1 Department of Ophthalmology, Miguel Servet University Hospital, Zaragoza, Isabel La Catolica 13, E-50009; 2 Department of Analytical Chemistry, 3 Department of Pharmacology and Physiology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Miguel Servet 177, E-50013, Spain
Received 23 November 2001; returned 25 March 2002; revised 15 July 2002; accepted 15 July 2002
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Abstract |
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Introduction |
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Fluoroquinolones have been found to be important therapeutic agents for the treatment of ocular infections, such as keratitis or conjunctivitis, when given by topical administration.1,7,10 However, only a few authors have reported the systemic use of fluoroquinolones in ocular infections. Keren et al.11 studied the ocular penetration of ciprofloxacin into both the vitreous and aqueous humour. Virgil-Alfaro et al.12 studied the ocular penetration of ciprofloxacin in pigs and reported the treatment of experimental endophthalmitis in rabbits with various fluoroquinolones. Gatti & Panozzo13 and Ng et al.,14 using experimental models of endophthalmitis in rabbits, studied the ocular penetration of trovafloxacin and ofloxacin. Hatano et al.15 compared the ocular penetration of lomefloxacin with that of other antimicrobial agents such as sulbenicillin and gentamicin, and finally, Cochereau-Massin et al.1,7,8 and Liu et al.16 compared the ocular penetration of various fluoroquinolones with that of imipenem, vancomycin and amikacin.
Grepafloxacin, although not currently marketed, exhibits good activity against many of the potential pathogens that may cause ocular infections.10,1720
The aim of this study was therefore to evaluate the pharmacokinetics of grepafloxacin in albino and pigmented rabbits following intravenous administration, and establish whether there were differences between the pharmacokinetics or ocular penetration in these two types of animal.
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Materials and methods |
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Chemicals
Grepafloxacin was kindly supplied by Otsuka Pharmaceutical (Barcelona, Spain). Other chemicals were obtained from various commercial sources. All reagents were of HPLC grade.
Animal housing
Ten New Zealand rabbits (albino) and 10 Gigante de España rabbits (pigmented) weighing 3.54 kg were obtained from the Animal Experimentation Service of the University of Zaragoza. They were housed in rooms with a controlled temperature (20°C) and light cycle (12/12 h). The animals were maintained on water and standard laboratory chow ad libitum throughout the study. Each animal was judged to be clinically normal by physical examination before beginning the experiment. During this procedure, the heart and respiratory rates and the rectal temperature were also recorded.
Pharmacokinetic studies
Grepafloxacin was administered intravenously (iv) in the marginal ear vein of five pigmented rabbits and five albino rabbits as a bolus dose of 10 mg/kg body weight. Serial blood samples (1 mL) were drawn from a catheter inserted in the contralateral central ear artery at 0, 5, 10, 15, 30,45, 60, 90, 120, 180, 240 and 360 min into heparinized tubes. The samples were centrifuged and the plasma stored at 80°C until analysis.
Intraocular penetration studies
Grepafloxacin was administered in the marginal ear vein of five pigmented rabbits and five albino rabbits as an iv continuous infusion (4.6 mg/h) using a Mod M312 Gilson infusion pump (Pacisa, Zaragoza, Spain). Serial blood samples were drawn from a catheter inserted in the contralateral ear artery. Blood levels were measured until 200 min, in order to ensure that steady-state plasma concentrations of grepafloxacin (expected concentration 1.5 µg/mL) were achieved. The animals were then killed by iv administration of pentobarbital (30 mg/kg). Aqueous and vitreous humour samples were aspirated by puncture with a 23-gauge needle. The eyes were then removed and stored at 80°C. Frozen eyes were dissected and the ocular tissues, cornea, iris, lens, chorioretina and sclera, were obtained and stored at 80°C until analysis.
HPLC assay of grepafloxacin
Grepafloxacin was assayed in plasma and ocular tissues according to an HPLC method described previously.21
Pharmacokinetic analysis
The time course of the grepafloxacin plasma concentration (C) in both types of rabbit, and after using the Akaike criterion,22 was described using the following equation:
where C1 and 1 and Cz and
z are, respectively, the compartmental kinetic variables of distribution and elimination phases obtained by non-linear least squares regression analysis, using a weighting factor of 1/C2. The half-life in the distribution and elimination phases was calculated as t1/2
1 = ln 2/
1 and, t1/2
z = ln 2/
z where
1 and
z are, respectively, the slope of the distribution and elimination phases.
Total body clearance was calculated as Clb = Dose/ AUCgrepafloxacin, where AUCgrepafloxacin is the area under the grepafloxacin blood concentration versus time curve, calculated by the trapezoidal rule to the last data point (Clast) and extrapolated to infinity by dividing Clast by the slope of the elimination phase. Finally, the volume of distribution of the central compartment (Vc) and the volume of distribution under the area (Vd) were calculated as Vc = Dose/Cz and Vd = Dose/(AUC grepafloxacin x z).
Statistical evaluation
The pharmacokinetic values obtained in the pigmented and albino rabbits are presented as mean ± S.D., except for the half-life, where the harmonic mean is shown. Comparisons between iv pharmacokinetic parameters from albino and pigmented rabbits were carried out using Students unpaired t-test. The differences between the respective tissue concentrations were obtained by one-way analysis of variance (ANOVA). In all instances, P < 0.05 was considered to be statistically significant.
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Results |
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Discussion |
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Following iv administration of grepafloxacin the best pharmacokinetic fit of the plasma concentration was obtained for a two-compartment open model. To date there are no other reports on the pharmacokinetics of grepafloxacin in rabbits, with all the published data for grepafloxacin obtained from humans or in rats.10,1720,2325 Our results are similar to the findings reported for the pharmacokinetics of other fluoroquinolones such as ciprofloxacin, sparfloxacin and enrofloxacin.5,16,2629 In our studies, the distribution kinetics of grepafloxacin were characterized by a large Vd, which agrees with findings reported in other animal species.5 In our studies, when the pharmacokinetic parameters were compared between albino and pigmented rabbits, the latter exhibited higher values of Vd, Clb and t1/2 than the former. Similar differences have also been described by other authors when the pharmacokinetics of pefloxacin was compared between albino and pigmented rabbits.1
In this article, we have also studied the penetration of grepafloxacin into ocular structures after continuous iv infusion of grepafloxacin, to steady-state plasma concentrations. We found clinically relevant concentrations of grepafloxacin in all ocular tissues, with the highest concentrations found in the iris and chorioretina of pigmented rabbits. Cochereau-Massin et al.1 described a similar result working with pefloxacin, and other authors have demonstrated that penetration of sparfloxacin or moxifloxacin in the iris and ciliary body is related to the melanin concentration of these tissues.3032 A similar influence of ocular pigments has been described for a number of other drugs, such as mydriatics, aminoglycosides or other fluoroquinolones.3335
For all ocular structures, the penetration of grepafloxacin was found to be higher in pigmented than in albino rabbits. The degree of penetration was influenced by tissue vascularization, in that vascular tissues, such as the chorioretina and the iris, showed grepafloxacin ratios > 1, while non-vascular tissues, such as the aqueous humour, vitreous humour and lens, showed grepafloxacin ratios < 1 (Table 2). However, in spite of the fact that the sclera is a non-vascular tissue it showed ratios > 1. This tissue is very difficult to separate from the chorioretina and contamination from chorioretina could explain this concentration finding. This may also explain the findings of Cochereau-Massin et al.,1 who found high concentrations in the sclera when studing the penetration of pefloxacin into ocular structures.
In conclusion, we have been able to demonstrate that grepafloxacin, a third-generation fluoroquinolone, has good capacity to cross the various ocular barriers after systemic administration, to achieve concentrations above those needed to inhibit bacteria that cause intraocular infections.
These results confirm the good penetration of fluoroquinolones into ocular tissues and highlight potential differences that may be observed when using pigmented or albino rabbits.
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Footnotes |
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References |
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