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Ocular Penetration of 14C-Labeled Chloramphenicol Following Subconjunctival or Sub-Tenon's Injection
OCULAR
PENETRATION
FOLLOWING RONALD
OF "C-LABELED
SUBCONJUNCTIVAL
G.
HARDY,
JR.,
M.D,
A N D
CHLORAMPHENICOL
O R SUB-TENON'S
CHRISTOPHER
A.
INJECTION
PATERSON,
PH.D.
Denver, Colorado
The subconjunctival method of introducing antibiotics into the eye is advocated by many for clinical use and is frequently compared with other routes of drug administration to study penetration into the eye. McPherson, Presley, and Crowford investigated the penetration of subconjunctival potassium penicillin G , ampicillin, chloramphenicol and streptomycin in animals and cataract patients. Primary aqueous samples were obtained 20 seconds to 24 hours after administration and quantitative analysis performed with a biological assay. They found that ampicillin and potassium penicillin G penetrated into primary aqueous in effective concentrations while chloramphenicol and streptomycin did not. Contrary to these investigators, Leopold, Nichols, and Vogel, and Abraham and Burnett found that subconjunctival chloramphenicol does penetrate into the aqueous. 1
2
3
Records and Ellis found that subconjunctivally administered penicillins penetrated primary aqueous well at one hour with variable persistence at latter times, oxacillin remaining in high concentration at four hours. Records - compared intravenous and subconjunctivally administered sodium cephalothin. Intravenously administered cephalothin failed to enter primary aqueous while subconjunctivally administered cephalothin penetrated well. In 1956, Swan, Crissman, and Bailey stressed the importance in differentiating subconjunctival from sub-Tenon's injection, or, as they termed it, "sub-epithelial versus 4
5
6
7
From the Division of Ophthalmology, University of Colorado Medical Center, Denver, Colorado. This study was supported in part by an unrestricted grant from Research to Prevent Blindness, Inc. Reprint requests to Christopher A. Paterson, Ph.D., Box 2404, 4200 East Ninth Avenue, Denver, Colorado 80220.
sub-capsular." On the basis of some qualitative experiments, they felt that drugs penetrated the eye better if administered by the sub-Tenon's rather than the subconjunctival •route. The present study was devised to determine if a drug, in this case C-labeled chloramphenicol, showed differential penetration into the eye when administered subconjunctivally as compared to sub-Tenon's administration. 14
MATERIALS A N D
METHODS
I N vivo
Penetration of chloramphenicol was studied using D-threo chloramphenicol (methyl e n e - ^ Amersham-Searle). An injection solution of 10% chloramphenicol (Chloromycetin, Parke-Davis) containing 1 u,c/ml of the labeled drug was prepared fresh daily. The vehicle used was sterile distilled water. Rabbits weighing between 2.5 and 3.5 kg were anesthetized with 25% urethane solution administered via an ear vein. Using a 27-gauge needle, 100 ul of the chloramphenicol solution was administered either by the subconjunctival or sub-Tenon's route. For topical administration, sutures were utilized to elevate the eyelids from the globe forming a cup to contain the radioactive antibiotic solution. 14
The rabbits were divided into seven groups as shown in Table 1. After the experimental time period had elapsed, samples of primary aqueous humor were aspirated using a 1-ml disposable syringe and 25-gauge needle, and these were assayed for radioactivity. Radioactivity measurements—Between 80 and 100 u,l aliquots of aqueous humor were weighed onto disks of lens paper on glass planchets. The samples were allowed to air dry and were counted in an automatic planchet counter (Picker Nuclear Magnachan-
1307
1308
AMERICAN JOURNAL OF OPHTHALMOLOGY
JUNE, 1971
TABLE 1 GROUPS OF ANIMALS AND ROUTE OF ADMINISTRATION OF CHLORAMPHENICOL
Group
No. Animals
Left Eye
Right Eye
1 2 3 4 5 6 7
4 5 7 11 10 5 5
Subconjunctival Sub-Tenon's Subconjunctival Subconjunctival Subconjunctival Subconjunctival Subconjunctival
Subconjunctival Sub-Tenon's Sub-Tenon's Sub-Tenon's Sub-Tenon's Sub-Tenon's Topical
ger 40 System). Suitably diluted aliquots of each injection solution were also counted. Radioactivity was expressed as counts per 10 minutes per gram. Assay for intact chloramphenicol—In experiments where the penetration of labeled drugs is being studied it is necessary to show that the labeled material recovered is in fact the intact drug and not a degradation product. Biological assay or chromatographic techniques were considered to be inconvenient, thus the method of Glasko, Wolf, and Dill was used to separate intact chloramphenicol from any breakdown products. The aqueous humor of three similar experiments (e.g., three subconjunctival administrations for the same time period) was pooled and weighed. This was shaken with a mixture of ethyl acetate and phosphate buffer (pH 6 ) . The two layers were allowed to separate. The ethyl
1 1 j 1 3 6 1
acetate layer was removed and washed with buffer. The final ethyl acetate and aqueous buffer extracts were evaporated to dryness, redissolved in 0.5 ml of distilled water and the radioactivity in each measured as described above. All intact chloramphenicol is taken up into the ethyl acetate while any breakdown products remain in the aqueous phase.
8
IN
VITRO
Several experiments were performed to principally compare the in vitro permeability of rabbit Tenon's capsule with that of the human. Both C-labeled chloramphenicol and N a were used. A simple lucite chamber was constructed which allowed a piece of Tenon's capsule to be clamped between two fluidfilled chambers. Each chamber contained an equal volume of 0.95 ml. One chamber con14
22
TABLE 2 RADIOACTIVITY (COUNTS TER 10 MINUTES PER GRAM) IN AQUEOUS HUMOR FOLLOWING SUBCONJUNCTIVAL OR SUB-TENON'S INJECTION OF "C-LABELED CHLORAMPHENICOL*
Groups
Left Eye
Right Eye
Ratio LE/RE
Controls Group 1 (STf vs ST, 1 hour) Group 2 (SC vs SC; 1 hour)
2041 (4) 3255 (5)
2102 (4) 3096 (5)
0.97 1.05
Experimental Group 3 (SC vs ST; i hour) Group 4 (SC vs ST; 1 hour)
SC 1290 (7) 2973 (11)
ST 850 (7) 1359 (11)
Ratio SC/ST 1.52 2.19
Group 5 (SC vs ST; 3 hours) Group 6 (SC vs ST; 6 hours) Group 7 (SC vs Topical)
2180 (10) 183 (5) 3199 (5)
1430 (10) 210 (5) 5758 (5)
1.52 0.87 0.56
* Counts represent the mean of the number of observation given in parentheses, t SC refers to subconjunctival and ST to sub-Tenon's administration.
VOL. 71, NO. 6
SUBCONJUNCTIVAL VS CHLORAMPHENICOL
1309
TIME (HOURS) Fig. 1 (Hardy and Paterson). Time course of penetration of C-labeled chloramphenicol into rabbit aqueous humor following subconjunctival (open circles) and sub-Tenon's (filled circles) injection. Constructed using data in Table 2. M
tained only saline while the other contained saline together with a trace amount of labeled chloramphenicol or Na. 10 or 20|A1 samples were taken from each chamber at 30-minute intervals and counted as described above. Stirring was achieved manually with flexible polyethylene paddles. Permeability coefficients were calculated by the method of Maffly, Hays, Lamdin, and Leaf. 22
9
RESULTS IN
VIVO
Controls—In Groups 1 and 2, identical degrees of penetration were found in two eyes of the same animal following either a subconjunctival or sub-Tenon's injection on each side (Table 2 ) . Subconjunctival versus sub-Tenon's administration—In Groups 3 to 5 at one half hour, one hour, and three hours, a greater amount of labeled chloramphenicol was
found in the aqueous of the eye given the drug subconjunctivally. At one half hour the ratio of penetration, comparing subconjunctival to sub-Tenon's capsule administration, was 1.52: 1, at one hour 2.19: 1 and at three hours (Group 5 ) , 1.52: 1. By paired statistics these differences were significant at the 95% or greater level. At six hours (Group 6) there was no difference in the degree of penetration (0.87: 1 ) . These results are depicted in Figure 1 and in Table 2. Subconjunctival versus topical administration—In Group 7, penetration of C-labeled chloramphenicol one hour following topical administration was some 1.8 times greater than when given subconjunctivally (Table 2 ) . Assay for chloramphenicol—In all instances the entire labeled fraction was recovered from the ethyl acetate extract. No radioactivity was found in the aqueous layer. 14
Thus, during the experimental period there was no detectable degradation of chloramphenicol. IN
JUNE, 1971
AMERICAN JOURNAL OF OPHTHALMOLOGY
1310
VITRO
All the in vitro experiments were performed at room temperature (23-25°C). Human Tenon's capsule was approximately 24 hour postmortem when used. No difference in permeability characteristics was found between rabbit Tenon's capsule dissected immediately and that dissected 24 hours postmortem. The permeability coefficient for Na was 5.11 X 10 cm/second in rabbit Tenon's capsule and 5.2 X 10~ cm/second in the human. Similarly for chloramphenicol, 4.35 X l O cm/second in rabbit and 4.9 X 10 cm/second in the human. Thus it would appear that the permeability characteristics of rabbit and human Tenon's capsule are quite similar. 22
-5
5
5
-5
DISCUSSION
The results of this study clearly indicate that in the rabbit, chloramphenicol penetrates the aqueous humor of the eye more rapidly and in greater quantity when given subconjunctivally than when given via sub-Tenon's capsule. Chloramphenicol is a highly lipid soluble drug and it would be expected that other lipid soluble drugs would behave similarly (e.g., steroids, epinephrine, atropine). Whether nonlipid soluble drugs behave similarly can only be conjectured from the present results. Direct correlation between the results in animals with the situation in human eyes is not possible, but the data does at least show that Tenon's capsule from rabbit eyes and human eyes exhibits almost identical permeability characteristics in vitro to chloramphenicol and sodium. On the basis of the present findings, then, it would seem wise to avoid injecting medication via sub-Tenon's capsule when such a significant penetration differential exists. This, however, is quite contrary to the rec-
ommendation of several authorities. Haverner writes : "Subconjunctival injection is actually a misnomer. Such an injection should be given beneath Tenon's capsule, since in this position the drug is effectively closer to the eye, is absorbed more rapidly, suffers less loss into the conjunctival circulation, and reaches greater intraocular concentrations." Also, Burns writes: "Subconjunctival and sub-Tenon's injection of medications is an obvious means of short circuiting the epithelial barrier (of the cornea). It is known that sub-Tenon's injection is more effective than subconjunctival in aiding drug penetration of intraocular tissues." 10
11
The original investigations that gave rise to these opinions were those of Swan, Chrisman, and Bailey. Subjects undergoing strabismus surgery were first made miotic with pilocarpine. A sub-Tenon's injection of 0.05 ml of 1: 1000 epinephrine was then given on one side of one eye and a similar injection given subconjunctivally on the other side of the same eye. Swan and his associates found that more rapid segmental pupil dilation occurred on the side receiving the sub-Tenon's injection. This was observed in 11 of 12 patients. The study was prompted by earlier experiments in rabbits which seemed to indicate that sub-Tenon's procaine penetrated the eye better than when given subconjunctivally. 7
12
In this laboratory, using rabbits and a more quantitive method utilizing C-labeled chloramphenicol we have shown quite the opposite to be true. Why the marked difference in ocular penetration following the two methods of administration exists poses quite a problem. Preliminary investigation has shown (Hardy and Paterson, unpublished data) that there is more radioactivity in the venous return from the eye receiving a sub-Tenon's injection than that receiving a subconjunctival injection. If labeled chloramphenicol penetrates the sclera from its sub-Tenon's location, encounters the large choroidal venous channels, then one would expect it to be largely carried 14
VOL. 71, NO. 6
SUBCONJUNCTIVAL VS CHLORAMPHENICOL
away from the eye. Would not the same be true for the subconjunctivally located drug? Most likely a fraction does penetrate the sclera after diffusing through Tenon's capsule and is flushed away from the eye. However, to explain the difference in penetration another route or routes must exist which bypass the venous channel in the choroid. One possible route is the limbus. At a finite distance from the limbus, the Tenon's capsule blends into scleral tissue thus possibly trapping the drug to some extent. The conjunctiva, however, continues over the limbus onto the cornea where conjunctiva blends with corneal epithelium. Thus, a relatively high concentration of drug may exist over the limbal region, subsequent penetration being across the corneal endothelium. Harris presents evidence for such a route using radiolabeled eserine. 13
Another possibility is that the drug passes from its subconjunctival location into the tears and thus penetration is directly across the cornea from the tears. Such a mechanism is favored by Richardson. In this context we have shown that at one hour chloramphenicol penetrates the aqueous humor faster following topical administration than subconjunctivally. (This does not necessarily mean that topical administration is more effective clinically, since our experimental situation was quite different in that the eye was effectively bathed in drug solution for one hour.) Also, Wine, Gornall, and Basu found evidence for this route of penetration utilizing C-hydrocortisone. In their investigation there was increased ocular penetration of drug when the conjunctiva was perforated in the usual manner of subconjunctival injection as opposed to nonperforating injections via the upper eyelid. Following a nonperforating subconjunctival administration no drug was found in the tears. The authors proposed that the drug is squeezed out of the injection hole into the tear film. Although the present study provides no further information to aid in elucidating the mechanism of penetration it does show that a 14
15
14
1311
drug, in this case chloramphenicol, penetrates the anterior segment of the eye more rapidly following a subconjunctival rather than a sub-Tenon's injection. S U M M A R Y
The level of radioactivity in the aqueous humor of albino rabbits was assayed at varying periods of time following C-labeled chloramphenicol administered subconjunctivally to one eye and via sub-Tenon's capsule to the contralateral eye. At 30 minutes, one hour, and three hours a greater quantity of the drug appeared to penetrate the eye receiving the subconjunctival injection. At six hours there was no difference in the degree of penetration. On the basis of these observations it is suggested that the use of sub-Tenon's injections could be avoided. The in vitro permeability characteristics of human and rabbit Tenon's capsule are virtually identical. 14
ACKNOWLEDGMENT
We thank Dr. Philip P. Ellis who suggested the project and for his continued interest and comment during the course of the study. REFERENCES
1. McPherson, S. D , Jr., Presley, G. D , and Crowford, J. R. : Aqueous humor assays of subconjunctival antibiotics. Am. J. Ophth. 66:430, 1968. 2. Leopold, I. H , Nichols, A. C , and Vogel, D. W. : Penetration of chloramphenicol U.S.P. into the eye. Arch Ophth. 44:706, 1950. 3. Abraham, R. K , and Burnett, H. H. : Tetracycline and chloramphenicol studies on rabbit and human eyes. Arch Ophth. 54:641, 1955. 4. Records, R. E , and Ellis, P. P. : The intraocular penetration of ampicillin, methicillin, and oxacillin. Am. J. Ophth. 64:135,1967. 5. Records, R. E. : Intraocular penetration of cephalothin, Part I. Animal studies. Am. J. Ophth. 66:436, 1968. 6. Records, R. E. : Intraocular penetration of cephalothin, Part II. Human studies. Am. J. Ophth. 66:441, 1968. 7. Swan, K. G., Crisman, H. R , and Bailey, P. E , Jr. : Subepithelial versus subcapsular injections of drugs. Arch. Ophth. 56:26, 1956. 8. Glazko, A. J., Wolf, L. M , and Dill, W. A. : Biochemical studies on chloramphenicol (Chloromycetin) I. Colorimetric methods for the determination of chloramphenicol and related vitro compounds. Arch Biochem. 23:411, 1949.
1312
AMERICAN JOURNAL OF OPHTHALMOLOGY
9. Maffly, R. H , Hays, R. M , Lamdin, E , and Leaf, A. : The effect of neurohypophyseal hormones on the permeability of the toad bladder to urea. J. Clin. Invest. 39:630, 19S9. 10. Havener, W. H. : Ocular Pharmacology. St. Louis, Mosby, 1966, p. 13. 11. Burns, R. P.: Pharmacology and toxicology of the conjunctiva. In Symposium on Ocular Pharmacology and Therapeutics. Transactions of the New Orleans Acadamy of Ophthalmology. St. Louis, Mosby, 1970, p. 97. 12. Schlegel, H. E , and Swan, K. G. : Ocular penetration of procaine following subconjunctival injection. Arch Ophth. 52 :774. 1954.
JUNE, 1971
13. Harris, J. E.: Problems in drug penetration. In Leopold, I. H.: Symposium in Ocular Therapy, vol. 3. St. Louis, Mosby 1968. 14. Richardson, K. T. : Ocular pharmacodynamics. In Symposium on Ocular Pharmacology and Therapeutics. Transaction of the New Orleans Academy of Ophthalmology. St. Louis, Mosby, 1970. 15. Wine, N. A., Gornall, A. G, and Basu, P. K. : The ocular uptake of subconjunctivally injected "C-hydrocortisone. I. Time and major route of penetration into the eye. Am. T. Ophth. 58:362, 1964.
O P H T H A L M I C MINIATURE
I noticed herfingersnervously twisting and untwisting a pleat of the chiffon of her dress. "We can state definitely now that your husband died as a result of eserine poisoning." "You mean those eyedrops killed him," "It seems quite certain that when you gave Mr. Leónides that last injection, it was eserine that you injected and not insulin." "But I didn't know that. I didn't have anything to do with it. Really I didn't, Inspector." "Then somebody must have deliberately replaced the insulin by the eyedrops." "What a wicked thing to do !" "Yes, Mrs. Leónides." "Do you think someone did it on purpose? Or by accident? It couldn't have been a joke, could it?" Taverner said smoothly, "We don't think it was a joke, Mrs. Leónides." Agatha Christie The Crooked House
PENETRATION
FOLLOWING RONALD
OF "C-LABELED
SUBCONJUNCTIVAL
G.
HARDY,
JR.,
M.D,
A N D
CHLORAMPHENICOL
O R SUB-TENON'S
CHRISTOPHER
A.
INJECTION
PATERSON,
PH.D.
Denver, Colorado
The subconjunctival method of introducing antibiotics into the eye is advocated by many for clinical use and is frequently compared with other routes of drug administration to study penetration into the eye. McPherson, Presley, and Crowford investigated the penetration of subconjunctival potassium penicillin G , ampicillin, chloramphenicol and streptomycin in animals and cataract patients. Primary aqueous samples were obtained 20 seconds to 24 hours after administration and quantitative analysis performed with a biological assay. They found that ampicillin and potassium penicillin G penetrated into primary aqueous in effective concentrations while chloramphenicol and streptomycin did not. Contrary to these investigators, Leopold, Nichols, and Vogel, and Abraham and Burnett found that subconjunctival chloramphenicol does penetrate into the aqueous. 1
2
3
Records and Ellis found that subconjunctivally administered penicillins penetrated primary aqueous well at one hour with variable persistence at latter times, oxacillin remaining in high concentration at four hours. Records - compared intravenous and subconjunctivally administered sodium cephalothin. Intravenously administered cephalothin failed to enter primary aqueous while subconjunctivally administered cephalothin penetrated well. In 1956, Swan, Crissman, and Bailey stressed the importance in differentiating subconjunctival from sub-Tenon's injection, or, as they termed it, "sub-epithelial versus 4
5
6
7
From the Division of Ophthalmology, University of Colorado Medical Center, Denver, Colorado. This study was supported in part by an unrestricted grant from Research to Prevent Blindness, Inc. Reprint requests to Christopher A. Paterson, Ph.D., Box 2404, 4200 East Ninth Avenue, Denver, Colorado 80220.
sub-capsular." On the basis of some qualitative experiments, they felt that drugs penetrated the eye better if administered by the sub-Tenon's rather than the subconjunctival •route. The present study was devised to determine if a drug, in this case C-labeled chloramphenicol, showed differential penetration into the eye when administered subconjunctivally as compared to sub-Tenon's administration. 14
MATERIALS A N D
METHODS
I N vivo
Penetration of chloramphenicol was studied using D-threo chloramphenicol (methyl e n e - ^ Amersham-Searle). An injection solution of 10% chloramphenicol (Chloromycetin, Parke-Davis) containing 1 u,c/ml of the labeled drug was prepared fresh daily. The vehicle used was sterile distilled water. Rabbits weighing between 2.5 and 3.5 kg were anesthetized with 25% urethane solution administered via an ear vein. Using a 27-gauge needle, 100 ul of the chloramphenicol solution was administered either by the subconjunctival or sub-Tenon's route. For topical administration, sutures were utilized to elevate the eyelids from the globe forming a cup to contain the radioactive antibiotic solution. 14
The rabbits were divided into seven groups as shown in Table 1. After the experimental time period had elapsed, samples of primary aqueous humor were aspirated using a 1-ml disposable syringe and 25-gauge needle, and these were assayed for radioactivity. Radioactivity measurements—Between 80 and 100 u,l aliquots of aqueous humor were weighed onto disks of lens paper on glass planchets. The samples were allowed to air dry and were counted in an automatic planchet counter (Picker Nuclear Magnachan-
1307
1308
AMERICAN JOURNAL OF OPHTHALMOLOGY
JUNE, 1971
TABLE 1 GROUPS OF ANIMALS AND ROUTE OF ADMINISTRATION OF CHLORAMPHENICOL
Group
No. Animals
Left Eye
Right Eye
1 2 3 4 5 6 7
4 5 7 11 10 5 5
Subconjunctival Sub-Tenon's Subconjunctival Subconjunctival Subconjunctival Subconjunctival Subconjunctival
Subconjunctival Sub-Tenon's Sub-Tenon's Sub-Tenon's Sub-Tenon's Sub-Tenon's Topical
ger 40 System). Suitably diluted aliquots of each injection solution were also counted. Radioactivity was expressed as counts per 10 minutes per gram. Assay for intact chloramphenicol—In experiments where the penetration of labeled drugs is being studied it is necessary to show that the labeled material recovered is in fact the intact drug and not a degradation product. Biological assay or chromatographic techniques were considered to be inconvenient, thus the method of Glasko, Wolf, and Dill was used to separate intact chloramphenicol from any breakdown products. The aqueous humor of three similar experiments (e.g., three subconjunctival administrations for the same time period) was pooled and weighed. This was shaken with a mixture of ethyl acetate and phosphate buffer (pH 6 ) . The two layers were allowed to separate. The ethyl
1 1 j 1 3 6 1
acetate layer was removed and washed with buffer. The final ethyl acetate and aqueous buffer extracts were evaporated to dryness, redissolved in 0.5 ml of distilled water and the radioactivity in each measured as described above. All intact chloramphenicol is taken up into the ethyl acetate while any breakdown products remain in the aqueous phase.
8
IN
VITRO
Several experiments were performed to principally compare the in vitro permeability of rabbit Tenon's capsule with that of the human. Both C-labeled chloramphenicol and N a were used. A simple lucite chamber was constructed which allowed a piece of Tenon's capsule to be clamped between two fluidfilled chambers. Each chamber contained an equal volume of 0.95 ml. One chamber con14
22
TABLE 2 RADIOACTIVITY (COUNTS TER 10 MINUTES PER GRAM) IN AQUEOUS HUMOR FOLLOWING SUBCONJUNCTIVAL OR SUB-TENON'S INJECTION OF "C-LABELED CHLORAMPHENICOL*
Groups
Left Eye
Right Eye
Ratio LE/RE
Controls Group 1 (STf vs ST, 1 hour) Group 2 (SC vs SC; 1 hour)
2041 (4) 3255 (5)
2102 (4) 3096 (5)
0.97 1.05
Experimental Group 3 (SC vs ST; i hour) Group 4 (SC vs ST; 1 hour)
SC 1290 (7) 2973 (11)
ST 850 (7) 1359 (11)
Ratio SC/ST 1.52 2.19
Group 5 (SC vs ST; 3 hours) Group 6 (SC vs ST; 6 hours) Group 7 (SC vs Topical)
2180 (10) 183 (5) 3199 (5)
1430 (10) 210 (5) 5758 (5)
1.52 0.87 0.56
* Counts represent the mean of the number of observation given in parentheses, t SC refers to subconjunctival and ST to sub-Tenon's administration.
VOL. 71, NO. 6
SUBCONJUNCTIVAL VS CHLORAMPHENICOL
1309
TIME (HOURS) Fig. 1 (Hardy and Paterson). Time course of penetration of C-labeled chloramphenicol into rabbit aqueous humor following subconjunctival (open circles) and sub-Tenon's (filled circles) injection. Constructed using data in Table 2. M
tained only saline while the other contained saline together with a trace amount of labeled chloramphenicol or Na. 10 or 20|A1 samples were taken from each chamber at 30-minute intervals and counted as described above. Stirring was achieved manually with flexible polyethylene paddles. Permeability coefficients were calculated by the method of Maffly, Hays, Lamdin, and Leaf. 22
9
RESULTS IN
VIVO
Controls—In Groups 1 and 2, identical degrees of penetration were found in two eyes of the same animal following either a subconjunctival or sub-Tenon's injection on each side (Table 2 ) . Subconjunctival versus sub-Tenon's administration—In Groups 3 to 5 at one half hour, one hour, and three hours, a greater amount of labeled chloramphenicol was
found in the aqueous of the eye given the drug subconjunctivally. At one half hour the ratio of penetration, comparing subconjunctival to sub-Tenon's capsule administration, was 1.52: 1, at one hour 2.19: 1 and at three hours (Group 5 ) , 1.52: 1. By paired statistics these differences were significant at the 95% or greater level. At six hours (Group 6) there was no difference in the degree of penetration (0.87: 1 ) . These results are depicted in Figure 1 and in Table 2. Subconjunctival versus topical administration—In Group 7, penetration of C-labeled chloramphenicol one hour following topical administration was some 1.8 times greater than when given subconjunctivally (Table 2 ) . Assay for chloramphenicol—In all instances the entire labeled fraction was recovered from the ethyl acetate extract. No radioactivity was found in the aqueous layer. 14
Thus, during the experimental period there was no detectable degradation of chloramphenicol. IN
JUNE, 1971
AMERICAN JOURNAL OF OPHTHALMOLOGY
1310
VITRO
All the in vitro experiments were performed at room temperature (23-25°C). Human Tenon's capsule was approximately 24 hour postmortem when used. No difference in permeability characteristics was found between rabbit Tenon's capsule dissected immediately and that dissected 24 hours postmortem. The permeability coefficient for Na was 5.11 X 10 cm/second in rabbit Tenon's capsule and 5.2 X 10~ cm/second in the human. Similarly for chloramphenicol, 4.35 X l O cm/second in rabbit and 4.9 X 10 cm/second in the human. Thus it would appear that the permeability characteristics of rabbit and human Tenon's capsule are quite similar. 22
-5
5
5
-5
DISCUSSION
The results of this study clearly indicate that in the rabbit, chloramphenicol penetrates the aqueous humor of the eye more rapidly and in greater quantity when given subconjunctivally than when given via sub-Tenon's capsule. Chloramphenicol is a highly lipid soluble drug and it would be expected that other lipid soluble drugs would behave similarly (e.g., steroids, epinephrine, atropine). Whether nonlipid soluble drugs behave similarly can only be conjectured from the present results. Direct correlation between the results in animals with the situation in human eyes is not possible, but the data does at least show that Tenon's capsule from rabbit eyes and human eyes exhibits almost identical permeability characteristics in vitro to chloramphenicol and sodium. On the basis of the present findings, then, it would seem wise to avoid injecting medication via sub-Tenon's capsule when such a significant penetration differential exists. This, however, is quite contrary to the rec-
ommendation of several authorities. Haverner writes : "Subconjunctival injection is actually a misnomer. Such an injection should be given beneath Tenon's capsule, since in this position the drug is effectively closer to the eye, is absorbed more rapidly, suffers less loss into the conjunctival circulation, and reaches greater intraocular concentrations." Also, Burns writes: "Subconjunctival and sub-Tenon's injection of medications is an obvious means of short circuiting the epithelial barrier (of the cornea). It is known that sub-Tenon's injection is more effective than subconjunctival in aiding drug penetration of intraocular tissues." 10
11
The original investigations that gave rise to these opinions were those of Swan, Chrisman, and Bailey. Subjects undergoing strabismus surgery were first made miotic with pilocarpine. A sub-Tenon's injection of 0.05 ml of 1: 1000 epinephrine was then given on one side of one eye and a similar injection given subconjunctivally on the other side of the same eye. Swan and his associates found that more rapid segmental pupil dilation occurred on the side receiving the sub-Tenon's injection. This was observed in 11 of 12 patients. The study was prompted by earlier experiments in rabbits which seemed to indicate that sub-Tenon's procaine penetrated the eye better than when given subconjunctivally. 7
12
In this laboratory, using rabbits and a more quantitive method utilizing C-labeled chloramphenicol we have shown quite the opposite to be true. Why the marked difference in ocular penetration following the two methods of administration exists poses quite a problem. Preliminary investigation has shown (Hardy and Paterson, unpublished data) that there is more radioactivity in the venous return from the eye receiving a sub-Tenon's injection than that receiving a subconjunctival injection. If labeled chloramphenicol penetrates the sclera from its sub-Tenon's location, encounters the large choroidal venous channels, then one would expect it to be largely carried 14
VOL. 71, NO. 6
SUBCONJUNCTIVAL VS CHLORAMPHENICOL
away from the eye. Would not the same be true for the subconjunctivally located drug? Most likely a fraction does penetrate the sclera after diffusing through Tenon's capsule and is flushed away from the eye. However, to explain the difference in penetration another route or routes must exist which bypass the venous channel in the choroid. One possible route is the limbus. At a finite distance from the limbus, the Tenon's capsule blends into scleral tissue thus possibly trapping the drug to some extent. The conjunctiva, however, continues over the limbus onto the cornea where conjunctiva blends with corneal epithelium. Thus, a relatively high concentration of drug may exist over the limbal region, subsequent penetration being across the corneal endothelium. Harris presents evidence for such a route using radiolabeled eserine. 13
Another possibility is that the drug passes from its subconjunctival location into the tears and thus penetration is directly across the cornea from the tears. Such a mechanism is favored by Richardson. In this context we have shown that at one hour chloramphenicol penetrates the aqueous humor faster following topical administration than subconjunctivally. (This does not necessarily mean that topical administration is more effective clinically, since our experimental situation was quite different in that the eye was effectively bathed in drug solution for one hour.) Also, Wine, Gornall, and Basu found evidence for this route of penetration utilizing C-hydrocortisone. In their investigation there was increased ocular penetration of drug when the conjunctiva was perforated in the usual manner of subconjunctival injection as opposed to nonperforating injections via the upper eyelid. Following a nonperforating subconjunctival administration no drug was found in the tears. The authors proposed that the drug is squeezed out of the injection hole into the tear film. Although the present study provides no further information to aid in elucidating the mechanism of penetration it does show that a 14
15
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drug, in this case chloramphenicol, penetrates the anterior segment of the eye more rapidly following a subconjunctival rather than a sub-Tenon's injection. S U M M A R Y
The level of radioactivity in the aqueous humor of albino rabbits was assayed at varying periods of time following C-labeled chloramphenicol administered subconjunctivally to one eye and via sub-Tenon's capsule to the contralateral eye. At 30 minutes, one hour, and three hours a greater quantity of the drug appeared to penetrate the eye receiving the subconjunctival injection. At six hours there was no difference in the degree of penetration. On the basis of these observations it is suggested that the use of sub-Tenon's injections could be avoided. The in vitro permeability characteristics of human and rabbit Tenon's capsule are virtually identical. 14
ACKNOWLEDGMENT
We thank Dr. Philip P. Ellis who suggested the project and for his continued interest and comment during the course of the study. REFERENCES
1. McPherson, S. D , Jr., Presley, G. D , and Crowford, J. R. : Aqueous humor assays of subconjunctival antibiotics. Am. J. Ophth. 66:430, 1968. 2. Leopold, I. H , Nichols, A. C , and Vogel, D. W. : Penetration of chloramphenicol U.S.P. into the eye. Arch Ophth. 44:706, 1950. 3. Abraham, R. K , and Burnett, H. H. : Tetracycline and chloramphenicol studies on rabbit and human eyes. Arch Ophth. 54:641, 1955. 4. Records, R. E , and Ellis, P. P. : The intraocular penetration of ampicillin, methicillin, and oxacillin. Am. J. Ophth. 64:135,1967. 5. Records, R. E. : Intraocular penetration of cephalothin, Part I. Animal studies. Am. J. Ophth. 66:436, 1968. 6. Records, R. E. : Intraocular penetration of cephalothin, Part II. Human studies. Am. J. Ophth. 66:441, 1968. 7. Swan, K. G., Crisman, H. R , and Bailey, P. E , Jr. : Subepithelial versus subcapsular injections of drugs. Arch. Ophth. 56:26, 1956. 8. Glazko, A. J., Wolf, L. M , and Dill, W. A. : Biochemical studies on chloramphenicol (Chloromycetin) I. Colorimetric methods for the determination of chloramphenicol and related vitro compounds. Arch Biochem. 23:411, 1949.
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9. Maffly, R. H , Hays, R. M , Lamdin, E , and Leaf, A. : The effect of neurohypophyseal hormones on the permeability of the toad bladder to urea. J. Clin. Invest. 39:630, 19S9. 10. Havener, W. H. : Ocular Pharmacology. St. Louis, Mosby, 1966, p. 13. 11. Burns, R. P.: Pharmacology and toxicology of the conjunctiva. In Symposium on Ocular Pharmacology and Therapeutics. Transactions of the New Orleans Acadamy of Ophthalmology. St. Louis, Mosby, 1970, p. 97. 12. Schlegel, H. E , and Swan, K. G. : Ocular penetration of procaine following subconjunctival injection. Arch Ophth. 52 :774. 1954.
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13. Harris, J. E.: Problems in drug penetration. In Leopold, I. H.: Symposium in Ocular Therapy, vol. 3. St. Louis, Mosby 1968. 14. Richardson, K. T. : Ocular pharmacodynamics. In Symposium on Ocular Pharmacology and Therapeutics. Transaction of the New Orleans Academy of Ophthalmology. St. Louis, Mosby, 1970. 15. Wine, N. A., Gornall, A. G, and Basu, P. K. : The ocular uptake of subconjunctivally injected "C-hydrocortisone. I. Time and major route of penetration into the eye. Am. T. Ophth. 58:362, 1964.
O P H T H A L M I C MINIATURE
I noticed herfingersnervously twisting and untwisting a pleat of the chiffon of her dress. "We can state definitely now that your husband died as a result of eserine poisoning." "You mean those eyedrops killed him," "It seems quite certain that when you gave Mr. Leónides that last injection, it was eserine that you injected and not insulin." "But I didn't know that. I didn't have anything to do with it. Really I didn't, Inspector." "Then somebody must have deliberately replaced the insulin by the eyedrops." "What a wicked thing to do !" "Yes, Mrs. Leónides." "Do you think someone did it on purpose? Or by accident? It couldn't have been a joke, could it?" Taverner said smoothly, "We don't think it was a joke, Mrs. Leónides." Agatha Christie The Crooked House