Ocular Penetration in Rabbits of Topically Applied Dexamethasone To the Editor.\p=m-\Thearticle
by
Kru-
al, which appeared in the October issue of the Archives (92:312, 1974), presents data purporting to
pin
et
demonstrate that substantial quantities of dexamethasone sodium phosphate penetrate into the cornea and anterior chamber following its topical administration to the eye. The authors point out that their results differ from the data obtained in our laboratories. Krupin et al attribute these differences in experimental results to the presumed greater sensitivity of their system. We feel that the differences cannot be explained so lightly and that the discrepancies merit further discussion. Krupin et al question the sensitivity of our method and its ability to detect radioactive corticosteroid of the specific activity used in our experiments. They imply that radioactive steroids with a specific activity of less than 10
microcuries/mg
cannot
produce
mea-
surable drug levels in the eye. However, these authors appear to have ignored the difference between a carbon 14 label (used in our experiments) and a tritium label (used in theirs). In our laboratory the counting efficiency of 14C is approximately 85%, while that of tritium is only about 30%. Thus, a direct comparison of the specific activity and counting sensi¬ tivity of the two types of preparations is inappropriate, and their calcula¬ tions referable to our system are incorrect. Krupin et al state that our experi¬ mental method is unable to detect a preparation labeled with 14C with a specific activity of only 0.1 microcu-
Yet our system detected measurable quantities of steroid in both cornea and aqueous humor when the corneal epithelium was absent, and in an inflamed eye with an intact epithelium when dexamethasone sodi¬ um phosphate was topically adminis¬ tered. It did so whether a solution or an ointment preparation (petrolatum vehicle) of dexamethasone sodium phosphate labeled with14C was used.'·2 Only in the uninflamed eye with intact epithelium did ..ur system fail to detect corticosteroid in either loca¬ tion. However, if in this same situa¬ tion (epithelium intact, uninflamed eye), dexamethasone or its acetate derivative were used instead of the phosphate, significant amounts of steroid were detectable in both cornea and aqueous humor despite the fact that these preparations (the acetate and the alcohol) also had a specific activity of only 0.1 microcurie/mg.3 Thus, to attack the sensitivity of our system is again inapj opriate, for it is clearly able to déte steroid at the specific activity ust.J. The data de¬ rived from our system iemonstrate that the lipophilic corneal epithelium impedes the penetration of watersoluble dexamethasone sodium phos¬ phate to a greater degree than other derivatives of dexamethasone, and therefore less steroid finds its way into the anterior segment of the eye when the phosphate derivative is used than when other derivatives of the same steroid base are used. The sensi¬ tivity of our system clearly was suffi¬ cient to obtain data that document these phenomena. Krupin et al state that they added radioactive-labeled dexamethasone to commercial 0.1% dexamethasone so¬ dium phosphate (Decadron). Used in
rie/mg.
the correct chemical sense, dexa¬ methasone is synonymous with dexa¬ methasone alcohol. This indicates that the labeled material was the free alcohol form of dexamethasone, not dexamethasone sodium phosphate. Thus, the material studied was a mixture of very small amounts of radioactive-labeled dexamethasone al¬ cohol and relatively large amounts of unlabeled dexamethasone sodium phosphate. It is highly unlikely that substantial quantities of the radioac¬ tive isotope would shift from the alcohol to the phosphate or that the phosphate group would shift to the alcohol, and the authors make no such claims. Therefore, we suggest that much of the radioactive-labeled ma¬ terial they detected in cornea and aqueous humor was dexamethasone alcohol, not dexamethasone sodium phosphate. Our investigations with the free alcohol form of dexametha¬ sone (0.1%) demonstrated a corneal drug level of 3.74µg/gm one hour after administration.4 Using a 0.1% solu¬ tion, Krupin et al report a corneal drug level of 6.53µg/gm one hour after drug administration. Comparable aqueous humor levels were 0.35µg/ml in our laboratories, 0.43µg/ml in Krupin's. The slightly higher levels obtained by Krupin et al could be attributable largely to a difference in experimental technique. The authors, using a technique of administration
designed to optimize drug penetra¬ tion, delivered a 0.025-ml drop to the eye and repeated the dose five minutes later, while we used a single 0.05-ml drop. Considering the differ¬ ence in experimental technique, the results are remarkably similar. Interestingly, the authors point out that the drug levels they obtained
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/19/2015
similar to those reported by Short et al.5 Short's article states that "a commercial preparation of dexa¬ methasone sodium phosphate (Maxidex)" was used in the study, and it indicates that Alcon Laboratories, Inc., of Port Worth, Tex, supplied the material. There is, therefore, an error in terminology here. Maxidex, the drug manufactured by Alcon Labora¬ tories, does indeed penetrate through the intact epithelium of the unin¬ flamed eye in substantial quantities, as we have confirmed in our recently published studies.4 However, Maxidex is dexamethasone alcohol, not dexa¬ methasone sodium phosphate. More¬ over, Short's article indicates that his material was labeled in much the same manner used by Krupin et al, by adding tritiated dexamethasone sup¬ plied by the New England Nuclear Corporation. Keates, co-author of Short's article, has verified that both the drug used and the labeled material were dexameLhasone alcohol (oral communication, November 1974). Thus, Short's studies support our contention that much of the material detected by Krupin et al in cornea and aqueous humor was dexamethasone alcohol, not dexamethasone sodium phosphate. Of additional importance is the fact that Short et al conducted their studies with a tritium-labeled steroid preparation whose specific activity was 6.945 microcuries/mg, below the 10.7 microcuries/mg of the dexamethasone alcohol preparation used by us,4 and below the minimal specific activity that Krupin et al claim is able to produce counts per minute that are statistically different from background. This statement at best is questionable, since techniques are available to count low levels of were
radioactivity. The primary evidence presented by Krupin et al that the material they
detected in the anterior chamber was dexamethasone sodium phosphate is purported to be provided by data from
thin-layer chromatography (TLC). The TLC procedure used by them
included the solvent system chloroform-methanol (9:1). Using the same
thin-layer chromatography system,
have found that dexamethasone sodium phosphate does not migrate at all in this solvent system (A. Kupferman and H. M. Leibowitz, unpublished data); virtually all of the dexametha¬ sone sodium phosphate remains on the starting line. However, dexametha¬ sone alcohol does migrate in this we
solvent system with relative flow rate (Rf) of about 0.45, essentially the same value reported by Krupin et al. They failed to detect the dexametha¬ sone alcohol after staining with blue tetrazolium, because the quantity of steroid present was below that detect¬ able by the technique. A blue tetra¬ zolium spot shows only when a minimum of ^g of steroid material is applied to the TLC plate. The labeled material used by Krupin et al had a specific activity of 31.2 curies/ millimol (1 mol of dexametha¬ sone 392.5 mg), and it therefore contained 79.5 microcuries^g. The final steroid mixture studied had a level of radioactivity of 200 microcu¬ ries/mg and therefore contained 2.52µg of tritium-labeled dexametha¬ sone per milliliter. The total amount of steroid in the 0.05 ml applied to the eye contained 0.126µg, and use of their penetration constant (4.27 x 10~4) in¬ dicates that only 0.537 x 10" ng or 54 pg of labeled dexamethasone entered the aqueous humor. Since eight eyes were used, the pooled aqueous humor sample contained only 432 pg. This material was dried, the steroid was extracted with chloroform-methanol (1:1), and the extract was spotted on thin-layer plates. Assuming that all of the extract was used and that there was 100% recovery of the steroid (which, of course, is impossible), less than 0.5 ng of tritiated dexametha¬ sone was spotted on each plate, well below the ^g necessary for a blue tetrazolium reaction. The spots with approximate Rf values of 0.45 obtained by Krupin et al after staining developed chromatograms with 2,4-dinitrophenylhydrazine provide no evidence that the material detected was dexamethasone sodium phosphate. As the authors point out, this material detects keto groups. However, the most active of the keto groups present in the basic steroid ring is at position 3 and is present in both dexamethasone and dexamethasone sodium phosphate. Staining with 2,4-dinitrophenylhydrazine, therefore, does not differentiate between the two forms of dexametha¬ sone. Moreover, our studies have demonstrated that a small percentage of radioactivity (up to 5%) will migrate when tritium-labeled dexa¬ methasone sodium phosphate derived from the commercial ophthalmic prep¬ aration is applied to a silica gel TLC plate. The material migrates at the same Rf value as dexamethasone and =
'
a breakdown product of dexamethasone sodium phosphate, not the steroid itself (A. Kupferman and H. M. Leibowitz, unpublished data). Experiments of Boltralik et al have yielded a similar finding.6 Additional data are available that are pertinent to this discussion. Bol¬ tralik et al have presented evidence that dexamethasone sodium phos¬ phate, applied topically to the eye, enters the anterior chamber largely as dexamethasone, not dexamethasone sodium phosphate.6 In its passage through the cornea, the phosphate form appears to be broken down to the free alcohol form. Boltralik et al
represents
experienced great difficulty getting dexamethasone sodium phosphate in¬ to the aqueous humor in sufficient to measure by
quantities thin-layer
chromatography and was forced to administer topically large quantities of the drug (1.2 mg to each of ten eyes) to obtain aqueous humor levels with
sufficient
quantities of steroid for Chromatographie analysis of the pooled specimen. Edelhauser studied the penetration through the cornea of several steroids with use of a sophisti¬ cated in vitro system. His technique confirmed that the rate of penetration of dexamethasone sodium phosphate through the intact cornea was much
less than that of the other steroids studied.7 The contention of Krupin et al that the system they used to study dexa¬ methasone sodium phosphate was more sensitive than the one used by us is, of course, fundamentally correct. As one increases the specific activity of a radioactive-labeled material, one should be able to detect smaller quan¬ tities of the material. However, this approach is not without pitfalls, par¬ ticularly when tritium is used. There is an inverse correlation between the level of specific activity of the isotope and the quantity that need exchange with water, etc, to produce a signifi¬ cant error. Obviously, therefore, one must determine the relative gain that
be achieved by increasing the absolute sensitivity of a system and this, of course, depends on the level of sensitivity needed to prove a given point. Among the several commer¬ cially available ophthalmic corticoste¬ roids whose ocular penetration we have studied,1·2·4·8·3 dexamethasone sodium phosphate was the first that was made available to us, and we obtained it labeled with 14C. Because of the greater certainty that the
might
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/19/2015
in ocular tis¬ and fluids is attached to the steroid, we had hoped to obtain the other steroid products labeled in a similar fashion. However, they proved to be unavailable and, like Krupin et
radioactivity measured sues
al,
our
subsequent experiments
were
carried out with tritium-labeled corti¬ costeroids. The 14C dexamethasone sodium phosphate is a synthesized preparation whose specific activity is difficult to increase. Although it prov¬ ed to be most satisfactory for the studies in which it was used, it has apparently generated some confusion in the comparison of results obtained with tritium-labeled steroid. We have therefore repeated our studies, using tritium-labeled dexamethasone sod¬ ium phosphate (specific activity 23.12
microcuries/mg). Administering a single 0.05-ml drop to the cornea and using the techniques described in our we obtained a publications, biphasic curve with peak values in the 1·2·4·8-9·
at 5 minutes and 90 minutes after drug application. The following concentrations were measured (arithemetic mean and standard error). Cornea: 5 minutes, 1.66 ( ± 0.23) µg/ gm; 90 minutes, 1.66 (±0.26) µg/gm. For purposes of comparison, our 60minute value was 1.23 (±0.23) µg/gm. These results are based on scintil¬ lation counting data only. We have not yet had the opportunity to repeat Boltralik's studies on the form of the drug that reaches various locations in the eye. Our data are very different from those reported by Krupin et al, but not significantly different from the radioactivity data obtained by Boltralik.6 Finally, it should be emphasized that absolute values of drug levels in the rabbit cornea and aqueous humor are of limited usefulness. Such values can be manipulated by the use of anesthesia, by the position of the animal, by the manner in which the lids are handled, by the quantity of drug administered, by the vehicle in which the drug is delivered, etc. We have felt that far more important to the ophthalmologist is the relative ability of the various commercially available corticosteroid preparations to gain access to the cornea and aqueous humor. We believe that the major contribution of our series of studies is that all of these prepara¬ tions have been compared under a standardized set of conditions. With cornea
reference to dexamethasone sodium phosphate, the absolute values are different when the 14C-labeled drug is compared to the tritiumlabeled drug. However, its ability to penetrate into and through the cornea in comparison to the other steroids studied remains the same, regardless of the label used. Ultimately, of course, it is the ability of the drug to suppress inflammation that has the greatest relevance. Here, too, dexa¬ methasone sodium phosphate fares poorly in comparison to the other oph¬ thalmic steroids studied.10"13 Howard M. Leibowitz, MD Allan Kupferman, PhD
specific
References 1. Cox
WV, Kupferman A, Leibowitz HM:
Topically applied
steroids in corneal disease: I. The role of inflammation in stromal absorption of dexamethasone. Arch Ophthalmol 88:308, 1972. 2. Cox WV, Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: II. The role of drug vehicle in stromal absorption of dexamethasone. Arch Ophthalmol 88:549, 1972. 3. Kupferman A, Pratt MV, Suckewer K, et al: Topically applied steroids in corneal disease: III. The role of drug derivative in stromal absorption of dexamethasone. Arch Ophthalmol 91:373, 1974. 4. Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: V. Dexamethasone alcohol. Arch Ophthalmol 92:329-330, 1974. 5. Short C, Keates RH, Donovan EF, et al: Ocular penetration studies: I. Topical administration of dexamethasone. Arch Ophthalmol 75:689\x=req-\ 692, 1966. 6. Boltralik JJ, et al: In vivo and in vitro hydrolysis of ophthalmic steroids using rabbit eyes. Read before the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla, April 1974. 7. Hull DS, et al: Permeability of the isolated rabbit cornea to corticosteroids. Invest Ophthalmol 13:457-459, 1974. 8. Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: IV. The role of drug concentration in stromal absorption of prednisolone acetate. Arch Ophthalmol 91:377, 1974. 9. Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: VI. Kinetics of prednisolone phosphate. Arch Ophthalmol 92:331-334, 1974. 10. Leibowitz HM, Kupferman A: Pharmacology of topically applied dexamethasone. Trans Am Acad Ophthalmol Otolaryngol, to be published. 11. Leibowitz HM, Kupferman A: Anti-inflammatory effectiveness in the cornea of topically administered prednisolone. Invest Ophthalmol 13:757-763, 1974. 12. Kupferman A, Leibowitz HM: Anti-inflammatory effectiveness of topically administered corticosteroids in the cornea without epithelium. Invest Ophthalmol, to be published. 13. Leibowitz HM, Kupferman A: Bioavailability and therapeutic effectiveness of topically administered corticosteroids. Trans Am Acad Ophthalmol Otolaryngol, to be published.
Reply To the Editor.\p=m-\We do not wish to engage in a literary debate with these investigators, and, therefore, do not have a formal reply. However, we do disagree with many of their comments.
We thank these investigators for showing a concern in our study. Large differences in results might be explained by our studies being performed in fully awake animals, while Leibowitz and Kupferman used anesthetized animals with altered physiologic and metabolic states. We were happy to see that these investigators provided references to show that dexamethasone sodium phosphate
does penetrate the cornea to enter the anterior chamber. While we do not agree with the comments of Drs. Leibowitz and Kupferman, we respect their right to defend their initial studies in their long line of papers on corneal drug penetrations. We are pleased that our article presented enough data and technical descriptions to allow these investigators to write a detailed "letter to the editor." We hope that all articles that appear in the Archives will contain enough data to allow interested parties the ability to criti¬ cally evaluate the presented work. Theodore Krupin, MD Stephen R. Waltman, MD
Complication
of the Use of
Ocuserts
To the Editor.\p=m-\During a 48-hour period, I saw two patients who had been using the new Ocuserts to control their glaucoma. They complained that while riding along expressways, they
almost involved in disastrous accidents. The Ocusert, which wandered under the upper lid, temporarily came over the cornea, while these persons were driving and blinking. One of them almost crashed into another car when he could not see were
temporarily.
I think this complication of the use of Ocuserts is worth bringing to the attention of our ophthalmologic col-
leagues.
Ira A. Abrahamson,
Cincinnati
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/19/2015
Jr., MD
by
Kru-
al, which appeared in the October issue of the Archives (92:312, 1974), presents data purporting to
pin
et
demonstrate that substantial quantities of dexamethasone sodium phosphate penetrate into the cornea and anterior chamber following its topical administration to the eye. The authors point out that their results differ from the data obtained in our laboratories. Krupin et al attribute these differences in experimental results to the presumed greater sensitivity of their system. We feel that the differences cannot be explained so lightly and that the discrepancies merit further discussion. Krupin et al question the sensitivity of our method and its ability to detect radioactive corticosteroid of the specific activity used in our experiments. They imply that radioactive steroids with a specific activity of less than 10
microcuries/mg
cannot
produce
mea-
surable drug levels in the eye. However, these authors appear to have ignored the difference between a carbon 14 label (used in our experiments) and a tritium label (used in theirs). In our laboratory the counting efficiency of 14C is approximately 85%, while that of tritium is only about 30%. Thus, a direct comparison of the specific activity and counting sensi¬ tivity of the two types of preparations is inappropriate, and their calcula¬ tions referable to our system are incorrect. Krupin et al state that our experi¬ mental method is unable to detect a preparation labeled with 14C with a specific activity of only 0.1 microcu-
Yet our system detected measurable quantities of steroid in both cornea and aqueous humor when the corneal epithelium was absent, and in an inflamed eye with an intact epithelium when dexamethasone sodi¬ um phosphate was topically adminis¬ tered. It did so whether a solution or an ointment preparation (petrolatum vehicle) of dexamethasone sodium phosphate labeled with14C was used.'·2 Only in the uninflamed eye with intact epithelium did ..ur system fail to detect corticosteroid in either loca¬ tion. However, if in this same situa¬ tion (epithelium intact, uninflamed eye), dexamethasone or its acetate derivative were used instead of the phosphate, significant amounts of steroid were detectable in both cornea and aqueous humor despite the fact that these preparations (the acetate and the alcohol) also had a specific activity of only 0.1 microcurie/mg.3 Thus, to attack the sensitivity of our system is again inapj opriate, for it is clearly able to déte steroid at the specific activity ust.J. The data de¬ rived from our system iemonstrate that the lipophilic corneal epithelium impedes the penetration of watersoluble dexamethasone sodium phos¬ phate to a greater degree than other derivatives of dexamethasone, and therefore less steroid finds its way into the anterior segment of the eye when the phosphate derivative is used than when other derivatives of the same steroid base are used. The sensi¬ tivity of our system clearly was suffi¬ cient to obtain data that document these phenomena. Krupin et al state that they added radioactive-labeled dexamethasone to commercial 0.1% dexamethasone so¬ dium phosphate (Decadron). Used in
rie/mg.
the correct chemical sense, dexa¬ methasone is synonymous with dexa¬ methasone alcohol. This indicates that the labeled material was the free alcohol form of dexamethasone, not dexamethasone sodium phosphate. Thus, the material studied was a mixture of very small amounts of radioactive-labeled dexamethasone al¬ cohol and relatively large amounts of unlabeled dexamethasone sodium phosphate. It is highly unlikely that substantial quantities of the radioac¬ tive isotope would shift from the alcohol to the phosphate or that the phosphate group would shift to the alcohol, and the authors make no such claims. Therefore, we suggest that much of the radioactive-labeled ma¬ terial they detected in cornea and aqueous humor was dexamethasone alcohol, not dexamethasone sodium phosphate. Our investigations with the free alcohol form of dexametha¬ sone (0.1%) demonstrated a corneal drug level of 3.74µg/gm one hour after administration.4 Using a 0.1% solu¬ tion, Krupin et al report a corneal drug level of 6.53µg/gm one hour after drug administration. Comparable aqueous humor levels were 0.35µg/ml in our laboratories, 0.43µg/ml in Krupin's. The slightly higher levels obtained by Krupin et al could be attributable largely to a difference in experimental technique. The authors, using a technique of administration
designed to optimize drug penetra¬ tion, delivered a 0.025-ml drop to the eye and repeated the dose five minutes later, while we used a single 0.05-ml drop. Considering the differ¬ ence in experimental technique, the results are remarkably similar. Interestingly, the authors point out that the drug levels they obtained
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/19/2015
similar to those reported by Short et al.5 Short's article states that "a commercial preparation of dexa¬ methasone sodium phosphate (Maxidex)" was used in the study, and it indicates that Alcon Laboratories, Inc., of Port Worth, Tex, supplied the material. There is, therefore, an error in terminology here. Maxidex, the drug manufactured by Alcon Labora¬ tories, does indeed penetrate through the intact epithelium of the unin¬ flamed eye in substantial quantities, as we have confirmed in our recently published studies.4 However, Maxidex is dexamethasone alcohol, not dexa¬ methasone sodium phosphate. More¬ over, Short's article indicates that his material was labeled in much the same manner used by Krupin et al, by adding tritiated dexamethasone sup¬ plied by the New England Nuclear Corporation. Keates, co-author of Short's article, has verified that both the drug used and the labeled material were dexameLhasone alcohol (oral communication, November 1974). Thus, Short's studies support our contention that much of the material detected by Krupin et al in cornea and aqueous humor was dexamethasone alcohol, not dexamethasone sodium phosphate. Of additional importance is the fact that Short et al conducted their studies with a tritium-labeled steroid preparation whose specific activity was 6.945 microcuries/mg, below the 10.7 microcuries/mg of the dexamethasone alcohol preparation used by us,4 and below the minimal specific activity that Krupin et al claim is able to produce counts per minute that are statistically different from background. This statement at best is questionable, since techniques are available to count low levels of were
radioactivity. The primary evidence presented by Krupin et al that the material they
detected in the anterior chamber was dexamethasone sodium phosphate is purported to be provided by data from
thin-layer chromatography (TLC). The TLC procedure used by them
included the solvent system chloroform-methanol (9:1). Using the same
thin-layer chromatography system,
have found that dexamethasone sodium phosphate does not migrate at all in this solvent system (A. Kupferman and H. M. Leibowitz, unpublished data); virtually all of the dexametha¬ sone sodium phosphate remains on the starting line. However, dexametha¬ sone alcohol does migrate in this we
solvent system with relative flow rate (Rf) of about 0.45, essentially the same value reported by Krupin et al. They failed to detect the dexametha¬ sone alcohol after staining with blue tetrazolium, because the quantity of steroid present was below that detect¬ able by the technique. A blue tetra¬ zolium spot shows only when a minimum of ^g of steroid material is applied to the TLC plate. The labeled material used by Krupin et al had a specific activity of 31.2 curies/ millimol (1 mol of dexametha¬ sone 392.5 mg), and it therefore contained 79.5 microcuries^g. The final steroid mixture studied had a level of radioactivity of 200 microcu¬ ries/mg and therefore contained 2.52µg of tritium-labeled dexametha¬ sone per milliliter. The total amount of steroid in the 0.05 ml applied to the eye contained 0.126µg, and use of their penetration constant (4.27 x 10~4) in¬ dicates that only 0.537 x 10" ng or 54 pg of labeled dexamethasone entered the aqueous humor. Since eight eyes were used, the pooled aqueous humor sample contained only 432 pg. This material was dried, the steroid was extracted with chloroform-methanol (1:1), and the extract was spotted on thin-layer plates. Assuming that all of the extract was used and that there was 100% recovery of the steroid (which, of course, is impossible), less than 0.5 ng of tritiated dexametha¬ sone was spotted on each plate, well below the ^g necessary for a blue tetrazolium reaction. The spots with approximate Rf values of 0.45 obtained by Krupin et al after staining developed chromatograms with 2,4-dinitrophenylhydrazine provide no evidence that the material detected was dexamethasone sodium phosphate. As the authors point out, this material detects keto groups. However, the most active of the keto groups present in the basic steroid ring is at position 3 and is present in both dexamethasone and dexamethasone sodium phosphate. Staining with 2,4-dinitrophenylhydrazine, therefore, does not differentiate between the two forms of dexametha¬ sone. Moreover, our studies have demonstrated that a small percentage of radioactivity (up to 5%) will migrate when tritium-labeled dexa¬ methasone sodium phosphate derived from the commercial ophthalmic prep¬ aration is applied to a silica gel TLC plate. The material migrates at the same Rf value as dexamethasone and =
'
a breakdown product of dexamethasone sodium phosphate, not the steroid itself (A. Kupferman and H. M. Leibowitz, unpublished data). Experiments of Boltralik et al have yielded a similar finding.6 Additional data are available that are pertinent to this discussion. Bol¬ tralik et al have presented evidence that dexamethasone sodium phos¬ phate, applied topically to the eye, enters the anterior chamber largely as dexamethasone, not dexamethasone sodium phosphate.6 In its passage through the cornea, the phosphate form appears to be broken down to the free alcohol form. Boltralik et al
represents
experienced great difficulty getting dexamethasone sodium phosphate in¬ to the aqueous humor in sufficient to measure by
quantities thin-layer
chromatography and was forced to administer topically large quantities of the drug (1.2 mg to each of ten eyes) to obtain aqueous humor levels with
sufficient
quantities of steroid for Chromatographie analysis of the pooled specimen. Edelhauser studied the penetration through the cornea of several steroids with use of a sophisti¬ cated in vitro system. His technique confirmed that the rate of penetration of dexamethasone sodium phosphate through the intact cornea was much
less than that of the other steroids studied.7 The contention of Krupin et al that the system they used to study dexa¬ methasone sodium phosphate was more sensitive than the one used by us is, of course, fundamentally correct. As one increases the specific activity of a radioactive-labeled material, one should be able to detect smaller quan¬ tities of the material. However, this approach is not without pitfalls, par¬ ticularly when tritium is used. There is an inverse correlation between the level of specific activity of the isotope and the quantity that need exchange with water, etc, to produce a signifi¬ cant error. Obviously, therefore, one must determine the relative gain that
be achieved by increasing the absolute sensitivity of a system and this, of course, depends on the level of sensitivity needed to prove a given point. Among the several commer¬ cially available ophthalmic corticoste¬ roids whose ocular penetration we have studied,1·2·4·8·3 dexamethasone sodium phosphate was the first that was made available to us, and we obtained it labeled with 14C. Because of the greater certainty that the
might
Downloaded From: http://archopht.jamanetwork.com/ by a University of British Columbia Library User on 06/19/2015
in ocular tis¬ and fluids is attached to the steroid, we had hoped to obtain the other steroid products labeled in a similar fashion. However, they proved to be unavailable and, like Krupin et
radioactivity measured sues
al,
our
subsequent experiments
were
carried out with tritium-labeled corti¬ costeroids. The 14C dexamethasone sodium phosphate is a synthesized preparation whose specific activity is difficult to increase. Although it prov¬ ed to be most satisfactory for the studies in which it was used, it has apparently generated some confusion in the comparison of results obtained with tritium-labeled steroid. We have therefore repeated our studies, using tritium-labeled dexamethasone sod¬ ium phosphate (specific activity 23.12
microcuries/mg). Administering a single 0.05-ml drop to the cornea and using the techniques described in our we obtained a publications, biphasic curve with peak values in the 1·2·4·8-9·
at 5 minutes and 90 minutes after drug application. The following concentrations were measured (arithemetic mean and standard error). Cornea: 5 minutes, 1.66 ( ± 0.23) µg/ gm; 90 minutes, 1.66 (±0.26) µg/gm. For purposes of comparison, our 60minute value was 1.23 (±0.23) µg/gm. These results are based on scintil¬ lation counting data only. We have not yet had the opportunity to repeat Boltralik's studies on the form of the drug that reaches various locations in the eye. Our data are very different from those reported by Krupin et al, but not significantly different from the radioactivity data obtained by Boltralik.6 Finally, it should be emphasized that absolute values of drug levels in the rabbit cornea and aqueous humor are of limited usefulness. Such values can be manipulated by the use of anesthesia, by the position of the animal, by the manner in which the lids are handled, by the quantity of drug administered, by the vehicle in which the drug is delivered, etc. We have felt that far more important to the ophthalmologist is the relative ability of the various commercially available corticosteroid preparations to gain access to the cornea and aqueous humor. We believe that the major contribution of our series of studies is that all of these prepara¬ tions have been compared under a standardized set of conditions. With cornea
reference to dexamethasone sodium phosphate, the absolute values are different when the 14C-labeled drug is compared to the tritiumlabeled drug. However, its ability to penetrate into and through the cornea in comparison to the other steroids studied remains the same, regardless of the label used. Ultimately, of course, it is the ability of the drug to suppress inflammation that has the greatest relevance. Here, too, dexa¬ methasone sodium phosphate fares poorly in comparison to the other oph¬ thalmic steroids studied.10"13 Howard M. Leibowitz, MD Allan Kupferman, PhD
specific
References 1. Cox
WV, Kupferman A, Leibowitz HM:
Topically applied
steroids in corneal disease: I. The role of inflammation in stromal absorption of dexamethasone. Arch Ophthalmol 88:308, 1972. 2. Cox WV, Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: II. The role of drug vehicle in stromal absorption of dexamethasone. Arch Ophthalmol 88:549, 1972. 3. Kupferman A, Pratt MV, Suckewer K, et al: Topically applied steroids in corneal disease: III. The role of drug derivative in stromal absorption of dexamethasone. Arch Ophthalmol 91:373, 1974. 4. Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: V. Dexamethasone alcohol. Arch Ophthalmol 92:329-330, 1974. 5. Short C, Keates RH, Donovan EF, et al: Ocular penetration studies: I. Topical administration of dexamethasone. Arch Ophthalmol 75:689\x=req-\ 692, 1966. 6. Boltralik JJ, et al: In vivo and in vitro hydrolysis of ophthalmic steroids using rabbit eyes. Read before the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla, April 1974. 7. Hull DS, et al: Permeability of the isolated rabbit cornea to corticosteroids. Invest Ophthalmol 13:457-459, 1974. 8. Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: IV. The role of drug concentration in stromal absorption of prednisolone acetate. Arch Ophthalmol 91:377, 1974. 9. Kupferman A, Leibowitz HM: Topically applied steroids in corneal disease: VI. Kinetics of prednisolone phosphate. Arch Ophthalmol 92:331-334, 1974. 10. Leibowitz HM, Kupferman A: Pharmacology of topically applied dexamethasone. Trans Am Acad Ophthalmol Otolaryngol, to be published. 11. Leibowitz HM, Kupferman A: Anti-inflammatory effectiveness in the cornea of topically administered prednisolone. Invest Ophthalmol 13:757-763, 1974. 12. Kupferman A, Leibowitz HM: Anti-inflammatory effectiveness of topically administered corticosteroids in the cornea without epithelium. Invest Ophthalmol, to be published. 13. Leibowitz HM, Kupferman A: Bioavailability and therapeutic effectiveness of topically administered corticosteroids. Trans Am Acad Ophthalmol Otolaryngol, to be published.
Reply To the Editor.\p=m-\We do not wish to engage in a literary debate with these investigators, and, therefore, do not have a formal reply. However, we do disagree with many of their comments.
We thank these investigators for showing a concern in our study. Large differences in results might be explained by our studies being performed in fully awake animals, while Leibowitz and Kupferman used anesthetized animals with altered physiologic and metabolic states. We were happy to see that these investigators provided references to show that dexamethasone sodium phosphate
does penetrate the cornea to enter the anterior chamber. While we do not agree with the comments of Drs. Leibowitz and Kupferman, we respect their right to defend their initial studies in their long line of papers on corneal drug penetrations. We are pleased that our article presented enough data and technical descriptions to allow these investigators to write a detailed "letter to the editor." We hope that all articles that appear in the Archives will contain enough data to allow interested parties the ability to criti¬ cally evaluate the presented work. Theodore Krupin, MD Stephen R. Waltman, MD
Complication
of the Use of
Ocuserts
To the Editor.\p=m-\During a 48-hour period, I saw two patients who had been using the new Ocuserts to control their glaucoma. They complained that while riding along expressways, they
almost involved in disastrous accidents. The Ocusert, which wandered under the upper lid, temporarily came over the cornea, while these persons were driving and blinking. One of them almost crashed into another car when he could not see were
temporarily.
I think this complication of the use of Ocuserts is worth bringing to the attention of our ophthalmologic col-
leagues.
Ira A. Abrahamson,
Cincinnati
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Jr., MD
