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 trova- Results: In vitro susceptibility study findings indicated floxacin mesylate, a newer-generation fluoroquinolone that the MIC of trovafloxacin was significantly lower than with an expanded spectrum of activity, in the treatment the MIC of ciprofloxacin and ofloxacin for S aureus, S of experimental bacterial keratitis. pneumoniae, and Haemophilus influenzae, lower than the MIC of ciprofloxacin and ofloxacin for Staphylococcus epi- Methods: Susceptibility studies were performed on vari- dermidis, and intermediate between ciprofloxacin and ous strains of ocular isolates to determine the minimum ofloxacin for P aeruginosa. Pharmacokinetic studies inhibitory concentration (MIC) of trovafloxacin com- showed a significant concentration of trovafloxacin in the pared with ciprofloxacin and ofloxacin, using the E-test treated corneas, especially in eyes with a denuded epi- method. Pharmacokinetic studies were performed by a thelium. All serum samples had undetectable trovafloxa- single topical administration of trovafloxacin to rabbit cin concentrations. Experimental keratitis studies showed eyes with either an intact or denuded corneal epithe- a statistically significant decrease of colony-forming units lium. Aqueous humor, vitreous, and corneal concentra- in trovafloxacin-treated eyes in the S aureus model and tions of trovafloxacin were determined at different time a similar decrease in the S pneumoniae and P aeruginosa points. Experimental bacterial keratitis studies were per- models. formed in rabbit eyes. Three identical studies were con- ducted using Staphylococcus aureus, Streptococcus pneu- Conclusions: Topical 0.5% trovafloxacin proved to be moniae,orPseudomonas aeruginosa. Therapy groups an effective ocular medication for the therapy of gram- included 0.5% trovafloxacin, 0.3% ciprofloxacin, 0.3% positive and gram-negative keratitis. ofloxacin, and isotonic sodium chloride solution. After 12 hours of drops administration, corneas were excised, Clinical Relevance: Trovafloxacin may provide an ex- homogenized, and serially plated. The main outcome mea- cellent therapeutic alternative in bacterial keratitis. sure was quantitative bacteriologic analysis for residual colony-forming units. Arch Ophthalmol. 2004;122:65-69

ICROBIAL KERATITIS travenous administration. Trovafloxacin continues to be a com- has broad spectrum antibacterial activity mon, potentially sight- against gram-negative, gram-positive, and threatening ocular anaerobic bacteria. In vitro and in vivo infection.1,2 Fluoroqui- studies8-13 in systemic diseases have dem- nolonesM are increasingly selected as ini- onstrated a greater activity against clini- tial, broad-spectrum agents for the topical cally important gram-positive organisms 3 From the Ocular Microbiology therapy of bacterial keratitis. The use of (most notably streptococci), while main- Laboratory, The Wilmer ofloxacin and ciprofloxacin, as single taining activity against gram-negative or- Ophthalmological Institute, agents, was shown in clinical trials to be ganisms, when compared with ciprofloxa- The Johns Hopkins University comparable to a combination of fortified an- cin and ofloxacin. School of Medicine, Baltimore, tibiotics in the treatment of acute bacte- The purpose of this study is to deter- Md (Drs Barequet, Tuli, and rial keratitis.4,5 However, these 2 quino- mine the microbiological efficacy of topi- O’Brien and Messrs Denton and lones, which currently are often used, have cal trovafloxacin for potential use in Osterhout); and Goldschleger limited in vitro activity against some gram- therapy of experimental bacterial kerati- Eye Institute, Sheba Medical positive organisms and various strains of tis. After performing susceptibility and Center, Tel Aviv University 6,7 Sackler School of Medicine, Pseudomonas aeruginosa. pharmacokinetic studies, we studied the Tel Hashomer, Israel Trovafloxacin mesylate, 7-(3- microbiological effect of topical trova- (Dr Barequet). The authors azabicyclo[3,1,0]hexyl)-naphthyridone, is floxacin on bacterial keratitis caused by have no relevant financial a newer-generation synthetic fluoroqui- either gram-positive organisms, such as interest in this article. nolone currently available for oral and in- Staphylococcus aureus and Streptococcus

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 pneumoniae, or a gram-negative organism (P aerugi- late of a methicillin-sensitive S aureus with a predetermined nosa) and compared its efficacy with that of ciprofloxa- MIC50 (inhibits 50% of strains) of 0.5 µg/mL for ciprofloxacin and ofloxacin. cin. 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 random- METHODS ized to 4 treatment groups, with 6 rabbits in each group: (1) ciprofloxacin, 3 mg/mL (Ciloxan 0.3%; Alcon Laboratories, Fort BACTERIAL STRAINS Worth, Tex); (2) ofloxacin, 3 mg/mL (Ocuflox 0.3%; Aller- gan, Irvine, Calif); (3) trovafloxacin mesylate, 5 mg/mL (Trovan- Bacterial strains used in this study were clinical ocular isolates IV; Pfizer, New York, NY); and (4) preservative-free 0.9% non- collected from corneal specimens at the Wilmer Ophthalmo- bacteriostatic isotonic sodium chloride solution for intravenous logical Institute, Baltimore, Md. use (control). Trovafloxacin eyedrops were prepared using the intravenous solution that was transferred into a dropper bottle ANTIMICROBIAL SUSCEPTIBILITY STUDIES under a sterile hood. The eyedrops were administered every hour for 12 hours. Minimum inhibitory concentrations (MICs) of trovafloxacin, One hour after the last dose of eyedrops, the rabbits were sys- ciprofloxacin, and ofloxacin were determined for various ocu- temically anesthetized and then humanely killed by intracar- lar isolates of S aureus, S pneumoniae, Staphylococcus epidermi- diac injection of pentobarbital sodium (Beuthanasia-D Spe- dis, P aeruginosa, and Haemophilus influenzae. The E-test method cial; Schering-Plough Animal Health Corp, Kenilworth, NJ). was used for determining the MIC for each strain. The bacte- The rabbits’ corneas were excised using a sterile, disposable, rial suspension was prepared by collecting the clinical isolates 7.5-mm corneal trephine and irrigated with 3 mL of phosphate- from a blood agar plate. The isolate sample was adjusted with buffered saline to eliminate any residual antibiotic or debris on 0.9% nonbacteriostatic isotonic sodium chloride solution to the surface of the corneas. The corneal buttons were immedi- achieve the same density as a 0.5 McFarland standard (1 ϫ 108 ately homogenized and then serially diluted before plating in colony-forming units/mL). The appropriate E-test strip was duplicate on blood agar for S aureus and S pneumoniae and on placed on a Mueller-Hinton II agar plate (BBL, Cockeysville, Mueller-Hinton II agar for P aeruginosa. The specimens were Md) inoculated with the suspension of bacteria. The plates were incubated at 35°C for 24 hours before quantitative bacterio- incubated for 16 to 24 hours at 37°C, and the MIC was read logic analysis. from the scale on the side of the strip at the point where the ellipse of growth inhibition intercepted the strip. All the tests RESULTS were performed in duplicate.

PHARMACOKINETIC STUDIES ANTIMICROBIAL SUSCEPTIBILITY STUDIES

The pharmacokinetic studies were performed on rabbit eyes. The MICs for the various ocular strains are given in Twelve rabbits were used for this study. Epithelial removal (to Table 1. The MIC for P aeruginosa strains was signifi- promote antibiotic entry and to simulate human ulcerative kera- cantly lower for ciprofloxacin compared with trova- titis) was evaluated. In each rabbit, one eye underwent re- floxacin (P=.008) and ofloxacin (P=.007) and signifi- moval of the central (7.5 mm) corneal epithelium, and in the cantly lower for trovafloxacin compared with ofloxacin fellow eye the epithelium remained intact. A single drop of trova- (P=.007). floxacin was applied to both eyes. Rabbits were humanely killed The MIC for S aureus strains was significantly lower at 15, 30, 60, 120, and 240 minutes (at each time point, 3 rab- Ͻ bits were euthanized), and samples were obtained immedi- for trovafloxacin compared with ciprofloxacin (P .001) ately thereafter from aqueous humor, vitreous, and the central and ofloxacin (PϽ.001), and no significant difference was cornea (7.5 mm). The tissue concentration of trovafloxacin was found between the MIC for ciprofloxacin and ofloxacin determined using a high-performance liquid chromatography (P=.10). The MIC for H influenzae strains was signifi- assay.14 cantly lower for trovafloxacin (P=.005) and ciprofloxacin (P=.005) compared with ofloxacin. No significant dif- BACTERIAL KERATITIS STUDIES ference was found between trovafloxacin and ciprofloxacin (P=.44). The MIC for S epidermidis was lower for trova- In these studies, 72 (24 in each experiment) New Zealand white floxacin than ciprofloxacin; however, this was not sta- rabbits, weighing 2.0 to 2.25 kg, were used in accordance with the guidelines for animal experimentation established by the tistically conclusive (P=.07). The MIC for S pneumonia Association of Research in Vision and Ophthalmology (Rock- strains was significantly lower for trovafloxacin (P=.006) ville, Md); approval from the appropriate institutional review and ciprofloxacin (P=.006) compared with ofloxacin. board was obtained for the study, and the institutional guidelines regarding animal experimentation were followed. All rab- PHARMACOKINETIC STUDIES bits were sedated and anesthetized with intramuscular injection of ketamine hydrochloride (60 mg/kg) and xylazine After the single trovafloxacin drop application, a sub- hydrochloride (12 mg/kg) and topical 0.5% proparacaine hy- stantial penetration was found in the corneas at all time drochloride before having only one eye of each animal in- points (Table 2). Mean (SD) trovafloxacin levels were jected with bacteria. higher in eyes with denuded epithelium (25.9 [22.1] µg/g) To produce keratitis, 103 colony-forming units (0.1 mL) of clinical bacterial isolates in logarithmic growth phase were compared with intact epithelium (3.7 [2.2] µg/g). The injected into the corneal stroma in one eye. The injection was aqueous levels were higher in eyes with denuded epithe- performed with a 30-gauge needle into the stroma in the cen- lium (2.5 [1.4] µg/mL) compared with intact epithe- tral part of the cornea under microscopic guidance. The first lium (0.3 [0.1] µg/mL). The vitreous concentrations af- experiment was performed with 24 rabbits, using a clinical iso- ter a single topical application were low in most of the

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 eyes. All serum samples for all the time points had undetectable trovafloxacin concentrations. Table 1. Comparative In Vitro Activity of Trovafloxacin Against Ocular Strains of Various Microorganisms BACTERIAL KERATITIS STUDIES MIC, µg/mL

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

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 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 Ͻ0.1 0.1 Ͻ0.1 30 4.6 35.2 0.4 4.2 Ͻ0.1 0.1 Ͻ0.1 60 1.5 5.2 0.25 1.5 0.1 0.1 Ͻ0.1 120 2.3 10.7 0.3 1.1 0.1 0.2 Ͻ0.1 240 Ͻ0.1 Ͻ0.1 Ͻ0.1 0.1 Ͻ0.1 Mean (SD) 3.7 (2.2) 25.9 (22.1) 0.3 (0.1) 2.5 (1.4) Ͻ0.1 0.1 (0.1) Ͻ0.1

*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 activ- tremely low, if detectable at all; moreover, the restricted ity in vivo as a result of undergoing rapid hydrolysis systemic use may offer an advantage with the topical use and is rapidly converted to trovafloxacin after intrave- due to a lesser chance of emergence of resistance. In con- nous administration. We used the intravenous formu- clusion, the high in vitro efficacy against ocular isolates lation for preparation of the eyedrops, and this dem- along with the high ocular tissue penetration and non- onstrated a good antibacterial activity in topical use detectable systemic levels and in vivo efficacy indicate for the keratitis model. that trovafloxacin may have an important clinical role in In vitro activity of trovafloxacin has been shown to the topical treatment of bacterial keratitis, including strep- be superior to both ciprofloxacin and ofloxacin against tococcal species. both the penicillin-susceptible and penicillin-resistant pneumococci.23,24 In our study the in vitro antibacterial Submitted for publication August 1, 2003; final revision re- activity of trovafloxacin against ocular isolates has been ceived August 12, 2003; accepted August 21, 2003. demonstrated by using the E-test, a modification of a disc Corresponding author and reprints: Terrence P. O’Brien, diffusion susceptibility testing.25 MD, Ocular Microbiology Laboratory, The Wilmer Oph- Pascual et al26 showed that trovafloxacin pen- thalmological Institute, Woods 255, 600 N Wolfe St, Bal- etrates into phagocytic and nonphagocytic cells, reach- timore, MD 21287-9121 (e-mail: [email protected]). ing intracellular concentrations several times greater than the extracellular ones, whereas it remained active intra- REFERENCES cellularly in human polymorphonuclear leukocytes; this fact may enhance the in vivo antibacterial activity. Lately, the systemic use of trovafloxacin was restricted to life- 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. or limb-threatening conditions due to several reports of Eye. 2002;16:608-618. 27,28 hepatic toxicity after systemic use of the drug. With 2. Scott IU, Flynn HW Jr, Feuer W, et al. Endophthalmitis associated with microbial topical ocular use, the expected systemic levels are ex- keratitis. Ophthalmology. 1996;103:1864-1870.

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