International Journal of Pediatric Otorhinolaryngology 74 (2010) 361364

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International Journal of Pediatric Otorhinolaryngology

journal homepage: www.elsevier.com/locate/ijporl

Ototoxicity caused by once- and twice-daily administration of amikacin in rabbits

Pavlos Pavlidis a,b,c, Vasilios Nikolaidis a, Haralampos Gouveris c, Elias Papadopoulos d, Georgios Kekes a, Dimitrios Kouvelas b,*
a

2nd Department of Otolaryngology, School of Medicine, Aristotle University, Papageorgiou G.H., Thessaloniki, Greece 2nd Laboratory of Pharmacology, School of Medicine, Aristotle University of Thessaloniki, Greece c Department of Otolaryngology, School of Medicine, University of Thessaly, Larissa, Greece d Veterinary School, Aristotle University of Thessaloniki, Greece
b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 July 2009 Received in revised form 21 December 2009 Accepted 22 December 2009 Available online 20 January 2010 Keywords: Ototoxicity Cochlear Amikacin Post-antibiotic effect Distortion Product Otoacoustic Emissions

Objective: The cochleotoxic effects of aminoglycosides, such as amikacin, are well-established. The aim of the present study was to investigate the possible differences in cochleotoxic effects between once-daily administration (ODA) and twice-daily administration (TDA) of amikacin simulating pediatric dosing. Methods: Twenty-one rabbits were used. Seven animals received intramuscularly amikacin once daily (ODA-group) and seven received the drug twice daily (TDA-group), for a total time period of 2 weeks. All the animals were subjected to Distortion Product Otoacoustic Emissions (DPOAEs) every 3 days since beginning of the experiment. The rest 7 animals did not receive any medication and served as controls (Control group). Two measurements (7 and 14 days) were obtained following the cease of drug administration. Results: Reduced cochlear activity (as depicted in the respective reduced DPOAE-amplitudes) compared to the pre-treatment state was found in both ODA- and TDA-groups. Cochlear activity was reduced at a wider range of frequencies (from 593 to 4031 Hz in TDA-group and from 593 to 1093 Hz in ODA-group) and to a higher degree in group B than in group A. Cochlear activity was reduced earlier in ODA-group than in TDA-group. No differences to the pre-treatment state were observed in the control group. Conclusions: The above ndings suggest that less frequent administration in higher dose of amikacin is associated with minimal cochleotoxicity. 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Aminoglycosides are bactericidal aminoglycosidic aminocyclitols. They were discovered in the 1940s and are the treatment of choice for tuberculosis and advanced bacterial infections. They were the rst class of drugs to call the attention to the problem of ototoxicity when streptomycin and dihydrostreptomycin were used to treat tuberculosis [1]. Dosing regimens for aminoglycosides, can be classied as conventional or pulse. The majority of clinicians is familiar with conventional dosing in which the antibiotic is administered in equal doses every 812 h. Pulse-dosing involves administering the drug in a single dose per dosing interval. Many authors prefer the term pulse-dosing to the term once-daily aminoglycoside because the interval may exceed 24 h [2]. This type of regimen

aims at achieving optimum concentration-dependent bactericidal activity to maximize efcacy, avoid rst-exposure adaptive bacterial resistance and use better post-antibiotic effect [37]. In the last two decades, animal experiments and clinical studies supported the efcacy of once-daily, high doses of aminoglycosides for severe gram-negative infections [8,9]. The aim of the present study was to investigate the possible differences in cochleotoxic effects between once-daily administration (ODA) and twice-daily administration (TDA) of amikacin with the use of DPOAE. 2. Methods Every effort was made in order to minimize pain and discomfort to the animals throughout the study. For this purpose, experiments have been conducted in accordance to the European Communities Council Directive of November 24 1986 (86/609/EEC) and approved by the Animal Care and Use Committee of the National Veterinary Institute and the Ethical Committee of our institution. Twenty-one, female, 3-month old, New Zealand, rabbits, weighting 10001500 g, were studied prospectively every day, for 14 days. All the animals had free access to food and water.

* Corresponding author at: P.O. Box 1532, 54006 Thessaloniki, Greece. Tel.: +30 2310 999335; fax: +30 2310 999335. E-mail addresses: pavlof@otenet.gr (P. Pavlidis), athk@med.auth.gr (V. Nikolaidis), hagouve@yahoo.de (H. Gouveris), eliaspap@vet.auth.gr (E. Papadopoulos), mariakeke@yahoo.gr (G. Kekes), kouvelas@auth.gr (D. Kouvelas). 0165-5876/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2009.12.018

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Initially, the cochlear activity of the animals was examined with DPOAEs in a sound-proof booth using the GSI 60 equipment (Grason Stadler, Milford, New Hampshire, USA) for DPOAE recording. Exclusion criterion from the experiment was an abnormal pre-treatment cochlear activity. The rabbits were divided in three equal groups. The members of the rst two groups were injected with 15 mg/kg (i.m.) of amikacin, every day, for 14 days. The dosage, approved for veterinary purposes is 816 mg/kg/day (i.m.). The remaining 7 animals served as Control group. The rabbits of the rst group (ODA group) were injected with amikacin 15 mg/kg (i.m.), once-daily and those of the second group (TDA group) were injected the same total dose divided in two equal doses. The rabbits of the Control group received a saline injection twice daily. Body weights were monitored daily and the administered dose of amikacin was adjusted accordingly. The cochlear activity of the right ear of all rabbits was examined every 4 days using DPOAEs, in conscious animals. The Vanderbilt protocol for DPOAE-measurements was used. The DPOAE test protocol included an f2/f1 ratio of 1.22, stimulus intensity levels of L1 = L2 = 65 dB SPL, f2 values from 593 to 6781 Hz (3 test frequencies/octave), and 16 averages/frequency [10]. Two additional DPOAE-measurements were also obtained on days 7 and 14 after the discontinuation of the drug, in order to detect any possible delayed deterioration or improvement of the cochlear activity. The numerical values of the intensities of the DP recorded on day 14 of drug administration were compared between the ODAand the TDA-groups. For this purpose KruskalWallis and Mann Whitney U tests were used. The level of statistical signicance was set at p < 0.05. The Bonferroni correction was used as needed. The results were analyzed with SPSS 12 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results Differences in DPOAE-amplitudes, and therefore in cochlear activity, between the two experimental groups were revealed. The decrease of cochlear activity in ODA-group involved frequencies between 593 and 1187 Hz (lower and higher frequency, respectively). All frequencies refer to F2. No other frequencies were affected during the 28-day period of the experiment. The cochlear activity in the TDA-group has shown increased deterioration compared to the respective activity of the ODAgroup. The deterioration involved frequencies between 593 and 4031 Hz. Cochlear activity was reduced earlier in the ODA-group compared to the TDA-group, since a reduction in DPOAEamplitude was already observed in the rst DPOAE-measurement. In two of the rabbits of the TDA-group, reduction of cochlear activity was observed in the second DPOAE-measurement (day 8 of the experiment). DPOAEs did not shown any additional change (deterioration or improvement) since the cease of drug administration, indicating that the reduction of the cochlear activity was due to injury of the cochlear outer hair cells. Moreover, no improvement was observed in DPOAE-measurements obtained 7 and 14 days after the discontinuation of amikacin. The mean of DPs intensities, as they were recorded on the 14th day, is presented in Fig. 1. Reduction of DPs intensities was observed mainly in the frequencies affected. Statistical signicant difference between the ODA- and the TDA-groups was measured in F2-frequencies 1687 Hz (p = 0.02), 2031 Hz (p = 0.02), 2404 Hz (p = 0.021), 2843 Hz (p = 0.018), 3406 Hz (p = 0.03) and 4031 Hz (p = 0.038) (Figs. 13). No changes in cochlear activity were found at the Control group.

Fig. 1. The reduction of the DPs intensity, in the ODA- and TDA-groups, on the day 14. The black boxes correspond to the Control group, the white to the ODA- and those with the white and black lines to the TDA-group. Distortion Products intensity (dB SPL), in normal subjects is positive. Values are means S.D., n = 7 rabbits/group. *p < 0.05 (KruskalWallis and MannWhitney U tests). Statistical signicant difference between the ODA- and the TDA-groups was detected in F2frequencies 1687 Hz (p = 0.02), 2031 Hz (p = 0.02), 2404 Hz (p = 0.021), 2843 Hz (p = 0.018), 3406 Hz (p = 0.03) and 4031 Hz (p = 0.038). n = 7, *p < 0.05, **p < 0.01.

Fig. 2. The range of affected frequencies (mean S.D.) in the ODA-group in relation to time (each frequency corresponds to the F2 primary frequency of the respective DPOAE-gram). Reduction of cochlear activity is observed between 593 and 1187 Hz. The continuous line and the black boxes correspond to the higher affected frequencies. The dashed line and the black circles correspond to lower affected frequencies. The frequencies with the asterisks correspond to the higher frequencies F2 in which the DP differed signicantly compared to that of the TDA group in the same frequencies. n = 7, *p < 0.05, **p < 0.01.

Fig. 3. The range of affected frequencies (mean S.D.) in the TDA-group in relation to time (each frequency corresponds to the F2 primary frequency of the respective DPOAE-gram). Reduction of cochlear activity is observed between 593 and 4031 Hz. The continuous line and the black boxes correspond to the higher affected frequencies. The dashed line and the black circles correspond to lower affected frequencies. The frequencies with the asterisks correspond to the higher frequencies F2 in which the DP differed signicantly compared to that of the TDA group in the same frequencies. n = 7, *p < 0.05, **p < 0.01.

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The ranges of frequencies, in which a reduction in cochlear activity was observed, are shown in Fig. 2, for ODA and in Fig. 3, for TDA-group, respectively. 4. Discussion The aim of the present study was to examine potential functional differences, between one and two doses of daily amikacin administration. The ODA regimen was designed to simulate pediatric dosing. For this purpose a rabbit model was used in order to investigate hearing impairment. Rabbit models have been used in the past in order to investigate hearing impairment after drug administration [11], closed head injury [12] and noise-induced changes in suppression in DPOAEs [13,14]. Ototoxicity is a severe side effect of aminoglycoside-administration, because it is, in most cases, permanent. Ototoxic phenomena are associated with factors such as the duration of treatment, the number of treatment courses with aminoglycosides and the total quantity of administered drug and the pre-existence or not of hearing impairment [15]. A number of different hypotheses have been proposed to explain the ototoxic effects of aminoglycosides. Some investigators suggest that aminoglycosides disrupt mitochondrial protein synthesis in hair cells due to the molecular similarities between human mitochondrial and bacterial ribosome [16]. Others claimed the idea of reversible inhibition of the sensory transduction by blocking Ca2+-sensitive K+-channels of the tubulo-vesicular cell system [17]. It has also been suggested that aminoglycosides act as agonists of the Nmethyl-D-aspartate (NMDA) subtype of glutamate receptor resulting in excitotoxicity [18]. Individual aminoglycosides differ in their ability to produce cochlear versus vestibular toxicity. Cochlear damage is generally observed when amikacin, kanamycin and neomycin are used. The aminoglycosides enter the cochlea after systematic administration, but the distribution within inner ear tissues does not correlate with their preferential toxicity to particular cells in the cochlea and vestibular system. It has been suggested that the aminoglycosides persist in the inner ear tissues for 6 months or longer after administration [19]. Identication of patients at a high risk for ototoxic hearing loss is important to avoid such a complication. The risk of ototoxic effects can be reduced by monitoring hearing with repeated puretone audiometry [4]. One of the methods used for monitoring aminoglycoside ototoxicity is the Distortion Product Otoacoustic Emissions (DPOAEs). DPOAEs are responses generated when the cochlea is stimulated simultaneously by two pure-tone frequencies (F1 and F2). They are an objective, non-invasive and rapid method for the evaluation of the outer hair cell function. Clinical studies in humans and experimental studies in animal models have established a link between drug-induced changes in DPOAEs and changes in the outer hair cells [20]. In many patients, such as children with cystic brosis treated with aminoglycoside antibiotics [21] and adults with cancer receiving cisplatin [22], ototoxic effects in behavioral audiometry occurred later than in otoacoustic emissions (OAE). Advantages of using DPOAEs as objective measures of ototoxicity include their frequency specicity and their measurability over a wide frequency range. Many experimental [22] and clinical studies [23,24] have taken advantage of these parameters. The results of the present study show that both ODA and TDA are responsible for reduction of cochlear activity. There are signicant differences between the two regimens concerning the range of the affected frequencies. Another interesting clue, which needs further investigation, is the fact that the reduction of cochlear activity begun earlier in TDA- than in ODA-group.

It is known that damage to hair cells progresses from the base of the cochlea (an area for high frequency sound detection) to the apex (an area for low frequency sound detection) [25]. In the present study, cochleotoxic effects at high frequencies in rabbits given the ODA regimen as it is observed in human were not revealed. This may be due to the differences of hearing acuity between rabbits and human. The signicance of animal experiments for predicting therapeutic outcome and toxicity in patients may be limited by various factors. Pharmacokinetic differences between humans and small animals may interfere with direct clinical application of the results for optimal dosing schedule [26]. Once-daily administration of aminoglycosides is advantageous, because not only of their concentration-dependent bactericide ability but also of two other important factors, the time-dependent bactericide toxicity and a more prolonged post-antibiotic effect. ODA regimens enhance concentration-dependent killing by maximizing the peak concentration/minimal inhibitory concentration (MIC) ratio for the infecting organisms. Clinical trials showed no differences in efcacy of amikacin between the once and the twice-a-day groups with respect to drug dosage, duration of therapy and concomitant treatment [26,27]. The ODA administration of aminoglycosides is an attractive issue. The results from clinical trials are promising and the potential benets to the patient, concerning ototoxicity, are quite satisfactory [27,28]. It seems that less frequent dosing reduces the contact time between host tissues and the drug. The once-daily administration of aminoglycosides is a concept which needs further investigation. Conict of interest None. References
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