Ocular Penetration of Topically Applied Gentamicin Bernd
Ellerhorst, MD; Bruce Golden, MD; Nabil Jarudi,
The biologically available concentration of gentamicin sulfate in eight ocular tissues of 140 rabbits and 96 humans was measured by a modified disc diffusion technique. If enough topically administered gentamicin was applied, this bioassay detected it in all ocular tissues tested except the lens nucleus. The decay of antibiotic activity was faster in some tissues, eg, aqueous, than in others. A higher level of antibiotic was found in corneas altered by sutures than in virgin tissue. The degree of inflammation, the frequency of application, and the interval between the drops are directly proportional to the antibiotic level in the tissues. The antibiotic free vehicle of commercial 0.3% gentamicin drops possesses an antibacterial potential.
Gentami cin important aminoglycoside
sulfate is
of the of antibi¬ group otics that is in ophthal¬ mology because of its broad spec¬ trum and particular efficacy for Pseudomonas and penicillinase pro¬ ducing Staphylococcus. There have been a number of contributions to the ophthalmologic literature on this one
drug. Furgiuele,1
Litwack et al,2 Mathalone and Harden,3 Goulstine and Submitted for publication Nov 19, 1973. From the Department of Ophthalmology, University Hospitals, Iowa City, (Dr. Ellerhorst); Department of Ophthalmology, University of Witwatersrand, Johannesburg, South Africa (Dr. Golden); and Doctor's Center, Beirut, Lebanon
(Dr. Jarudi). Reprint requests to Department of Ophthalmology, Irminen-Hospital, 5500 Trier, West Germany (Dr. Ellerhorst).
MD
Marmion,4 Golden and Coppel,5 and Golden11 reported on ocular penetra¬ tion of gentamicin after subconjunc¬ tival, sub-Tenon intramuscular ad¬ ministration or both. Furgiuele7 and Sloan et als discussed gentamicin in the aqueous humor and Baum et al
(oral communication, September 1972) measured corneal concentra¬ tions after topical administration; they found that these concentrations do not necessarily reflect that the an¬ tibiotic is available against infection
in other ocular tissues. The purpose of this study was to determine the con¬ centration in each of the ocular tis¬ sues after topical administration of the commercially available gen¬ tamicin using various protocols in a controlled manner, both in humans and in rabbits. We used special tech¬ niques to measure minute quantities in tissues such as lens and vitreous. Materials and Methods
Approximately 40 human specimens and 50 rabbits were used in a pilot study prior to the investigation. This report is the re¬ sult of a study using 145, 2 to 3-kg (4.4- to 6.6-lb), New Zealand male rabbits' eyes and 96 human eyes. Using aseptic technique, half of the ex¬ perimental rabbits were infected with a 6-0 silk intracorneal suture soaked in a human strain of Pseudomonas aeruginosa isolated in our laboratory. To maintain a constant level of 5x10" Pseudomonas per milliliter, the concentration of the culture was moni¬ tored with a spectrophotometer. The other half received a sterile suture in the same manner. Animals were anesthetized with
50 mg of
pentobarbital
and
topically
ap¬
plied proparacaine hydrochloride before placement of the sutures. Commercially available 0.3% gentamicin solution was ap¬ plied to the right eye, while the left received the same number of drops of the antibiotic free vehicle, containing benzalkonium chloride, disodium hydrogen phosphate, and a mixture of monosodium phosphate, sodium bicarbonate, and citric acid. The rabbits' eyes were enucleated ei¬ ther one or 25 hours after the last appli¬ cation of medication. Immediately after enucleation, the eyes were rinsed with sa¬ line, aqueous was withdrawn with a 27gauge needle and vitreous with an 18gauge needle attached to disposable syringes. The syringes and the globes were placed at -30 C for several days until as¬ says were performed. Previous experi¬ ments showed no change in assay using frozen or fresh material." The human studies were performed on 96 eyes undergoing cataract extraction, corneal transplantation, or enucleation. In patients subjected to anterior segment surgery, aqueous was obtained using a 27gauge disposable needle and 1-ml tubercu¬ lin syringe before opening the anterior chamber as well as additional tissue de¬ pending on the procedure. Iris from iridectomies is obviously smaller than the 6-mm trephine obtained in the animal experi¬ ments; however, both were treated the same way in the calculation of drug tissue concentrations. This means that the de¬ rived tissue concentration for human iris was underestimated and did not represent values comparable with the animal data. Enucleated eyes underwent the same com¬ prehensive tissue analysis as rabbit eyes and all tissues were frozen for later assay. The bioassay technique has been re-
Downloaded From: http://archopht.jamanetwork.com/ by a Michigan State University User on 06/11/2015
-·
-—·
30
•-·
ported in detail previously.9 '" It consists of cutting solid tissue with a 6-mm trephine and absorbing 25 of the fluid tissues into
Bacillus subtilis (low range) S aureus—ATCC 6 to 638 (midrange) High Salt Plate With S aureus—ATCC 6 to 638
(high range)
a
_
-* -"· 22 o e
o
c
6.35-mm Alter paper disc. These
are
placed onto various media after inoculation with either Staphylococcus aureus, Ameri¬ can Type Culture Collection (ATCC) No. 6638, or Bacillus subtilis spores, refrig¬ erated together with standards at 4 C for
26
24 hours and then incubated at 37 C for 18 to 24 hours. The zones of inhibition were read with a zone reader to 0.1 mm. Stan¬
18
14
10 0.05
"0
2
.01
0.15
.02
-I-1_I_I_I_I_L_
4
6
10
8
12
16
14
18
20
22
Gentamicin, /xg/ml
Fig 1.—Standardized
inhibition
zones
of
gentamicin, expansion
of lower values.
dards were always run simultaneously with each assay. Three methods of assay were used depending on the anticipated level of antibiotic. A curve was drawn relating known con¬ centrations of gentamicin to the size of the inhibition zone. The concentration of gen¬ tamicin in both the rabbit and human eyes was determined by measuring the zones of inhibition and relating them to the curve and portion of its slope that most accu¬ rately represented the level of gentamicin
(Fig 1).
for Low Levels of Gentamicin
Assay
Table 1.—Humans—Protocol 1 No. of Specimens Taken tast drops five to 30 min before removal of tissue 12 Aqueous Iris 14 11 Lens Vitreous 1 tast drops 31 to 60 min before removal of tissue 17 Aqueous Iris 10 Lens 12 1 Vitreous Last drops 61 to 90 min before removal of tissue 4 Aqueous Iris 12 Lens 13 tast drops 91 to 120 min before removal of tissue
Aqueous Iris Lens
Last
drops
'
Assay Measurable (Above 0.03 mg)* Aqueous, 0.06/ig (15 min after last drops) Aqueous, 0.24^g (25 min) Aqueous, 0.42,ug (30 min)4:
Aqueous, 0.17¿ig (35 min) Aqueous, 0.08,[¿g (35 min)
Aqueous,
Aqueous, 0.05/xg (110 min)
None
removal of tissue Iris Lens
2 5 2
Total eyes in protocol 1 50 Total specimens assayed_128 of
Brain-heart infusion agar of pH was used along with the same S aureus (ATCC No. 6638) suspension used for the intermediate levels. Plating procedures were as follows: from three to six filter paper discs or tissue samples were placed onto each agar plate according to the concen¬ trations involved. Fluid samples were placed onto the discs with a 0.025-ml micropipette. The same 6.35-mm size discs and micropipettes were used to assay the standards. All specimens were run at least in duplicate, but standards were assayed simultane7.25
4 6
Aqueous
for Intermediate Levels of Gentamicin
Assay
µß (70 min)
2
121 to 180 min before
Bacillus subtilis was grown in a Roux bottle containing (Difco) anti¬ biotic medium No. 1 for one week and the resulting washed spore suspen¬ sion maintained at 4 C as a stock solu¬ tion. A 1:10,000 dilution of this suspen¬ sion was made with pH 8.0, 0.1 M phosphate buffered saline. Pour plates were made using 0.2 ml of this dilution to each 100 ml of (Difco) antibiotic medium No. 5, pH 7.95 held at 45 C. The minimum level of biologically available gentamicin that this assay could detect accurately was 0.03^g/ ml.
* Two drops every five minutes for five applications. Normal cataract patients. t Of aqueous specimens, 18.9% had measurable levels of gentamicin after five applications
drops (normal eyes).
Í Same patient different eyes.
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Fig 2.—Topical application of gen¬ tamicin to rabbit eyes every 15 minutes for eight hours. in triplicate or more and the results plotted on semilog paper to determine the concentration equiva¬ lents. Solid tissues were taken from specific areas measured from the limbus and rinsed with saline. When undesired material was adherent to a tissue to be assayed, it was cleaned with forceps, scissors, and alginate swabs. In the rabbit experiments, seven treatment protocols (1 to 7) and one control protocol (8) were used: 1. Two drops every 15 minutes for eight hours. 2. Two drops every 15 minutes for six hours. 3. Two drops every 30 minutes for six hours. 4. Two drops every hour for seven hours. 5. Two drops every hour for 24 hours. 6. Two drops every three hours for 24 hours. 7. Two drops every six hours for 48 hours. 8. The control protocol. Treatment protocols comprised four groups of at least three animals (six eyes) each: group I—noninflamed eyes removed one hour after the last drop of gentamicin; group II—in¬ flamed eyes obtained one hour after the last drop; group III—noninflamed eyes enucleated 25 hours after the last application; group IV—inflamed eyes taken 25 hours after the last
ously
Fig 3.—Topical application of gentamicin
six hours.
drop.
The control protocol was composed of nine groups (each with at least six eyes): group I—one eye received only a sterile intracorneal suture, the other eye was not modified or treated; group II—one eye received a suture contaminated with Pseudomonas, the other eye a sterile suture; neither eye was treated; group III—the same as group II, but treated with normal sa¬ line; group IV—both eyes received a sterile suture, one eye treated with gentamicin every 15 minutes for six Fig 4.—Topical application of gen¬ tamicin to rabbit eyes every 30 minutes for six hours.
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to rabbit
eyes every 15 minutes for
hours; the other eye untreated;
Table 2.—Human—Protocol 2*
Assay Measurable (Above 0.03 mg)t Aqueous, 0.25/jg (30 min after last drops) Aqueous, 0.29/xg (30 min)
No. of Specimens Taken Last drops 5 to 30 min before removal of tissues 5 Aqueous Iris 2 1 Lens Last drops 31 to 60 min before removal of tissues
Aqueous Iris Lens
8 9 11
Last drops 61 to 90 min before removal of tissues 4 Aqueous Iris 5 Lens 6 Last drops 91 to 120 min before removal of tissues 4 Aqueous Iris 4 Lens 4 Last drops 121 to 180 min before removal of tissues Iris 1 1 Lens Total eyes in protocol 2 26 Total specimens assayed 65
Aqueous, 0.05/ig (35 Aqueous, 0.08/tg (32 Aqueous, 0.07/xg (45 Aqueous, 0.24/jg (35 Aqueous, 0.35¿ig (32 lris,0.03/tg (35 min) Iris, 0.03/ig (35 min) Aqueous, 0.13µ (70
min) min) min) min) min)
min)
Aqueous, 0.06/ig (110 min) Aqueous, 0.36/jg (110 min)
None
ten
*
Two drops every five minutes for ten applications. Normal cataract patients. t Of aqueous specimens, 47.6% and 9% of iris specimens had measurable levels of genta¬ micin after ten applications of drops (normal eyes).
group
V—both eyes received infected su¬ tures, one eye treated with vehicle, the other eye untreated; group VI— both eyes received sterile sutures, one eye treated with vehicle, the other eye was untreated; group VII—both eyes infected with Pseudomonas, one eye treated with vehicle, the other eye untreated; group VIII—no modi¬ fication of the corneas, treated every three hours in the one eye with gen¬ tamicin (Garamycin) and with the ve¬ hicle in the other eye; group IX—un¬ modified and untreated animals. In the human studies five protocols were used (Tables 1 to 5): 1. Two drops every five minutes for five applications—normal cataract pa¬ tients. 2. Two drops every five minutes for
applications—normal
cataract pa¬
tients. 3. Two drops hourly (eight hours) on the night before surgery and every five minutes for ten applications im¬ mediately before surgery—normal
cataract patients. 4. Two drops every five minutes be¬ fore surgery for five applications—in¬ flamed or otherwise abnormal eyes.
Table 3.—Human—Protocol 3* No. of Specimens Taken Last drops 5 to 60 min before removal of tissues 4 Aqueous Iris 4 Lens 4 Vitreous 1 Last drops 61 to 120 min before removal of tissues 1 Aqueous Iris 1 Lens 1 Last drops 121 to 180 min before removal of tissues 2 Aqueous Iris 2 Lens 2
Assay Measurable (Above 0.03 mg) t Aqueous, 0.4,ug (35 min after last drops) Aqueous, 0.1^g (60 min) Aqueous, 1.0^g (50 min)
Special cases—drops given day
Aqueous, 4. µß (65 min) Lens Capsule, 0.07/ug (75 min)
before
surgery as
*
Results
Confirming
the work of Barza et 6.35-mm filter paper disc was found to absorb 0.025 ml of fluid. The average weight of at least ten sam¬ ples in each group (noninflamed and inflamed eyes) of 6-mm trephine tis¬ sues shows that many are less in weight than the fluid tissue weight of 0.025 gm and hence the antibiotic concentration is underestimated. In the noninflamed tissue group, con¬ junctiva weight was 0.0130 gm; cor¬ nea, 0.0160 gm; iris, 0.0133 gm; lens capsule, 0.0396 gm; choroid-retina, 0.0025 gm; sclera, 0.0128 gm. In the inflamed tissue group, the weights were 0.0165 gm, 0.0352 gm, 0.0140 gm, 0.0336 gm, 0.0028 gm, and 0.0125 gm, respectively, for the conjunctiva, cor¬ nea, iris, lens capsule, choroid-retina, and sclera. The ordinate of Fig. 2 and
al,1"
Aqueous, 0.5/ig (65 min)
Aqueous, 0.06/ig (180 min)
above, but given every
day of surgery 2.5 min for 10 applications 1 Aqueous Iris 1 Lens 1 Total eyes in protocol 3 Total specimens assayed on
5. Two drops every five minutes be¬ fore surgery for ten applications. Tis¬ sues removed at various timed inter¬ vals after the last applicationinflamed or otherwise abnormal eyes.
8
25
drops hourly (eight times) from 1,700 to 2,400 hours on the night before surgery and five minutes immediately before surgery for ten applications. Normal cataract patients. t Of aqueous specimens, 75% had measurable levels of gentamicin after the protocol 3 regimen (normal eyes). Two
every
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a
Fig 5.—Topical application of gen¬ tamicin to rabbit eyes every hour for seven hours. of the following figures do not have an arithmetic progression for the biologi¬ cally available gentamicin; rather, the lower values are expanded. Indi¬ vidual variations between rabbits and replicate samples of the same tissue have been averaged. Note in Fig 2 the substantial levels of biologically ac¬ tive gentamicin found in the conjunc¬ tiva, cornea, aqueous, and sclera. There is a larger difference in this protocol between the concentration in the inflamed vs the noninflamed groups than is seen in the next proto¬ col (Fig 3) that uses the same fre¬ quency of application, but for a shorter interval of time (six hours in¬ stead of eight). Higher levels of anti¬ biotic are found, as expected, one hour after the last drop than at 25 hours. This difference is magnified with a more frequent application. Figure 4 (application every 30 min¬ utes for six hours) points out that de¬ creasing the frequency of application proportionately lessens the level of antibiotic in ocular tissues. Figure 5 (application every hour for seven hours) continues to show the trend that less frequent application will give reduced levels of gentamicin, but it is important to recognize in com¬ parison with Fig 4 that although the application is cut in half, intraocu¬ lar levels are only slightly diminished. As hourly application is continued for 24 hours (Fig 6), intraocular levels increase, approaching those of more frequent application, even to the point of being measurable in the lens capsule. Reducing the application to every three hours, (Fig 7) puts the intraocular levels in all cases below lug, which is the minimum inhibitory concentration (MIC) for the Pseudo¬ monas strain used in these experi¬ ments. Only the cornea in this proto¬ col has levels higher than tyg/ml. Extending the time between each ap¬ plication but continuing the treat¬ ment for 48 hours (Fig 8) again dem¬ onstrates the ability of tissues to be loaded with antibiotic and thereby to maintain a biologically active level sufficiently higher than the MIC for a
Fig 6.—Topical application of gentamicin
Fig 7.—Topical application
of
to rabbit eyes every hour for 24 hours.
gentamicin to rabbit eyes every three hours for 24
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hours.
ter five
applications of drops at five Only seven of 37 aqueous specimens (18.9%) showed de¬ tectable levels of gentamicin. The minute intervals.
greater the time intervals between
:ig 8.—Topical application
of
gentamicin
to rabbit eyes every six hours for 48 hours.
Table 4.—Human—Protocol 4* No. of Specimens Taken Last drop 5 to 30 min before removal of tissues Corneas 3 1 Aqueous Last drop 31 to 60 min before removal of tissues Corneas 4 1 Aqueous 1 Vitreous
Total eyes in protocol 4 Total specimens assayed
7 10
Assay Measurable (Above 0.03 mg)t Cornea, 0.5/ig (30 min after last drops) Cornea, 1.5^g (25 min after last drops) Cornea, 4.0^g (15 min) Aqueous, 0.21/ig (15 min)_ Cornea, 0.78µ (60 min) Cornea, 1 .O^g (60 min) Cornea, 0.5^g (30 min) Cornea, 2.0/ig (45 min) Aqueous, 0.33,ug (40 min) Vitreous,
Ellerhorst, MD; Bruce Golden, MD; Nabil Jarudi,
The biologically available concentration of gentamicin sulfate in eight ocular tissues of 140 rabbits and 96 humans was measured by a modified disc diffusion technique. If enough topically administered gentamicin was applied, this bioassay detected it in all ocular tissues tested except the lens nucleus. The decay of antibiotic activity was faster in some tissues, eg, aqueous, than in others. A higher level of antibiotic was found in corneas altered by sutures than in virgin tissue. The degree of inflammation, the frequency of application, and the interval between the drops are directly proportional to the antibiotic level in the tissues. The antibiotic free vehicle of commercial 0.3% gentamicin drops possesses an antibacterial potential.
Gentami cin important aminoglycoside
sulfate is
of the of antibi¬ group otics that is in ophthal¬ mology because of its broad spec¬ trum and particular efficacy for Pseudomonas and penicillinase pro¬ ducing Staphylococcus. There have been a number of contributions to the ophthalmologic literature on this one
drug. Furgiuele,1
Litwack et al,2 Mathalone and Harden,3 Goulstine and Submitted for publication Nov 19, 1973. From the Department of Ophthalmology, University Hospitals, Iowa City, (Dr. Ellerhorst); Department of Ophthalmology, University of Witwatersrand, Johannesburg, South Africa (Dr. Golden); and Doctor's Center, Beirut, Lebanon
(Dr. Jarudi). Reprint requests to Department of Ophthalmology, Irminen-Hospital, 5500 Trier, West Germany (Dr. Ellerhorst).
MD
Marmion,4 Golden and Coppel,5 and Golden11 reported on ocular penetra¬ tion of gentamicin after subconjunc¬ tival, sub-Tenon intramuscular ad¬ ministration or both. Furgiuele7 and Sloan et als discussed gentamicin in the aqueous humor and Baum et al
(oral communication, September 1972) measured corneal concentra¬ tions after topical administration; they found that these concentrations do not necessarily reflect that the an¬ tibiotic is available against infection
in other ocular tissues. The purpose of this study was to determine the con¬ centration in each of the ocular tis¬ sues after topical administration of the commercially available gen¬ tamicin using various protocols in a controlled manner, both in humans and in rabbits. We used special tech¬ niques to measure minute quantities in tissues such as lens and vitreous. Materials and Methods
Approximately 40 human specimens and 50 rabbits were used in a pilot study prior to the investigation. This report is the re¬ sult of a study using 145, 2 to 3-kg (4.4- to 6.6-lb), New Zealand male rabbits' eyes and 96 human eyes. Using aseptic technique, half of the ex¬ perimental rabbits were infected with a 6-0 silk intracorneal suture soaked in a human strain of Pseudomonas aeruginosa isolated in our laboratory. To maintain a constant level of 5x10" Pseudomonas per milliliter, the concentration of the culture was moni¬ tored with a spectrophotometer. The other half received a sterile suture in the same manner. Animals were anesthetized with
50 mg of
pentobarbital
and
topically
ap¬
plied proparacaine hydrochloride before placement of the sutures. Commercially available 0.3% gentamicin solution was ap¬ plied to the right eye, while the left received the same number of drops of the antibiotic free vehicle, containing benzalkonium chloride, disodium hydrogen phosphate, and a mixture of monosodium phosphate, sodium bicarbonate, and citric acid. The rabbits' eyes were enucleated ei¬ ther one or 25 hours after the last appli¬ cation of medication. Immediately after enucleation, the eyes were rinsed with sa¬ line, aqueous was withdrawn with a 27gauge needle and vitreous with an 18gauge needle attached to disposable syringes. The syringes and the globes were placed at -30 C for several days until as¬ says were performed. Previous experi¬ ments showed no change in assay using frozen or fresh material." The human studies were performed on 96 eyes undergoing cataract extraction, corneal transplantation, or enucleation. In patients subjected to anterior segment surgery, aqueous was obtained using a 27gauge disposable needle and 1-ml tubercu¬ lin syringe before opening the anterior chamber as well as additional tissue de¬ pending on the procedure. Iris from iridectomies is obviously smaller than the 6-mm trephine obtained in the animal experi¬ ments; however, both were treated the same way in the calculation of drug tissue concentrations. This means that the de¬ rived tissue concentration for human iris was underestimated and did not represent values comparable with the animal data. Enucleated eyes underwent the same com¬ prehensive tissue analysis as rabbit eyes and all tissues were frozen for later assay. The bioassay technique has been re-
Downloaded From: http://archopht.jamanetwork.com/ by a Michigan State University User on 06/11/2015
-·
-—·
30
•-·
ported in detail previously.9 '" It consists of cutting solid tissue with a 6-mm trephine and absorbing 25 of the fluid tissues into
Bacillus subtilis (low range) S aureus—ATCC 6 to 638 (midrange) High Salt Plate With S aureus—ATCC 6 to 638
(high range)
a
_
-* -"· 22 o e
o
c
6.35-mm Alter paper disc. These
are
placed onto various media after inoculation with either Staphylococcus aureus, Ameri¬ can Type Culture Collection (ATCC) No. 6638, or Bacillus subtilis spores, refrig¬ erated together with standards at 4 C for
26
24 hours and then incubated at 37 C for 18 to 24 hours. The zones of inhibition were read with a zone reader to 0.1 mm. Stan¬
18
14
10 0.05
"0
2
.01
0.15
.02
-I-1_I_I_I_I_L_
4
6
10
8
12
16
14
18
20
22
Gentamicin, /xg/ml
Fig 1.—Standardized
inhibition
zones
of
gentamicin, expansion
of lower values.
dards were always run simultaneously with each assay. Three methods of assay were used depending on the anticipated level of antibiotic. A curve was drawn relating known con¬ centrations of gentamicin to the size of the inhibition zone. The concentration of gen¬ tamicin in both the rabbit and human eyes was determined by measuring the zones of inhibition and relating them to the curve and portion of its slope that most accu¬ rately represented the level of gentamicin
(Fig 1).
for Low Levels of Gentamicin
Assay
Table 1.—Humans—Protocol 1 No. of Specimens Taken tast drops five to 30 min before removal of tissue 12 Aqueous Iris 14 11 Lens Vitreous 1 tast drops 31 to 60 min before removal of tissue 17 Aqueous Iris 10 Lens 12 1 Vitreous Last drops 61 to 90 min before removal of tissue 4 Aqueous Iris 12 Lens 13 tast drops 91 to 120 min before removal of tissue
Aqueous Iris Lens
Last
drops
'
Assay Measurable (Above 0.03 mg)* Aqueous, 0.06/ig (15 min after last drops) Aqueous, 0.24^g (25 min) Aqueous, 0.42,ug (30 min)4:
Aqueous, 0.17¿ig (35 min) Aqueous, 0.08,[¿g (35 min)
Aqueous,
Aqueous, 0.05/xg (110 min)
None
removal of tissue Iris Lens
2 5 2
Total eyes in protocol 1 50 Total specimens assayed_128 of
Brain-heart infusion agar of pH was used along with the same S aureus (ATCC No. 6638) suspension used for the intermediate levels. Plating procedures were as follows: from three to six filter paper discs or tissue samples were placed onto each agar plate according to the concen¬ trations involved. Fluid samples were placed onto the discs with a 0.025-ml micropipette. The same 6.35-mm size discs and micropipettes were used to assay the standards. All specimens were run at least in duplicate, but standards were assayed simultane7.25
4 6
Aqueous
for Intermediate Levels of Gentamicin
Assay
µß (70 min)
2
121 to 180 min before
Bacillus subtilis was grown in a Roux bottle containing (Difco) anti¬ biotic medium No. 1 for one week and the resulting washed spore suspen¬ sion maintained at 4 C as a stock solu¬ tion. A 1:10,000 dilution of this suspen¬ sion was made with pH 8.0, 0.1 M phosphate buffered saline. Pour plates were made using 0.2 ml of this dilution to each 100 ml of (Difco) antibiotic medium No. 5, pH 7.95 held at 45 C. The minimum level of biologically available gentamicin that this assay could detect accurately was 0.03^g/ ml.
* Two drops every five minutes for five applications. Normal cataract patients. t Of aqueous specimens, 18.9% had measurable levels of gentamicin after five applications
drops (normal eyes).
Í Same patient different eyes.
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Fig 2.—Topical application of gen¬ tamicin to rabbit eyes every 15 minutes for eight hours. in triplicate or more and the results plotted on semilog paper to determine the concentration equiva¬ lents. Solid tissues were taken from specific areas measured from the limbus and rinsed with saline. When undesired material was adherent to a tissue to be assayed, it was cleaned with forceps, scissors, and alginate swabs. In the rabbit experiments, seven treatment protocols (1 to 7) and one control protocol (8) were used: 1. Two drops every 15 minutes for eight hours. 2. Two drops every 15 minutes for six hours. 3. Two drops every 30 minutes for six hours. 4. Two drops every hour for seven hours. 5. Two drops every hour for 24 hours. 6. Two drops every three hours for 24 hours. 7. Two drops every six hours for 48 hours. 8. The control protocol. Treatment protocols comprised four groups of at least three animals (six eyes) each: group I—noninflamed eyes removed one hour after the last drop of gentamicin; group II—in¬ flamed eyes obtained one hour after the last drop; group III—noninflamed eyes enucleated 25 hours after the last application; group IV—inflamed eyes taken 25 hours after the last
ously
Fig 3.—Topical application of gentamicin
six hours.
drop.
The control protocol was composed of nine groups (each with at least six eyes): group I—one eye received only a sterile intracorneal suture, the other eye was not modified or treated; group II—one eye received a suture contaminated with Pseudomonas, the other eye a sterile suture; neither eye was treated; group III—the same as group II, but treated with normal sa¬ line; group IV—both eyes received a sterile suture, one eye treated with gentamicin every 15 minutes for six Fig 4.—Topical application of gen¬ tamicin to rabbit eyes every 30 minutes for six hours.
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to rabbit
eyes every 15 minutes for
hours; the other eye untreated;
Table 2.—Human—Protocol 2*
Assay Measurable (Above 0.03 mg)t Aqueous, 0.25/jg (30 min after last drops) Aqueous, 0.29/xg (30 min)
No. of Specimens Taken Last drops 5 to 30 min before removal of tissues 5 Aqueous Iris 2 1 Lens Last drops 31 to 60 min before removal of tissues
Aqueous Iris Lens
8 9 11
Last drops 61 to 90 min before removal of tissues 4 Aqueous Iris 5 Lens 6 Last drops 91 to 120 min before removal of tissues 4 Aqueous Iris 4 Lens 4 Last drops 121 to 180 min before removal of tissues Iris 1 1 Lens Total eyes in protocol 2 26 Total specimens assayed 65
Aqueous, 0.05/ig (35 Aqueous, 0.08/tg (32 Aqueous, 0.07/xg (45 Aqueous, 0.24/jg (35 Aqueous, 0.35¿ig (32 lris,0.03/tg (35 min) Iris, 0.03/ig (35 min) Aqueous, 0.13µ (70
min) min) min) min) min)
min)
Aqueous, 0.06/ig (110 min) Aqueous, 0.36/jg (110 min)
None
ten
*
Two drops every five minutes for ten applications. Normal cataract patients. t Of aqueous specimens, 47.6% and 9% of iris specimens had measurable levels of genta¬ micin after ten applications of drops (normal eyes).
group
V—both eyes received infected su¬ tures, one eye treated with vehicle, the other eye untreated; group VI— both eyes received sterile sutures, one eye treated with vehicle, the other eye was untreated; group VII—both eyes infected with Pseudomonas, one eye treated with vehicle, the other eye untreated; group VIII—no modi¬ fication of the corneas, treated every three hours in the one eye with gen¬ tamicin (Garamycin) and with the ve¬ hicle in the other eye; group IX—un¬ modified and untreated animals. In the human studies five protocols were used (Tables 1 to 5): 1. Two drops every five minutes for five applications—normal cataract pa¬ tients. 2. Two drops every five minutes for
applications—normal
cataract pa¬
tients. 3. Two drops hourly (eight hours) on the night before surgery and every five minutes for ten applications im¬ mediately before surgery—normal
cataract patients. 4. Two drops every five minutes be¬ fore surgery for five applications—in¬ flamed or otherwise abnormal eyes.
Table 3.—Human—Protocol 3* No. of Specimens Taken Last drops 5 to 60 min before removal of tissues 4 Aqueous Iris 4 Lens 4 Vitreous 1 Last drops 61 to 120 min before removal of tissues 1 Aqueous Iris 1 Lens 1 Last drops 121 to 180 min before removal of tissues 2 Aqueous Iris 2 Lens 2
Assay Measurable (Above 0.03 mg) t Aqueous, 0.4,ug (35 min after last drops) Aqueous, 0.1^g (60 min) Aqueous, 1.0^g (50 min)
Special cases—drops given day
Aqueous, 4. µß (65 min) Lens Capsule, 0.07/ug (75 min)
before
surgery as
*
Results
Confirming
the work of Barza et 6.35-mm filter paper disc was found to absorb 0.025 ml of fluid. The average weight of at least ten sam¬ ples in each group (noninflamed and inflamed eyes) of 6-mm trephine tis¬ sues shows that many are less in weight than the fluid tissue weight of 0.025 gm and hence the antibiotic concentration is underestimated. In the noninflamed tissue group, con¬ junctiva weight was 0.0130 gm; cor¬ nea, 0.0160 gm; iris, 0.0133 gm; lens capsule, 0.0396 gm; choroid-retina, 0.0025 gm; sclera, 0.0128 gm. In the inflamed tissue group, the weights were 0.0165 gm, 0.0352 gm, 0.0140 gm, 0.0336 gm, 0.0028 gm, and 0.0125 gm, respectively, for the conjunctiva, cor¬ nea, iris, lens capsule, choroid-retina, and sclera. The ordinate of Fig. 2 and
al,1"
Aqueous, 0.5/ig (65 min)
Aqueous, 0.06/ig (180 min)
above, but given every
day of surgery 2.5 min for 10 applications 1 Aqueous Iris 1 Lens 1 Total eyes in protocol 3 Total specimens assayed on
5. Two drops every five minutes be¬ fore surgery for ten applications. Tis¬ sues removed at various timed inter¬ vals after the last applicationinflamed or otherwise abnormal eyes.
8
25
drops hourly (eight times) from 1,700 to 2,400 hours on the night before surgery and five minutes immediately before surgery for ten applications. Normal cataract patients. t Of aqueous specimens, 75% had measurable levels of gentamicin after the protocol 3 regimen (normal eyes). Two
every
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a
Fig 5.—Topical application of gen¬ tamicin to rabbit eyes every hour for seven hours. of the following figures do not have an arithmetic progression for the biologi¬ cally available gentamicin; rather, the lower values are expanded. Indi¬ vidual variations between rabbits and replicate samples of the same tissue have been averaged. Note in Fig 2 the substantial levels of biologically ac¬ tive gentamicin found in the conjunc¬ tiva, cornea, aqueous, and sclera. There is a larger difference in this protocol between the concentration in the inflamed vs the noninflamed groups than is seen in the next proto¬ col (Fig 3) that uses the same fre¬ quency of application, but for a shorter interval of time (six hours in¬ stead of eight). Higher levels of anti¬ biotic are found, as expected, one hour after the last drop than at 25 hours. This difference is magnified with a more frequent application. Figure 4 (application every 30 min¬ utes for six hours) points out that de¬ creasing the frequency of application proportionately lessens the level of antibiotic in ocular tissues. Figure 5 (application every hour for seven hours) continues to show the trend that less frequent application will give reduced levels of gentamicin, but it is important to recognize in com¬ parison with Fig 4 that although the application is cut in half, intraocu¬ lar levels are only slightly diminished. As hourly application is continued for 24 hours (Fig 6), intraocular levels increase, approaching those of more frequent application, even to the point of being measurable in the lens capsule. Reducing the application to every three hours, (Fig 7) puts the intraocular levels in all cases below lug, which is the minimum inhibitory concentration (MIC) for the Pseudo¬ monas strain used in these experi¬ ments. Only the cornea in this proto¬ col has levels higher than tyg/ml. Extending the time between each ap¬ plication but continuing the treat¬ ment for 48 hours (Fig 8) again dem¬ onstrates the ability of tissues to be loaded with antibiotic and thereby to maintain a biologically active level sufficiently higher than the MIC for a
Fig 6.—Topical application of gentamicin
Fig 7.—Topical application
of
to rabbit eyes every hour for 24 hours.
gentamicin to rabbit eyes every three hours for 24
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hours.
ter five
applications of drops at five Only seven of 37 aqueous specimens (18.9%) showed de¬ tectable levels of gentamicin. The minute intervals.
greater the time intervals between
:ig 8.—Topical application
of
gentamicin
to rabbit eyes every six hours for 48 hours.
Table 4.—Human—Protocol 4* No. of Specimens Taken Last drop 5 to 30 min before removal of tissues Corneas 3 1 Aqueous Last drop 31 to 60 min before removal of tissues Corneas 4 1 Aqueous 1 Vitreous
Total eyes in protocol 4 Total specimens assayed
7 10
Assay Measurable (Above 0.03 mg)t Cornea, 0.5/ig (30 min after last drops) Cornea, 1.5^g (25 min after last drops) Cornea, 4.0^g (15 min) Aqueous, 0.21/ig (15 min)_ Cornea, 0.78µ (60 min) Cornea, 1 .O^g (60 min) Cornea, 0.5^g (30 min) Cornea, 2.0/ig (45 min) Aqueous, 0.33,ug (40 min) Vitreous,
