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Lincomycin Levels in Rabbit Ocular Fluids and Serum
LINCOMYCIN LEVELS IN RABBIT OCULAR FLUIDS AND SERUM ROBERT
S.
COLES,
M.D.,
GERARD L. BOYLE, F.I.M.L.T., IRVING AND S. STANLEY SCHNEIERSON, M.D.
H.
LEOPOLD,
M.D.,
New York, New York
Lincomycin hydrochloride monohydrate is derived from the actinomycete, Streptomyces lincolensis, var. lincolnensis (Mason, Dietz, and DeBoer 1 ). The mechanism of its antibacterial activity is probably through in hibition of bacterial protein synthesis. The antibiotic is active in vitro primarily against Gram-positive coccal microorganisms with the exception of the enterococci. Kohn, Hewitt, and Fraser, 2 as well as Saunders, Foster, and Scott,3 report that lincomycinresistant strains of Group A streptococci have been isolated in some patients undergo ing treatment with this antibiotic. Lincomycin does penetrate the blood-brain barrier, producing significant detectable cerebrospinal levels in the presence of inflamed meninges, according to Kaplan, Chew, and Weinstein." These authors also report that, in the presence of meningeal infection, the level of the drug in the cerebrospinal fluid is about 40% of that in the blood. Becker5 re ports that in 14 patients given lincomycin before routine eye surgery, significant levels were observed in all samples except one taken from a patient in whom the drug had been administered orally. The other 13 pa tients were given the drug parenterally; 11 received 2.4 g intramuscularly in multiple doses, and two patients received the drug in travenously, all in the same dosage. Follow ing the last injection in patients with noninflamed eyes, the average lincomycin level of the aqueous humor was 1.3 («.g/ml after one and one-half to two hours. After intramusFrom the Departments of Ophthalmology and Microbiology, Mount Sinai School of Medicine of the City University of New York. This study was supported in part by Public Health Service Grant EY-00330-05 from the National Eye Institute and a grant from the Up John Company. Reprint requests to Irving H. Leopold, M.D., 11 East 100th Street and Fifth Avenue, New York, New York 10029.
cular injection, the aqueous humor level in the affected eye (glaucoma, staphyloma) was 21 (/.g/ml, and in the same time period the serum levels ranged from 10.2 to 23 [ig/ml. Boyle, Lichtig, and Leopold8 report that sig nificant concentrations appeared in the aque ous humor as early as two minutes after lin comycin was injected subconjunctivally (0.25 ml of a solution containing 300 mg/ ml) in 20 patients with noninflamed eyes who were hospitalized for elective ocular surgery (usually cataract extraction). Peak levels of the antibiotic in the aqueous humor occurred between one and three hours, while minimal levels persist in the se rum and aqueous humor for 12 hours after injection. In the study reported here, we measured the concentration of lincomycin in the aque ous humor, vitreous humor, and serum of normal rabbits after administration by vari ous routes—intramuscular, intravenous, and subconjunctival—and the results were com pared. MATERIALS AND METHODS
All rabbits used in this study weigh 1.5 to 4.0 kg. The average dose per animal was 15 to 20 mg/kg. Lincomycin HC1 monohy drate, 300 mg/ml, was diluted to 50 mg/ml with sterile sodium chloride and then admin istered intramuscularly or intravenously; another series of rabbits received 0.1 ml of a 300 mg/ml solution of lincomycin subcon junctivally to each eye. Samples of aqueous humor, vitreous humor, and serum were ob tained at one-half, two-, four-, and six-hour intervals, following the intramuscular and intravenous administrations, and one- and four-hour intervals after subconjunctival in jection. The ocular fluids and serum were assayed for lincomycin by the tube dilution method. 464
465
LINCOMYCIN LEVELS
VOL. 72, N O . 2
This method consists of an arithmetic pro concentration of lincomycin that could be de gression of aliquots of standard antibiotic tected by this procedure was 0.3 [Jig/ml. with the addition of media in inverse RESULTS amounts to achieve a constant total volume of 1.0 ml per tube in all tubes. The levels of lincomycin in the aqueous The inoculum of the test organism, Sar- humor, vitreous humor, and serum of nor cina lutea (FDA No. 1001), was prepared mal rabbits following intramuscular, intra by inoculating glucose broth from a stock venous and subconjunctival administration agar slant maintained at 4 to 6°C. After in are indicated in Table 1. cubation at room temperature for 24 hours, Intramuscular administration—Lincomy extent of growth was determined and ad cin was present in detectable concentra justed on a LAimetron colorimeter with a tion in the aqueous humor of all of the test number 650 mu, filter until an optical density rabbits but was not detectable in the vitreous reading of 0.15 was obtained. This suspen humor of most of the animals. At one-half sion was then diluted 10~3. Fresh 1.5% tryp- hour the highest concentration in the aque ticase soy broth, pH 7.0, was employed as ous humor was 1.0 |J.g/ml and in the serum the medium and diluent. 10.3 jAg/ml. Aqueous humor levels tapered The minimum inhibitory concentration of off gradually with no marked decline up to the lincomycin standard was determined. six hours. As opposed to this situation, the The standardized Sarcina lutea test inoculum serum levels declined rapidly between onewas found to be consistently inhibited by half and two hours; between two and six 0.15 ixg/ml of the lincomycin standard solu hours the decline was more gradual. tion. Intravenous administration—Lincomycin The volume of test sample and volume of was detected in the aqueous humor, serum, standard inoculum employed in all assays and in the vitreous humor only after the onewas 0.5 ml of each per tube to obtain a total half and the two-hour aspiration. At onevolume in each tube of 1.0 ml. Appropriate half hour, the highest concentration found in controls were also prepared to verify the ste the aqueous humor was 1.19 l^g/ml and in rility of samples and medium, and also the serum 6.44 (xg/ml. Decline in the aqueous viability of the test organism. The minimum humor and serum levels followed the same TABLE 1 LINCOMYCIN LEVELS IN THE SERUM, AQUEOUS HUMOR, AND VITREOUS HUMOR OF NORMAL RABBITS AT VARYING INTERVALS AFTER INTRAMUSCULAR ( I M ) , INTRAVENOUS ( I V ) , AND SUBCONJUNCTIVAL ( S C ) INJECTIONS
Time (in hours)
Route of Adminis tration
1/2
IM IV SC IM IV IM IV SC IM IV
1 2 4 6
Serum Levels (/ig/ml) Average
Range
10.3(10)* 3.2 -15 6.44(7) 3.3 - I S Not detectable 4.16(13) 2.25-7.S 1.96(7) 0.5 - 3 1.78(8) 0.75-2.25 1.25(6) 0.5 - 2 . 2 5 Not detectable 1.64(8) 0.5 - 2 . 2 5 0.62(11) 0.37-0.75
Aqueous Levels (jug/ml) Average 1.0(7) 1.19(9) >15(2) 0.92(9) 0.87(5) 0.75(5) 0.83(3) >0.75(2) 0.64(6) 0.44(2)
Range 0.75-1.5 0.75-1.5 0.5 0.6 0.5 0.5
-1.5 -1.5 -1.25 -1.0
0.5 - 0 . 8 3 0.37-0.5
* Figure in parenthesis represents the number of rabbits in each group.
Average
Range
0.39(3)
0.33-0.5
0.47(4)
0.37-0.5
466
AMERICAN JOURNAL OF OPHTHALMOLOGY
pattern of decline observed after intramus cular injection. The aqueous level tapered off gradually, whereas the serum level rapidly declined between one-half and two hours, and then tapered down slowly between two and six hours. The concentration of lincomycin in the vitreous humor was slightly greater at two hours than at one-half hour, but the antibiotic could not be detected at the four- and six-hour assays. Subconjunctival administration—Lincomycin penetrated the aqueous humor of the test rabbits, but no perceptible level of lincomycin was found in the serum or vitre ous humor. The aqueous humor lincomycin concentration after subconjunctival injection exceeded 15 (xg/ml after one hour but at four hours the level was found to be greater than 0.75 |xg/nil. COM MENT
The design of the assay was planned to ac commodate the small volume of each sample that could be aspirated from the eye of a rabbit—approximately 0.2 ml of aqueous hu mor and 0.75 ml vitreous humor. It was nec essary to pool the aqueous from both eyes of each animal, at each time interval, in order to have enough sample to perform the assay. The vitreous humor from each eye was as sayed individually. Comparison of the serum levels, following intramuscular and intravenous administra tion, revealed similar curves. The serum lev els after intramuscular administration were found to be higher than the serum levels af ter intravenous administration at all time in tervals tested. It is possible that the intrave nous concentration peaked prior to the 30minute specimen. Kaplan and associates4 re ported finding no significant differences in serum levels in humans when intramuscular and intravenous injections are compared. The aqueous humor level after intravenous administration was found to be somewhat higher than after intramuscular injection at one-half hour but, after one and one-half hours, it was slightly lower than the intra
AUGUST, 1971
muscular injection. With both intramuscular and intravenous injections a gradual straight line decline was noted. Lincomycin did pene trate the vitreous humor after intravenous administration up to at least two hours, but beyond two hours no lincomycin could be de tected by the assay method employed. Sub conjunctival administration produced the highest intraocular concentrations obtained in this study. Kaplan and associates4 showed that concentrations of lincomycin required to inhibit 60 strains of Staphylpcoccus aureus ranged from 6.2 to 0.75 pig/ml, against eight strains of Streptococcus pyogenes from 1.5 to 0.09 |xg/ml, against six strains of Strep tococcus viridans from 0.75 to 0.19 [Ag/ml, and against five strains of Diplococcus pneumoniae from 1.5 to 0.19 (Ag/ml. It is evident that the required antibacterial concentration for the above bacteria is read ily achievable in the serum after both intra muscular and intravenous injection after one-half hour, and for some strains up to six hours (Fig. 1). Concentration of lincomycin obtained in the aqueous humor is adequate
Time ( Hours) Fig. 1 (Coles, Boyle, Leopold, and Schneierson). Average lincomycin levels (in ng/ml) in aqueous humor and serum at varying time intervals follow ing intramuscular and intravenous injections.
VOL. 72, NO. 2
LINCOMYCIN LEVELS
against all strains with a minimal inhibitory concentration below 1.19 ng/ml after intra venous and 1.0 [ig/ml after intramuscular injection at one-half hour. Some but not all of the above bacterial strains will be inhib ited by the aqueous humor concentrations. Intravenous injection of lincomvcin will achieve antibacterial concentration in the vit reous at one-half and two hours against some strains of Streptococcus pyogenes, viridans streptococci, and Diplococcus pneumoniae. The concentration of lincomvcin in the aqueous humor one hour following subconjunctival administration is theoretically ade quate to inhibit all strains with minimal in hibitory concentrations below IS [/.g/ml, and most strains will still be inhibited by the con centrations observed up to four hours after administration. SUMMARY
A series of experiments were performed to determine the level of lincomycin in the aqueous and vitreous humor of noninflamed eyes and in the serum of rabbits at varying periods following intramuscular, intrave nous, or subconjunctival injection. Detect
467
able levels were achieved by all routes of ad ministration within one-half hour and up to six hours after injection. The concentrations found in the serum and aqueous humor were sufficient to be effective against a number of Gram-positive coccal pathogens. Absorption into the vitreous humor was irregular and was of questionable significance in the noninflamed eye. ACKNOWLEDGMENT
The lincomycin used in this study was supplied as Lincocin by the Upjohn Company. REFERENCES
1. Mason, D. J., Dietz, A., and DeBoer, C.: Lin comycin, a new antibiotic. I. Discovery and biologi cal properties. Antimicrob. Chemotherap., 1962, p. 554. 2. Kohn, J., Hewitt, J. H., and Fraser, C. A. M.: G'-otip A streptocci resistant to lincomycin. Brit. Med. J. 1:703, 1968. 3. Sanders, E., Foster, M. T., and Scott, D.: Group A beta hemolytic streptococci resistant to erythromycin and lincomycin. New Eng. J. Med. 278: 538, 1968. 4. Kaplan, K., Chew, W. H., and Weinstein, L.: Microbiological, pharmacological and clinical stud ies of lincomycin. Am. J. Med. Sci. 250:137, 1965. 5. Becker, E. F.: The intraocular penetration of lincomycin. Am. J. Ophth. 67:963, 1969. 6. Boyle, G. L., Lichtig, M. L., and Leopold, I. H.: Lincomycin levels in human ocular fluids and serum following subconjunctival injection. In press.
S.
COLES,
M.D.,
GERARD L. BOYLE, F.I.M.L.T., IRVING AND S. STANLEY SCHNEIERSON, M.D.
H.
LEOPOLD,
M.D.,
New York, New York
Lincomycin hydrochloride monohydrate is derived from the actinomycete, Streptomyces lincolensis, var. lincolnensis (Mason, Dietz, and DeBoer 1 ). The mechanism of its antibacterial activity is probably through in hibition of bacterial protein synthesis. The antibiotic is active in vitro primarily against Gram-positive coccal microorganisms with the exception of the enterococci. Kohn, Hewitt, and Fraser, 2 as well as Saunders, Foster, and Scott,3 report that lincomycinresistant strains of Group A streptococci have been isolated in some patients undergo ing treatment with this antibiotic. Lincomycin does penetrate the blood-brain barrier, producing significant detectable cerebrospinal levels in the presence of inflamed meninges, according to Kaplan, Chew, and Weinstein." These authors also report that, in the presence of meningeal infection, the level of the drug in the cerebrospinal fluid is about 40% of that in the blood. Becker5 re ports that in 14 patients given lincomycin before routine eye surgery, significant levels were observed in all samples except one taken from a patient in whom the drug had been administered orally. The other 13 pa tients were given the drug parenterally; 11 received 2.4 g intramuscularly in multiple doses, and two patients received the drug in travenously, all in the same dosage. Follow ing the last injection in patients with noninflamed eyes, the average lincomycin level of the aqueous humor was 1.3 («.g/ml after one and one-half to two hours. After intramusFrom the Departments of Ophthalmology and Microbiology, Mount Sinai School of Medicine of the City University of New York. This study was supported in part by Public Health Service Grant EY-00330-05 from the National Eye Institute and a grant from the Up John Company. Reprint requests to Irving H. Leopold, M.D., 11 East 100th Street and Fifth Avenue, New York, New York 10029.
cular injection, the aqueous humor level in the affected eye (glaucoma, staphyloma) was 21 (/.g/ml, and in the same time period the serum levels ranged from 10.2 to 23 [ig/ml. Boyle, Lichtig, and Leopold8 report that sig nificant concentrations appeared in the aque ous humor as early as two minutes after lin comycin was injected subconjunctivally (0.25 ml of a solution containing 300 mg/ ml) in 20 patients with noninflamed eyes who were hospitalized for elective ocular surgery (usually cataract extraction). Peak levels of the antibiotic in the aqueous humor occurred between one and three hours, while minimal levels persist in the se rum and aqueous humor for 12 hours after injection. In the study reported here, we measured the concentration of lincomycin in the aque ous humor, vitreous humor, and serum of normal rabbits after administration by vari ous routes—intramuscular, intravenous, and subconjunctival—and the results were com pared. MATERIALS AND METHODS
All rabbits used in this study weigh 1.5 to 4.0 kg. The average dose per animal was 15 to 20 mg/kg. Lincomycin HC1 monohy drate, 300 mg/ml, was diluted to 50 mg/ml with sterile sodium chloride and then admin istered intramuscularly or intravenously; another series of rabbits received 0.1 ml of a 300 mg/ml solution of lincomycin subcon junctivally to each eye. Samples of aqueous humor, vitreous humor, and serum were ob tained at one-half, two-, four-, and six-hour intervals, following the intramuscular and intravenous administrations, and one- and four-hour intervals after subconjunctival in jection. The ocular fluids and serum were assayed for lincomycin by the tube dilution method. 464
465
LINCOMYCIN LEVELS
VOL. 72, N O . 2
This method consists of an arithmetic pro concentration of lincomycin that could be de gression of aliquots of standard antibiotic tected by this procedure was 0.3 [Jig/ml. with the addition of media in inverse RESULTS amounts to achieve a constant total volume of 1.0 ml per tube in all tubes. The levels of lincomycin in the aqueous The inoculum of the test organism, Sar- humor, vitreous humor, and serum of nor cina lutea (FDA No. 1001), was prepared mal rabbits following intramuscular, intra by inoculating glucose broth from a stock venous and subconjunctival administration agar slant maintained at 4 to 6°C. After in are indicated in Table 1. cubation at room temperature for 24 hours, Intramuscular administration—Lincomy extent of growth was determined and ad cin was present in detectable concentra justed on a LAimetron colorimeter with a tion in the aqueous humor of all of the test number 650 mu, filter until an optical density rabbits but was not detectable in the vitreous reading of 0.15 was obtained. This suspen humor of most of the animals. At one-half sion was then diluted 10~3. Fresh 1.5% tryp- hour the highest concentration in the aque ticase soy broth, pH 7.0, was employed as ous humor was 1.0 |J.g/ml and in the serum the medium and diluent. 10.3 jAg/ml. Aqueous humor levels tapered The minimum inhibitory concentration of off gradually with no marked decline up to the lincomycin standard was determined. six hours. As opposed to this situation, the The standardized Sarcina lutea test inoculum serum levels declined rapidly between onewas found to be consistently inhibited by half and two hours; between two and six 0.15 ixg/ml of the lincomycin standard solu hours the decline was more gradual. tion. Intravenous administration—Lincomycin The volume of test sample and volume of was detected in the aqueous humor, serum, standard inoculum employed in all assays and in the vitreous humor only after the onewas 0.5 ml of each per tube to obtain a total half and the two-hour aspiration. At onevolume in each tube of 1.0 ml. Appropriate half hour, the highest concentration found in controls were also prepared to verify the ste the aqueous humor was 1.19 l^g/ml and in rility of samples and medium, and also the serum 6.44 (xg/ml. Decline in the aqueous viability of the test organism. The minimum humor and serum levels followed the same TABLE 1 LINCOMYCIN LEVELS IN THE SERUM, AQUEOUS HUMOR, AND VITREOUS HUMOR OF NORMAL RABBITS AT VARYING INTERVALS AFTER INTRAMUSCULAR ( I M ) , INTRAVENOUS ( I V ) , AND SUBCONJUNCTIVAL ( S C ) INJECTIONS
Time (in hours)
Route of Adminis tration
1/2
IM IV SC IM IV IM IV SC IM IV
1 2 4 6
Serum Levels (/ig/ml) Average
Range
10.3(10)* 3.2 -15 6.44(7) 3.3 - I S Not detectable 4.16(13) 2.25-7.S 1.96(7) 0.5 - 3 1.78(8) 0.75-2.25 1.25(6) 0.5 - 2 . 2 5 Not detectable 1.64(8) 0.5 - 2 . 2 5 0.62(11) 0.37-0.75
Aqueous Levels (jug/ml) Average 1.0(7) 1.19(9) >15(2) 0.92(9) 0.87(5) 0.75(5) 0.83(3) >0.75(2) 0.64(6) 0.44(2)
Range 0.75-1.5 0.75-1.5 0.5 0.6 0.5 0.5
-1.5 -1.5 -1.25 -1.0
0.5 - 0 . 8 3 0.37-0.5
* Figure in parenthesis represents the number of rabbits in each group.
Average
Range
0.39(3)
0.33-0.5
0.47(4)
0.37-0.5
466
AMERICAN JOURNAL OF OPHTHALMOLOGY
pattern of decline observed after intramus cular injection. The aqueous level tapered off gradually, whereas the serum level rapidly declined between one-half and two hours, and then tapered down slowly between two and six hours. The concentration of lincomycin in the vitreous humor was slightly greater at two hours than at one-half hour, but the antibiotic could not be detected at the four- and six-hour assays. Subconjunctival administration—Lincomycin penetrated the aqueous humor of the test rabbits, but no perceptible level of lincomycin was found in the serum or vitre ous humor. The aqueous humor lincomycin concentration after subconjunctival injection exceeded 15 (xg/ml after one hour but at four hours the level was found to be greater than 0.75 |xg/nil. COM MENT
The design of the assay was planned to ac commodate the small volume of each sample that could be aspirated from the eye of a rabbit—approximately 0.2 ml of aqueous hu mor and 0.75 ml vitreous humor. It was nec essary to pool the aqueous from both eyes of each animal, at each time interval, in order to have enough sample to perform the assay. The vitreous humor from each eye was as sayed individually. Comparison of the serum levels, following intramuscular and intravenous administra tion, revealed similar curves. The serum lev els after intramuscular administration were found to be higher than the serum levels af ter intravenous administration at all time in tervals tested. It is possible that the intrave nous concentration peaked prior to the 30minute specimen. Kaplan and associates4 re ported finding no significant differences in serum levels in humans when intramuscular and intravenous injections are compared. The aqueous humor level after intravenous administration was found to be somewhat higher than after intramuscular injection at one-half hour but, after one and one-half hours, it was slightly lower than the intra
AUGUST, 1971
muscular injection. With both intramuscular and intravenous injections a gradual straight line decline was noted. Lincomycin did pene trate the vitreous humor after intravenous administration up to at least two hours, but beyond two hours no lincomycin could be de tected by the assay method employed. Sub conjunctival administration produced the highest intraocular concentrations obtained in this study. Kaplan and associates4 showed that concentrations of lincomycin required to inhibit 60 strains of Staphylpcoccus aureus ranged from 6.2 to 0.75 pig/ml, against eight strains of Streptococcus pyogenes from 1.5 to 0.09 |xg/ml, against six strains of Strep tococcus viridans from 0.75 to 0.19 [Ag/ml, and against five strains of Diplococcus pneumoniae from 1.5 to 0.19 (Ag/ml. It is evident that the required antibacterial concentration for the above bacteria is read ily achievable in the serum after both intra muscular and intravenous injection after one-half hour, and for some strains up to six hours (Fig. 1). Concentration of lincomycin obtained in the aqueous humor is adequate
Time ( Hours) Fig. 1 (Coles, Boyle, Leopold, and Schneierson). Average lincomycin levels (in ng/ml) in aqueous humor and serum at varying time intervals follow ing intramuscular and intravenous injections.
VOL. 72, NO. 2
LINCOMYCIN LEVELS
against all strains with a minimal inhibitory concentration below 1.19 ng/ml after intra venous and 1.0 [ig/ml after intramuscular injection at one-half hour. Some but not all of the above bacterial strains will be inhib ited by the aqueous humor concentrations. Intravenous injection of lincomvcin will achieve antibacterial concentration in the vit reous at one-half and two hours against some strains of Streptococcus pyogenes, viridans streptococci, and Diplococcus pneumoniae. The concentration of lincomvcin in the aqueous humor one hour following subconjunctival administration is theoretically ade quate to inhibit all strains with minimal in hibitory concentrations below IS [/.g/ml, and most strains will still be inhibited by the con centrations observed up to four hours after administration. SUMMARY
A series of experiments were performed to determine the level of lincomycin in the aqueous and vitreous humor of noninflamed eyes and in the serum of rabbits at varying periods following intramuscular, intrave nous, or subconjunctival injection. Detect
467
able levels were achieved by all routes of ad ministration within one-half hour and up to six hours after injection. The concentrations found in the serum and aqueous humor were sufficient to be effective against a number of Gram-positive coccal pathogens. Absorption into the vitreous humor was irregular and was of questionable significance in the noninflamed eye. ACKNOWLEDGMENT
The lincomycin used in this study was supplied as Lincocin by the Upjohn Company. REFERENCES
1. Mason, D. J., Dietz, A., and DeBoer, C.: Lin comycin, a new antibiotic. I. Discovery and biologi cal properties. Antimicrob. Chemotherap., 1962, p. 554. 2. Kohn, J., Hewitt, J. H., and Fraser, C. A. M.: G'-otip A streptocci resistant to lincomycin. Brit. Med. J. 1:703, 1968. 3. Sanders, E., Foster, M. T., and Scott, D.: Group A beta hemolytic streptococci resistant to erythromycin and lincomycin. New Eng. J. Med. 278: 538, 1968. 4. Kaplan, K., Chew, W. H., and Weinstein, L.: Microbiological, pharmacological and clinical stud ies of lincomycin. Am. J. Med. Sci. 250:137, 1965. 5. Becker, E. F.: The intraocular penetration of lincomycin. Am. J. Ophth. 67:963, 1969. 6. Boyle, G. L., Lichtig, M. L., and Leopold, I. H.: Lincomycin levels in human ocular fluids and serum following subconjunctival injection. In press.