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Comparison of Therapeutic Routes in Experimental Pseudomonas Keratitis
COMPARISON O F T H E R A P E U T I C ROUTES IN EXPERIMENTAL PSEUDOMONAS KERATITIS STARKEY D. D A V I S , M.D.,
L A R R I E D. S A R F F ,
AND R O B E R T A. H Y N D I U K , Milwaukee,
Subconjunctival injection of antibiotics has been used for many years in the treatment of bacterial keratitis. Objective proof of the effectiveness of antibiotics by the subconjunctival injection route is lacking both in experimental and human infections. 1 - 3 Little is known about the effectiveness of antibiotics by the intra muscular route. 2 ' 3 We devised an experimental model of Pseudomonas keratitis suitable for objec tive evaluation" of therapy. 4 ' 5 Results were assessed quantitatively by determining the number of bacteria surviving in the cornea. 5 We report herein results of stud ies comparing the effectiveness of antibi otic administered by subconjunctival in jection, intramuscular, or topical routes in the treatment of experimental Pseudomo nas keratitis in guinea pigs and rabbits. M A T E R I A L AND M E T H O D S
Pseudomonas aeruginosa strains 107 and 113 were isolated from human corneal ulcers and were maintained by routine bacteriologic methods. 6 The strains were susceptible by in vitro tests to tobramycin. 5 Tobramycin was supplied in vials containing solutions of 40 mg/ml. Dilution of antibiotic for topical applica tion were made with sterile 0.15M NaCl. From the Departments of Pediatrics (Drs. Davis and Sarff), and Ophthalmology (Dr. Hyndiuk), Med ical College of Wisconsin, Milwaukee Children's Hospital, and the Cornea-External Eye Disease Unit (Dr. Hyndiuk), the Eye Institute, Milwaukee Coun ty Medical Complex, Milwaukee, Wisconsin. This study was supported in part by Grant EY01665, National Institute of Health, and by Ophthalmic Research Center Grant 1-P30-Ey01931; by Eli Lilly and Co., Indianapolis, Indiana; and by Research to Prevent Blindness, Inc., New York. Reprint requests to Starkey D. Davis, M.D., Sec tion of Infectious Diseases, Milwaukee Children's Hospital, 1700 W. Wisconsin Ave., Milwaukee, WI 53233. 710
M.D.,
M.D.
Wisconsin
Male Hartley-strain guinea pigs weigh ing approximately 300 g and New Zea land white rabbits weighing approximate ly 2 to 3 kg were used in this study. Animals were anesthetised and were then infected intracorneally with about ten vi able bacteria in a volume of 20 u.1 with a 30-gauge needle and microsyringe. Treat ments were begun 24 hours later when all animals had frank keratitis. All topical therapy consisted of two drops every 30 minutes. Animals were killed either six or 24 hours after the beginning of treat ment. Corneas were removed and ground in broth. Quantitative subcultures were made from serial dilutions in broth. Re sults were expressed as numbers of viable bacteria in the cornea in common loga rithms (base 10). Results were evaluated statistically by analysis of variance. 7 Con centrations of tobramycin in serum were assayed with a modification of an enzy matic technique. 8 For the therapy trials, all animals were infected bilaterally. Subconjunctival in jections of antibiotic were made only in the right eyes. The right eyes were re ferred to as the ipsilateral eyes, and the left uninjected eyes were identified as the contralateral eyes. RESULTS
Pharmacokinetic studies with intramus cular injection of tobramycin indicated that 1 mg/kg of body weight was the ap propriate parenteral dose for rabbits. Ab sorption of tobramycin by the subcon junctival injection route was more rapid than by the intramuscular route but was less rapid than by the intravenous route (Table 1). Serum concentrations after administra tion of 20 mg of tobramycin (approxi-
AMERICAN JOURNAL O F OPHTHALMOLOGY 87:710-716, 1979
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TABLE 1 SERUM LEVELS OF TOBRAMYCIN IN RABBITS AFTER ADMINISTRATION OF 1 MG/KG OF BODY WEIGHT, BY INTRAMUSCULAR, INTRAVENOUS, AND SUBCONJUNCTIVAL ROUTES
Time (hrs) >/4
V2 1 2 3 4
Intramuscular (No.=5) (M-g/ml) x±S.D.
Intravenous (No.=5) (ng/ml) x + S.D.
Subconjunctival Injection (No.=4) (M-g/ml) x ± S.D.
6.3±2.3 Not done 6.5±1.2 3.6±0.9 2.3+1.4 1.1±0.7
4.5+0.8 3.2±0.3 2.1±0.4 1.9±1.2 0.2±0.2 None
3.6±1.5 5.0±1.3 4.0±0.6 2.4±0.7 1.8+0.2 1.2±0.7
mately 7 to 10 mg/kg of body weight) by the subconjunctival injection route were also determined (Table 2). The mean 30-minute concentration was approxi mately five times the serum concentration achieved after 1 mg/kg of body weight by the subconjunctival injection route. The efficacy of tobramycin by the in tramuscular and subconjunctival injec tion routes in combination with topical therapy was determined in guinea pigs (Table 3). Previous studies indicated that 2 mg/kg of body weight of tobramycin injected intramuscularly in guinea pigs achieved appropriate blood levels. 5 In this trial, topical therapy was effective alone. Neither intramuscular nor subcon junctival injection administration of anti biotic significantly enhanced the effec tiveness of topical antibiotic during a six-hour trial. In the next study, the efficacy of to bramycin, 2 mg/kg of body weight, by TABLE 2 SERUM CONCENTRATIONS I N T H R E E RABBITS AFTER SUBCONJUNCTIVAL INJECTION OF 20 MG OF TOBRAMYCIN
Time (min) 30 60 120 180
Serum Concentrations (f^g/ml) x ± S.D. 24.5 21.7 10.3 6.2
± + ± ±
0.3 1.0 3.1 1.3
subconjunctival injection or intramuscu lar administration alone without topical therapy was evaluated (Table 4). All groups treated with antibiotic had sig nificantly lower corneal colony counts than the group treated with saline alone (P < .001). The effectiveness of one or two doses of antibiotic by the intramus cular route was not significantly different from that of one dose of antibiotic by the subconjunctival injection route. Two doses of tobramycin by the subconjunc tival injection route did achieve a greater therapeutic effect in the ipsilateral eyes than in the contralateral eyes or in the other treatment groups. The efficacy of tobramycin by topical, intramuscular, and subconjunctival injec tion routes was determined in rabbits with experimental Pseudomonas keratitis (Table 5). Tobramycin, 1 mg/kg of body weight, by the intramuscular or subcon junctival injection route was not signifi cantly more effective than topical saline. Topical tobramycin, 40 mg/ml, was effec tive. Tobramycin, 20 mg, by the subcon junctival injection route (Table 6) had a significant therapeutic effect in the ipsi lateral injected eyes as compared to the saline treatment group, the intramuscular treatment group, or the left contralateral eyes (P = .002). However, tobramycin, 20 mg/ml, by the topical route was more effective than tobramycin, 20 mg, by the subconjunctival injection route.
TABLE 3 SIX-HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L , INTRAMUSCULAR, AND SUBCONJUNCTIVAL INJECTION R O U T E S IN E X P E R I M E N T A L KERATITIS IN PIGS W I T H PSEUDOMONAS 107*
Corneal Colony Counts Treatment 0.15M NaCl, topical and one subconjunctival injection dose at 0 hr Tobramycin, 20 mg/ml Topical Topical and 2 mg/kg of body weight, intramuscular, at 0 hr Topical and 2 mg/kg of body weight, subconjunctival injection, at 0 hr, right eye Right eyes Left eyes
x ± S.D.
(No.)
5.80 ± 0.45 (6) 3.90 ± 0.58 (6) < .001 3.42 ± 0.79 (6) .61 3.41 ± 0.36 (6) 3.64 ± 1.15 (6)
"Topical therapy consisted of two drops every 30 minutes. TABLE 4 SIX-HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN, 2 MG/KG O F BODY W E I G H T , BY INTRAMUSCULAR AND SUBCONJUNCTIVAL I N J E C T I O N R O U T E S IN E X P E R I M E N T A L KERATITIS IN G U I N E A P I G S W I T H PSEUDOMONAS 107
Corneal Colony Counts
x ± S.D.
Treatment 0.15M NaCl, subconjunctival injection, 0 and 2 hrs Tobramycin intramuscular injection, Ohr 0 and 2 hrs Tobramycin subconjunctival injection, 0 hr, right eye Right eyes Left eyes Tobramycin subconjunctival injection, 0 and 2 hrs, right eye Right eyes Left eyes
(No.)
6.26 ± 0.27
(6)
0.24 0.49
(6) (6)
5.50 ± 0.39 5.89 ± 0.42
(6) (6)
4.73 ± 0.60 5.51 ± 0.58
(6) (6)
5.69 5.45
.32
.005
TABLE 5 S K - H O U R THERAPY TRIAL WITH TOBRAMYCIN BY INTRAMUSCULAR, SUBCONJUNCTIVAL INJECTION, AND TOPICAL ROUTES IN EXPERIMENTAL KERATITIS IN RABBITS WITH PSEUDOMONAS 1 0 7 *
Treatment 0.15M NaCl, topical Tobramycin, 1 mg/kg of body weight, intramuscular, 0 hr Tobramycin, 1 mg/kg of body weight, subconjunctival injection, 0 hr, right eye Right eyes Left eyes Tobramycin, 40 mg/ml, topical
Corneal Colony Counts x ± S.D. (No.) 7.04 ± 0.44
(4)
7.02 ± 0.22
(6)
6.71 ± 0.35 7.03 ± 0.22 3.32 ± 1.75
(5) (5) (6)
.92
*Topical therapy consisted of two drops every 30 minutes. 712
< .001
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TABLE 6 S I X - H O U R T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L , INTRAMUSCULAR, O R SUBCONJUNCTIVAL I N J E C T I O N R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 107*
Treatment 0.15M NaCl, topical Tobramycin, 20 mg, intramuscular, at Ohr Tobramycin, 20 mg, subconjunctival injection, at 0 hr, right eye Right eyes Left eyes Tobramycin, 20 mg/ml, topical
Corneal Colony Counts x ± S.D. (No.) 7.25 ± 0.29
(6)1
6.94 ± 0.26
(6)
5.48 ± 1.50 6.91 ± 0.19 3.93 ± 1.01
(6) (6). (6)
.007 < .005
"Topical therapy consisted of two drops every 30 minutes.
The numbers of bacteria in the eyes of the group treated with 20 mg of tobram ycin intramuscularly or in the left, contralateral eyes of the subconjunctival injection treatment group were not signif icantly different from those of the saline group (P = .74). Thus, there was no sys temic therapeutic effect of this dose by either intramuscular or subconjunctival injection routes. The next trial was designed to deter mine whether antibiotic by the subcon junctival injection route would improve the effectiveness of topical therapy in the rabbit (Table 7). Tobramycin, 20 mg, by the subconjunctival injection route did not significantly enhance the effective ness of 20 mg/ml of topical tobramycin.
In a trial with Pseudomonas strain 113, the effectiveness of topical tobramycin was compared with that of tobramycin by the subconjunctival injection route in rabbits (Table 8). Tobramycin by the sub conjunctival injection route was not sig nificantly more effective than topical sa line. Topical tobramycin, 20 mg/ml, was effective. A 24-hour trial was carried out compar ing topical therapy alone with a single dose by the subconjunctival injection route at the beginning of therapy (Table 9). Numbers of bacteria in the left eyes of the subconjunctival injection group were not different from the saline-treatment group (P = .79). Antibiotic by the sub conjunctival injection route was more ef-
TABLE 7 SIX-HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L AND SUBCONJUNCTIVAL I N J E C T I O N R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 107*
Treatment 0.15M NaCl topical Tobramycin, 20 mg/ml, topical Tobramycin 20, mg/ml, topical and tobramycin 20 mg, subconjunctival injection, right eye Right eyes Left eyes
Corneal Colony Counts x ± S.D. (No.) 7.18 ± 0.14 3.41 ± 1.75
(6) (6)
<.001 .29
3.21 ± 1.27 4.24 ± 0.76
*Topical therapy consisted of two drops every 30 minutes.
(5) (6)
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MAY, 1979
TABLE 8 S I X - H O U R T H E R A P Y TRIAL W I T H TOBRAMYCIN BY SUBCONJUNCTIVAL I N J E C T I O N AND TOPICAL R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 113*
Treatment 0.15M NaCl, topical Tobramycin, 20 mg, subconjunctival injection, right eye Right eyes Left eyes Tobramycin, 20 mg/ml, topical
Corneal Colony Counts x + S.D. (No.) 7.06 ± 0.09
(6)1
6.49 ± 0.63 6.69 ± 0.23 3.37 ± 1.48
(6)
(6) J
.48
< .001
(6)
"Topical therapy consisted of two drops every 30 minutes.
fective than saline but was much less effective than by the topical route. In the final study, (Table 10), the effica cy of a single dose of tobramycin 20 mg by the subconjunctival injection route alone was determined by comparing re sults in right and left eyes. DISCUSSION
In experimental Pseudomonas keratitis in guinea pigs and rabbits, topical thera py was consistently superior to adminis tration of antibiotic by the subconjunc tival injection or intramuscular routes. In one comparison in guinea pigs (Table 4) and four comparisons in rabbits (Tables 5, 6, 8, and 9) topical therapy was more effective than therapy by the intramuscu lar or subconjunctival injection routes. In two other trials, therapy by the intramus
cular or subconjunctival injection routes did not improve efficacy of topical thera py (Tables 3 and 7). Comparable results were obtained both in guinea pigs and in rabbits. Because the properties in vitro and in vivo are so similar, we expect that other aminoglycosides would also be more ef fective by the topical route than by the intramuscular or subconjunctival injec tion routes in this experimental model. Data presented in Table 1 show more rapid absorption of antibiotic into the blood after subconjunctival injection ad ministration than after intramuscular ad ministration. We previously observed similar results in guinea pigs. 5 This ob servation may be because the periorbital tissues were very vascular. One way to estimate the degree of effi-
TABLE 9 T W E N T Y - F O U R HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L AND SUBCONJUNCTIVAL INJECTION R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 107*
Treatment 0.15M NaCl, topical Tobramycin, 20 mg/ml, topical Tobramycin, 20 mg, subconjunctival injection, at 0 hr, right eye Right eyes Left eyes
Corneal Colony Counts x ± S.D. (No.) 6.74 ± 0.31 0.79 ± 0.94
(6) (6)
5.48 ± 1.08 6.60 ± 0.79
(6) (6)
< .001
*Topical therapy was given as two drops every 30 minutes.
.03
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TABLE 10 TWENTY-FOUR HOUR THERAPY TRIAL OF 20 MG OF TOBRAMYCIN BY THE SUBCONJUNCTIVAL INJECTION ROUTE ALONE IN EXPERIMENTAL KERATITIS IN RABBITS WITH PSEUDOMONAS 107*
Treatment Tobramycin, 20 mg, subconjunctival injection, right eye Right eyes Left eyes
Corneal Colony Counts x ± S.D. (No.)
4.78 ± 1.94 6.55 ± 0.50
(6) (6)
P
„
Ue
*A single dose of tobramycin was injected in the right eye 24 hours after infection was established. No other therapy was given. Animals were killed 24 hours later.
cacy of 20 mg of tobramycin by the sub conjunctival injection route was to com pare numbers of bacteria in the right eyes that were injected with those of the left eyes which were not. In one trial (Table 6), the difference was highly signif icant (P = .002). In two other trials with the same organism (Tables 9 and 10), the difference was of marginal significance (P = .03; P = .06). In a trial with another strain (Table 8), there was no difference (P = .63). Topical therapy consistently had greater efficacy. We have previously shown in guinea pigs that therapy by the intramuscular route had less variability of therapeutic effect than therapy by the topical route. 5 Specifically, the standard deviations of the means were smaller for intramuscular treatment groups than for topical treat ment groups. The greater variability for the topical treatment groups is probably related to variables that are difficult to control, such as the interval between ap plication of a drop and a blink. In our studies in which therapy by the subcon junctival injection route was effective, the standard deviations were large (Tables 6, 9, and 10). We do not believe the variabil ity is related to the techniques of the injections. Our results directly conflict with many reports that subconjunctival therapy yields high intraocular concentrations of
d r u g . 1 - 3 Some differences may be the result of the method used. Baum and associates 9 reported that concentrations of gentamicin in the rabbit cornea were consistently higher with administration by the subconjunctival injection route than with the topical route in rabbits with Pseudomonas ulcers. However, these in vestigators compared topical therapy with 3 mg/ml of gentamicin, one drop every 30 minutes, with subconjunctival injection of 20 mg of gentamicin. In contrast, we compared 20 mg of tobramycin by the subconjunctival injection route with topi cal tobramycin, 20 mg/ml, two drops every 30 minutes. This higher concentra tion was used topically because it was more effective. 10 Because of differences in design and method, it was difficult to directly compare our results with those of Baum and associates. 9 We are not aware of any studies in vivo that provide objective quantitative proof of the effectiveness of administration of antibiotics by the subconjunctival injec tion route. Rabbits and guinea pigs with experi mental Pseudomonas keratitis differ in response to parenteral antibiotic therapy. We previously showed in guinea pigs that intramuscular therapy with carbenicillin, gentamicin, or tobramycin was more ef fective than intramuscular saline. 5 In this study, administration of tobramycin by
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AMERICAN JOURNAL OF OPHTHALMOLOGY
the subconjunctival injection route in guinea pigs was effective in the contralateral, uninjected eye as well as in the ipsilateral injected eye (Table 4). The doses of antibiotics used in these trials achieve appropriate blood levels. 5 In sharp contrast, intramuscular thera py with tobramycin in rabbits was not more effective than saline (Tables 5 and 6). Furthermore, 20 mg of tobramycin by subconjunctival injection, which achieves high blood levels, did not have a thera peutic effect in the contralateral, unin jected eye (Tables 6, 8, and 9). The phys iologic or anatomic mechanisms that account for this difference in response to therapy are unknown. Whether infection in the human cornea would more clearly resemble the guinea pig or rabbit is un certain. SUMMARY
We determined the efficacy of tobram ycin administered by topical, intramuscu lar, and subconjunctival routes in guinea pigs and rabbits with experimental Pseudomonas keratitis. The topical route of administration was consistently more ef fective than either subconjunctival or in tramuscular routes. Subconjunctival in jection of antibiotic did not enhance the effectiveness of topical therapy in either guinea pigs or rabbits. Intramuscular tobramycin was more ef
MAY, 1979
fective than saline in guinea pigs with keratitis but not in rabbits with keratitis. ACKNOWLEDGMENT
Tobramycin was supplied by Eli Lilly and Co.
REFERENCES 1. Ellis, P. P.: Subconjunctival therapy. In Leo pold, I. H. (ed.): Symposium on Ocular Therapy, vol. 5. St. Louis, C. V. Mosby Co., 1972, p p . 36-42. 2. Baum, J. L., Barza, M., and Weinstein, L.: Preferred routes of antibiotic administration in treatment of bacterial ulcers of the cornea. Int. Ophthalmol. Clin. 13:31, 1973. 3. Havener, W. H.: Ocular Pharmacology, 3rd ed. St. Louis, C. V. Mosby Co., 1974. 4. Davis, S. D., and Chandler, J. W.: Experimen tal keratitis due to Pseudomonas aeruginosa. Model for evaluation of antimicrobial drugs. Angimicrob. Agents Chemother. 8:350, 1975. 5. Davis, S. D., Sarff, L. D., and Hyndiuk, R. A.: Antibiotic therapy of experimental Pseudomonas keratitis in guinea pigs. Arch. Ophthalmol. 95:1638, 1977. 6. Lenette, E. H., Spaulding, E. H., and Truant, J. P. (eds.): Manual of Clinical Microbiology, 2nd ed. Washington, American Society for Microbiolo gy, 1974. 7. Snedecor, G. W., and Cochran, W. G.: Statisti cal Methods, 6th ed. Ames, Iowa State University Press, 1976. 8. Smith, D. H., Van Otto, B., and Smith, A. L.: A rapid chemical assay for gentamicin. N. Engl. J. Med. 286:583, 1972. 9. Baum, J. L., Barza, M., Shushan, D., and Wein stein, L.: Concentration of gentamicin in experi mental corneal ulcers. Arch. Ophthalmol. 92:315, 1974. 10. Davis, S. D., Sarff, L. D., and Hyndiuk, R. A.: Topical tobramycin therapy of experimental Pseu domonas keratitis. An evaluation of some factors that potentially enhance efficacy. Arch. Ophthalmol. 96:123, 1978.
L A R R I E D. S A R F F ,
AND R O B E R T A. H Y N D I U K , Milwaukee,
Subconjunctival injection of antibiotics has been used for many years in the treatment of bacterial keratitis. Objective proof of the effectiveness of antibiotics by the subconjunctival injection route is lacking both in experimental and human infections. 1 - 3 Little is known about the effectiveness of antibiotics by the intra muscular route. 2 ' 3 We devised an experimental model of Pseudomonas keratitis suitable for objec tive evaluation" of therapy. 4 ' 5 Results were assessed quantitatively by determining the number of bacteria surviving in the cornea. 5 We report herein results of stud ies comparing the effectiveness of antibi otic administered by subconjunctival in jection, intramuscular, or topical routes in the treatment of experimental Pseudomo nas keratitis in guinea pigs and rabbits. M A T E R I A L AND M E T H O D S
Pseudomonas aeruginosa strains 107 and 113 were isolated from human corneal ulcers and were maintained by routine bacteriologic methods. 6 The strains were susceptible by in vitro tests to tobramycin. 5 Tobramycin was supplied in vials containing solutions of 40 mg/ml. Dilution of antibiotic for topical applica tion were made with sterile 0.15M NaCl. From the Departments of Pediatrics (Drs. Davis and Sarff), and Ophthalmology (Dr. Hyndiuk), Med ical College of Wisconsin, Milwaukee Children's Hospital, and the Cornea-External Eye Disease Unit (Dr. Hyndiuk), the Eye Institute, Milwaukee Coun ty Medical Complex, Milwaukee, Wisconsin. This study was supported in part by Grant EY01665, National Institute of Health, and by Ophthalmic Research Center Grant 1-P30-Ey01931; by Eli Lilly and Co., Indianapolis, Indiana; and by Research to Prevent Blindness, Inc., New York. Reprint requests to Starkey D. Davis, M.D., Sec tion of Infectious Diseases, Milwaukee Children's Hospital, 1700 W. Wisconsin Ave., Milwaukee, WI 53233. 710
M.D.,
M.D.
Wisconsin
Male Hartley-strain guinea pigs weigh ing approximately 300 g and New Zea land white rabbits weighing approximate ly 2 to 3 kg were used in this study. Animals were anesthetised and were then infected intracorneally with about ten vi able bacteria in a volume of 20 u.1 with a 30-gauge needle and microsyringe. Treat ments were begun 24 hours later when all animals had frank keratitis. All topical therapy consisted of two drops every 30 minutes. Animals were killed either six or 24 hours after the beginning of treat ment. Corneas were removed and ground in broth. Quantitative subcultures were made from serial dilutions in broth. Re sults were expressed as numbers of viable bacteria in the cornea in common loga rithms (base 10). Results were evaluated statistically by analysis of variance. 7 Con centrations of tobramycin in serum were assayed with a modification of an enzy matic technique. 8 For the therapy trials, all animals were infected bilaterally. Subconjunctival in jections of antibiotic were made only in the right eyes. The right eyes were re ferred to as the ipsilateral eyes, and the left uninjected eyes were identified as the contralateral eyes. RESULTS
Pharmacokinetic studies with intramus cular injection of tobramycin indicated that 1 mg/kg of body weight was the ap propriate parenteral dose for rabbits. Ab sorption of tobramycin by the subcon junctival injection route was more rapid than by the intramuscular route but was less rapid than by the intravenous route (Table 1). Serum concentrations after administra tion of 20 mg of tobramycin (approxi-
AMERICAN JOURNAL O F OPHTHALMOLOGY 87:710-716, 1979
VOL. 87, NO. 5
EXPERIMENTAL PSEUDOMONAS KERATITIS
711
TABLE 1 SERUM LEVELS OF TOBRAMYCIN IN RABBITS AFTER ADMINISTRATION OF 1 MG/KG OF BODY WEIGHT, BY INTRAMUSCULAR, INTRAVENOUS, AND SUBCONJUNCTIVAL ROUTES
Time (hrs) >/4
V2 1 2 3 4
Intramuscular (No.=5) (M-g/ml) x±S.D.
Intravenous (No.=5) (ng/ml) x + S.D.
Subconjunctival Injection (No.=4) (M-g/ml) x ± S.D.
6.3±2.3 Not done 6.5±1.2 3.6±0.9 2.3+1.4 1.1±0.7
4.5+0.8 3.2±0.3 2.1±0.4 1.9±1.2 0.2±0.2 None
3.6±1.5 5.0±1.3 4.0±0.6 2.4±0.7 1.8+0.2 1.2±0.7
mately 7 to 10 mg/kg of body weight) by the subconjunctival injection route were also determined (Table 2). The mean 30-minute concentration was approxi mately five times the serum concentration achieved after 1 mg/kg of body weight by the subconjunctival injection route. The efficacy of tobramycin by the in tramuscular and subconjunctival injec tion routes in combination with topical therapy was determined in guinea pigs (Table 3). Previous studies indicated that 2 mg/kg of body weight of tobramycin injected intramuscularly in guinea pigs achieved appropriate blood levels. 5 In this trial, topical therapy was effective alone. Neither intramuscular nor subcon junctival injection administration of anti biotic significantly enhanced the effec tiveness of topical antibiotic during a six-hour trial. In the next study, the efficacy of to bramycin, 2 mg/kg of body weight, by TABLE 2 SERUM CONCENTRATIONS I N T H R E E RABBITS AFTER SUBCONJUNCTIVAL INJECTION OF 20 MG OF TOBRAMYCIN
Time (min) 30 60 120 180
Serum Concentrations (f^g/ml) x ± S.D. 24.5 21.7 10.3 6.2
± + ± ±
0.3 1.0 3.1 1.3
subconjunctival injection or intramuscu lar administration alone without topical therapy was evaluated (Table 4). All groups treated with antibiotic had sig nificantly lower corneal colony counts than the group treated with saline alone (P < .001). The effectiveness of one or two doses of antibiotic by the intramus cular route was not significantly different from that of one dose of antibiotic by the subconjunctival injection route. Two doses of tobramycin by the subconjunc tival injection route did achieve a greater therapeutic effect in the ipsilateral eyes than in the contralateral eyes or in the other treatment groups. The efficacy of tobramycin by topical, intramuscular, and subconjunctival injec tion routes was determined in rabbits with experimental Pseudomonas keratitis (Table 5). Tobramycin, 1 mg/kg of body weight, by the intramuscular or subcon junctival injection route was not signifi cantly more effective than topical saline. Topical tobramycin, 40 mg/ml, was effec tive. Tobramycin, 20 mg, by the subcon junctival injection route (Table 6) had a significant therapeutic effect in the ipsi lateral injected eyes as compared to the saline treatment group, the intramuscular treatment group, or the left contralateral eyes (P = .002). However, tobramycin, 20 mg/ml, by the topical route was more effective than tobramycin, 20 mg, by the subconjunctival injection route.
TABLE 3 SIX-HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L , INTRAMUSCULAR, AND SUBCONJUNCTIVAL INJECTION R O U T E S IN E X P E R I M E N T A L KERATITIS IN PIGS W I T H PSEUDOMONAS 107*
Corneal Colony Counts Treatment 0.15M NaCl, topical and one subconjunctival injection dose at 0 hr Tobramycin, 20 mg/ml Topical Topical and 2 mg/kg of body weight, intramuscular, at 0 hr Topical and 2 mg/kg of body weight, subconjunctival injection, at 0 hr, right eye Right eyes Left eyes
x ± S.D.
(No.)
5.80 ± 0.45 (6) 3.90 ± 0.58 (6) < .001 3.42 ± 0.79 (6) .61 3.41 ± 0.36 (6) 3.64 ± 1.15 (6)
"Topical therapy consisted of two drops every 30 minutes. TABLE 4 SIX-HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN, 2 MG/KG O F BODY W E I G H T , BY INTRAMUSCULAR AND SUBCONJUNCTIVAL I N J E C T I O N R O U T E S IN E X P E R I M E N T A L KERATITIS IN G U I N E A P I G S W I T H PSEUDOMONAS 107
Corneal Colony Counts
x ± S.D.
Treatment 0.15M NaCl, subconjunctival injection, 0 and 2 hrs Tobramycin intramuscular injection, Ohr 0 and 2 hrs Tobramycin subconjunctival injection, 0 hr, right eye Right eyes Left eyes Tobramycin subconjunctival injection, 0 and 2 hrs, right eye Right eyes Left eyes
(No.)
6.26 ± 0.27
(6)
0.24 0.49
(6) (6)
5.50 ± 0.39 5.89 ± 0.42
(6) (6)
4.73 ± 0.60 5.51 ± 0.58
(6) (6)
5.69 5.45
.32
.005
TABLE 5 S K - H O U R THERAPY TRIAL WITH TOBRAMYCIN BY INTRAMUSCULAR, SUBCONJUNCTIVAL INJECTION, AND TOPICAL ROUTES IN EXPERIMENTAL KERATITIS IN RABBITS WITH PSEUDOMONAS 1 0 7 *
Treatment 0.15M NaCl, topical Tobramycin, 1 mg/kg of body weight, intramuscular, 0 hr Tobramycin, 1 mg/kg of body weight, subconjunctival injection, 0 hr, right eye Right eyes Left eyes Tobramycin, 40 mg/ml, topical
Corneal Colony Counts x ± S.D. (No.) 7.04 ± 0.44
(4)
7.02 ± 0.22
(6)
6.71 ± 0.35 7.03 ± 0.22 3.32 ± 1.75
(5) (5) (6)
.92
*Topical therapy consisted of two drops every 30 minutes. 712
< .001
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TABLE 6 S I X - H O U R T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L , INTRAMUSCULAR, O R SUBCONJUNCTIVAL I N J E C T I O N R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 107*
Treatment 0.15M NaCl, topical Tobramycin, 20 mg, intramuscular, at Ohr Tobramycin, 20 mg, subconjunctival injection, at 0 hr, right eye Right eyes Left eyes Tobramycin, 20 mg/ml, topical
Corneal Colony Counts x ± S.D. (No.) 7.25 ± 0.29
(6)1
6.94 ± 0.26
(6)
5.48 ± 1.50 6.91 ± 0.19 3.93 ± 1.01
(6) (6). (6)
.007 < .005
"Topical therapy consisted of two drops every 30 minutes.
The numbers of bacteria in the eyes of the group treated with 20 mg of tobram ycin intramuscularly or in the left, contralateral eyes of the subconjunctival injection treatment group were not signif icantly different from those of the saline group (P = .74). Thus, there was no sys temic therapeutic effect of this dose by either intramuscular or subconjunctival injection routes. The next trial was designed to deter mine whether antibiotic by the subcon junctival injection route would improve the effectiveness of topical therapy in the rabbit (Table 7). Tobramycin, 20 mg, by the subconjunctival injection route did not significantly enhance the effective ness of 20 mg/ml of topical tobramycin.
In a trial with Pseudomonas strain 113, the effectiveness of topical tobramycin was compared with that of tobramycin by the subconjunctival injection route in rabbits (Table 8). Tobramycin by the sub conjunctival injection route was not sig nificantly more effective than topical sa line. Topical tobramycin, 20 mg/ml, was effective. A 24-hour trial was carried out compar ing topical therapy alone with a single dose by the subconjunctival injection route at the beginning of therapy (Table 9). Numbers of bacteria in the left eyes of the subconjunctival injection group were not different from the saline-treatment group (P = .79). Antibiotic by the sub conjunctival injection route was more ef-
TABLE 7 SIX-HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L AND SUBCONJUNCTIVAL I N J E C T I O N R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 107*
Treatment 0.15M NaCl topical Tobramycin, 20 mg/ml, topical Tobramycin 20, mg/ml, topical and tobramycin 20 mg, subconjunctival injection, right eye Right eyes Left eyes
Corneal Colony Counts x ± S.D. (No.) 7.18 ± 0.14 3.41 ± 1.75
(6) (6)
<.001 .29
3.21 ± 1.27 4.24 ± 0.76
*Topical therapy consisted of two drops every 30 minutes.
(5) (6)
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TABLE 8 S I X - H O U R T H E R A P Y TRIAL W I T H TOBRAMYCIN BY SUBCONJUNCTIVAL I N J E C T I O N AND TOPICAL R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 113*
Treatment 0.15M NaCl, topical Tobramycin, 20 mg, subconjunctival injection, right eye Right eyes Left eyes Tobramycin, 20 mg/ml, topical
Corneal Colony Counts x + S.D. (No.) 7.06 ± 0.09
(6)1
6.49 ± 0.63 6.69 ± 0.23 3.37 ± 1.48
(6)
(6) J
.48
< .001
(6)
"Topical therapy consisted of two drops every 30 minutes.
fective than saline but was much less effective than by the topical route. In the final study, (Table 10), the effica cy of a single dose of tobramycin 20 mg by the subconjunctival injection route alone was determined by comparing re sults in right and left eyes. DISCUSSION
In experimental Pseudomonas keratitis in guinea pigs and rabbits, topical thera py was consistently superior to adminis tration of antibiotic by the subconjunc tival injection or intramuscular routes. In one comparison in guinea pigs (Table 4) and four comparisons in rabbits (Tables 5, 6, 8, and 9) topical therapy was more effective than therapy by the intramuscu lar or subconjunctival injection routes. In two other trials, therapy by the intramus
cular or subconjunctival injection routes did not improve efficacy of topical thera py (Tables 3 and 7). Comparable results were obtained both in guinea pigs and in rabbits. Because the properties in vitro and in vivo are so similar, we expect that other aminoglycosides would also be more ef fective by the topical route than by the intramuscular or subconjunctival injec tion routes in this experimental model. Data presented in Table 1 show more rapid absorption of antibiotic into the blood after subconjunctival injection ad ministration than after intramuscular ad ministration. We previously observed similar results in guinea pigs. 5 This ob servation may be because the periorbital tissues were very vascular. One way to estimate the degree of effi-
TABLE 9 T W E N T Y - F O U R HOUR T H E R A P Y TRIAL W I T H TOBRAMYCIN BY T O P I C A L AND SUBCONJUNCTIVAL INJECTION R O U T E S IN E X P E R I M E N T A L KERATITIS IN RABBITS W I T H PSEUDOMONAS 107*
Treatment 0.15M NaCl, topical Tobramycin, 20 mg/ml, topical Tobramycin, 20 mg, subconjunctival injection, at 0 hr, right eye Right eyes Left eyes
Corneal Colony Counts x ± S.D. (No.) 6.74 ± 0.31 0.79 ± 0.94
(6) (6)
5.48 ± 1.08 6.60 ± 0.79
(6) (6)
< .001
*Topical therapy was given as two drops every 30 minutes.
.03
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TABLE 10 TWENTY-FOUR HOUR THERAPY TRIAL OF 20 MG OF TOBRAMYCIN BY THE SUBCONJUNCTIVAL INJECTION ROUTE ALONE IN EXPERIMENTAL KERATITIS IN RABBITS WITH PSEUDOMONAS 107*
Treatment Tobramycin, 20 mg, subconjunctival injection, right eye Right eyes Left eyes
Corneal Colony Counts x ± S.D. (No.)
4.78 ± 1.94 6.55 ± 0.50
(6) (6)
P
„
Ue
*A single dose of tobramycin was injected in the right eye 24 hours after infection was established. No other therapy was given. Animals were killed 24 hours later.
cacy of 20 mg of tobramycin by the sub conjunctival injection route was to com pare numbers of bacteria in the right eyes that were injected with those of the left eyes which were not. In one trial (Table 6), the difference was highly signif icant (P = .002). In two other trials with the same organism (Tables 9 and 10), the difference was of marginal significance (P = .03; P = .06). In a trial with another strain (Table 8), there was no difference (P = .63). Topical therapy consistently had greater efficacy. We have previously shown in guinea pigs that therapy by the intramuscular route had less variability of therapeutic effect than therapy by the topical route. 5 Specifically, the standard deviations of the means were smaller for intramuscular treatment groups than for topical treat ment groups. The greater variability for the topical treatment groups is probably related to variables that are difficult to control, such as the interval between ap plication of a drop and a blink. In our studies in which therapy by the subcon junctival injection route was effective, the standard deviations were large (Tables 6, 9, and 10). We do not believe the variabil ity is related to the techniques of the injections. Our results directly conflict with many reports that subconjunctival therapy yields high intraocular concentrations of
d r u g . 1 - 3 Some differences may be the result of the method used. Baum and associates 9 reported that concentrations of gentamicin in the rabbit cornea were consistently higher with administration by the subconjunctival injection route than with the topical route in rabbits with Pseudomonas ulcers. However, these in vestigators compared topical therapy with 3 mg/ml of gentamicin, one drop every 30 minutes, with subconjunctival injection of 20 mg of gentamicin. In contrast, we compared 20 mg of tobramycin by the subconjunctival injection route with topi cal tobramycin, 20 mg/ml, two drops every 30 minutes. This higher concentra tion was used topically because it was more effective. 10 Because of differences in design and method, it was difficult to directly compare our results with those of Baum and associates. 9 We are not aware of any studies in vivo that provide objective quantitative proof of the effectiveness of administration of antibiotics by the subconjunctival injec tion route. Rabbits and guinea pigs with experi mental Pseudomonas keratitis differ in response to parenteral antibiotic therapy. We previously showed in guinea pigs that intramuscular therapy with carbenicillin, gentamicin, or tobramycin was more ef fective than intramuscular saline. 5 In this study, administration of tobramycin by
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the subconjunctival injection route in guinea pigs was effective in the contralateral, uninjected eye as well as in the ipsilateral injected eye (Table 4). The doses of antibiotics used in these trials achieve appropriate blood levels. 5 In sharp contrast, intramuscular thera py with tobramycin in rabbits was not more effective than saline (Tables 5 and 6). Furthermore, 20 mg of tobramycin by subconjunctival injection, which achieves high blood levels, did not have a thera peutic effect in the contralateral, unin jected eye (Tables 6, 8, and 9). The phys iologic or anatomic mechanisms that account for this difference in response to therapy are unknown. Whether infection in the human cornea would more clearly resemble the guinea pig or rabbit is un certain. SUMMARY
We determined the efficacy of tobram ycin administered by topical, intramuscu lar, and subconjunctival routes in guinea pigs and rabbits with experimental Pseudomonas keratitis. The topical route of administration was consistently more ef fective than either subconjunctival or in tramuscular routes. Subconjunctival in jection of antibiotic did not enhance the effectiveness of topical therapy in either guinea pigs or rabbits. Intramuscular tobramycin was more ef
MAY, 1979
fective than saline in guinea pigs with keratitis but not in rabbits with keratitis. ACKNOWLEDGMENT
Tobramycin was supplied by Eli Lilly and Co.
REFERENCES 1. Ellis, P. P.: Subconjunctival therapy. In Leo pold, I. H. (ed.): Symposium on Ocular Therapy, vol. 5. St. Louis, C. V. Mosby Co., 1972, p p . 36-42. 2. Baum, J. L., Barza, M., and Weinstein, L.: Preferred routes of antibiotic administration in treatment of bacterial ulcers of the cornea. Int. Ophthalmol. Clin. 13:31, 1973. 3. Havener, W. H.: Ocular Pharmacology, 3rd ed. St. Louis, C. V. Mosby Co., 1974. 4. Davis, S. D., and Chandler, J. W.: Experimen tal keratitis due to Pseudomonas aeruginosa. Model for evaluation of antimicrobial drugs. Angimicrob. Agents Chemother. 8:350, 1975. 5. Davis, S. D., Sarff, L. D., and Hyndiuk, R. A.: Antibiotic therapy of experimental Pseudomonas keratitis in guinea pigs. Arch. Ophthalmol. 95:1638, 1977. 6. Lenette, E. H., Spaulding, E. H., and Truant, J. P. (eds.): Manual of Clinical Microbiology, 2nd ed. Washington, American Society for Microbiolo gy, 1974. 7. Snedecor, G. W., and Cochran, W. G.: Statisti cal Methods, 6th ed. Ames, Iowa State University Press, 1976. 8. Smith, D. H., Van Otto, B., and Smith, A. L.: A rapid chemical assay for gentamicin. N. Engl. J. Med. 286:583, 1972. 9. Baum, J. L., Barza, M., Shushan, D., and Wein stein, L.: Concentration of gentamicin in experi mental corneal ulcers. Arch. Ophthalmol. 92:315, 1974. 10. Davis, S. D., Sarff, L. D., and Hyndiuk, R. A.: Topical tobramycin therapy of experimental Pseu domonas keratitis. An evaluation of some factors that potentially enhance efficacy. Arch. Ophthalmol. 96:123, 1978.