Purpose
To evaluate the ocular pharmacokinetics of azithromycin and moxifloxacin in human conjunctiva and aqueous humor in subjects undergoing cataract surgery.
Design
Multicenter, open-label, randomized study.
Methods
Subjects scheduled for routine cataract surgery and with normal-appearing conjunctiva were eligible. One conjunctival biopsy sample and 1 aqueous humor sample were obtained from subjects randomly assigned to 1 of 10 prespecified time points (1 to 312 hours) after treatment initiation of azithromycin ophthalmic solution 1% or moxifloxacin ophthalmic solution 0.5%. Samples were assayed using liquid chromatography tandem mass spectrometry.
Results
Azithromycin 1% provided high concentrations (peak level, 559.7 μg/g) in human conjunctiva that were sustained at levels 1 to 2 orders of magnitude higher than those of moxifloxacin 0.5% throughout the 7-day dosing period and for at least 7 days thereafter. Azithromycin also showed an extended half-life (65.7 hours) in conjunctiva relative to that of moxifloxacin (28.6 hours). Accordingly, the concentration of azithromycin was maintained well above the minimum inhibitory concentration required for inhibition of growth of 90% of tested bacterial isolates for at least 7 days, whereas moxifloxacin conjunctival levels fell to levels at or less than the minimum inhibitory concentration required for inhibition of growth of 90% of tested bacterial isolates approximately 24 hours after the last dose. Peak aqueous humor concentration of moxifloxacin was higher (0.77 μg/mL) than that of azithromycin (0.053 μg/mL). No clinically relevant safety findings were observed.
Conclusions
Azithromycin 1% demonstrated high, therapeutic levels in the conjunctiva that were maintained up to 7 days after completion of a 1-week dosing regimen. Aqueous humor levels, however, were subtherapeutic with this dosing regimen. In comparison, moxifloxacin achieved lower conjunctival tissue levels, but higher aqueous humor levels.
Azithromycin is a broad-spectrum macrolide antibiotic of the azalide subclass that has good antimicrobial activity against gram-positive cocci (including staphylococci and streptococci), moderate activity against Haemophilus species, and variable activity against Enterobacteriaceae . The mechanism of azithromycin’s antibacterial activity is the inhibition of protein synthesis by reversible binding to the 50S subunit of the bacterial ribosome. Azithromycin ophthalmic solution 1% (AzaSite; Inspire Pharmaceuticals, Inc, Durham, North Carolina, USA) is approved by the United States Food and Drug Administration for the treatment of bacterial conjunctivitis caused by susceptible isolates, including Centers for Disease Control and Prevention group G coryneform bacteria, Haemophilus influenzae , Staphylococcus aureus , mitis group streptococci, and Streptococcus pneumoniae . Azithromycin ophthalmic solution is formulated for topical dosing with an aqueous, polymer-based ocular drug delivery vehicle (DuraSite; InSite Vision, Inc, Alameda, California, USA) that has been shown to increase azithromycin delivery to ocular tissues.
The pharmacokinetic properties of azithromycin in human ocular tissues and fluids after topical administration of azithromycin 1% have not been well characterized. This open-label study was designed to evaluate the aqueous humor and conjunctival pharmacokinetic parameters of topically applied azithromycin ophthalmic solution 1%, both during and after a 7-day dosing regimen that corresponds to the labeled dosing regimen for azithromycin 1% for the treatment of bacterial conjunctivitis. For comparison purposes, the pharmacokinetic characteristics of moxifloxacin HCl ophthalmic solution 0.5% (Vigamox; Alcon Laboratories, Forth Worth, Texas, USA) were studied in a parallel cohort, both during and after a 7-day dosing regimen that represented the labeled dosing regimen for moxifloxacin 0.5% for the treatment of bacterial conjunctivitis. Moxifloxacin was selected as representative of the fluoroquinolone class of antimicrobials, which is in widespread use for the treatment and prophylaxis of ophthalmic infections. Moxifloxacin is an 8-methoxyfluoroquinolone with enhanced activity against gram-positive cocci in particular.
Methods
Subjects
Subjects were enrolled at 11 clinical centers in the United States between December 2007 and April 2008. Eligible subjects were male or female, 21 years or older, who were scheduled for routine cataract surgery using the investigator’s preferred technique. The subject’s operative eye had to have normal-appearing, freely mobile conjunctiva in the inferior temporal bulbar portion of the conjunctival cul de sac. Exclusion criteria included concurrent infectious or noninfectious conjunctivitis, keratitis, or uveitis in either eye. Subjects also were excluded if cataract surgery was planned in the contralateral eye within 2 weeks of the study-related surgical procedure or sample collection time point, or if cataract surgery was combined with another procedure (e.g., combined trabeculectomy and cataract extraction).
Subjects were randomized in a 1:1 ratio to receive azithromycin ophthalmic solution or moxifloxacin HCl ophthalmic solution and were randomized further to 1 of 10 aqueous humor and conjunctiva biopsy sample collection time points ( Table 1 ). Conjunctival biopsy and aqueous humor samples were obtained at the start of cataract surgery from subjects at time points that varied from 1 to 14 days (1 to 312 hours) after the onset of a 7-day dosing regimen of azithromycin or moxifloxacin ophthalmic solution, administered in accordance with each antibiotic’s respective labeled dosing regimen for the treatment of bacterial conjunctivitis. These sampling time points were selected to enable evaluation of the earliest feasibly measured peak level after instillation, the trough concentrations, and the elimination profile after completion of a 7-day treatment that corresponded to the respective per-label dosing regimen for each of the 2 antimicrobials.
| Azithromycin 1% ophthalmic solution sampling time points a |
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| Moxifloxacin HCl 0.5% ophthalmic solution sampling time points a |
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a The sample collection time point to which the subject was assigned determined how many doses of study drug the subject received. Samples were collected from subjects at various timepoints during dosing (days 1 to 7) and after completion of dosing (days 7 to 14). Human conjunctiva was collected by biopsy.
Materials
Azithromycin ophthalmic solution was obtained from commercial samples and supplied as a sterile aqueous ophthalmic formulation of 1% azithromycin. Moxifloxacin HCl ophthalmic solution (Alcon Laboratories, Forth Worth, Texas, USA) also was provided from commercial supplies as a sterile ophthalmic formulation of 0.5% moxifloxacin. This was an open-label study.
Treatment Methods
Depending on the sampling time point assigned to a given subject, the treatment regimen for that given a subject ranged from a single dose to a full 7-day regimen for treatment of bacterial conjunctivitis, as described in the package insert for each drug. For azithromycin 1% ophthalmic solution, the regimen consisted of 1 drop twice daily for 2 days and 1 drop once daily for the next 5 days. The regimen for moxifloxacin HCl ophthalmic solution was 1 drop 3 times daily for 7 days. The dosing regimen for each subject was determined by the assigned treatment group (azithromycin vs moxifloxacin) and assigned sample collection time point (0 to 312 hours; Table 1 ). Each subject was sampled only once, and assignments of treatment group and sampling interval were performed at random. Study drug was instilled only in the study eye (operative eye).
The study consisted of 5 clinic visits for all subjects: visit 1 (screening and randomization), visit 2 (in-clinic dosing), visit 3 (aqueous humor and conjunctiva biopsy sample and surgery), and visits 4 and 5 (follow-up and postoperative examinations). Conjunctival biopsy samples (2 to 3 mg of tissue) were obtained before the start of the cataract surgical procedure, immediately after the surgeon’s routine presurgical procedures, which included draping and periocular application of povidone–iodine. The aqueous humor sample (aspiration, 20 μL) was collected after conjunctiva collection and immediately before the cataract surgical procedure itself using a 1-mL tuberculin syringe or acceptable alternative syringe that had a sampling accuracy of at least 0.01 mL (10 μL).
Aqueous humor and conjunctiva concentrations of azithromycin and moxifloxacin were determined using qualified liquid chromatography tandem mass spectrometry. The lower limit of quantitation for each compound in conjunctiva was 1 ng of drug per gram of tissue (1 ng/g), whereas the aqueous humor lower limit of quantitation was 1 ng/mL of humor. Recovery of drug from matrix and stability under collection and test conditions also was assessed to ensure robustness and accuracy of quantitative data. Slit-lamp biomicroscopy of the study eye (operative eye) and the fellow eye (nonstudy) was performed at the screening and follow-up postoperative examinations.
Statistical Methods
The pharmacokinetic parameters of highest concentration, area under the curve, and half-life were derived using noncompartmental methods with WinNonlin Professional version 5. 2 (Pharsight Corp, Mountain View, California, USA) for both azithromycin and moxifloxacin. The pharmacokinetic model for extravascular administration (model 200) was used with a sparse sampling approach. The standard error of the mean concentration also was calculated for each nominal time point.
The final analysis was conducted on a subset of subjects from the pharmacokinetic population who met the study requirements for adequacy of sampling and adherence to the assigned sampling time point defined as the per protocol pharmacokinetic population ( Table 2 ). Data from male and female subjects were combined for the analysis because there were insufficient numbers for a gender comparison to be performed. The number of subjects analyzed for each drug was 3 to 6 per time point. Conjunctival and aqueous humor samples were obtained ±15 minutes from the scheduled time point.
| Population (All Randomized Subjects) | Azithromycin 1% (n = 64) | Moxifloxacin 0.5% (n = 63) | Total (n = 127) |
|---|---|---|---|
| Safety a | 58 (91%) | 58 (92%) | 116 (91%) |
| Pharmacokinetic (conjunctiva) b | 57 (89%) | 54 (86%) | 111 (87%) |
| Pharmacokinetic (aqueous humor) c | 56 (88%) | 56 (89%) | 112 (88%) |
| Per protocol pharmacokinetic (conjunctiva) d | 47 (73%) | 46 (73%) | 93 (73%) |
| Per protocol pharmacokinetic (aqueous humor) e | 55 (86%) | 53 (84%) | 108 (85%) |
a All subjects who received at least 1 dose of study drug.
b Subjects treated with study drug who provided concentration data for conjunctiva.
c Subjects treated with study drug who provided concentration data for aqueous humor.
d Subset of the pharmacokinetic population for conjunctiva that included those subjects who achieved reasonable compliance with the study protocol and had no major protocol deviations.
e Subset of the pharmacokinetic population for aqueous humor that included those subjects who achieved reasonable compliance with the study protocol and had no major protocol deviations.
It was assumed that the drug concentration for all subjects at the 0 time point was nil (0). Concentrations less than the limit of quantitation were handled as 0 in the analysis. Concentrations more than the limit of quantitation were increased by 1 at the last level of precision. Based on the individual concentration data, using the nominal sampling times, the pharmacokinetic parameters of azithromycin or moxifloxacin were determined from the bioanalytic results when concentrations were measurable. Analyses included computing pharmacokinetic parameters for both azithromycin and moxifloxacin using a sparse data analysis approach.
Estimates and confidence intervals for the minimum inhibitory concentration (MIC) required for inhibition of growth of 50% (MIC 50 ) and 90% (MIC 90 ) of tested bacterial isolates were calculated for azithromycin and moxifloxacin using aggregate values from 496 ocular bacterial isolates at visit 1 from a previously reported study. Two-sided nonparametric 95% confidence intervals were calculated for the percentiles (fiftieth and ninetieth) of either drug using methods detailed in Hahn and Meeker.
Tolerability was monitored by noting the incidence of adverse events and the results of the slit-lamp biomicroscopy. Because of the small sample size, statistical comparisons were not performed for any of the tolerability analyses.
Results
Subject Demographics
Of the 127 subjects undergoing routine cataract surgery and randomized to open-label study drug, 64 were to receive azithromycin ophthalmic solution and 63 were to receive moxifloxacin HCl ophthalmic solution. Fifty-eight subjects in each arm received study drug (116 of subjects = safety population). The per protocol pharmacokinetic population included 47 and 46 azithromycin and moxifloxacin subjects, respectively, from the conjunctiva pharmacokinetic population and included 55 and 53 azithromycin and moxifloxacin subjects, respectively, from the aqueous humor pharmacokinetic Population. Demographics and treatment groups were comparable across the study populations ( Tables 2 and 3 ).
| Conjunctiva | Azithromycin 1% (n = 57) | Moxifloxacin 0.5% (n = 54) | Total (n = 111) |
|---|---|---|---|
| Mean age (yrs) ± SD | 70.9 ± 7.57 | 66.1 ± 9.73 | 68.6 ± 8.97 |
| Age range (minimum, maximum) | 52 to 85 | 36 to 84 | 36 to 85 |
| Female, n (%) | 34 (60) | 26 (48) | 60 (54) |
| Male, n (%) | 23 (40) | 28 (52) | 51 (46) |
| White, n (%) | 48 (84) | 52 (96) | 100 (90) |
| Aqueous Humor | Azithromycin 1% (n = 56) | Moxifloxacin 0.5% (n = 56) | Total (n = 112) |
|---|---|---|---|
| Mean age (yrs) ± SD | 70.8 ± 7.60 | 66.3 ± 10.01 | 68.5 ± 9.13 |
| Age range (minimum, maximum) | 52 to 85 | 36 to 85 | 36 to 85 |
| Female, n (%) | 34 (61) | 26 (46) | 60 (54) |
| Male, n (%) | 22 (39) | 30 (54) | 52 (46) |
| White, n (%) | 47 (84) | 54 (96) | 101 (90) |
A total of 57 (89%) of 64 azithromycin subjects and 55 (87%) of 63 moxifloxacin subjects completed the study. The most common reason for discontinuation in both treatment groups was withdrawal of consent (5 azithromycin subjects and 4 moxifloxacin subjects). Only 2 subjects discontinued because of an adverse event, one in each treatment group. One subject in the azithromycin group discontinued the study before taking study drug because of a sprained knee, and 1 subject in the moxifloxacin group discontinued because of influenza. Both events were considered not related to administration of study drug.
Conjunctiva and Aqueous Humor Concentrations (Per Protocol Pharmacokinetic Population)
The pharmacokinetic characteristics of topically applied azithromycin 1% and moxifloxacin 0.5% in human conjunctiva and aqueous humor, based on the per protocol pharmacokinetic population, are summarized in Table 4 . The profile of the concentrations of azithromycin and moxifloxacin at various nominal time points (as described in Table 1 ) are plotted in Figure 1 (conjunctiva) and in Figure 2 (aqueous humor).
