Aspects of the Chronic Toxicity of Gentamicin Sulfate in Cats

THE JOURNAL OF INFECTIOUS DISEASES VOL. 124, SUPPLEMENT DECEMBER 1971 1971 by the University of Chicago. All rights reserved. Aspects of the Chronic Toxicity of Gentamicin Sulfate in Cats J. Allan Wait, Eugene L. Moss, Jr., and Marvin J. Weinstein From the Microbiology Division, Schering Corporation, Bloomfield, New Jersey For determination of the importance of several factors affecting the vestibular toxicity of gentamicin in cats, several therapeutic regimens, routes of administration, and levels of dosage were examined. Cats were given high levels of gentamicin daily and observed for appearance of ataxia and impairment of the righting reflex. The time of appearance of ataxia was related to dosage. Death, attributed to renal toxicity, closely follows the appearance of ataxia after a dose of 60 mg/kg per day. At lower doses, there is a temporal separation between vestibular and renal toxicity. The renal damage is reversible, but the vestibular damage is not. Total dosage and duration of treatment appear to be more important factors than peak levels of drug in the serum with reference to time of appearance of ataxia at the dosage levels studied. The two major, potentially toxic effects associated with the aminoglycoside group of antibiotics are renal damage and damage to the vestibular or auditory functions of the eighth cranial nerve. Studies have shown that chronic administration of high doses (40-120 mg/kg) of gentamicin produces nephrotoxicity in dogs [1] and vestibular dysfunction in cats [1-5]. In guinea pigs given high doses for several weeks, both labyrinthine and cochlear damage were noted [6-9]. This report examines the importance of some aspects of the vestibular toxicity of gentamicin in cats. Methods All preparations of gentamicin used were in the form of the sulfate dissolved in sterile distilled water and were given in terms of the base in a volume of 0.5 nil/kg. Cats were male mongrels obtained from a local supplier and weighed 2.5 5.0 kg. All cats were conditioned for at least four weeks before use. Cats with indwelling cannulae in the carotid artery were prepared by Dr. W. Zeman, formerly of these laboratories, by procedures to be described elsewhere. During tests for ataxia, each cat was weighed daily and dropped from an inverted position 2 ft above a sponge-rubber mat for observation of impairment of righting reflex; cats were given gentamicin subcutaneously (except for those cats that received the drug intraarterially as discussed below). Animals were then observed for signs of ataxia as they traversed the room. Each cat was weighed, dropped, treated with drug, and walked daily, seven days each week (except as indicated). Each dose was given within 1 hr of 24 hr after the previous dose. Administration of gentamicin continued until frank ataxia or death occurred, except as noted below where treatment was reinstated after a rest period of one month. Assays of antibiotic were performed on I-ml samples of serum as described by Oden et al. [10]. Bacillus subtilis with a sensitivity of O.04g of gentamicin/ml was used. We thank Dr. William Zeman for preparing cannulated cats, Dr. Frank Fielder for histopathological evaluation, Mr. Edwin Oden for microbiological assays, and Mr. David Loebenberg for assistance with tests for ataxia. Please address requests for reprints to Dr. J. Allan Wait, Chemotherapy Department, Schering Corporation, Bloomfield, New Jersey 07003. Results One possible factor affecting the appearance of vestibular toxicity is level of the dosage. The results of a typical experiment with three levels of gentamicin sulfate are shown in table 1. The appearance of ataxia was obviously related to the S125

8126 Wait, Moss, and Weinstein Table 1. Chronic toxicity of gentamicin sulfate in cats given daily subcutaneous doses. Day righting Day Dose Day reflex of Cat (mg/kg/day) ataxic impaired death 3697 60 10 11 12 3482 60 10 11 11 5021 60 14 14 27 5273 60 14 14 23 3855 60 13 14 14 Mean 12.2 12.8 17.4 3923 40 18 17 28 5250 40 19 19 >70 3602 40 15 14 16 5188 40 15 15 15 5310 40 19 19 23 Mean 17.2 16.8 >30.4 5390 20 27 27 64 21549 20 34 34 >70 5385 20 35 34 >70 5298 20 35 35 >70 5137 20 26 26 40 Mean 31.4 31.2 >62.8 NOTE. Days given are number of days elapsed after beginning of treatment with gentamicin. dose. Ataxia was observed after an average of 12.2 days at a level of 60 rug/kg per day, 17.2 days at a level of 40 mg/kg per day, and 31.4 days at a level of 20 rug/kg per day. Impairment of the righting reflex always occurred within one day of the appearance of ataxia. It has been suggested that ataxia occurs after renal damage is sufficient to result in increased levels of gentamicin in serum. The cats in this experiment were treated continuously until death or until day 70, when the experiment was terminated. At the highest dosage level, animals died 1-13 days (average, 5.2) after the appearance of ataxia. Death was attributable to the effects of renal tubular necrosis on the basis of histopathologic examination. Of the five cats receiving 40 rug/kg per day, four died within one to 10 days after becoming ataxic. As with the group that received the highest dose,.this group had necrosis of renal tubules. The remaining cat in this group survived more than 50 days of treatment with gentamicin after becoming ataxic. When this cat was sacrificed on day 70, mild renal tubular damage was observed. Of the group that received 20 mg/kg per day, one died 14 days after becoming ataxic, while three became ataxic on day 35 and survived another 35 days of treatment. Histopathologic examination of these cats killed on day 70 revealed mild or no renal tubular damage, thus showing a temporal separation of renal and vestibular toxicities. These results suggested an experiment to determine the reversibility of vestibular and renal aspects of toxicity. Three cats were given gentamicin subcutaneously at a dose of 40 mg/kg per day until ataxia appeared in 16, 18, and 19 days respectively. Treatment of all three cats was stopped at the time ataxia appeared. Based on the data in table 1, continued treatment might be expected to result in death within a few days. All animals were observed daily and remained ataxic until day 45 (29-32 days without treatment), when treatment was started again at the same level of gentamicin. The three cats died 19-20 days after treatment recommenced. Histopathologic examination revealed the presence of renal tubular necrosis. The kidneys of these animals apparently were repaired sufficiently in the month of rest to permit another 20-day course of treatment before death. The ataxia was irreversible. The possibility that the appearance of ataxia might contain an idiosyncratic component was considered. Over the past two years, we have treated approximately 21 cats with gentamicin at a reference level of 40 rug/kg and fewer cats at other doses. Distribution curves for the day of appearance of ataxia are shown in figure 1. The frequency distribution as well as 100 r---"'---.--:.,--.,-----.---r--r----., 12 16 20 24 28 DAYS OF DOSING Figure 1. Percentage distribution curves of time of appearance of ataxia in cats given various amounts of gentamicin sulfate. Numbers of cats are given in parentheses. 32 40

Chronic Toxicity of Gentamicin in Cats 8127 the cumulative distribution curves follow those of a normal distribution for the levels of 40 and 60 mg/kg per day. There was no evidence of clustering or a bimodal type of distribution which might be expected if some cats were significantly more or less sensitive to vestibular toxicity. The distribution for the 20 mg/kg per day level is flatter and suggests a range of responses. This may be due to the small number of animals studied and needs confirmation. The relationship between level of dose and time required to produce ataxia suggested an examination of total dose as a factor in vestibular toxicity. Figure 2 shows plots of total dose and number of days required for production of ataxia against the daily dose. Ataxia occurred after a smaller average total dose at lower dosage levels than at higher ones. This inverse relationship suggests that the length of treatment may be an important factor at the dosage levels studied. Two other experiments were performed for evaluation of the importance of total dose. In the first, two groups of five cats each were given 40 rug/kg per day. One group was given 40 mg/ kg five days a week, with rests on the weekend, and the other group was given 40 mg/kg per day seven days a week. Although the group treated five days each week took 25 days to become ataxic compared to 18 days for the group treated seven days a week, the total number of doses, 18, was the same for both groups, suggesting the importance of total dose and total number of days treated. loool: : J 800...J 600 g 500 4 S:2 30 x I- >- 0 20 1 In another study, one group of six cats was treated once a day with 40 mg/kg, while another group of five cats was given 20 mg/kg twice a day. The single daily treatment produced ataxia in an average of 17 days, while the group treated twice a day became ataxic in an average of 19.4 days. This slight difference was not significant as indicated by the range of 16 to 21 days in both groups. The average total dose for production of ataxia was higher with the split doses (776 mg/ kg) than with a single daily dose (680 mg/kg), Since gentamicin is rapidly excreted in the urine, it was thought that monitoring of levels of gentamicin in serum might provide a measure of renal function during the course of a study of ataxia. To provide an easy means of obtaining multiple samples of serum from cats, and also to permit evaluation of the relationship between peak serum levels and the appearance of ataxia, we prepared two cats with carotid artery cannulae. Initially, levels of gentamicin in serum were determined after a single subcutaneous dose of 40 mg/kg per day and were found to peak at approximately 100 ug/rnl one-half hour after treatment, with a rapid decline to less than 1 ug/ml 24 hr after treatment. The two cats were then started on a course of treatment of 40 mg/ kg per day, injected intraarterially. Levels of gentamicin in serum were monitored daily. Average serologic levels after intraarterial administration of gentamicin during the first two days of treatment are shown in figures 3 and 4. Peak levels after this route were two to six times Ẹ...! 100 ::J cr W Ul 10 <3 ;:! C) w 10 20 30 40 50 60 70 DOSE (mg/kg) Figure 2. Relationship between dosage of gentamicin, time required to produce ataxia, and total dosage in cats. Eight cats received 60 mg/kg per day; 21, 40 mg/kg per day; and five, 20 mg/kg per day. 0.1 I I I o 2 :3 4 5 TIME (HOURS) Figure 3. Average levels of gentamicin in serum of cat 955 at various times after a single dose of 40 mg of gentamicin sulfate/ kg, given intraarterially (I.A.) or subcutaneously (S.C.).

S12 Walt, Moss, and Weinstein E <, 100 ::J cr W (fj w C> 0.1 "'----'--..._-.L.._--L...l-.._-l.-_---L..--rj"I---J o 3 4 24 TIME (HOURS) Figure 4. Average levels of gentamicin in serum of cat 5314 at various times after a single dose of 40 mg of gentamicin sulfate/kg, given intraarterially (la.) or subcutaneously (S.C.). as high as after subcutaneous administration and declined rapidly to less than 1 ug/ml 24 hr after treatment. Levels in serum of these two cats given 40 mg/kg per day intraarterially daily for 20 days are shown in tables 2 and 3. Ataxia was noted in both cats on day 16. At the time ataxia appeared, a substantial decrease in renal function, as measured by the ability to excrete gentamicin, also occurred. This was most evident as an increase in levels in serum after 24 hr and progressed until the cats were sacrificed on day 20. Table 2. Levels of gentamicin in serum of cat 955 treated intraarterially with 40 mg of gentamicin/kg per day. Gentamicin in serum (ug/rnl) Day of at hr after injection treatment 1A Y:2 1 2 4 6 7 24 1 195 145 105 80 14 7 0.2 2 580 210 125 80 22 10 0.7 4 0.6 5 250 176 25 0.5 7 1.0 8 220 182 37 0.7 12 2.3 13 166 128 55 3.5 15 24 Ataxia 16 60 12 17 200 154 155 19 Complete ataxia 18 120 28.5 19 67.5 42 20 310 340 220 150 Table 3. Levels of gentamicin in serum of cat 5314 treated intraarterially with 40 mg of gentamicin/kg per day. Gentamicin in serum (ug/ml) at hr Day of after injection treatment 1A Y:2 1 2 4 6 7 24 1 170 150 130 105 17.5 7.3 0.2 2 950 220 180 125 19 7.3 0.3 4 3.7 5 280 180 32.5 0.9 8 22 12 1.0 13 240 122 26.3 5.7 15 3.5 Ataxia 16 34.5 3.3 17 200 180 75 7.8 Complete ataxia 18 135 24 19 60 21 20 360 190 210 130 Histopathologic examination revealed renal tubular necrosis. Discussion The data demonstrate that high levels of gentamicin sulfate administered daily to normal male cats produce ataxia and that the time of appearance of ataxia is related to dose. At a dose of 60 mg/kg per day (20-60 times the recommended daily dose for humans of 1-3 mg/kg per day), death, apparently as a result of renal tubular damage, follows the appearance of ataxia quite closely. At lower doses, a temporal separation of renal damage from vestibular damage becomes apparent. In a small number of cats, cessation of treatment after the appearance of ataxia permitted recovery from the renal damage. The vestibular damage, however, appeared to be irreversible. Administration by the intraarterial route demonstrated that at the high doses used, the magnitude of peak levels in serum was not solely responsible for the time of appearance of ataxia, since ataxia occurred after the same length of time whether the doses of 40 rug/kg were given subcutaneously or intraarterially, or whether it was given as 40 mg/kg once a day or 20 mg/kg twice a day. Total dose is important but is not the only factor involved. Cats given 60 rug/kg per day became ataxic in an average of 13.5 days; this

Chronic Toxicity of Gentamicin in Cats S129 represents an average total dose of 810 mg/kg, At 40 rag/kg per day, the total dose required to produce ataxia was 700 mg/kg, while at 20 mg/kg per day it was 628 rug/kg. This slight inverse relationship suggests that length of treatment is also an important factor in determining when ataxia will occur. References 1. Black, J., Calesnick, B., Williams, D., Weinstein, M. J. Pharmacology of gentamicin, a new broadspectrum antibiotic. Antimicrobial Agents and Chemotherapy-1963, p. 138-147, 1964. 2. Weinstein, M. J., Wagman, G. H., Taber, R. I. Toxicity of acetylated gentamicin and neomycin. Antimicrobial Agents and Chemotherapy-1965, p. 227-231, 1966. 3. Wagman, G. H., Oden, E. M., Weinstein, M. J., Irwin, S. Effect of calcium on the toxicity of gentamicin. Antimicrobial Agents and Chemotherapy-1966, p. 175-181, 1967. 4. Hawkins, J. E., Jr., Johnsson, L.-G., Aran, J.-M. Comparative tests of gentamicin ototoxicity. J. Infect. Dis. 119:417-426, 1969. 5. McGee, T. M., Webster, J., Williams, M. Histological and functional changes in the ears of cats after subcutaneous administration of gentamicin. J. Infect. Dis. 119:432-439, 1969. 6. Lundquist, P.-G., Wersall, J. The ototoxic effect of gentamicin-an electron microscopical study. In First international symposium on gentamicin, Paris, Jan., 1967. Schwabe, Basel, 1967, p. 25-46. 7. Bernabei, L., Pierangeli, C. E., DiBrino, M., Consalvo, P. Contributo allo studio del danno labirintico sperimentale da gentamicina. 1. Rilievi funionali. Atti Accad. Fisiocr. Siena, Series 13, 16:445-467, 1967. 8. Bernabei, L., DiBrino, M., Consalvo, P., Pierangeli, C. E. Contributo allo studio del danno labirintico sperimentale da gentamicina. 2. Rilievi istopatologici. Atti Accad. Fisiocr. Siena, Series 13, 16:1221-1249, 1967. 9. Wright, I. Investigation of ototoxicity of an antibiotic from Micromonospora purpurea (gentamicin). J. Path. 98:129-136, 1969. to. Oden, E. M., Stander, H., Weinstein, M. J. Microbiological assay of gentamicin. Antimicrobial Agents and Chemotherapy-1963, p. 8-13, 1964.