Epileptogenic Activity of Methicillin-Resistant Staphylococcus aureus (MRSA) Antibiotics in Rats

Transcription

1 October 2006 Biol. Pharm. Bull. 29(10) (2006) 2035 Epileptogenic Activity of Methicillin-Resistant Staphylococcus aureus (MRSA) Antibiotics in Rats Md. Ashequr RAHMAN, Jun AGO, Naotaka MATSUMOTO, Takashi ISHIKAWA, and Chiaki KAMEI* Department of Medicinal Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences; Tsushima-naka, Okayama , Japan. Received May 29, 2006; accepted July 18, 2006; published online July 20, 2006 The present study was undertaken to clarify the epileptogenic activity induced by intracerebroventricular injection (i.c.v.) of antibiotics effective in methicillin-resistant Staphylococcus aureus (MRSA) in chronically electrode implanted rats. Teicoplanin ( mg, i.c.v.) caused dose-related electroencephalographic (EEG) seizure characterized by an uninterrupted high voltage and wave complex. At the same time, the rats showed forelimb clonus, head nodding, jumping and severe convulsion. At a high dose (100 mg, i.c.v.), the drug caused a severe twisting immediately after the intracerebroventricular injection (i.c.v.) followed by jumping and violent convulsion with a continuous rhythmic spike and wave complex in EEG. On the other hand, vancomycin ( mg, i.c.v.) caused no or almost no epileptogenic activity in terms of behavior and in EEG. However, at a high dose (1000 mg, i.c.v.), the drug caused an occasional spike from the hippocampus without showing any behavioral changes in the rats. Fosfomycin ( mg, i.c.v.), cefazolin ( mg, i.c.v.) and penicillin G ( mg, i.c.v.), used as reference drugs, caused dose-dependent epileptogenic activity in both EEG. From these findings, it was found that teicoplanin caused a potent epileptogenic activity, different to vancomycin. Therefore, it can be concluded that vancomycin may be safety on epileptogenic activity used for the clinical purpose of infections caused by MRSA. Key words methicillin-resistance Staphylococcus aureus (MRSA); vancomycin; teicoplanin; epileptogenic activity; electroencephalographic (EEG); intracerebroventricular injection (i.c.v.) It is well known that some strains of Staphylococcus aureus have developed resistance to methicillin, and this has become an important public-health problem in all over the world. 1,2) Methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening infections, such as meningitis, bone marrow destruction, sepsis, endocarditis, septic pulmonary emboli and extensive soft tissue infection in humans; and for these, people seek an effective and safe treatment. 3 5) Therefore, extensive research has been ongoing to find an effective and safe antibiotic to treat the infections caused by MRSA. Now, there exist some MRSA antibiotics, such as vancomycin, teicoplanin and arbekacin that have been widely used in clinical practice for the treatment of infections caused by MRSA. Zetola et al. 2) reported that vancomycin could be considered as a first line therapy for the treatment of infections caused by MRSA. In addition, Boger 6) demonstrated that vancomycin is used for the treatment of infections in patients allergic to beta-lactam antibiotics. Furthermore, Losonsky et al. 7) found the safety and potential usefulness of teicoplanin in meningitis caused by Enterococcus faecium. On the other hand, we have reported that a large number of beta-lactam antibiotics, such as penicillin G and cephalosporins, caused a potent epileptogenic activity in rats when administered intravenously or intracerebroventricularly. 8,9) Therefore, in the present study, we investigated to clarify whether or not MRSA antibiotics elicit an epileptogenic activity in rats. MATERIALS AND METHODS Animals Six-eight week old male Wistar rats (body weight g) were obtained from Japan SLC, Shizuoka, Japan. The animals were housed in an air-conditioned room maintained at 24 2 C with a relative humidity of 55 15%. They were kept in aluminum cages with sawdust. The rats were given standard laboratory rodent chow (Oriental Yeast, Tokyo, Japan) and water ad libitum. All procedures involving the animals were conducted in accordance with the Guidelines for Animal Experiments at Okayama University Advanced Science Research Centers and all procedures were licensed by the Animal Research Control Committee of Okayama University. Drugs The drugs used were cefazolin sodium (Cefamezin, Fujisawa), penicillin G potassium (Banyu Co.), vancomycin hydrochloride (Sigma, St. Louis, MO, U.S.A.) and fosfomycin sodium (Fosmicin-S, Meiji Seika Kaisha, Ltd., Tokyo, Japan). Teicoplanin (Targocid) was kindly provided by Hoechst Marion Roussel Ltd., Japan. All drugs were dissolved in distilled water. Intracerebroventricular injection was performed through an injection cannula, which was fitted inside the guide cannula, and for all drugs 10 ml per head was given within 15 s. Drugs were administered at intervals of 7 d when the same animals were used for repeated experiments. Groups of 5 rats were used for each dose of the test drugs. Implantation of Electrodes and Cannula The animals were anesthetized with sodium pentobarbital (Nembutal, 35 mg/kg, i.p., Abbott Laboratories, North Chicago, IL, U.S.A.), then fixed to a stereotaxic apparatus (Narishige, Type SR-5, Tokyo, Japan). For EEG recording, monopolar electrodes were chronically implanted into the right frontal cortex (area 3, A: 6.9, L: 3.0), right hippocampus (A: 3.0, L: 2.5, H: 2.0), and right amygdala (A: 5.0, L: 5.0, H: 3.5) according to the stereotaxic atlas of Krieg 10) or de Groot. 11) An additional stainless steel screw electrode was also placed at the posterior end of the skull to serve as the reference electrode. Sub-cortical electrodes were made of stainless steel To whom correspondence should be addressed. kamei@pheasant.pharm.okayama-u.ac.jp 2006 Pharmaceutical Society of Japan

2 2036 Vol. 29, No. 10 wire, 200 mm in diameter, and insulated except for 0.5 mm of the tip. Small screws serving as recording electrodes were inserted into the skull. A guide cannula made of stainless steel tubing, 700 mm in outside diameter, was implanted into the right lateral ventricle (A: 0.5, L: 2.0, H: 3.4). Electrodes were connected to a miniature receptacle, and the whole assembly was fixed to the skull together with guide cannula with dental cement. At least 10 d were allowed for recovery from the surgery. After the experiments, the animals were killed with sodium pentobarbital (100 mg/kg, i.p.) and the localization of the electrodes in the brain was verified histologically. In this experiment, the gross localization of the electrodes in the brain was checked during the dissection of the brain region. Measurement of EEG and Behavior The animals were placed in a soundproof observation chamber. EEG was recorded monopolarly with an electroencephalograph (Model EEG-7314, Nihon Kohden, Tokyo, Japan), and at the same time behavioral observation was made. The extent of the EEG changes and behavioral seizures induced by the drugs were estimated using the scoring system shown in Table 1 and the ED 50 was calculated according to the method of probit. Statistical Analysis The statistical significance of differences in the EEG and behavior was determined using the Mann-Whitney U test. p values of 0.05 were considered statistically significant. RESULTS Figure 1 and Table 2 show the epileptogenic activity induced by intracerebroventricular administration of teicoplanin. Teicoplanin caused some EEG activation in the hippocampus and amygdala with occasional wet dog behavior at a dose of 10 mg. However, at a dose of 30 mg, the drug showed an uninterrupted high voltage spike and wave complex in EEG and a potent epileptogenic activity in behavior characterized by forelimb clonus and head nodding within a few minutes after injection. Upon the intracerebroventricular injection of teicoplanin 100 mg, the animal immediately showed a severe twitching within a few seconds, and this lasted for few minutes, followed by violent convulsions and muscle relaxation. The animals also showed jumping and violent convulsions, and consistently died of dyspnea under status epilepticus within approximately 30 min. In parallel with the violent convulsion, rhythmic spikes or high voltage spike and wave complexes in EEG were observed for an initial 10 min and the gradual disappearance of spikes was also found after 15 min and all the rats died within 30 min due to severe dyspnea. The epileptogenic activity induced by the intracerebroventricular administration of vancomycin is shown in Fig. 2 Table 1. Scoring System Used for Estimation of Seizure Intensity Score EEG Behavior 0 No change No convulsions 1 Spike Twitching and wet dog shaking 2 Occasional appearance Forelimb clonus and of spike-and-wave complex head nodding 3 Burst of spike-and-wave complex with Kangaroo posture short duration appearing no more and falling back than twice 4 Uninterrupted burst of spike-and-wave Jumping and violent complex with high amplitude and convulsions high frequency Fig. 1. Epileptogenic Activity Induced by Intracerebroventricular Injection of Teicoplanin

3 October and Table 2. At a dose of 100 mg, vancomycin caused no remarkable seizures on either EEG or in behavior. Occasional spikes after 15 min of injection were found in the hippocampus at a dose of 300 mg without any change in behavior. At a dose of 1000 mg, the spikes were found to propagate to the frontal cortex and amygdala; and in behavior, twitching was also observed. Figure 3 and Table 2 show the epileptogenic activity induced by the intracerebroventricular injection of fosfomycin. Animals showed a spike, spike wave complex in EEG from the dose of 100 mg; and in behavior, twitching, wet dog shakes, forelimb clonus, head nodding, Table 2. Epileptogenic Activity Induced by Intracerebroventricular Injections of MRSA and Reference Antibiotics in Rats Drugs Dose (mg) N EEG Score Behavior Lethality (%) Teicoplanin * * * 100 Vancomycin Fosfomycin * * 60 Cefazolin * * * * 80 Penicillin G * * * 60 Significantly different from the control group; p kangaroo posture and falling back were observed. Moreover, with a dose of 1000 mg, fosfomycin evoked potent epileptogenic activity. After 5 min of intracerebroventricular injection, a spike and wave complex with a high amplitude was observed in association with forelimb clonus, head nodding, kangaroo posture and falling back, and violent convulsion. These EEG patterns and physical behaviors lasted for about 30 min, and these signs disappeared after 60 min. Similar changes in EEG and behavior were observed after the administration of penicillin G (Fig. 4, Table 2) and cefazolin (Fig. 5, Table 2). Cefazolin, at the doses of 30 and 100 mg elicited severe seizures in animals. Initially, it produced a spike, spike and wave complex in the frontal cortex and after 5 min it propagated to other regions of the brain, the hippocampus and the amygdala and showed an uninterrupted burst of spike with high amplitude and high frequency. Behaviorally, it also evoked twitching, forelimb clonus, head nodding, kangaroo posture and falling back, jumping and violent convulsions. All seizures lasted for 60 min and four of the 5 animals died after 1 h. Severe signs of seizures were noticed both in EEG and behavior after the intracerebroventricular injection of penicillin G in a dose related manner. Penicillin G at doses of 100 and 300 mg induced a repeated seizure characterized by a spike and wave complex in EEG and jumping and violent convulsions in behavior. Three out of 5 animals died from dyspnea within 60 min at a dose of 300 mg. The ED 50 values of epileptogenic activity induced by MRSA and reference antibiotics are shown in Table 3. As shown in Table 3, cefazolin exhibited the strongest epileptic activity among the antibiotics tested in both EEG and physical behavior. Teicoplanin also showed severe seizures in both EEG and behavior, but this effect was almost the same as those of cefazolin and penicillin G. Fig. 2. Non-epileptogenic Activity Induced by Intracerebroventricular Injection of Vancomycin

4 2038 Vol. 29, No. 10 Fig. 3. Epileptogenic Activity Induced by Intracerebroventricular Injection of Fosfomycin Fig. 4. Epileptogenic Activity Induced by Intracerebroventricular Injection of Penicillin G DISCUSSION In the present study, it was found that the intracerebroventricular injection of teicoplanin caused a potent epileptic seizure both in EEG and behavior in rats in a dose-related fashion. On the other hand, vancomycin showed no remarkable epileptogenic activity in both EEG and behavior in rats, even at a dose of 1000 mg. Grondahl and Langmoen 12) reported that vancomycin had no epileptic effects in humans, which is in analogous with our present study. In addition,

5 October Fig. 5. Epileptogenic Activity Induced by Intracerebroventricular Injection of Cefazolin Table 3. ED 50 Values of Epileptogenic Activity-Induced by MRSA and Reference Antibiotics in Rats Drugs ED 50 (95% confidence limits) mg/head, i.c.v. EEG Behavior Teicoplanin ( ) ( ) Vancomycin Fosfomycin ( ) ( ) Penicillin G ( ) ( ) Cefazolin ( ) ( ) Moellering 13) demonstrated that the treatment of MRSA meningitis with vancomycin is limited due to its low penetration into the cerebrospinal fluid (CSF). Therefore, it may be that vancomycin caused no epileptic seizures in rats due to its low penetration into the CSF to induce seizures. On the other hand, the intracerebroventricular injection of teicoplanin caused potent seizures in behavior and spikes in EEG in a dose-dependent manner. Furthermore, the animals showed a remarkable muscle relaxant activity, and 4 of the 5 rats died within 30 min. On the other hand, a spike and wave complex in EEG was observed at a dose of 100 mg for 10 min, and the gradual disappearance of the spikes was also noticed due to severe dyspnea after 15 min. In association with this, Bambeke 14) reported that teicoplanin has hydrophobic chain in its chemical structure and showed high penetration to the CSF, which may responsible for its potent epileptic activity in rats. Although there are some reports that the epileptogenic activity induced by penicillins and cephalosporins are considered to be due to the structural resemblance of beta-lactam and GABA antagonist, bicucculine. 15) On the other hand, in the present study it has been found that teicoplanin and fosfomycin caused a severe epileptic seizure in rats, but none of these antibiotics possess beta-lactam ring in their chemical structure. Therefore, it may be assumed that a beta-lactam ring is not the only contributor to the epileptogenic properties. Van Gelder et al. 16) demonstrated that the release of excitatory amino acids has been found to increase during seizures induced by penicillin G. Moreover, some excitatory amino acid antagonists inhibited the seizures induced by imipenem, which suggest the participation of excitatory amino acids in the genesis of seizures caused by carbapenem derivatives. 17) From the above observation, it may be considered that excitatory amino acid or other mechanisms may be involved in seizures induced by teicoplanin and fosfomycin, but further elucidation of the mechanisms is needed. In the present study, it was also found that the severity of epileptic seizures induced by teicoplanin was almost the same as cefazolin in terms of behavior (Table 3). On the hand, Morocutti et al. 18) reported that the intravenous injection of calcium antagonist, nimodipine, showed the significant inhibition of the cefazolin-induced epileptogenic foci in rabbits. In addition, nimodipine prevented penicillin-induced seizures in rats. Walden et al. 19) also demonstrated that the intracerebroventricular injection of verapamil reduced the amplitude and frequency of focal interictal epileptiform discharges (FIEDs) induced by penicillin in rats. On the basis of the reports mentioned above, it is quite reasonable to consider that the epileptic seizures induced by cefazolin and penicillin G are also associated with the influx of calcium ions. From these results, it is concluded that in comparison with the other antibiotics studied in this experiment, the epileptic

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