Effects of the Des-F(6)-Quinolone Garenoxacin (BMS-284756), in Comparison to Those of Ciprofloxacin and Ofloxacin, on Joint Cartilage in Immature Rats

Abstract

We did not observe signs of chondrotoxicity in immature rats treated orally with garenoxacin (BMS-284756) at doses up to five times 600 mg/kg of body weight or with ciprofloxacin, whereas ofloxacin induced typical cartilage lesions. The peak plasma garenoxacin concentration was 25.5 mg/liter after administration of a dose of 600 mg/kg once daily for 5 days. Assuming that this model is predictive of human risk, BMS-284756 and ciprofloxacin should be more suitable for pediatric use than ofloxacin.

Garenoxacin (BMS-284756, T-3811ME) is a des-(6)-fluoroquinolone with antipneumococcal activity. It was selected for clinical development from a series of related quinolones because it exhibited less cytotoxicity in an in vitro screening test than other quinolones, e.g., the corresponding 6-fluoro-derivative (L. E. Lawrence, P. Wu, L. Fran, K. Gouveia, A. Card, K. Denbleyker, and J. F. Barrett, 40th Intersci. Conf. Antimicrob. Agents Chemother., poster 751, 2000). Furthermore, compared to some other quinolones, the chondrotoxic potential of this drug in dogs seems to be rather low (Y. Kawamura, A. Nagai, M. Miyazaki, T. Sanzen, H. Fukumoto, H. Hayakawa, Y. Todo, N. Terashima, Y. Watanabe, and H. Narita, 40th Intersci. Conf. Antimicrob. Agents Chemother., poster 277, 2000). When given orally to rats in routine toxicological studies for 1 or 3 months, garenoxacin at daily doses of 400 mg/kg of body weight or greater induced erosions, blisters, and chondrocyte degeneration in joint cartilage; animals were 6 weeks of age at the beginning of the treatment (3). Since a comparative study with immature rats providing histological data is not available (3), we performed experiments with juvenile rats, which can be used as a model for quinolone-induced chondrotoxicity, if species differences in kinetic behavior are considered (1, 2, 4, 5, 7).

Groups of 4- to 5-week-old Wistar rats bred at our institute were treated by oral intubation once with garenoxacin at doses of 200, 400, and 600 mg/kg or once daily on five consecutive days with garenoxacin, ofloxacin, or ciprofloxacin. Garenoxacin was supplied by Bristol-Myers Squibb [mesylate salt; lot no. 807 T-3 (A)]. For treatment with ofloxacin and ciprofloxacin, commercially available fluoroquinolone-containing tablets (ofloxacin [Tarivid 200] from Hoechst, Frankfurt/Main, Germany, and ciprofloxacin [Ciprobay 500] from Bayer Vital, Leverkusen, Germany) were used. All drugs were suspended in a 2% starch solution and were administered by gastric intubation at a volume of 10 ml per kg. For histological examination, knee joints were prepared within 24 h after dosing and fixed in formalin (10%), decalcified in an EDTA solution (10%, pH 7.4), dehydrated in an alcohol series, and embedded in paraffin. Series of 40 to 50 slices (6 μm thick) were prepared from the predilection sites of one knee joint of each animal and stained with an aqueous 1% solution of toluidine blue (Merck, Darmstadt, Germany).

Satellite groups of rats (total n = 330) were treated once or five times at 4 to 5 weeks of age by oral intubation with garenoxacin at doses of 200, 400, and 600 mg/kg. Concentrations in plasma were analyzed by high-performance liquid chromatography at 11 different time points after treatment. Animals (five per group) were decapitated after 0.25, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8, and 24 h, and blood samples were collected with hematocrit capillaries coated with sodium heparinate. Blood was centrifuged, and plasma was stored at −20°C until high-performance liquid chromatography analysis. After deproteinization, separation was performed on a reversed-phase column (Nucleosil-100-5 C18; length, 125 mm; diameter, 4 mm). The mobile phase consisted of 84% 5 mM tetrabutylammonium dihydrogen-phosphate (Sigma)-16% acetonitrile, with a pH of 3.6 (regulated with citric acid). Garenoxacin was determined by spectrofluorometry (excitation wavelength, 280 nm; emission wavelength, 406 nm). Noncompartmental pharmacokinetic analysis of plasma concentration-time data was performed by using WinNonlin (version 1.5, 1984-1998; Pharsight Corporation).

Results of the histological examination of the knee joints from immature rats treated for 1 or 5 days with garenoxacin and the two other quinolones are summarized in Table 1. No joint cartilage lesions were detected histologically in any knee joint from immature rats after treatment with garenoxacin or ciprofloxacin; with both quinolones the knee joint cartilages of treated animals were not distinguishable from those of controls (Fig. 1A). Treatment with ofloxacin, however, caused typical lesions in articular cartilage in five of seven rats (Fig. 1B). Besides the lesions in immature articular cartilage, a cleft was also observed in the epiphyseal growth plate of one knee joint (Fig. 1C).

TABLE 1.

Results of the histological examination of knee joints from immature rats after treatment with quinolones from day 32 to day 36 and concentrations in plasma measured under corresponding conditions

Treatment	No. of doses × amt of dose (mg/kg)	No. of rats with lesions/ no. of rats treated	Cmaxd (mg/liter)	AUC (TAU) (mg · h/liter)
Vehicle	5	0/7
Garenoxacin	1 × 200	0/5	12.5	34
	1 × 400	0/5	18.5	67
	1 × 600	0/5	18.0	133
	5 × 200	0/7	12.0	33
	5 × 400	0/16	18.1	58
	5 × 600	0/16	25.5	108
Ofloxacin	5 × 600	5/7	—a,b
Ciprofloxacin	5 × 600	0/7	—a,c

^aConcentrations in plasma were not determined in the experiments described in this paper, because data had been established before in our laboratory.

^bAfter a single oral dose of 600 or 1,200 mg of ofloxacin/kg, peak concentrations were 33.5 ± 9.8 and 45.4 ± 12.4 mg/liter, respectively.

^cAfter a single oral dose of 1,200 mg of ciprofloxacin/kg, peak concentrations were 5.1 ± 2.3 mg/liter in immature rats (6).

^dC_max, maximum concentration of drug in plasma.

FIG. 1.

Knee joint cartilage from juvenile rats (age, 37 days) stained with toluidine blue. (A) Treatment from day 32 to day 36 with daily oral doses of 600 mg of BMS-284756/kg. Normal cartilage layer, characteristic chondrocyte distribution, and regular staining of the matrix with toluidine blue are observed, with no difference from controls. m, menisci; asterisk, bone. Magnification, ×75. (B) Treatment from day 32 to day 36 with daily oral doses of 600 mg of ofloxacin/kg. Multiple lesions in the immature articular-epiphyseal cartilage complex are observed (arrows). (C) Treatment from day 32 to day 36 with daily oral doses of 600 mg of ofloxacin/kg. A pronounced lesion in the epiphyseal growth plate was observed (arrows).

Garenoxacin was rapidly absorbed from the gastrointestinal tracts of the immature rats. Peak concentrations were measured 15 to 60 min after dosing. Peak concentrations and area under the concentration-time curve (AUC) values determined after one or five doses are presented in Table 1; the calculated elimination half-lives ranged from 3.8 to 6.4 h.

In this paper we describe the lack of histological changes in knee joints from juvenile rats treated once daily for a period of 5 days, resembling the length of a possible treatment period. Data on the pharmacokinetic behavior of the drug in pediatric patients are not available, but first results for adults show that after an oral dose of 400 mg peak concentrations in plasma reached approximately 5 to 6 mg/liter and the AUC was 84 mg · h/liter (D. A. Gajjar, D. M. Grasela, A. Bello, Z. Ge, and L. Christopher, 40th Intersci. Conf. Antimicrob. Agents Chemother., poster 2259, 2000). Peak concentrations measured in 4-week-old rats were three to five times higher, but only after a dose of 600 mg/kg was the AUC value in rats higher than the value in humans after a single oral dose of 400 mg. In this context it is of interest that in a further study with 4-day-old rats we obtained peak concentrations in plasma of more than 75 mg/liter, probably due to the immature metabolic capacity of these very young animals. Under these conditions, too, we observed no histological changes in knee joint cartilage, but on an ultrastructural level effects on chondrocytes and tenocytes were present in rats treated with garenoxacin or other quinolones (S. Fassheber, E.-M. Kappel, K. Riecke, U. Rahm, M. Shakibaei, and R. Stahlmann, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2207, 2001).

In summary, garenoxacin was not chondrotoxic when given to 4-week-old rats at doses that lead to peak concentrations in plasma higher than those expected during therapy. Similar results were obtained for ciprofloxacin. On the assumption that this model is predictive of human risk, garenoxacin and ciprofloxacin should be more suitable for pediatric use than ofloxacin.