Abstract

The pharmacokinetics of grepafloxacin were determined in albino and pigmented rabbits following a single 10 mg/kg intravenous administration. The penetration of grepafloxacin into various ocular tissues was also determined after continuous intravenous infusion in both types of animal. Grepafloxacin showed a bicompartmental model of distribution in both pigmented and albino rabbits with significant differences in the pharmacokinetics between the two types of animal. After continuous intravenous infusion, significantly greater penetration of grepafloxacin was found in the iris, cornea and chorioretina of pigmented rabbits compared with albino rabbits.

Received 23 November 2001; returned 25 March 2002; revised 15 July 2002; accepted 15 July 2002

Introduction

Fluoroquinolones are important therapeutic agents for the treatment of bacterial ocular infections,13 with good bactericidal activity against many of the Gram-negative aerobes, as well as limited activity against the Gram-positive bacteria that cause these infections.48 Moreover, they show excellent pharmacokinetic properties, such as long half-life, good tissue distribution, low protein binding, high antimicrobial activity and relatively few side-effects. The newer fluoroquinolones show good penetration into ocular tissues but with a spectrum of activity that includes coagulase-negative staphylococci.9

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.

Materials and methods

Animals were obtained and cared for in accordance with the recommendations of the European Community guidelines for the use of experimental animals.

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 3.5–4 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:

graphic (Equation 1)

where C1 and λ1 and Cz and λz 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 t1/2λ1 = ln 2/λ1 and, t1/2λz = ln 2/λz where λ1 and λz 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 × λz).

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 Student’s 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.

Results

The grepafloxacin plasma concentration–time plot following bolus injection for the two types of rabbit is shown in Figure 1, and the mean values for the grepafloxacin pharmacokinetic parameters are shown in Table 1. In all instances, the best fit for the pharmacokinetic data was obtained with a two-compartment model, as determined by the use of the Akaike criterion.22 Following iv administration, grepafloxacin had a distribution phase in both albino and pigmented rabbits of ∼40 min. When the pharmacokinetic parameters of both albino and pigmented rabbits were compared, significant differences were observed in C1, Cz, λz, Vc, Vd, AUC and Clb (P < 0.05) (Table 1).

Figure 2 shows the mean plasma levels of grepafloxacin during continuous iv infusion of grepafloxacin in both albino and pigmented rabbits. After iv infusion of grepafloxacin, we found that the steady-state plasma concentrations of this drug were ∼1.5 mg/L in both albino and pigmented rabbits.

In general, there were differences in the penetration of grepafloxacin into all ocular tissues, except aqueous humour, between albino and pigmented rabbits, with these differences reaching statistical significance for the chorioretina, cornea and iris (Table 2).

Discussion

Drugs used to treat eye infections must have a broad spectrum of activity and must reach effective concentrations in ocular tissue, especially in the vitreous and aqueous humours. However, when administered by the systemic route, many antibiotics show poor ocular penetration and the levels reached are often insufficient to cure virulent endophthalmitis.1 Thus, new drugs that offer both good ocular penetration by the systemic route and a broad spectrum of activity against the potential pathogens in ocular infections are required.

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.

*

Corresponding author. Tel: +34-976-761-627; Fax: +34-976-761-612; E-mail: Bregante@posta.unizar.es

Figure 1. Mean ± s.d. plasma concentrations of grepafloxcin following an iv bolus dose of 10 mg/kg grepafloxacin. Filled triangles, pigmented rabbits; open triangles, albino rabbits.

Figure 1. Mean ± s.d. plasma concentrations of grepafloxcin following an iv bolus dose of 10 mg/kg grepafloxacin. Filled triangles, pigmented rabbits; open triangles, albino rabbits.

Figure 2. Mean ± s.d. plasma concentrations of grepafloxacin during continuous iv infusion in albino and pigmented rabbits. Filled triangles, pigmented rabbits; open triangles, albino rabbits.

Figure 2. Mean ± s.d. plasma concentrations of grepafloxacin during continuous iv infusion in albino and pigmented rabbits. Filled triangles, pigmented rabbits; open triangles, albino rabbits.

Table 1.

 Pharmacokinetic parameters of grepafloxacin in albino and pigmented rabbits given a single iv administration of 10 mg/kg body weight

Parameter (units) Albino (mean ± s.d.Pigmented (mean ± s.d.
C1a(mg/L)   13.9 ± 1.5    3.0 ± 0.8 
λ1 (min) 0.0737 ± 0.0162 0.0591 ± 0.0431 
Cza (mg/L)    4.3 ± 1.0    0.5 ± 0.2 
λza (min) 0.0098 ± 0.0018 0.0054 ± 0.0028 
t1/2λ1 (min) 9b 11b 
t1/2λz (min) 71b 126b 
Vca (mL/kg)  723.9 ± 82.0 3576.0 ± 969.2 
Vda (mL/kg) 2469.5 ± 305.5 2067.7 ± 11350.3 
AUCa (µg/mL/min)  615.2 ± 151.1 139.44 ± 19.20 
Clba (mL/min.kg)  17.06 ± 4.16  72.74 ± 9.40 
Parameter (units) Albino (mean ± s.d.Pigmented (mean ± s.d.
C1a(mg/L)   13.9 ± 1.5    3.0 ± 0.8 
λ1 (min) 0.0737 ± 0.0162 0.0591 ± 0.0431 
Cza (mg/L)    4.3 ± 1.0    0.5 ± 0.2 
λza (min) 0.0098 ± 0.0018 0.0054 ± 0.0028 
t1/2λ1 (min) 9b 11b 
t1/2λz (min) 71b 126b 
Vca (mL/kg)  723.9 ± 82.0 3576.0 ± 969.2 
Vda (mL/kg) 2469.5 ± 305.5 2067.7 ± 11350.3 
AUCa (µg/mL/min)  615.2 ± 151.1 139.44 ± 19.20 
Clba (mL/min.kg)  17.06 ± 4.16  72.74 ± 9.40 

These terms are related to rate constants for the specific two-compartment pharmacokinetic model. C1 and Cz are the coefficients and λ1 and λz are the the exponents derived by non-linear least squares regression of concentration data to equation graphic . t1/2λ1 = distribution half-life; t1/2λz, elimination half-life; Vc, volume of the central compartment; Vd, apparent volume of distribution calculated by the area method; AUC, area under the blood concentration time curve; Clb, body clearance.

aSignificantly different.

bHarmonic mean.

Table 2.

 Concentration and tissue to serum ratio of grepafloxacin following continuous iv infusion

 Albino  Pigmented 
Tissue concentration (ng/g or ng/mL) ratio  concentration (ng/g or ng/mL) ratio 
Plasma 1830 –  1314  – 
Chorioretina 3367 1.84  40248 30.63a 
Sclera 2580 1.41  4336  3.30 
Iris 2361 1.29  53940 41.05a 
Cornea 1116 0.61  2037  1.55a 
Lens  293 0.16  526  0.40 
Vitreous h.  366 0.20  762  0.58 
Aqueous h.  458 0.25  276  0.21 
 Albino  Pigmented 
Tissue concentration (ng/g or ng/mL) ratio  concentration (ng/g or ng/mL) ratio 
Plasma 1830 –  1314  – 
Chorioretina 3367 1.84  40248 30.63a 
Sclera 2580 1.41  4336  3.30 
Iris 2361 1.29  53940 41.05a 
Cornea 1116 0.61  2037  1.55a 
Lens  293 0.16  526  0.40 
Vitreous h.  366 0.20  762  0.58 
Aqueous h.  458 0.25  276  0.21 

Data are mean of five albino and five pigmented rabbits.

aSignificantly different.

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Author notes

1Department of Ophthalmology, Miguel Servet University Hospital, Zaragoza, Isabel La Catolica 1–3, E-50009; 2Department of Analytical Chemistry, 3Department of Pharmacology and Physiology, Faculty of Veterinary Sciences, University of Zaragoza, Zaragoza, Miguel Servet 177, E-50013, Spain