Pharmacokinetics of Sparffoxacin in the Serum and Vitreous Humor of ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1998, p. 1417–1423 0066-4804/98/$04.0010 Copyright © 1998, American Society for Microbiology

Vol. 42, No. 6

Pharmacokinetics of Sparfloxacin in the Serum and Vitreous Humor of Rabbits: Physicochemical Properties That Regulate Penetration of Quinolone Antimicrobials WEIGUO LIU,1 QING FENG LIU,1 RUTH PERKINS,1 GEORGE DRUSANO,1,2 ARNOLD LOUIE,1 ASSUMPTA MADU,3 UMAR MIAN,3,4 MARTIN MAYERS,3,4 AND MICHAEL H. MILLER1* Divisions of Infectious Diseases1 and Clinical Pharmacology,2 Departments of Medicine and Pharmacology, Albany Medical College, Albany, and Department of Ophthalmology, Montefiore Medical Center, University Hospital for the Albert Einstein College of Medicine,3 and Department of Ophthalmology, Bronx Lebanon Medical Center, Albert Einstein College of Medicine,4 Bronx, New York Received 29 May 1997/Returned for modification 11 December 1997/Accepted 19 March 1998

We have used a recently described animal model to characterize the ocular pharmacokinetics of sparfloxacin in vitreous humor of uninfected albino rabbits following systemic administration and direct intraocular injection. The relationships of lipophilicity, protein binding, and molecular weight to the penetration and elimination of sparfloxacin were compared to those of ciprofloxacin, fleroxacin, and ofloxacin. To determine whether elimination was active, elimination rates following direct injection with and without probenecid or heat-killed bacteria were compared. Sparfloxacin concentrations were measured in the serum and vitreous humor by a biological assay. Protein binding and lipophilicity were determined, respectively, by ultrafiltration and oilwater partitioning. Pharmacokinetic parameters were characterized with RSTRIP, an iterative, nonlinear, weighted, least-squares-regression program. The relationship between each independent variable and mean quinolone concentration or elimination rate in the vitreous humor was determined by multiple linear regression. The mean concentration of sparfloxacin in the vitreous humor was 59.4% 6 12.2% of that in serum. Penetration of sparfloxacin, ciprofloxacin, fleroxacin, and ofloxacin into, and elimination from, the vitreous humor correlated with lipophilicity (r 2 > 0.999). The linear-regression equation describing this relationship was not improved by including the inverse of the square root of the molecular weight and/or the degree of protein binding. Elimination rates for each quinolone were decreased by the intraocular administration of probenecid. Heat-killed Staphylococcus epidermidis decreased the rate of elimination of fleroxacin. Penetration of sparfloxacin into the noninflamed vitreous humor was greater than that of any quinolone previously examined. There was an excellent correlation between lipophilicity and vitreous entry or elimination for sparfloxacin as well as ciprofloxacin, fleroxacin, and ofloxacin. There are two modes of quinolone translocation into and out of the vitreous humor: diffusion into the eye and both diffusion and carrier-mediated elimination out of the vitreous humor. developed and validated an animal model in which sequential vitreous humor samples can be obtained from a small number of rabbits. Based upon the comparison of pharmacokinetic parameters in single and serially sampled eyes, we have shown that serial sampling does not alter ocular pharmacokinetic parameters. By this approach, the pharmacokinetic parameter estimates from as few as three animals give more accurate data than it is possible to obtain with more than 20 times this number of animals by the approach of combining single datum points from different animals (23, 35, 41, 43, 51). Our method provides more-robust parameter estimates that permit the characterization of ocular pharmacokinetics which are difficult to address by the older approach (23, 35, 40, 41, 43, 51). Studies in our laboratory (23, 41, 43) and by others (16, 39) have shown that quinolones penetrate into the noninflamed vitreous better than beta-lactams, aminoglycosides, or vancomycin (5, 31, 34, 36, 38, 59, 60). Based primarily upon these penetration data, systemically administered ciprofloxacin has been used to treat patients with bacterial endophthalmitis (32). However, the activity of ciprofloxacin against ocular pathogens, particularly coagulase-negative staphylococci, is marginal and its penetration is poor relative to that of fleroxacin (43) or ofloxacin (51). Sparfloxacin, a recently introduced quinolone antimicrobial (14, 54, 58), is more active against staphylococci and appears to penetrate into the noninflamed vitreous better

Bacterial endophthalmitis is a severe and often blinding condition (2, 22, 48, 52). While the direct injection of antimicrobials into the vitreous humor is known to improve visual outcome, the roles of systemic antibiotics are less well understood (7, 21, 48, 52). Systemically administered antimicrobials commonly used in the therapy of endophthalmitis do not penetrate into the noninflamed vitreous humor (24, 48, 52). Following cataract surgery, the intravitreal injection of antimicrobial agents in the therapy of endophthalmitis, which is primarily due to Staphylococcus epidermidis, is currently considered the treatment of choice for most patients (24). However, the potential role of systemically administered agents that exhibit better penetration into the vitreous humor has not been studied. Moreover, neither therapy nor prophylaxis of endophthalmitis of other causes (e.g., posttraumatic and hematogenous) or microbial etiologies (e.g., Streptococcus pneumoniae, Bacillus spp., and Pseudomonas aeruginosa) has been well characterized. Since accurate pharmacokinetic data have fundamental implications for outcome studies of animals and humans, we have * Corresponding author. Mailing address: Department of Medicine, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Phone: (518) 262-5343. Fax: (518) 262-6727. E-mail: michael_miller @ccgateway.amc.edu. 1417

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than ciprofloxacin (16, 23, 41). However, the existing pharmacokinetic data are based upon studies which combine single samples from different subjects to generate pharmacokinetic estimates. This method is unreliable when used to describe pharmacokinetic data in humans (55). The primary goals of the current study were threefold. We wanted to (i) characterize the ocular pharmacokinetics of sparfloxacin, (ii) compare the relationships of protein binding, lipophilicity, and molecular weight (MW) to the vitreous translocation (entry and elimination) of sparfloxacin with those of other quinolones, and (iii) determine if the elimination of these drugs was blocked by probenecid or heat-killed bacteria. MATERIALS AND METHODS Animal model. Adult male, New Zealand White rabbits (Milbrook Farms, Amherst, Mass.) weighing 2 to 3 kg were used. Animals were obtained and cared for in accordance with Association for Research in Vision and Ophthalmology guidelines. The care, anesthesia, and vitreous sampling methods were similar to those described previously (43). The animals were anesthetized with an intramuscular dose of diazepam (2.5 mg) and a subcutaneous dose of urethane (1.62 g/kg of body weight) given approximately 45 min prior to antibiotic administration. Anesthesia was maintained throughout the sampling period, with administration of supplemental intramuscular ketamine (10 mg/kg) and xylazine (0.6 mg/kg) as needed. Following anesthesia, a 24-gauge angiocatheter was inserted into a marginal ear vein to facilitate antibiotic administration and a second catheter was inserted into the central artery of the contralateral ear to obtain serum samples. A solution of sparfloxacin (obtained from Rhone-Poulenc Rorer Pharmaceuticals, Inc., Collegeville, Pa.) for intravenous injection was prepared with 5 ml of a 5% dextrose in water solution and 0.5 ml of lactic acid (pH 3.6) and heated by means of a hot tap water bath. After the sparfloxacin was dissolved, another 5 ml of 5% dextrose in water was added to obtain a final concentration of 9.5 mg/ml. The solution was administered by a rapid (1-min) intravenous infusion (40 mg/kg) through a marginal ear vein, followed by a 1-ml flush with 0.9% NaCl. Serial samples (blood and vitreous humor) were taken at 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h after drug administration as previously described (41). For the determination of sparfloxacin ocular pharmacokinetics following systemic administration, six animals were used. For direct injection studies with quinolone with and without probenecid, 20 animals in four groups were used. Animals in each group received either sparfloxacin, ofloxacin, ciprofloxacin, or fleroxacin; one eye received both probenecid and a quinolone, and the other eye received quinolone alone. For direct-injection experiments, solutions of quinolones alone or in combination with probenecid or heat-killed bacteria were injected into the midvitreous. Five additional animals were used in the heatkilled-bacterium experiments. Probenecid was dissolved in 1 N NaOH and adjusted to pH 8.6 prior to injection. Probenecid was diluted in balanced salt to a final concentration of 2.86 mg/ml. The concentration of ciprofloxacin (Miles Pharmaceutical Division, West Haven, Conn.), fleroxacin (Roche Pharmaceuticals, Nutley, N.J.), ofloxacin (RWG Pharmaceutical Research Institute, Raritan, N.J.), and sparfloxacin in the direct-injection experiments was 5 mg/ml. Heat-killed S. epidermidis (ATCC 155) was prepared with an overnight inoculum following three cycles of centrifugation and washing with 0.9% saline. Thereafter, cells were spectrophotometrically adjusted to a final inoculum of 109 with 0.9% saline and then heated to 80°C for 20 min. One hundred microliters of 109 heat-killed S. epidermidis organisms was injected via a 30-gauge needle into the midvitreous cavity of one eye; the contralateral eye received the same volume of 0.9% saline. For direct-injection experiments, 100 ml of each quinolone was injected into the midvitreous as previously described (43). Following the designated sampling period, animals were sacrificed with pentobarbital sodium solution (125 mg/kg) and bilateral pneumothoraces. Antibiotic assays. To determine sparfloxacin concentrations in the serum and vitreous, a well-diffusion microbiological assay was used. Prior to analysis, all samples were stored at 220°C. Blood samples were allowed to clot and were immediately centrifuged at 1,000 3 g for 15 min. The test organism was Escherichia coli KL16. An inoculum of 107 organisms/ml diluted 1:10 in 3% brain heart infusion agar mixed with Mueller-Hinton broth (Difco) adjusted to pH 8.0 with 1 N NaOH was used. Wells (4-mm-diameter) were cut and 10-ml aliquots of serum or vitreous humor were then pipetted into the wells. The agar was incubated overnight at 37°C in an ambient-air incubator. Zones of inhibition were read to the nearest 0.1 mm with a vernier caliper. Sparfloxacin standards were prepared by dissolving 100 mg of drug per ml in 1 mmol of NaOH per liter; this solution was then diluted with either rabbit serum (for serum standards, 24, 12, 8, 4, and 2 mg/ml) or balanced salt solution (for vitreous standards, 12, 6, 3, 1.5, 0.75, 0.375, and 0.1875 mg/ml). The sensitivity of the biological assay was 1.6 ng. The coefficients of variation in the biological assay for the high and low standards were 4.3 to 7.5% and 0.4 to 3.1%, respectively, with an assay linearity of 0.99. There is little or no metabolism of sparfloxacin with no biologically active metabolites (11, 30, 45, 50). To compare the sensitivity of the biological assay to that of high-pressure

ANTIMICROB. AGENTS CHEMOTHER. liquid chromatography (HPLC), sparfloxacin concentrations were also measured by HPLC according to the method of Borner et al. (11). Samples were run at 25°C in a C18, 5-mm column (220 by 2.1 mm) packed with Nucleosil. Sample preparation was performed by mixing 20 ml of serum with 130 ml of mobile phase to acid precipitate proteins and by filtering. The mobile phase (75% acetonitrile– 25% 0.1 M H3PO4 adjusted to pH 3.82 with concentrated phosphoric acid) was delivered to the column at a rate of 0.2 ml/min with a Hewlett-Packard (Wilmington, Del.) series 1050 pump. Serum samples were prepared in pooled rabbit serum. Vitreous samples could not be assessed by HPLC because of the low sensitivity (sparfloxacin does not fluoresce) of the assay. One hundred microliters of sample was injected by a Hewlett-Packard series 1050 autosampler and run serially through a Hewlett-Packard 1040A UV detector (240- to 280-nm wavelengths) and a Hewlett-Packard 1046A fluorescence detector (excitation, 280 nm; emission, 445 nm). Data were collected on a Hewlett-Packard Chemstation. Quantitation of the antibiotic concentrations used peak heights. Antibiotic concentrations in the serum and vitreous following systemic drug administration were determined by HPLC (51); concentrations following direct injection were determined by the microbiological assay. The coefficients of variation for the high and low standards were 2.4 and 2.2%, respectively. Protein quantitation and characterization. Protein concentrations in the vitreous humor samples were determined with Coomassie protein assay reagent (Pierce, Rockford, Ill.). The Coomassie protein assay was performed by placing 1 ml of sample, 9 ml of distilled water (dH2O), and 240 ml of Coomassie reagent into each well of a 96-well microtiter plate. The plate was read on an EL 312e Biokinetics Reader (Bio-Tek Instruments, Winooski, Vt.) at a filter width of 630 nm. To prevent overloading of the sodium dodecyl sulfate (SDS)-polyacrylamide gels, samples were diluted to a final concentration of ,4 mg/ml. Albumin standards (rabbit albumin; Sigma, St. Louis, Mo.) were run at concentrations of 0.5, 1, 2, 4, 6, 8, and 10 mg/ml. Identification and quantitation of proteins in the vitreous humor were performed by SDS-polyacrylamide gel electrophoresis Mini-Protean II cell, model with 1000/500 power supply; Bio-Rad, Hercules, Calif.) and densitometry (model 60S video densitometer; BioImage, Ann Arbor, Mich.). Minigels were run according to the method of Laemmli (33). We used a 12% running gel, a 4.5% stacking gel, and a Tris (0.25 M)–glycine (1.92 M)–SDS (1%) buffer. Samples were prepared by using 1 ml of sample, 4 ml of dH2O, and 5 ml of sample solubilizer. Eight microliters of sample was loaded onto the gel, which was run at 175 V for 40 to 45 min. The gel was stained with Coomassie brilliant blue (J. T. Baker, Inc., Danvers, Mass.) for 30 min and destained with a 5% acetic acid solution. Standards included rabbit serum albumin (0.5, 2, and 4 mg/ml), rabbit lens protein, and rabbit hemoglobin. Rabbit lens protein was obtained by homogenizing surgically resected rabbit lenses after the capsules had been removed. Rabbit hemoglobin was obtained from rabbit erythrocytes that had been washed three times in phosphate-buffered saline (PBS) and lysed in dH2O; cell fragments were removed by centrifugation at 8,000 3 g (Micro Centrifuge model 5415C; Brinkmann Instruments Inc., Westbury, N.Y.). An MW standard (midrange kit; Enprotech, New York, N.Y.) and lens protein (diluted 403) were also run with each gel. Albumin concentrations in vitreous samples and sera were determined by densitometry. Protein binding. The protein binding was determined by ultrafiltration of 4-ml standards at several concentrations of sparfloxacin and other quinolones (1.0, 5.0, 10, and 20 mg/ml) through Centriflo CF25 (MW cutoff, 25,000) membrane cones (Amicon, Inc. Beverly, Mass.) according to the specifications of the manufacturer. Standard solutions for each quinolone were prepared with rabbit serum (Sigma). Briefly, cones were moistened with dH2O, placed into their supports, and dried by centrifugation at 1,000 3 g for 3 min. Ultrafiltration was performed at 780 3 g for 10 min. Filter binding was determined by comparing drug concentrations in ultrafiltrates prepared with PBS with those in spiked PBS. Protein binding was adjusted to account for binding to the filter. Concentrations of free drug in ultrafiltrates were determined by the bioassay described above. Lipophilicity. The lipophilicities of the quinolones were characterized by determining their partitioning ratios into octanol and PBS by standard methods (15). Briefly, solutions containing 10 mg/ml in 0.1 M phosphate buffer (pH 7.2) were agitated with an equal volume of n-octanol at 25°C for 48 h and subsequently centrifuged at 1,870 3 g for phase separation. The concentrations of quinolones in the aqueous phase were then determined by the microbiological assay. Partition coefficients were expressed as the ratio of the amount of the compound in the n-octanol phase to that in the aqueous phase. Mathematical modeling and statistics. Pharmacokinetic analyses of the plasma and vitreous humor concentration-time data following systemic administration were performed with RSTRIP (Micromath Scientific Software, Salt Lake City, Utah), an iterative, nonlinear, weighted, least-squares-regression program. The most appropriate pharmacokinetic models were determined by using the coefficient of determination and the RSTRIP model selection criterion, which is a modified form of the Akaike (1) information criterion. Noncompartmental parameters were estimated by the statistical-moment theory. Estimations for each exponential coefficient and time constant were computed with the standard deviations of each estimate, along with its 95% confidence range, which was calculated by using both univariate and support-plane approximations for the bounds of the 95% confidence range. Other standard pharmacokinetic parameters were determined with computer-generated primary coefficients and standard pharmacokinetic equations (26, 27). Parameters were calculated for each

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PHARMACOKINETICS OF SPARFLOXACIN IN RABBITS

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FIG. 1. Mean concentrations of sparfloxacin in the serum and vitreous humor of six rabbits following a single intravenous dose (40 mg/kg). The left graph shows the data plotted arithmetically, and the right graph shows the data plotted semilogarithmically.

animal; population pharmacokinetic parameters were then calculated by a standard two-step technique (27). To determine the relative contribution of MW, protein binding, and hydrophobicity (independent variables) to the penetration of quinolones into the vitreous humor, we used multiple linear regression (SYSTAT, Evanston, Ill.). Penetration was expressed as a percentage by dividing the area under the concentration-time curve (AUC) from 0 h to infinity in the vitreous by that in the serum following a single dose of each quinolone. To determine the relative importance of protein binding, levels of penetration were expressed as both total and free fractions; the latter were calculated as percent penetration (free) 5 percent penetration (total) 3 (1% of protein bound). Since protein concentrations in the vitreous humor are less than 1% of those in serum and since animals with any breakdown of the blood-ocular barrier (BOB) were excluded from analysis, for these calculations we assumed that there was no binding in the vitreous humor. The logarithms of the mean penetration and of the mean free penetration were the dependent variables (33, 50) in systemic-administration experiments. In direct-injection experiments, the first-order elimination rate half-lives were compared with the logarithm of the partition coefficient (3). We performed univariate-linear-regression analysis, employing the octanol-water partition coefficient (the permeability coefficient [p]), the square root of the MW, the fraction of protein bound, and a hybrid variable (p/√MW) as independent variables. (46, 49) The statistical significance of each of the variables was determined univariately (6, 30, 46, 57). For the multiple linear regressions, each of the independent variables was allowed to step in (P , 0.05) or step out (P , 0.15).

RESULTS Determination of sparfloxacin concentrations in serum and vitreous humor. Because of the small sample sizes (5 to 10 ml) used when serial samples were obtained from the vitreous humor in our ocular pharmacokinetic model (40, 41, 43), very sensitive assay methods were required. As a result, we compared the sensitivities and reproducibilities of results of HPLC and microbiological assays for sparfloxacin using modifications of standard assays previously described by others (11). The sensitivities of the microbiological and HPLC assays were 1.9 and 25 ng, respectively. Thus, the biological assay was 14-fold more sensitive than HPLC. For the biological assay, the coefficients of variation for the high and low standards were 1 and 4.5%, respectively. No metabolites were found in serum samples by HPLC. Ocular pharmacokinetics of sparfloxacin. Data from six animals with no breakdown of the blood-vitreous barrier, as determined by SDS-polyacrylamide gel electrophoresis, were analyzed (Fig. 1). Results are plotted arithmetically and semilogarithmically to better demonstrate the relative levels of penetration and terminal elimination slopes, respectively. Modelpredicted and actually observed drug concentrations in the serum and vitreous were similar (Table 1). Both hybrid and derived microconstants are given in Table 2. Model-dependent analysis gave excellent fit with coefficients of determination for the serum and vitreous of 0.999 and 0.997, respectively. The AUCs in the vitreous humor and serum were 14.43 and 22.03 mg z h/liter, respectively. Penetration into the vitreous humor was 59.4% 6 12.2% of that in the serum. The terminal elim-

ination rate constants in the vitreous humor and serum were 0.28 and 0.24, respectively. The elimination half-life in the vitreous humor was 2.99 h, and that in serum was 2.39 h (P . 0.05). On the basis of the coefficient of determination and model selection criterion, vitreous humor and serum antibiotic concentration-time data following intravenous administration were best-fitted to a two-compartment model. Correlation between physicochemical properties and protein binding and ocular translocation. The second goal of this ocular pharmacokinetic study was to determine the relationship of lipophilicity, MW, and protein binding to translocation across the blood-ocular barrier of the quinolone antimicrobial following systemic and direct injections. The translocation of sparfloxacin was compared to those of three other quinolones (ciprofloxacin, fleroxacin, and ofloxacin) for which we have previously shown significant differences in levels of ocular penetration (Fig. 2) (23, 41, 43, 51). Among the four quinolones studied, levels of penetration differed by an order of magnitude; levels of ciprofloxacin and sparfloxacin penetration were 5.5 and 59%, respectively. The effects of three independent variables on ocular penetration were considered in the multiple-linear-regression model: lipophilicity, MW, and protein binding. Table 3 shows the ocular penetration of each drug along with its MW, level of protein binding, and partition coefficient. Only the lipophilicities were statistically significant when examined univariately. This relationship is described by the equation log(mean percent vitreal penetration) 5 2.739(p) 1 0.59, where p is the octanol-water partition coefficient (r 2 . 0.999, P , 0.001). Multiple linear regression was then undertaken after considering additional variables, including MW and the

TABLE 1. Comparison of measured and pharmacokinetic-modelpredicted sparfloxacin concentrations in serum and vitreous humor following systemic administration Sparfloxacin level (mg/ml) in: Time (h)

0.25 0.5 1.0 2.0 3.0 4.0 6.0 8.0

Serum

Vitreous humor

Measured

Predicted

Measured

Predicted

12.42 6 3.86 7.89 6 1.86 4.708 6 0.72 3.429 6 0.54 2.059 6 0.27 1.589 6 0.12 0.961 6 0.045 0.611 6 0.085

12.355 7.9837 4.6642 3.0106 2.2635 1.7190 0.9928 0.5734

1.638 6 0.844 2.702 6 0.765 2.836 6 0.484 2.706 6 0.496 2.267 6 0.462 1.909 6 0.424 1.056 6 0.224 0.781 6 0.235

1.6642 2.4495 2.9741 2.7177 2.2234 1.7928 1.1605 0.7509

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ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Kinetic parameters of sparfloxacin following intravenous administration

Sample

Serum Vitreous a

Mean time (h)a 6 SD for: A

B

a

b

bt1/2

18.85 6 11.97 24.80 6 1.54

5.19 6 1.19 4.83 6 1.59

3.33 6 1.63 2.01 6 1.57

0.28 6 0.04 0.24 6 0.06

2.39 6 0.29 2.99 6 0.76

% Penetration 6 SD

AUC (mg z h/liter)

59.38 6 12.26

22.31 6 3.70 13.06 6 2.38

A, zero time intercept for a phase; B, zero time intercept for b phase; a, distribution phase; b, elimination phase; bt1/2, terminal elimination half-life.

free fraction of drug in the serum available for transport; these additional variables did not improve model fit. To determine if carrier-independent translocation across the blood-ocular barrier following direct injection also correlated with the physicochemical properties of quinolones, we also determined the association between lipophilicity and drug elimination following direct injection into the vitreous humor in 20 animals. One eye received the quinolone alone, and the other eye received both the quinolone and probenecid. The elimination half-lives for ciprofloxacin, fleroxacin, ofloxacin, and sparfloxacin were 4.41, 3.35, 3.04, and 2.78 h, respectively (Table 4). As with systemic-injection experiments, there was an excellent correlation between lipophilicity and efflux (r 2 . 0.99, P , 0.01); MW and the free fraction did not improve model fit. The relationship is described by the equation t1/2b 5 (21.8172) (log10p) 1 2.1239, where t1/2b is the half-life at beta phase. Effects of probenecid and heat-killed bacteria on quinolone elimination following direct injection. Since the renal elimination of quinolones and beta-lactam antibiotics in humans and rabbits is blocked by probenecid and since the ocular elimination of the carrier-mediated export of beta-lactams from the vitreous humor is blocked by both probenecid and heat-killed bacteria (8, 25, 37), we examined the effects of each on the elimination of quinolones following direct injection. As shown in Fig. 3 and Table 4, probenecid significantly increased the elimination half-lives of ciprofloxacin, fleroxacin, and sparfloxacin (P , 0.05). While probenecid also increased the elimination half-life of ofloxacin (4.15 h with probenecid versus 3.04 h without), this difference was not significant (P 5 0.15). Heat-killed bacteria also increased the elimination half-life of fleroxacin 1.42-fold (P , 0.01). The effects of inflammation on the elimination rates of ciprofloxacin, ofloxacin, and sparfloxacin were not tested. DISCUSSION Because of the small sample sizes obtained from the vitreous humor, very sensitive assay methods were required. When we compared the sensitivities of HPLC and microbiological assays for sparfloxacin, the latter method proved to be 14-fold more

sensitive than HPLC with coefficients of variation for the high and low standards of 1 and 4.5%, respectively. No metabolites were found in serum samples by HPLC. Recent studies in our laboratory with the quinolone ciprofloxacin have shown that, in general, the sensitivities and reproducibilities of results of HPLC and biological assays are equivalent. However, the activities of quinolones differ in the presence and absence of microbiologically active metabolites and quinolones differ in their capacities to fluoresce. For compounds with active metabolites (50) (e.g., ciprofloxacin and ofloxacin), HPLC is the preferred assay method when drugs are administered systemically. On the other hand, for compounds like fleroxacin, for which there are no active metabolites (57), the biological assay is preferred (43). Like fleroxacin, sparfloxacin differs from ciprofloxacin and ofloxacin by not having biologically active metabolites. However, unlike other quinolones, sparfloxacin does not fluoresce; the sensitivity of HPLC assays with quinolones is increased by at least an order of magnitude when fluorescent compounds are used. As a result, when doses of sparfloxacin that mimic those achieved in the sera of humans were used, the HPLC assay was not sufficiently sensitive to measure drug concentrations in ocular fluid. Sparfloxacin showed excellent penetration into the vitreous humor, with mean concentrations in the vitreous humor of uninflamed eyes of 59.4% 6 12.2% of that in the serum. Following systemic administration, the elimination half-life from the vitreous in rabbits was 3.34 h and that from the serum was 2.2 h. The terminal-elimination half-life and maximum concentration of sparfloxacin in human serum were 17.6 h and 1.6 mg/ml, respectively. (30) The maximum concentrations in the serum and vitreous of rabbits following a 40-mg/kg bolus, achieved at approximately 1 hour after intravenous administration, were 12.43 and 2.84 mg/ml, respectively. While albino rabbits were used in this study, previous experiments in our laboratory have shown that the levels of penetration of other quinolones, namely, ofloxacin and ciprofloxacin, into the vitreous humor are identical in pigmented and nonpigmented animals (51). Recent pharmacokinetic studies by Cochereau-Massin and colleagues with pigmented, uninfected rabbits showed the

FIG. 2. (A) Relationship between the partition coefficients for ciprofloxacin (F), fleroxacin (}), ofloxacin (Œ), and sparfloxacin (■) and levels of penetration into the vitreous humor. (B) Relationship between the partition coefficients for these quinolones (same symbols) and the elimination rate half-lives following direct intravitreal injection.

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TABLE 3. Relationship of lipophilicity, protein binding, and MW to the penetration of four quinolones into the vitreous humor Quinolone

Ocular penetration (%)

Partition coefficient

Protein binding (%)

MW

Ciprofloxacin Fleroxacin Ofloxacin Sparfloxacin

5.5 14 30 59

0.056 0.200 0.330 0.431

23 31 33 42

331.3 369.3 360.4 392.4

maximum vitreal concentration to be 5.6 mg/ml, with a level of penetration of 54% following systemic injection of 50 mg/kg (16). Those authors also showed that sparfloxacin was more efficacious in the therapy of staphylococcal endophthalmitis in rabbits than systemically administered vancomycin or amikacin (37). Importantly, newer quinolones such as sparfloxacin (35) and ofloxacin (51) not only show better penetration into the vitreous humor than other quinolones such as ciprofloxacin (P , 0.05) but also are more active against gram-positive bacteria commonly isolated from patients with endophthalmitis. Using multiple linear regression, we have shown that there was an excellent correlation between lipophilicity and penetration into or elimination from the eye following systemic or intravitreal injection in nonpigmented, uninfected rabbits. Both lipophilicity and protein binding were measured under conditions that mimic those in ocular tissue and serum; lipophilicity was measured at physiological pH, and protein binding was measured with rabbit sera. The penetration of quinolones across the outer lipid membrane of E. coli is also propor-

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TABLE 4. Comparison of the effects of probenecid on the half-lives for elimination from the vitreous humor of quinoline antimicrobials Regimen

t1/2b 6 SD (h)

Median (h)

Ciprofloxacin Ciprofloxacin 1 probenecid Fleroxacin Fleroxacin 1 probenecid Ofloxacin Ofloxacin 1 probenecid Sparfloxacin Sparfloxacin 1 probenecid

4.41 6 1.69 9.66 6 0.81 335 6 0.26 5.52 6 0.83 3.04 6 0.70 4.15 6 3.93 2.78 6 0.63 4.60 6 1.34

4.064 9.53 3.35 5.52 3.30 2.59 4.37

P value

0.014 0.002 0.15 0.05

tional to lipophilicity. (28) Additionally, studies using artificial lipid membranes show that lipophilicity rather than molecular size best correlates with penetration (49). Carrier-independent penetration of antibiotics and other drugs into the eye (10, 13, 19, 44, 53, 61), like that at other anatomical sites (3, 13, 19, 30, 44, 46, 53, 61), correlates with physicochemical properties, including lipophilicity and MW as well as protein binding. These independent variables were considered in the multiple-linear-regression model. The log of the penetration of compounds across planar lipid bilayers and tissue correlates with the oil/water partition ratio, the inverse of the square route of the MW (17, 20, 46, 50), and the free fraction of drug (44, 62). As shown in our studies, lipophilicity is generally the most important variable.

FIG. 3. Effect of probenecid on the elimination of sparfloxacin (A), fleroxacin (B), ofloxacin (C), and ciprofloxacin (D) with (F) and without (Œ) the coadministration of probenecid; both probenecid and the quinolones were given intravitreally. The inset shows the effect of heat-killed S. epidermidis (F) on the elimination of fleroxacin alone (Œ).

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Our inability to discern the potential role of protein binding was limited because of the restricted range examined for this independent variable. Since protein binding clearly affects antibiotic penetration into tissue (30, 44, 46), studies with quinolones which show greater differences in these properties are planned. It has been difficult to establish rigorous models characterizing the importance of these properties in antibiotic penetration with single samples (56) which are pooled from different animals (10, 13, 19, 44, 53, 61). While we showed an excellent correlation between lipophilicity and translocation, modeling of quinolone elimination rates following systemic injection compared to that following direct injection suggested that drug elimination from the eye was more rapid than drug entry. This finding, in conjunction with results of studies showing that the renal and ocular eliminations of both quinolones and beta-lactam antibiotics in humans and rabbits are blocked by probenecid (4, 5, 9, 12, 18, 28) and inflammation (8, 25), suggested that more than one mechanism may be involved in elimination from the vitreous humor. To test this hypothesis, the elimination rates of four quinolones were examined following direct injection. Both probenecid and heat-killed bacteria prolonged the elimination rates of quinolones following direct injection. These observations suggest that the elimination of quinolones from the eye likely involves carrier-independent translocation via biological membranes with tight capillary and/or retinal cell barriers as well as carrier-dependent elimination blocked by both probenecid and heat-killed bacteria. In vivo and in vitro studies characterizing the rates of renal elimination of zwitterionic quinolones suggest the presence of separate and distinct carrier-mediated systems (29, 47). As a result, while the experiments with probenecid and heat-killed organisms suggest carrier-mediated export from the eye, the nature of this carrier(s) is unknown. The excellent correlation between lipophilicity and penetration or elimination also demonstrates the strength of our animal model in comparison to that in which one sample obtained from different animals is pooled to generate single-subject estimates. In addition to characterizing the effects of physicochemical properties on ocular pharmacokinetics, using this animal model we have characterized the effects of different modes of drug administration on ocular penetration (35), established a model that permits the determination of robust ocular pharmacokinetic parameters in the serum and vitreous humor using sparse datum sets (47), and described the ocular pharmacokinetics of quinolones following direct and systemic drug administration in pigmented and albino rabbits (23, 49). These experiments would have been difficult if we had used the single-sample methods employed in most ocular pharmacokinetic studies of animals. We have developed mathematical models that are highly explanatory of the penetration of and elimination from the vitreous of fluoroquinolones which differ mostly in their octanol-water partition coefficients. We have also shown that quinolones, like the beta-lactam antibiotics, are exported from the vitreous humor via a pump which is blocked by both probenecid and inflammation. It will be important in future studies to examine accuracy of prediction of penetration into the vitreous for additional quinolones as well as other drugs whose efficacies are dependent upon penetration into the vitreous humor. This will allow this model either to be further validated or to be shown to be only locally predictive for fluoroquinolones, in which case, other independent variables may be needed to make the model more globally predictive.

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