Download Drug reservoirs in topical therapy.

Drug Reservoirs in Topical Therapy
Joel 5. Mindel,*t Harry Smith,:}: Miriam Jacobs,* Alex B. Kharlamb,* and Alan H. Friedman"
The nictitating membrane, corneal epithelium, and corneal stroma were investigated as drug reservoirs.
A hydrophilic drug, D,L-epinephrine HC1, or a lipophilic drug, chloramphenicol, was applied topically
to rabbit eyes. Tissue levels of radioactive drug-plus-metabolites and unmetabolized epinephrine were
assayed up to 24 hrs later. On a per-mg-tissue basis, concentrations of epinephrine-plus-metabolites
in the stroma-endothelium were similar to or higher than those in the epithelium. The percentages of
radioactivity representing unmetabolized epinephrine in the stroma-endothelium were found to be
similar to or higher than those in the epithelium. On a per-mg-tissue basis, concentrations of cltiloramphenicol-plus-metabolites were significantly higher in the epithelium than in the stroma-endothelium
during the first 130 min after drug application. While the physical properties of these drugs determined
whether a higher concentration was found in the epithelium or stroma-endothelium, the ninefold greater
mass of the stroma-endothelium made it the major drug reservoir on a per-whole-tissue basis. The
presence or absence of a nictitating membrane had little effect on the level of either drug in the
epithelium, stromal-endothelium, or aqueous humor. Invest Ophthalmol Vis Sci 25:346-350, 1984
mans. Salem and Ellison4 found that the greatest concentration of epinephrine bitartrate during the 24 hrs
following a single topical application was in the nictitating membrane. Anderson5 reported three times as
much epinephrine bitartrate in the nictitating membrane as in the cornea 1 hr after application. The ducts
of the Hardarian and nictitans glands empty onto the
nictitating membrane. Miller and O'Connor6 pointed
out that all three potential reservoirs could be eliminated by amputating the nictitating membrane.
The present investigations dealt with the relative
importance of the corneal epithelium and stroma-endothelium as drug reservoirs and with the effect produced by amputating the nictitating membrane.
The cornea is a barrier that retards the penetration
of topically applied drugs. However, there is evidence
that the cornea may act as a drug reservoir for those
molecules that succeed in penetrating the epithelium.
Van Hoose and Leaders1 found that as external pilocarpine concentrations were increased progressively,
the cornea appeared to accumulate pilocarpine. Lazare
and Horlington2 reported that pilocarpine concentrations in the rabbit cornea remained higher than those
in the aqueous humor for at least 4 hrs. Van Hoose
and Leaders' believed that the stroma served as the
corneal reservoir for pilocarpine. However, Sieg and
Robinson3 found that 2 hrs after topical administration,
approximately 70% of the corneal pilocarpine was in
the epithelial layer.
Rabbits possess several potential drug reservoirs that
are not found in humans: the nictitans gland, the Harderian gland, and the nictitating membrane. These
structures may alter the corneal penetration and distribution of drugs and may invalidate attempts to extrapolate experimental pharmacokinetic data to hu-
Materials and Methods
Albino rabbits were used. Unilateral or bilateral amputation of the nictitating membrane was performed
at least 17 days prior to the experiment. Amputation
followed subconjunctival injection of lidocaine 1%.
The nictitating membrane was grasped with a forceps
and cut off at its base. The corneas of two rabbits with
unilateral removal of their nictitating membranes were
examined in a masked manner, using electron microscopy, to determine whether the amputation had
altered the corneal epithelium.
D,L-epinephrine HC1, 50 mM, pH 6 contained D,L[7-l4C]-epinephrine HC1. Chloramphenicol, 7.7 mM,
pH 6 contained [dichloroacetyl-l,2-14C]-chloramphenicol. The pH was adjusted using HC1 and NaOH.
The rabbits received a 50 ix\ drop of the same drug to
both eyes. After application of the drops, the lids were
opposed and held away from the globe for several sec-
From the Ophthalmology Section (JSM) of the Bronx Veterans
Administration Hospital and the Departments of Ophthalmology,*
Pharmacology,! and Biomathematical Sciences}: of the Mount Sinai
School of Medicine, New York.
Supported by the Medical Research Service of the Veterans Administration and National Eye Institute Grants EY 03203 and EY 01867.
Dr. Jacobs was supported in part by National Institute of Arthritis,
Metabolism and Digestive Diseases Grant 5T35AM07420-04.
Submitted for publication: December 23, 1982.
Reprint requests: Joel S. Mindel, MD, PhD, Department of Ophthalmology, Annenberg 22-14, the Mount Sinai School of Medicine,
One Gustave L. Levy Place, New York, NY 10029.
346
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DRUG RESERVOIRS IN TOPICAL THERAPY / Mindel er ol.
No. 3
Table 1. Cornea and aqueous humor drug levels, mean ± standard error without nictitating membrane
pmol Drug + metabolites (number of eyes)
Per mg Corneal epithelium
Minutes*
10
40
70
130
190
370
1450
Epinephrine
37.7 ±
19.4 ±
20.8 ±
4.2 ±
16.8 ±
4.5 ±
1.9 ±
8.2 (5)
3.4(4)$
4.9 (5)$
1.1 (6)
8.8 (8)
2.4 (4)
0.4 (6)
Chloramphenicol
214.3 ±65.1 (6)f
126.7 ± 36.2 (6)t
70.8 ± 16.2 (6)t
30.2 ± 9.9 (8)t
8.8 ± 4.0 (4)
3.5 ± 0.6 (4)$
2.3 ± 0.8 (4)
Per mg Stroma-endothelium
Epinephrine
86.2 ±
47.2 ±
38.6 ±
3.0 ±
9.5 ±
1.8 ±
1.4 ±
30.5 (5)
11.9(4)$
8.5 (5)$
1.3(6)
2.1 (8)
0.6(4)
0.3(6)
Per nl Aqueous humor
Chloramphenicol
Epinephrine
Chloramphenicol
68.5 ±
39.3 ±
23.1 ±
11.2 ±
8.8 ±
6.2 ±
3.9 ±
2.2 ± 0.7 (6)
2.8 ± 0.9 (4)
2.2 ± 0.6 (6)
0.6 ± 0.2 (6)
1.3 ±0.4(8)
0.4 ± 0.2 (4)
0.1 ±0.0(6)
3.2 ± 0.7 (6)
6.7 ± 2.1 (6)
5.1 ±0.5 (6)
2.5 ± 0.5 (8)
1.3 ±0.5 (4)
0.2 ± 0.0 (4)
0.0 ± 0.0 (4)
8.4 (6)t
6.7 (6)t
1.8 (6)t
2.2 (8)t
3.0 (4)
1.1 (4)$
0.3 (4)
* After topical application of 50 /tl: D,L-epinephrine HO, 50 mM, pH 6
or chloramphenicol 7.7 mM, pH 6.
t Epithelial drug level significantly (P < 0.05) higher than stromal-endothelial
drug level at this point in time.
% Stromal-endothelial drug level significantly (P< 0.05) higher than epithelial
drug level at this point in time.
onds to prevent overflow. The rabbits were killed 10,
40, 70, 130, 190, 370 and 1,450 min later with an
intravenous injection of pentobarbitol. Each eye
quickly was enucleated and dipped once in a fresh
solution of NaCl, 0.9% to remove any drug remaining
in the tear film. The aqueous humor was removed as
completely as possible using a 26-gauge needle inserted
at the corneal limbus. The volume of aqueous humor
aspirate was recorded, and the fluid was placed in a
scintillation vial. The anterior chamber was refilled
with air to restore the shape of the anterior segment
of the eye and facilitate dissection. The cornea was
removed at its limbus, weighed, and placed epitheliumside down for 1 min on an n-heptanol-soaked filter
paper disc approximately 15 mm in diameter.7 The
filter paper disc then was placed in a scintillation vial.
The remainder of the cornea, ie, the stroma-endothelium, was reweighed and placed in a scintillation
vial. Tissue solubilizer was added to all vials; after the
tissues were dissolved, their radioactivity in Bray's solution was determined with the use of a scintillation
counter. In 20 additional rabbits, the tissues were handled similarly, except that unmetabolized epinephrine
was assayed after being separated from epinephrine
metabolites.8'9
To estimate the amount of drug transferred from
epithelium to stroma-endothelium during the 1-min
period on the filter paper disc, 10 ^1 of the epinephrine
(seven determinations) or chloramphenicol (five determinations) solution were placed on the surface of
an n-heptanol-soaked paper disc immediately before
an untreated cornea was placed epthelium-side down
on the disc. After 1 min of contact the stroma-endothelium was lifted from the filter paper disc. The
relative amounts of radioactivity remaining in the filter
paper and transferred to the stroma-endothelium were
determined by dissolving the samples and assaying
them as previously described.
Four corneas were examined histologically to confirm that 1 min of contact with n-hepatanol removed
the corneal epithelium.
Results
Rabbits tolerated the absence of their nictitating
membranes for the 2.5-5-week period prior to drug
application without objective evidence of discomfort.
The corneas remained clear and lustrous. Masked
evaluation of electron micrographs at 7500 X magnification failed to indicate any consistant differences
between corneas with nictitating membranes and those
without.
Light-microscopic examination showed that 1 min
of contact with the n-hepatanol-soaked filter paper disc
removed the corneal epithelium, leaving the basement
membrane and stroma intact.
The mean ± SE percent of drug transferred to the
stroma-endothelium during the 1-min period of epithelial dissolution was 5.1 ± 1.4% using the epinephrine solution and 6.0 ± 2.9% using the chloramphenicol
solution.
The mean ± SE weights of the corneal epithelium
and stroma-endothelium, determined by weighing 70
corneas before and after exposure to n-heptanol, were
6.07 ± 0.32 mg and 56.32 ± 1.82 mg, respectively.
The average agreement of the weights of the two corneas of a given rabbit was 92 ± 0.5%.
Tables 1 and 2 give the mean ± SE drug-plus-metabolite concentrations per mg corneal epithelium, per
mg stroma-endothelium, and per n\ aqueous humor
in rabbits with and without nictitating membranes.
Table 3 gives the percent of radioactivity representing
unmetabolized epinephrine in these three tissues. The
role of the nictitating membrane as a reservoir was
determined by comparing the drug-plus-metabolite
levels in corneal epithelia of animals with and without
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1984
Table 2. Cornea and aqueous humor drug levels, mean ± standard error* with nictitating membrane
pmol Drug + metabolites
Per mg Corneal epithelium
Minutes •f
10
40
70
130
190
Epinephrine
62.9
140.0
22.5
17.9
7.2
±21.4
± 54.0
± 5.6
± 5.8
± 1.0
Per mg Stroma-endothelium
Chloramphenicol
375.3
209.0
48.5
23.0
19.0
± 25.4$
± 51.0$
± 10.6$
± 3.5$
± 1.8$
Epinephrine
42.7
129.8
19.8
20.4
5.8
± 62
± 55.2
± 5.4
± 9.2
± 1.3
Chloramphenicol
86.5
51.6
18.2
8.9
7.0
± 13.8$
± 10.0$
± 5.2$
± 2.3$
± 2.1$
Per nl Aqueous humor
Epinephrine
0.9 ± 0.2
14.4 ± 6.8
2.1 ±0.4
4.2 ± 2.4
1.2 ±0.3
Chloramphenicol
4.0
10.4
4.2
2.2
1.9
± 0.4
± 2.0
± 1.3
± 0.2
±0.4
* Each value determined from four eyes.
t After topical application of 50 pi: D,L-epinephrine HC1, 50 mM, pH 6
or chloramphenicol 7.7 mM, pH 6.
$ Epithelial drug level significantly (P «£ 0.05) higher than stromal-endothelial
drug level at this point in time.
nictitating membranes and stroma-endothelia of animals with and without nictitating membranes. Each
point in time was tested using a student's /-test. Values
were assumed significant if P < 0.05. For a given drug,
there were no significant differences at any point in
time between eyes with and without nictitating membranes. The roles of the epithelium and stroma-endothelium as reservoirs were determined by comparing
their drug-plus-metabolite levels at each point in time
on a per-mg-tissue basis. In rabbits with nictitating
membranes, the per mg epithelial and stromal-endothelial levels were not significantly different after
epinephrine application. However, the chloramphenicol-plus-metabolite levels were significantly higher per
mg epithelium than per mg stroma-endothelium at
all points in time for rabbits with nictitating membranes. In rabbits without nictitating membranes, the
epinephrine-plus-metabolite levels per mg stroma-endothelium were significantly higher than those per mg
epithelium only at 40 and 70 min after drug application
(Table 2). After chloramphenicol administration to
rabbits without nictitating membranes, the per-mgepithelium drug-plus-metabolite levels were significantly higher for at least 130 min after application,
but not from 190 min to 1,450 min after application.
The percentages of unmetabolized epinephrine (Table 3) were compared in eyes without nictitating membranes to contralateral eyes with nictitating membranes
using a paired t-test. The percents of unmetabolized
epinephrine in the epithelium, stroma-endothelium,
and aqueous humor were not altered significantly by
the presence or absence of a nictitating membrane,
with one exception: 10 min after epinephrine application, corneal epithelia of eyes without nictitating
membranes had a slightly higher percent of unmetabolized epinephrine than epithelia of eyes with nictitating membranes (40 ± 6% vs 36 ± 4%). The percents
of unmetabolized epinephrine in the stroma-endothelium were not significantly different from those in
the corresponding epithelium, with one exception: 10
min after epinephrine application to eyes without nictitating membranes, the percent of unrnetabolized epinephrine was significantly higher in the stroma-endothelium than in the epithelium.
Discussion
Epinephrine and chloramphenicol were chosen for
this study because both are in clinical use and because
their pharmacokinetics have been studied in rabbits.2'4'5
The physical properties of the two drugs differ: epi-
Table 3. Percent radioactivity (mean ± SE) as unmetabolized epinephrine
Time after D,L-epinephrine HCl applied*
Tissue
Nictitating membrane absent (OD)
Corneal epithelium
Corneal stroma-endothelium
Aqueous humor
Nictitating membrane present (OS)
Corneal epithelium
Corneal stroma-endothelium
Aqueous humor
10 min
70 min
190 min
1,450 min
40 ± 6% (4)f$
68 ± 0% (4)$
54 ± 2% (4)
23 ± 7% (5)
27 ± 8% (5)
32 ± 4% (4)
24 ± 6% (5)
24 ± 1% (4)
27 ± 6% (5)
18 ± 2% (4)
19 ± 1% (4)
16 ± 1% (4)
36 ± 6% (4)f
65 ± 5% (4)
53 ± 3% (4)
17 ± 1% (5)
20 ± 4% (5)
27 ± 10% (4)
19 ± 1% (5)
24 ± 2% (4)
30 ± 12% (5)
16 ± 1% (4)
18 ± 0% (4)
17 ± 2% (4)
* Numbers in parentheses indicate number of eyes assayed.
t Significant difference between eyes with and without nictitating membranes
(P < 0.02).
X Significant difference between epithelial and stromal levels (P < 0.02).
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DRUG RESERVOIRS IN TOPICAL THERAPY / Mindel er ol.
nephrine HC1 is readily soluble in water, while chloramphenicol is more soluble in lipids, eg, chloramphenicol is 150 times more soluble in ethanol than in
water.l0 Thus, at pH 6, there was no difficulty in making
a solution of 50 mM, D,L-epinephrine HC1, while the
maximum chloramphenicol concentration was 7.7
mM. These concentrations are used clinically, ie, 50
mM epinephrine HC1 is a 1% concentration and 7.7
mM chloramphenicol is a 0.25% concentration.
The corneal penetration of drugs increases in proportion to lipid solubility," presumably because the
major barrier to drug penetration is the lipids in the
epithelial cell membrane. The stroma is relatively acellular and consists primarily of collagen.12 On the basis
of the physical properties of the drugs and the cornea,
we predicted that chloramphenicol would penetrate
into the epithelium more readily than epinephrine.
This expectation was fulfilled. Ten minutes after application, the chloramphenicol-plus-metabolite levels
in the corneal epithelium were six times higher than
the epinephrine-plus-metabolite levels, despite that the
concentration of the epinephrine drop was six times
greater. The presence or absence of a nictitating membrane did not alter this ratio significantly (Tables 1
and 2). We also predicted that with the passage of time,
the corneal epithelium would remain the primary reservoir for chloramphenicol, while the corneal stroma
would become the primary reservoir for the more hydrophilic epinephrine molecules. To a large extent,
this prediction was also correct. On a per-mg-tissue
basis, the epithelial levels of chloramphenicol-plusmetabolites remained significantly higher than those
in the stroma for at least 130 min. This was true for
rabbits with and without nictitating membranes. The
stromal levels of epinephrine-plus-metabolites tended
to be higher than those in the epithelium, but these
differences achieved significance only at 40 and 70 min
after application and only in rabbits without nictitating
membranes. The proportions of radioactivity representing unmetabolized epinephrine also tended to be
higher in the stroma-endothelium than in the epithelium (Table 3), but this difference was significant only
at 10 min after epinephrine application and only in
rabbits without nictitating membranes.
Although the presence of the nictitating membrane
increased the relative importance of the corneal epithelium as a depot for epinephrine, this effect was not
especially marked, because epinephrine-plus-metabolite levels in the epithelia of rabbits with nictitating
membranes were not significantly different from those
in rabbits without nictitating membranes. Nor were
significant differences found for stromal-endothelium
and aqueous humor epinephrine-plus-metabolite levels
when rabbits with and without nictitating membranes
were compared. When corresponding corneal epithe-
349
lial, stromal-endothelial, and aqueous humor levels
of chloramphenicol-plus-metabolites were compared
between rabbits with and without nictitating membranes, these too failed to show significant differences.
Thus, the results of the present study do not support
the theory that the nictitating membrane acts as a
major drug depot.
Which was the more important drug depot, the corneal epithelium or the corneal stroma? When data
were analyzed on a per-mg-tissue basis, the answer
varied, depending on the drug and the time after application. However, the mean stromal weight, 56.32
mg, was more than nine times greater than the mean
epithelial weight, 6.07 mg. As a result, on a ptv-wholetissue basis, the corneal stroma was the major drug
depot. This was so for epinephrine and chloramphenicol at all points in time. Thus, a distinction should
be made when discussing drug reservoirs between total
tissue content and tissue concentration.
When epinephrine was separated from its metabolites, the importance of the stroma as a reservoir for
unmetabolized molecules was increased only minimally. Presumably, the greater metabolic activity of
the epithelium13 diminished its proportion of unmetabolized epinephrine, but this disadvantage was largely
offset by the hydrophilic nature of epinephrine metabolites. These metabolites would tend to accumulate
in the stroma-endothelium. No attempt was made to
separate chloramphenicol from its metabolites. There
is evidence that chloramphenicol is metabolized within
the cornea. For example, Green and MacKeen14 reported that 2 hrs after topical chloramphenicol application, about 45% of rabbit aqueous humor radioactivity was due to chloramphenicol metabolites. Therefore, the present data may not accurately reflect the
relationship of unmetabolized chloramphenicol in the
epithelium to that in the stroma-endothelium.
Lippert and co-workers15 reported results similar to
ours. Two and three hours after the topical administration of tritiated dexamethasone to rabbits, the epithelial radioactivity was higher, on a per-mg-tissue
basis, than that in the stroma. But the stroma remained
the major depot on a per-whole-tissue basis. Sieg and
Robinson316 reported that the highly lipophilic drug
fluorometholone was similarly distributed to the lipophilic chloramphenicol used in the present study.
Ten minutes after application of fluorometholone, the
major corneal reservoir was the epithelium, even when
calculations were on a per-whole-tissue basis. Thereafter, from 20 to 120 min following application, the
stroma was the major drug reservoir. Surprisingly, for
pilocarpine, which was less lipid-soluble than fluorometholone, the corneal epithelium was the primary
reservoir during the entire 2 hrs of their study. Perhaps
pilocarpine exerted pharmacologic effects that altered
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1984
its distribution. Sieg and Robinson removed the corneal
epithelium by scraping with a scalpel blade. The present
authors used n-heptanol.7 Both techniques suffered
from a common defect: drug was released from disrupted epithelial cells into the stroma. Estimates of
the amounts of drug released into the stroma during
dissolution with n-heptanol yielded mean values well
under 10%. These errors were considered acceptable.
An assumption in the preceding analysis was that
epinephrine and its metabolites distributed by passive
diffusion. This may have been partially incorrect. Epinephrine has multiple pharmacologic effects. Among
the known actions of adrenergic agonists are: increasing
lacrimal gland tear production, 17 activating a cyclicAMP sensitive corneal epithelial transport system,18
being accumulated by corneal and nictitating membrane nerve endings (especially of the adrenergic
type19), and increasing aqueous humor outflow facility.20 A second assumption concerned the use of a
racemic mixture of D- and L-epinephrine. We believed
that the corneal distributions of both epinephrine isomers and their mebabolites were similar because both
isomers are substrates for the degradative enzymes
monoamine oxidase and catechol-O-methyl transferase.21'22 A third assumption was that an insignificant
amount of corneal and aqueous humor radioactivity
resulted from systemically absorbed drug molecules.
Systemic absorption does occur, eg, Anderson5 found
8 picograms epinephrine per ml blood 1 hr after topical
application of 0.1 % epinephrine bitartrate.
In summary, on a per-mg-tissue basis, the corneal
epithelium appeared to be the major drug reservoir
for highly lipophilic drugs, such as chloramphenicol,
while the corneal stroma appeared to be the major
drug reservoir for more hydrophilic drugs, such as epinephrine. On a per-whole-tissue basis, because of its
greater mass, the corneal stroma appeared to be the
major drug reservoir for many lipophilic and possibly
all hydrophilic drugs. The presence or absence of a
nictitating membrane did not significantly alter drug
pharmacokinetics.
Key words: epinephrine, chloramphenicol, pharmacokinetics,
cornea, nictitating membrane, eye, drug
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