Author for correspondence: Sung-Chul Lim, Department of Pathology, Chosun University Hospital, #588, Seosuk-Dong, Dong-Ku, Gwangju City, 501-140, Republic of Korea (fax +(82 62)234 4584, e-mail firstname.lastname@example.org).
Abstract: Gemifloxacin is a synthetic fluoroquinolone antimicrobial agent that exhibits potent activity against most Gram-negative and Gram-positive organisms, and has a comparatively low chondrotoxic potential in immature animals. This study examined the effects of gemifloxacin on the Achilles tendons in immature Sprague-Dawley rats treated by oral intubation once daily for 5 consecutive days from postnatal week 4 onward at doses of 0 (vehicle), and 600 mg/kg body weight. Ofloxacin or ciprofloxacin were used for comparison. The Achilles tendon specimens were examined by electron microscopy. In comparison with the vehicle-treated controls, there were ultrastructural changes in all samples from the gemifloxacin-, ofloxacin-, and ciprofloxacin-treated rats. Degenerative changes were observed in the tenocytes, and the cells that detached from the extracellular matrix were recognizable. The degree of degenerative changes and the number of degenerated cells in the Achilles tendon were significantly higher in the treated group than in the control group. Moreover, among the quinolone-treated groups, these findings were most significant in the ofloxacin-treated group, and least significant in the gemifloxacin-treated group. It is unclear what these findings mean with respect to the possible risk in juvenile patients treated with gemifloxacin or other quinolones. However, these results show that gemifloxacin causes less changes in the connective tissue structures.
Gemifloxacin is one of the recently developed fluoroquinolones (Factive, LB20304a). It is a potent, novel quinolone with broad-spectrum antibacterial activity against both Gram-negative and Gram-positive pathogens (Oh et al. 1996). The side effect profiles such as diarrhoea, rash, nausea, and headache are similar to those of the older members of this class (Saravolatz & Leggett 2003).
Musculoskeletal side-effects represent a small but significant fraction of the side-effects observed after treatment with fluoroquinolones (Stahlmann 2002). Besides arthralgia and myalgia, cases of tendinitis and tendon rupture have been described. As with quinolone-induced arthropathy, most cases of tendon disorders have occurred with the use of pefloxacin. Tendinitis associated with other drugs such as ciprofloxacin, ofloxacin, norfloxacin, and enoxacin has also been reported but the incidence appears to be much lower (Christ & Esch 1994; Carrasco et al. 1997; Stahlmann & Lode 2003).
There is little clinical information on quinolone-induced tendopathy. Selective clinical studies regarding the side-effects have not been published, and there is limited toxicological data. Some studies in rats do not appear to reflect the clinical situation because the alterations are detectable only in juvenile rats and can be prevented by co-administration of dexamethasone and N-nitro-L-arginine methyl ester (Kato et al. 1995; Kashida & Kato 1997a & b).
Tendopathy is one of the major adverse effects caused by quinolone antibacterial agents, but the incidence of tendopathy is generally low at the clinical doses of the quinolones used (Hooper & Wolfson 1993; Takayama et al. 1995; Stahlmann & Lode 1999). This study examined the effects of gemifloxacin on the Achilles tendon in immature rats with ofloxacin and ciprofloxacin used for comparison. A relatively high dose of quinolones, which can induce tendopathy and/or arthropathy in juvenile rats, was used in the study.
Materials and Methods
Animals. Male Sprague-Dawley rats aged 3 weeks were obtained from a specific pathogen-free colony at Bio Genomics Inc. (Seoul, Korea) and were used after 1 week of quarantine and acclimatization. The animals were housed in a room maintained at a temperature of 23±3° and a relative humidity of 50±10% with artificial lighting from 8 a.m.–8 p.m. and 13–15 air changes/hr. The rats were housed in clear polycarbonate cages with stainless steel wire lids and were provided with sterilized tap water and commercial rodent chow (Jeil Feed Co., Daejeon, Korea) ad libitum. The animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NRC 1996). The Ethics Committee of the Chosun University approved the protocol for this study.
Test chemical and treatment. The test chemical, gemifloxacin (Factive, LB20304a, LG Life Sciences Ltd., Daejon, Korea) as supplied by LG Life Sciences, was dissolved in saline solution and administered by gavage. The individual dose volume (10 ml/kg body weight) was determined according to the body weight immediately before administration. The ofloxacin (Ofcoxin inj., Samu Chem. Ind. Co. Ltd., Yesan, Korea) and ciprofloxacin (New-Floxin Liq., Yoonee Chemical Co. Ltd., Yesan, Korea) were suspended in saline solution and administered by gavage at a volume of 10 ml/kg body weight.
Although the molecular weights and bioavailability of three quinolones are somewhat different, a constant dose was used because there is no accurate bioavailability data available for these three quinolones, particularly regarding juvenile rats. In addition, these quinolones tested have been used at clinically similar doses of 300 to 600 mg/person/day.
The rats were treated once daily for 5 days by oral intubation with gemifloxacin at 0 (vehicle), and 600 mg/kg body weight. The animals were treated from postnatal day 30 to day 34 before being sacrificed, and samples were collected within 24 hr after the final dose. Other rats of the same age were treated with either ofloxacin or ciprofloxacin at 600 mg/kg (once daily for 5 days).
Transmission electron microscopy. The Achilles tendon samples were prepared from the right foot of five rats from each dosage group. Tangential sections were made from the distal part of the tendon using a razor blade. Subsequently, these tendons were cut crosswise to prepare ultrathin sections. Five sections of 1 mm3 were obtained from each tendon. All the samples were immersion-fixed in 1% glutaraldehyde plus 1% tannic acid in 0.1 M phosphate buffer (pH 7.4), and then fixed in 1% OsO4 in a phosphate buffer. The samples were embedded in Epon resin (Plano, Marburg, Germany) after rinsing and dehydrating them in ethanol. Eighty nm thick sections were cut using an Ultracut E (Reichert), and stained with 2% uranyl acetate/lead citrate. The specimens were examined by transmission electron microscopy (TEM, Hitachi H-7600, Japan).
Quantitative evaluation of tenocytes with pathological alterations. Ultrathin sections of the tendons from the control and treated rats from each group were prepared and evaluated using electron microscopy. Two independent observers (S. C. Lim and C. S. Bae), who were blinded to the sample sources, examined the pathological alterations. Consensus scores were assigned for each case by reviewing those cases with large discrepancies in scoring. The number of degenerated cells was determined by scoring a total 60 cells from 20 different microscopic fields.
There were no adverse clinical signs, body weight changes and feed intake in any of the treated groups throughout the observation period.
Characteristic changes at the ultrastructural level were found in all tendon samples studied from all gemifloxacin-, ciprofloxacin- or ofloxacin-treated rats.
Control group. The tenocytes of the Achilles tendon with a smooth well-demarcated cell border were embedded in an organized regular matrix with thick collagenous fibrils. The tenocytes formed plate- and finger-like long processes that were embedded between the fibers. The cells contained a well-developed rough endoplasmic reticulum, a Golgi apparatus and a large nucleus with loosely packed euchromatin. The pericellular unorganized matrix was closely attached to the cell membrane of the tenocyte (fig. 1).
In comparison with the controls, cellular and matrix alterations were observed in the quinolone-treated rats. Typical findings were degenerative changes such as vacuoles and vesicle formation in the cytoplasm of the tenocytes, which resulted from swelling and dilatation of the cell organelles.
Gemifloxacin treated group. Only slight changes were found in the tendon. The tenocytes of the Achilles tendon showed a relatively smooth cell border with a dilatation of intracellular micro-organelles such as the mitochondria and endoplasmic reticulum. The tenocytes detached focally from the extracellular matrix and had an indistinct cell border (fig. 2). Alterations, such as a dilatation of the intracellular micro-organelles and an indistinct cell border, were less evident in this group.
Ciprofloxacin-treated group. The tenocytes of the Achilles tendon showed an irregular cell border with degenerative changes, such as indented irregular cytoplasmic borders with disruption of the cell membrane, swelling and dilatation of the cell organelles (mitochondria and endoplasmic reticulum), densified nuclei with clumped chromatin, and irregular arrangement of extracellular collagenous fibrils. The tenocytes were detached from the surrounding matrix and contained cell debris. Some parts of the degenerated cytoplasmic processes of the tenocytes were embedded between the surrounding collagenous fibrils. In addition to the cellular alterations, there were some changes in the extracellular matrix (fig. 3).
Ofloxacin-treated group. Rather strong changes were obvious in the animals treated with ofloxacin. The tenocytes of the Achilles tendon showed degenerative changes, such as deeply indented irregular cytoplasmic borders, densified nuclei with clumped chromatin, multiple vacuoles and large vesicles in the cytoplasm, swelling and dilatation of the cell organelles (mitochondria and endoplasmic reticulum), and a decrease in the distance between the collagenous fibrils. Dilatations and vacuoles developed in the cytoplasm of the tenocytes due to swelling of the rough endoplasmic reticulum and mitochondria. The tenocytes detached from the extracellular matrix and had an indistinct cell border suggesting cell membrane disruption. Besides the cellular alterations, there were some changes in the extracellular matrix (fig. 4).
Quantitative evaluation. Quantitative analysis revealed that the number of pathologically altered cells increased in the samples obtained from the gemifloxacin-, ciprofloxacin- and ofloxacin-treated groups, particularly in those of the ofloxacin-treated group. The differences between these groups and controls were statistically significant (Student's t-test, P<0.05) (table 1).
Table 1. Quantitative analysis of the tenocytes with pathological alterations in the gemifloxacin, ciprofloxacin and ofloxacin treated groups.
Quinolones are antibacterial agents that have the potential to cause Achilles tendon disorders such as tendinitis or even ruptures (Shakibaei et al. 2001b). Quinolone treatment is contraindicated in juveniles and are only used in paediatric patients in rare cases (Schaad 2000). This study show that there are no adverse effects on clinical signs, body weight changes and feed intake in any of the treated groups throughout the observation period. This could be expected because the toxic potencies of these quinolones are low. The oral LD50 values for rats and mice are >2,000 and 5,000 mg/kg body weight, respectively.
In addition to clinical experience, a number of toxicological studies have confirmed that quinolone-induced tendopathy is a drug-induced, dose-dependent toxic effect of these agents (Stahlmann 2002). Kato et al. (1995) described quinolone-induced tendopathy after single oral administration of pefloxacin or ofloxacin. Tendon lesions were induced in immature rats (4 weeks of age) but not in 12-week-old rats. Tendon lesions were inhibited by co-administration of dexamethasone and N-nitro-L-arginine methyl ester. Phenidone (1-phenyl-3-pyrazolidinone) and 2-(12-hydroxydodeca-5,10-diynyl)3,5,6-trimethyl-1,4-benzoquinone (AA861) also decreased the incidence of tendon lesions. In contrast, catalase, dimethyl sulfoxide, indomethacin, pyrilamine, and cimetidine did not modify these tendon lesions. This suggests that nitric oxide and 5-lipoxygenase products partly mediate fluoroquinolone-induced tendon lesions (Kashida & Kato 1997a).
A group of French toxicologists examined the effect of pefloxacin on Achilles tendon proteoglycans and collagen in rodents, and provided convincing evidence that quinolone-induced oxidative stress on the Achilles tendon altered the proteoglycan anabolism and oxidized collagen. Biphasic changes in proteoglycan synthesis were observed after a single dose of pefloxacin, which consisted of an early inhibition followed by a repair-like phase. Pefloxacin treatment for several days induced oxidative damage to collagen type I, with the alterations being identical to those observed in an experimental tendinous ischaemia and reperfusion model. Oxidative damage was prevented by co-administration of N-acetylcysteine (Simonin et al. 2000).
Recent experiments have shown that ultrastructural alterations in tenocytes can be observed in immature and adult rats after treatment with quinolones. These effects were more pronounced when the animals were simultaneously given a magnesium-deficient diet, suggesting that the pathophysiology of tendopathy resembles that of arthropathy (Shakibaei et al. 2000). When Shakibaei & Stahlmann (2001) examined the Achilles tendons from quinolone-treated adult rats by electron microscopy 4–12 weeks after treatment with single oral doses of ofloxacin, levofloxacin or fleroxacin, they could detect specific, pathological alterations already at the lowest dose (30 mg/kg), which increased in severity with increasing dose. The tenocytes detached from the extracellular matrix and showed degenerative changes such as multiple vacuoles and large vesicles in the cytoplasm, which resulted from swelling and dilatation of the cell organelles (mitochondria, endoplasmic reticulum). Other findings were a general decrease in the fibril diameter and an increase in the distance between the collagenous fibrils (Shakibaei et al. 2001a).
The effects of gemifloxacin are of special interest because of the low chondrotoxic potential. It is unclear how ultrastructural changes in the Achilles tendons from immature rats relate to the potential risk in juvenile patients treated with gemifloxacin. However, these results underline the fact that, in principle, this new fluoroquinolone with a pyrrolidine derivative at the C-7 position has less potential to cause changes in the connective tissue structures. Further toxicological and clinical studies will be needed to characterize the conditions under which quinolone-induced tendon lesions develop.
This work was supported in part by a grant from the KOSEF project (R01-2004-000-10013-0), Ministry of Science and Technology, Republic of Korea.