper and lower limits of quantification for the assay (based on the lowest and highest known concentrations accurately predicted within 15%) were 6.7 and 5400 ng/ml for enrofloxacin and 29 and 6100 ng/ml for ciprofloxacin. Precision was 96.4% for enrofloxacin and 95.7% for ciprofloxacin. Accuracy was 108.5% for enrofloxacin and 100.9% for ciprofloxacin. Recovery of both compounds approximated 100%. Standards of enrofloxacin were frozen in saline with each set of samples collected from study animals and were assayed in conjunction with the tissue samples to verify the absence of drug degradation during cold storage. Data Analysis Enrofloxacin and ciprofloxacin concentrations were reported for each tissue; when concentrations were available in the same type of tissue for two or more animals, an average was reported along with the standard deviation. Inhibitory quotients (IQs; C max :MIC) 11,17 19 were calculated for enrofloxacin, ciprofloxacin, and enrofloxacin plus ciprofloxacin, based on an MIC value greater than the MIC 90 for the majority of organisms previously reported for enrofloxacin (0.5 µg/ml) and the MIC ranges listed on the package insert for Baytril. 20 Tissue:serum ratios were calculated for enrofloxacin and ciprofloxacin based on mean tissue concentration to 120-minute mean plasma concentration. RESULTS Enrofloxacin, but not ciprofloxacin, was detected in all samples submitted (Table 1). Tissues with the highest concentration of enrofloxacin were (in descending order) the gall bladder (wall), urine, bile, stomach, and liver. Tissues with the lowest concentration of enrofloxacin were the aqueous humor, articular capsule, fat, trachea (cartilage), and tendon. Concentrations of enrofloxacin did not achieve 4 µg/ml (the current MIC above which organisms are considered resistant to enrofloxacin) 15 in these tissues. Tissues with the highest ciprofloxacin concentration included the gall bladder, urine, bile, and liver (Table 1). Tissues with the lowest ciprofloxacin concentrations included amniotic fluid, aqueous humor, peritoneal fluid, and CSF. Ciprofloxacin was not detected in the articular capsule, cerebral cortex, colon, cornea, ear cartilage, fat, muscle, omentum, pericardium, skin, spinal cord, subcutaneous tissues, synovial fluid, tendon, testicle, and trachea. Tissue:serum drug concentrations were greater for enrofloxacin than for ciprofloxacin for all tissues except gall bladder and urine. DISCUSSION Physiologic responses to general anesthesia may have impacted the results in this study, specifically the distribution of drug to tissues. The impact of general anesthesia on tissue drug distribution appears to have been largely ignored. 21 Among the physiologic responses to general anesthetics, including pentobarbital, decreased cardiac contractility, redistribution of blood flow from splanchnic organs to the brain and heart, and hypoxia and hypercapnia are particularly pertinent to drug distribution. 21 Assuming these responses did occur, pentobarbital anesthesia is likely to have decreased drug distribution in this study, particularly to splanchnic organs, resulting in an underestimation of drug concentrations. Continuous capillaries such as those found in the central nervous system (and other blood tissue barriers), muscle, adipose, and other somatic tissues present the greatest barrier to drug penetration and as such should contain the lowest drug concentration. 9,22 24 Fenestrated capillaries of splanchnic organs (e.g., kidney, endocrine glands) contain pores connecting endothelial cells and should achieve 122

4 D. M. Boothe, A. Boeckh, H. W. Boothe, and S. Wilkie TABLE 1. Enrofloxacin and Ciprofloxacin Concentrations 2 Hours Following Single Intravenous Administration of Enrofloxacin at 20 mg/kg Enrofloxacin Ciprofloxacin No. of Mean Tissue: No. of Mean Tissue: Tissue Samples (µg/ml) SD IQ Serum* Samples (µg/ml) SD Serum* Amniotic fluid Aqueous humor Articular capsule NQ NQ Bile Bladder Bone marrow NQ NQ Cerebral cortex NQ NQ Colon NQ NQ Cornea NQ NQ Cerebrospinal fluid Diaphragm Ear cartilage NQ NQ Fat NQ NQ Gallbladder (wall) Jejunum Kidney (cortex) Large intestine Ligament NQ NQ Liver Lung Lymph node Muscle NQ NQ Myocardium Omentum NQ NQ Pancreas Pericardium NQ NQ Peritoneal fluid Prostate Salivary gland Serum (30 min) Serum (60 min) Serum (120 min) Skin NQ NQ Small intestine Spinal cord NQ NQ Spleen Stomach Subcutaneous tissue NQ NQ Synovial fluid NQ NQ Tendon NQ NQ (continues on next page) 123

5 TABLE 1. (Continued) Enrofloxacin Ciprofloxacin No. of Mean Tissue: No. of Mean Tissue: Tissue Samples (µg/ml) SD IQ Serum* Samples (µg/ml) SD Serum* Testicle NQ NQ Trachea NQ NQ Urine Uterus Vein (wall) NQ NQ *Tissue:serum ratio determined 120 minutes after treatment. IQ = peak serum concentration:0.5 µg/ml ratio; NQ = concentrations were below the lower limit of quantitation. higher drug concentrations. Sinusoidal capillaries offer minimal barrier and tend to have the highest concentration of drug. 22 Highly lipid-soluble (unbound) drugs also move through capillary endothelial cell membranes and basal lamina. 9 Despite the presence of general anesthesia, drug distribution of enrofloxacin in this study tends to follow the expected pattern based on vascular capillary anatomy. Concentrations of enrofloxacin in this study exceeded those reported on the package insert (for a single oral dose at 2.5 mg/kg) by at least fourfold, with the exception of bone marrow (3.5-fold increase) and urine (no increase). Tissues with the lowest concentration of enrofloxacin in this study (aqueous humor, articular capsule, and fatty tissues) are characterized by either a blood barrier (i.e., aqueous humor) or poor blood flow (cartilage and fat) and would, therefore, be expected to have the lowest drug concentration compared with plasma. Despite the presence of a blood barrier, both the brain and CSF had higher concentrations of enrofloxacin (approaching unity with plasma). The prostate and testicles, two other tissues characterized by barriers to drug penetration, had very high concentrations of drug compared with serum. These high concentrations suggest that factors other than simple passive diffusion play a role in enrofloxacin movement into and out of tissues. Organs capable of solute concentration (e.g., liver, kidney) had the highest concentrations of both enrofloxacin and the metabolite. De- Manuelle and coworkers 8 reported that enrofloxacin concentrations in skin homogenates increased following multiple oral dosing. Similar findings were reported for enrofloxacin in tissue cage fluid, 4 suggesting that higher concentrations might have been achieved in solid tissues with multiple daily dosing. Infecting bacteria located in extracellular fluid are exposed to drug that passes from plasma into the fluid. Although plasma provides a convenient, practical sampling compartment, drug concentrations in plasma have been described as having no value in determining the concentration of antibiotic at the level of infection. 9 However, measurement of drug in interstitial fluid is impractical in clinical patients and difficult in the experimental setting. A number of direct and indirect methods have been used to describe the relationship between plasma and interstitial fluid antibiotic concentrations, although each is characterized by drawbacks that preclude ideal application to clinical patients. 9,20,25 Homogenate data reported in this study have been among the most commonly 124

6 D. M. Boothe, A. Boeckh, H. W. Boothe, and S. Wilkie used methods to describe tissue drug concentrations. However, in addition to interstitial (extracelluar) fluid, homogenate data include drug located in lymph, blood, and intracellular fluids of the tissue. 22 Although animals were not exsanguinated in this study, previous studies have documented that levels of enrofloxacin present in erythrocytes are less than or equal to those in serum. 25 In this study, drug present in homogenates at concentrations that markedly exceeded plasma concentrations is likely to have been located in serum intracellular, rather than extracellular, fluid simply because the mechanisms by which a drug might accumulate are more likely to be found within cells. Passive transport alone cannot explain accumulation against a concentration gradient. However, the mechanism of accumulation of enrofloxacin in this study is not clear. Accumulation of a number of fluorinated quinolones including enrofloxacin has been documented in both circulating and tissue-phagocytic leukocytes. A contributing factor for accumulation of enrofloxacin and its metabolite in nonphagocytic cells is ion trapping within the cytoplasm of the cell. 31,32 Because both compounds are weak bases, a decrease in intracellular ph (compared with pka) should increase ionization and thus the intracellular concentration of nondiffusible drug. However, the pka of enrofloxacin is 7.7, suggesting that the amount of drug likely to be ionized and therefore trapped in the cell is not sufficient to explain the magnitude of accumulation. Active rather than passive transport has also been proposed as a mechanism for intracellular phagocytic accumulation of the fluorinated quinolones. 27,28,31,32 A number of nonphagocytic tissues in the body have the capacity for active transport of drugs, most notably organs of elimination such as the kidney and liver, which may contribute to higher drug concentrations that occur in urine and bile. Other organs and tissues capable of active transport include the central nervous system, eyes, and excretory glands such as the prostate, salivary glands, and pancreas. 9 Active transport might partially explain the higher concentrations that occurred in this study for enrofloxacin in the salivary gland and pancreas and may have contributed to movement of enrofloxacin into the central nervous system. Regardless of the mechanism of intracellular accumulation, infections caused by organisms that are intracellular may be more amenable to therapy with fluorinated quinolones. Because the fluorinated quinolones appear to remain active inside cells and are not located in subcellular organelles that preclude microbial exposure to drug, the high intracellular concentrations of enrofloxacin should facilitate antimicrobial efficacy against obligate and facultative intracellular organisms such as Staphylococcus, Salmonella, Nocardia, Chlamydia, Mycoplasma, Brucella, and atypical Mycobacterium. The clinical relevance of intracellular accumulation of enrofloxacin to infections located in extracellular tissues is less apparent. Higher intracellular concentrations may serve as a pool of diffusible drug, replenishing drug removed from extracellular tissues by hepatic or renal clearance. This could prolong the exposure of the organism to antibacterial drug. However, the clinical relevance of longer exposure of a microbe to a fluorinated quinolone is not clear. The relationship between drug concentration at the site of infection and the MIC of an infecting microbe has been described as concentration dependent for fluorinated quinolones. Based on experimental studies, the ratio of plasma drug concentration to MIC (the IQ) should be 8 or higher. 1,12,33 36 Although the clinical impact of such recommendations is not clear, they do provide basic guidelines for targeting drug concentrations. Achieving effective antimicrobial drug concentrations that is, avoiding sub- 125

7 therapeutic concentrations is critical for diminishing the development of antimicrobial resistance. 9 More recent studies suggest that the area under the curve (AUC) divided by the MIC of the infecting organism (area under the inhibitory curve [AUIC]) is an alternative and perhaps better pharmacokinetic/pharmacodynamic parameter for predicting antimicrobial efficacy Parameters based on AUC (e.g., AUIC) could not be calculated for enrofloxacin in this study because serial samples were not collected. However, based on tissue concentrations measured at 2 hours following intravenous administration of enrofloxacin at 20 mg/kg and an MIC of 0.5 µg/ml, which incorporates most organisms considered susceptible to enrofloxacin, 15 an IQ of 8 or more was generated in all tissues, with the exception of tracheal cartilage, aqueous humor, articular cartilage, fat, and tendon. This suggests that enrofloxacin, a concentration-dependent drug, can reach most tissues at concentrations that support efficacy against most susceptible bacteria. Metabolism of enrofloxacin to ciprofloxacin in dogs is often overlooked when considering the efficacy of enrofloxacin; however, the two compounds have an additive antibacterial effect. 16 Laboratory studies indicate that ciprofloxacin may comprise more than 30% of total drug concentrations in plasma and greater than 50% of the total plasma AUC. 6 Serum concentrations of ciprofloxacin represented 34% of the total drug concentrations in this study. Culture and susceptibility cannot take into account the presence of ciprofloxacin and thus tend to underestimate drug efficacy. Assessment of tissue to serum ciprofloxacin concentrations reveals some potentially disturbing considerations. The tissue:serum drug concentration was lower (sometimes much lower) for ciprofloxacin than for enrofloxacin in all tissues except urine and gall bladder. Interestingly, concentrations were not quantifiable in several tissues, including the skin. Although data in this report were generated after single-dose administration, the lack of quantifiable ciprofloxacin in many tissues and lower tissue: plasma drug concentrations ratios for tissues in which both drugs were quantifiable may indicate less tissue penetrability for ciprofloxacin than for enrofloxacin. These findings are in contrast to two reports of penetration of ciprofloxacin into inflamed tissues. 37,38 Using tissue cages, Walker and coworkers 37 found serum:tissue cage fluid ratios of 0.55 for ciprofloxacin at 8 hours (based on C max and time to C max ) following a single dose. This compares to a ratio of 0.51 for enrofloxacin at 4.8 hours. In humans, Wise and Donovan 38 found a serum:blister fluid ratio for ciprofloxacin of 0.6 at 2.6 hours. These authors also summarized other ciprofloxacin distribution studies, including a report of tissues (normal and inflamed CSF, aqueous humor, normal and infected bone, fat, and pleural fluid) with tissue:serum ratios less than 1.0. Tissues whose concentrations exceeded levels in serum included sputum, muscle, prostate, lungs, and normal and cortical bone. Several points might be considered when comparing ciprofloxacin findings in this study with previous studies. The lower limit of quantitation of the assay for ciprofloxacin in the present study was fourfold higher than in other studies (29 versus 7 ng/ml), which may have precluded detection of ciprofloxacin at lower concentrations in some tissues. The report by Walker and coworkers 37 reflects multiple sampling over a 12-hour period; serum and tissue cage concentrations reflect predicted rather than actual concentration. For both drugs, the peak concentration occurred well beyond the 2-hour sampling time in the study reported by Walker and coworkers 37 (9 hours for ciprofloxacin and less than 5 hours for enrofloxacin). Peak concentrations of both enro- 126

8 D. M. Boothe, A. Boeckh, H. W. Boothe, and S. Wilkie floxacin and ciprofloxacin in the present study may have been undetected by sampling too early. Ciprofloxacin is the deethylated metabolite of enrofloxacin. As such, it should be expected to be less lipophilic and therefore less able to penetrate cell membranes than the parent compound. Indeed, the octanyl:water partition coefficient of ciprofloxacin is approximately 100-fold less than that of enrofloxacin. 1 The marked difference in this measure of lipid solubility supports the premise that ciprofloxacin may not move into tissues as easily as enrofloxacin. 32 Regardless of the reasons for differences in tissue concentrations, this study suggests that ciprofloxacin may not distribute into tissues as efficiently as enrofloxacin. CONCLUSIONS A single intravenous dose of enrofloxacin at 20 mg/kg achieved concentrations equal to or above the IQ of 8 recommended for efficacy against most susceptible microbes in all tissues. However, absolute drug concentrations and tissue:serum concentrations were less than the current breakpoint MIC (4 µg/ml or less) for enrofloxacin in the trachea, articular cartilage, aqueous humor, tendon, fat, and subcutaneous tissue, suggesting that drug concentrations may not be sufficient for organisms with a high MIC causing infection in these tissues. Concentrations of enrofloxacin were greatest in the gall bladder, urine, bile, stomach, and liver. In contrast, ciprofloxacin was not quantifiable in many tissues; however, highest concentrations were detected in the gall bladder, urine, bile, and liver. ACKNOWLEDGMENT The author wishes to acknowledge Dr. Murl Bailey for his willingness to allow us access to animals and the collection of this data. REFERENCES 1. Papich MG: Comparison of fluoroquinolone antibacterials. Proc ACVIM 17:53 55, Stegemann M, Scheer M: Pharmacokinetics of Baytril in dogs and cats after oral and parenteral application:concentrations in serum, skin and saliva. Leverkusen, Germany, Bayer Communications, Bayer AG, Kung K, Riond JL, Wanner M: Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther 16: , Walker RD, Stein GE, Hauptman G, et al: Pharmacokinetic evaluation of enrofloxacin administered orally to healthy dogs. Am J Vet Res 53: , Monlouis JD, De Jong A, Richez P: Plasma pharmacokinetics and urine concentrations after oral administration of enrofloxacin to dogs. J Vet Pharmacol Ther 20(Suppl 1):61 62, Boothe DM, Boeckh A, Wilkie B, et al: Plasma concentrations of enrofloxacin in dogs following administration of single oral doses of enrofloxacin. Vet Ther In Press. 7. Duval JM, Budsberg SC: Cortical bone concentrations of enrofloxacin in dogs. Am J Vet Res 56: , DeManuelle TC, Ihrke PJ, Brand CM, et al: Determination of skin concentrations of enrofloxacin in dogs with pyoderma. Am J Vet Res 59: , Bergan T: Pharmacokinetics and tissue penetration of antibiotics. Rev Infect Dis 3(1):45 66, Whelton A, Stout RL:. An overview of antibiotic tissue penetration. In: Ristuccia AM, Cunha BA, eds. Antimicrobial Therapy. New York: Raven Press; 1984: Vogelman B, Gudmundsson S, Leggett J, et al: Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. J Infect Dis 158: , Nix DE, Goodwin SD, Peloquin CA, et al: Antibiotic tissue penetration and its relevance: Impact of tissue penetration on infection response. Antimicrob Agents Chemother 35: , Johnson CC: In vitro testing: Correlations of bacterial susceptibility, body fluid levels and effectiveness of antibacterial therapy. In: Lorian V, ed. Antibiotics in Laboratory Medicine. 4 th ed. Baltimore: Williams & Wilkins; 1996: Bergogne-Berezin E: Who or what is the source of an- 127

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