LABORATORY SCIENCES. Treatment of Experimental Bacterial Keratitis With Topical Trovafloxacin

LABORATORY SCIENCES Treatment of Experimental Bacterial Keratitis With Topical Trovafloxacin Irina S. Barequet, MD; Paul Denton, BS; Gerard J. Osterhout, MS; Suhas Tuli, MD; Terrence P. O Brien, MD Objective: To investigate the therapeutic role of trovafloxacin mesylate, a newer-generation fluoroquinolone with an expanded spectrum of activity, in the treatment of experimental bacterial keratitis. Methods: Susceptibility studies were performed on various strains of ocular isolates to determine the minimum inhibitory concentration (MIC) of trovafloxacin compared with ciprofloxacin and ofloxacin, using the E-test method. Pharmacokinetic studies were performed by a single topical administration of trovafloxacin to rabbit eyes with either an intact or denuded corneal epithelium. Aqueous humor, vitreous, and corneal concentrations of trovafloxacin were determined at different time points. Experimental bacterial keratitis studies were performed in rabbit eyes. Three identical studies were conducted using Staphylococcus aureus, Streptococcus pneumoniae, orpseudomonas aeruginosa. Therapy groups included 0.5% trovafloxacin, 0.3% ciprofloxacin, 0.3% ofloxacin, and isotonic sodium chloride solution. After 12 hours of drops administration, corneas were excised, homogenized, and serially plated. The main outcome measure was quantitative bacteriologic analysis for residual colony-forming units. Results: In vitro susceptibility study findings indicated that the MIC of trovafloxacin was significantly lower than the MIC of ciprofloxacin and ofloxacin for S aureus, S pneumoniae, and Haemophilus influenzae, lower than the MIC of ciprofloxacin and ofloxacin for Staphylococcus epidermidis, and intermediate between ciprofloxacin and ofloxacin for P aeruginosa. Pharmacokinetic studies showed a significant concentration of trovafloxacin in the treated corneas, especially in eyes with a denuded epithelium. All serum samples had undetectable trovafloxacin concentrations. Experimental keratitis studies showed a statistically significant decrease of colony-forming units in trovafloxacin-treated eyes in the S aureus model and a similar decrease in the S pneumoniae and P aeruginosa models. Conclusions: Topical 0.5% trovafloxacin proved to be an effective ocular medication for the therapy of grampositive and gram-negative keratitis. Clinical Relevance: Trovafloxacin may provide an excellent therapeutic alternative in bacterial keratitis. Arch Ophthalmol. 2004;122:65-69 From the Ocular Microbiology Laboratory, The Wilmer Ophthalmological Institute, The Johns Hopkins University School of Medicine, Baltimore, Md (Drs Barequet, Tuli, and O Brien and Messrs Denton and Osterhout); and Goldschleger Eye Institute, Sheba Medical Center, Tel Aviv University Sackler School of Medicine, Tel Hashomer, Israel (Dr Barequet). The authors have no relevant financial interest in this article. MICROBIAL KERATITIS continues to be a common, potentially sightthreatening ocular infection. 1,2 Fluoroquinolones are increasingly selected as initial, broad-spectrum agents for the topical therapy of bacterial keratitis. 3 The use of ofloxacin and ciprofloxacin, as single agents, was shown in clinical trials to be comparable to a combination of fortified antibiotics in the treatment of acute bacterial keratitis. 4,5 However, these 2 quinolones, which currently are often used, have limited in vitro activity against some grampositive organisms and various strains of Pseudomonas aeruginosa. 6,7 Trovafloxacin mesylate, 7-(3- azabicyclo[3,1,0]hexyl)-naphthyridone, is a newer-generation synthetic fluoroquinolone currently available for oral and intravenous administration. Trovafloxacin has broad spectrum antibacterial activity against gram-negative, gram-positive, and anaerobic bacteria. In vitro and in vivo studies 8-13 in systemic diseases have demonstrated a greater activity against clinically important gram-positive organisms (most notably streptococci), while maintaining activity against gram-negative organisms, when compared with ciprofloxacin and ofloxacin. The purpose of this study is to determine the microbiological efficacy of topical trovafloxacin for potential use in therapy of experimental bacterial keratitis. After performing susceptibility and pharmacokinetic studies, we studied the microbiological effect of topical trovafloxacin on bacterial keratitis caused by either gram-positive organisms, such as Staphylococcus aureus and Streptococcus 65

pneumoniae, or a gram-negative organism (P aeruginosa) and compared its efficacy with that of ciprofloxacin and ofloxacin. METHODS BACTERIAL STRAINS Bacterial strains used in this study were clinical ocular isolates collected from corneal specimens at the Wilmer Ophthalmological Institute, Baltimore, Md. ANTIMICROBIAL SUSCEPTIBILITY STUDIES Minimum inhibitory concentrations (MICs) of trovafloxacin, ciprofloxacin, and ofloxacin were determined for various ocular isolates of S aureus, S pneumoniae, Staphylococcus epidermidis, P aeruginosa, and Haemophilus influenzae. The E-test method was used for determining the MIC for each strain. The bacterial suspension was prepared by collecting the clinical isolates from a blood agar plate. The isolate sample was adjusted with 0.9% nonbacteriostatic isotonic sodium chloride solution to achieve the same density as a 0.5 McFarland standard (1 10 8 colony-forming units/ml). The appropriate E-test strip was placed on a Mueller-Hinton II agar plate (BBL, Cockeysville, Md) inoculated with the suspension of bacteria. The plates were incubated for 16 to 24 hours at 37 C, and the MIC was read from the scale on the side of the strip at the point where the ellipse of growth inhibition intercepted the strip. All the tests were performed in duplicate. PHARMACOKINETIC STUDIES The pharmacokinetic studies were performed on rabbit eyes. Twelve rabbits were used for this study. Epithelial removal (to promote antibiotic entry and to simulate human ulcerative keratitis) was evaluated. In each rabbit, one eye underwent removal of the central (7.5 mm) corneal epithelium, and in the fellow eye the epithelium remained intact. A single drop of trovafloxacin was applied to both eyes. Rabbits were humanely killed at 15, 30, 60, 120, and 240 minutes (at each time point, 3 rabbits were euthanized), and samples were obtained immediately thereafter from aqueous humor, vitreous, and the central cornea (7.5 mm). The tissue concentration of trovafloxacin was determined using a high-performance liquid chromatography assay. 14 BACTERIAL KERATITIS STUDIES In these studies, 72 (24 in each experiment) New Zealand white rabbits, weighing 2.0 to 2.25 kg, were used in accordance with the guidelines for animal experimentation established by the Association of Research in Vision and Ophthalmology (Rockville, Md); approval from the appropriate institutional review board was obtained for the study, and the institutional guidelines regarding animal experimentation were followed. All rabbits were sedated and anesthetized with intramuscular injection of ketamine hydrochloride (60 mg/kg) and xylazine hydrochloride (12 mg/kg) and topical 0.5% proparacaine hydrochloride before having only one eye of each animal injected with bacteria. To produce keratitis, 10 3 colony-forming units (0.1 ml) of clinical bacterial isolates in logarithmic growth phase were injected into the corneal stroma in one eye. The injection was performed with a 30-gauge needle into the stroma in the central part of the cornea under microscopic guidance. The first experiment was performed with 24 rabbits, using a clinical isolate of a methicillin-sensitive S aureus with a predetermined MIC50 (inhibits 50% of strains) of 0.5 µg/ml for ciprofloxacin. The other 2 experiments were performed in an identical fashion by inoculating either S pneumoniae or P aeruginosa. Twelve hours after the injection, the rabbits were randomized to 4 treatment groups, with 6 rabbits in each group: (1) ciprofloxacin, 3 mg/ml (Ciloxan 0.3%; Alcon Laboratories, Fort Worth, Tex); (2) ofloxacin, 3 mg/ml (Ocuflox 0.3%; Allergan, Irvine, Calif); (3) trovafloxacin mesylate, 5 mg/ml (Trovan- IV; Pfizer, New York, NY); and (4) preservative-free 0.9% nonbacteriostatic isotonic sodium chloride solution for intravenous use (control). Trovafloxacin eyedrops were prepared using the intravenous solution that was transferred into a dropper bottle under a sterile hood. The eyedrops were administered every hour for 12 hours. One hour after the last dose of eyedrops, the rabbits were systemically anesthetized and then humanely killed by intracardiac injection of pentobarbital sodium (Beuthanasia-D Special; Schering-Plough Animal Health Corp, Kenilworth, NJ). The rabbits corneas were excised using a sterile, disposable, 7.5-mm corneal trephine and irrigated with 3 ml of phosphatebuffered saline to eliminate any residual antibiotic or debris on the surface of the corneas. The corneal buttons were immediately homogenized and then serially diluted before plating in duplicate on blood agar for S aureus and S pneumoniae and on Mueller-Hinton II agar for P aeruginosa. The specimens were incubated at 35 C for 24 hours before quantitative bacteriologic analysis. RESULTS ANTIMICROBIAL SUSCEPTIBILITY STUDIES The MICs for the various ocular strains are given in Table 1. The MIC for P aeruginosa strains was significantly lower for ciprofloxacin compared with trovafloxacin (P=.008) and ofloxacin (P=.007) and significantly lower for trovafloxacin compared with ofloxacin (P=.007). The MIC for S aureus strains was significantly lower for trovafloxacin compared with ciprofloxacin (P.001) and ofloxacin (P.001), and no significant difference was found between the MIC for ciprofloxacin and ofloxacin (P=.10). The MIC for H influenzae strains was significantly lower for trovafloxacin (P=.005) and ciprofloxacin (P=.005) compared with ofloxacin. No significant difference was found between trovafloxacin and ciprofloxacin (P=.44). The MIC for S epidermidis was lower for trovafloxacin than ciprofloxacin; however, this was not statistically conclusive (P=.07). The MIC for S pneumonia strains was significantly lower for trovafloxacin (P=.006) and ciprofloxacin (P=.006) compared with ofloxacin. PHARMACOKINETIC STUDIES After the single trovafloxacin drop application, a substantial penetration was found in the corneas at all time points (Table 2). Mean (SD) trovafloxacin levels were higher in eyes with denuded epithelium (25.9 [22.1] µg/g) compared with intact epithelium (3.7 [2.2] µg/g). The aqueous levels were higher in eyes with denuded epithelium (2.5 [1.4] µg/ml) compared with intact epithelium (0.3 [0.1] µg/ml). The vitreous concentrations after a single topical application were low in most of the 66

eyes. All serum samples for all the time points had undetectable trovafloxacin concentrations. BACTERIAL KERATITIS STUDIES The bacterial counts for the 3 studies are given in Table 3. The S aureus keratitis study showed a statistically significant decrease in bacterial counts in the groups treated with trovafloxacin compared with the ciprofloxacin and ofloxacin treatment groups (P=.002). The S pneumoniae study showed a significant decrease in bacterial counts in the antibiotic-treated groups compared with the controls (P=.002) and a significant decrease in bacterial counts in the trovafloxacin groups compared with ciprofloxacin but not ofloxacin. The P aeruginosa study showed a significant decrease in all antibiotic-treated groups compared with the control group (P.001). COMMENT Until recently, the fluoroquinolones were primarily agents with excellent activity against gram-negative bacilli but with questionable activity against some important grampositive pathogens. Newer agents under development have an expanded profile of antibacterial activity against grampositive pathogens, including staphylococci, streptococci, enterococci, and anaerobic isolates. The fluoroquinolone evolutionary tree has 2 main branches. The first branch is the naphthyridine series, which have a nitrogen atom at the C-8 position (eg, nalidixic acid, enoxacin, tosufloxacin, trovafloxacin); all others are fluoroquinolone agents, with the C-7 position being the most adaptable site for chemical substitution. A major clinical challenge has been the worldwide emergence and spread of antimicrobial resistance, especially among gram-positive isolates, such as pneumococci. There is recent information on the mechanism of action of fluoroquinolone and how resistance develops. From this, it seems that the most efficient use of these fluoroquinolone compounds is to use a highly active agent with a favorable pharmacodynamic profile for the shortest treatment duration consistent with cure. Trovafloxacin is a newer-generation, expandedspectrum fluoroquinolone, which was shown in our study to have in vitro potency, favorable pharmacokinetics, and in vivo efficacy, suggesting a potential role in the therapy of bacterial keratitis. The use of topical fluorinated quinolones has become a frequent, initial, single-agent therapy for bacterial keratitis. 4,5 The mechanism of action of the quinolones is via inhibition of the type II topoisomerase DNA gyrase, an essential bacterial enzyme that alters the topology of double-stranded DNA within the cell. Inhibition of this activity by fluoroquinolones is associated with rapid killing of the bacterial cell. 15 The important advantages in the systemic use of trovafloxacin are the pharmacokinetic properties and its expanded antibacterial spectrum, especially against grampositive organisms, including Streptococcus species. Our results showed that trovafloxacin not only is effective against P aeruginosa, similar to other experimental keratitis studies, 16,17 but also is superior as far as activity against gram-positive bacteria. 18-22 Table 1. Comparative In Vitro Activity of Trovafloxacin Against Ocular Strains of Various Microorganisms MIC, µg/ml Trovafloxacin Bacteria and Strain Mesylate Ciprofloxacin Ofloxacin Pseudomonas aeruginosa OJ3143 92 0.38 0.094 0.75 4J4569 84 1.0 0.125 1.5 SN8756 0.5 0.094 0.75 3P3786 98 0.75 0.125 1.0 6N8196 1.0 0.094 1.5 3J465 0.5 0.094 0.75 25877 49 0.75 0.125 1.0 OJ3711 59 1.0 0.125 1.5 4J3619 75 0.75 0.094 1.5 Mean MIC 0.737 0.108 1.139 Staphylococcus aureus 4N1718 152 0.032 0.25 0.38 4J4569 119 0.064 0.5 1.0 OJ3692 106 0.047 0.38 0.5 4J3552 105 0.032 0.38 0.5 3P3626 120 0.047 0.5 0.5 ON1740 151 0.032 0.25 0.38 OJ3711 110 0.047 0.5 1.0 7E800 116 0.125 1.5 1.0 4J443 118 0.064 0.75 0.5 OP3555 117 0.032 0.38 0.5 4N1718 152 0.19 0.38 0.047 Mean MIC 0.065 0.525 0.573 Streptococcus pneumoniae 14 0.125 0.75 2.0 56 0.064 0.38 0.75 31 0.125 0.75 2.0 1 0.125 1.0 2.0 142 0.19 1.0 2.0 813 0.125 0.75 2.0 806 B28 0.094 0.75 1.5 797 0.19 1.0 3.0 2 0.125 0.75 1.5 49619 0.094 0.75 1.5 Mean MIC 0.129 0.792 1.861 Haemophilus influenzae 8570 0.008 0.023 0.047 16 0.032 0.016 0.047 894 0.012 0.023 0.032 14 0.012 0.016 0.047 719 0.047 0.032 0.094 8909 0.047 0.023 0.064 8932 0.032 0.032 0.064 8342 0.016 0.023 0.047 938 0.016 0.023 0.064 952 0.047 0.032 0.064 Mean MIC 0.027 0.024 0.057 Staphylococcus epidermidis 126 2 16 32 111 0.094 0.19 0.5 114 0.064 0.19 0.5 112 0.094 0.125 0.5 125 32 32 32 Mean MIC 6.850 9.70 13.1 Abbreviation: MIC, minimum inhibitory concentration. The current clinical use of trovafloxacin so far has been systemic, intravenous, intramuscular, or oral. The intravenous formulation of trovafloxacin is the prodrug alatrofloxacin mesylate, which has little intrinsic anti- 67

Table 2. Tissue Concentrations of Trovafloxacin Mesylate at Different Time Points After Application of a Single Drop of 0.5% Trovafloxacin* Corneal Button, µg/g Aqueous, µg/ml Vitreous, µg/ml Time Point, min Intact Denuded Intact Denuded Intact Denuded Serum, µg/ml 15 6.3 52.8 0.3 3.05 0.1 0.1 0.1 30 4.6 35.2 0.4 4.2 0.1 0.1 0.1 60 1.5 5.2 0.25 1.5 0.1 0.1 0.1 120 2.3 10.7 0.3 1.1 0.1 0.2 0.1 240 0.1 0.1 0.1 0.1 0.1 Mean (SD) 3.7 (2.2) 25.9 (22.1) 0.3 (0.1) 2.5 (1.4) 0.1 0.1 (0.1) 0.1 *Intact and denuded refer to the corneal epithelium. Table 3. Bacterial Counts for Each Antibiotic Treatment Group* Bacterial Count, CFU 10 4 /L Bacteria Trovafloxacin Mesylate Ciprofloxacin Ofloxacin Control Staphylococcus aureus Mean (SD) 1.45 (2.11) 51.15 (40.95) 102.35 (90.72) 859.58 (655.01) Median 0.57 47.0 92.5 622.5 25% Quartile 0.13 21.95 40.75 543.5 75% Quartile 1.67 64.25 141.25 1282.5 Streptococcus pneumoniae Mean (SD) 1.64 (3.56) 24.83 (57.4) 1.57 (1.26) 265.23 (333.79) Median 0.18 1.92 1.34 18.15 25% Quartile 0.01 0.87 1.29 106.37 75% Quartile 0.52 2.38 1.46 198.88 Pseudomonas aeruginosa Mean (SD) 0 0 0 181.17 (137.17) Median 0 0 0 132.5 25% Quartile 0 0 0 93.25 75% Quartile 0 0 0 246.75 Abbreviation: CFU, colony-forming units. *Corneas were excised 25 hours after the induction of keratitis (1 hour after the completion of the 12-hour therapy administration). bacterial activity in vitro but demonstrates good activity in vivo as a result of undergoing rapid hydrolysis and is rapidly converted to trovafloxacin after intravenous administration. We used the intravenous formulation for preparation of the eyedrops, and this demonstrated a good antibacterial activity in topical use for the keratitis model. In vitro activity of trovafloxacin has been shown to be superior to both ciprofloxacin and ofloxacin against both the penicillin-susceptible and penicillin-resistant pneumococci. 23,24 In our study the in vitro antibacterial activity of trovafloxacin against ocular isolates has been demonstrated by using the E-test, a modification of a disc diffusion susceptibility testing. 25 Pascual et al 26 showed that trovafloxacin penetrates into phagocytic and nonphagocytic cells, reaching intracellular concentrations several times greater than the extracellular ones, whereas it remained active intracellularly in human polymorphonuclear leukocytes; this fact may enhance the in vivo antibacterial activity. Lately, the systemic use of trovafloxacin was restricted to lifeor limb-threatening conditions due to several reports of hepatic toxicity after systemic use of the drug. 27,28 With topical ocular use, the expected systemic levels are extremely low, if detectable at all; moreover, the restricted systemic use may offer an advantage with the topical use due to a lesser chance of emergence of resistance. In conclusion, the high in vitro efficacy against ocular isolates along with the high ocular tissue penetration and nondetectable systemic levels and in vivo efficacy indicate that trovafloxacin may have an important clinical role in the topical treatment of bacterial keratitis, including streptococcal species. Submitted for publication August 1, 2003; final revision received August 12, 2003; accepted August 21, 2003. Corresponding author and reprints: Terrence P. O Brien, MD, Ocular Microbiology Laboratory, The Wilmer Ophthalmological Institute, Woods 255, 600 N Wolfe St, Baltimore, MD 21287-9121 (e-mail: tobrien@jhmi.edu). REFERENCES 1. Lam DS, Houang E, Fan DS, Lyon D, Seal D, Wong E. Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America. Eye. 2002;16:608-618. 2. Scott IU, Flynn HW Jr, Feuer W, et al. Endophthalmitis associated with microbial keratitis. Ophthalmology. 1996;103:1864-1870. 68

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