pH and Drug Ionization Affects Ocular Pressure Lowering
of Topical Carbonic Anhydrase Inhibitors
William F. Brechue and Thomas H. Maren
Purpose. To evaluate the effect of drug ionization on the ocular hypotensive activity of topical
carbonic anhydrase inhibitors.
Methods. Ocular normotensive New Zealand albino and ocular hypertensive Dutch Belted
pigmented rabbits were used. Tonometric intraocular pressure levels were taken after topical
application of 50 nl of drug (at various concentrations and pH values) to one eye with the
contralateral eye used as an untreated control. The drugs tested were MK-927, L-662,583, and
AHR-16329. Eye tissues were analyzed for drug by our enzymatic methods.
Results. In all cases, the more ionized the applied drug the greater the ocular hypotensive
activity. Tissue distribution studies showed that there was more drug found in the eye after the
ionized form of a drug was applied than that found after application of the less ionized forms.
Conclusions. Increasing the ionization of three ampholyte topical carbonic anhydrase inhibitors increases their ocular hypotensive activity. These data taken with ocular disposition data
suggest that ionized compounds of this type are more readily sequestered in the cornea, which
serves as a drug depot for prolonged drug delivery and activity. Invest Ophthalmol Vis Sci.
1993;34:2581-2587.
JVecently, several carbonic anhydrase (CA) inhibitors
were introduced that, when applied topically to the
cornea, significantly reduce intraocular pressure
(IOP) in normotensive and hypertensive animals1"5
and humans.6-7 In general, these compounds are ampholytes with a basic amine (pKj between 5.5 and 6.0)
and an acidic sulfonamide group (pK2 of 8.3-8.9). Delivered as their hydrochloride salts, these drugs have
relatively good water and lipid solubility in the pH
range of 4.5-6; such solutions elicit a significant reduction in IOP. In this pH range these compounds are
50-95% ionized. Similarly, ionization of the sulfonamide at the pH 9 level yields soluble solutions. We
originally supposed that at neutral pH, where ioniza-
tion was about 10% and lipid solubility was increased,
corneal permeability would increase and result in
greater IOP reduction. However, we were surprised to
find that decreasing drug ionization and increasing
lipid solubility actually reduced ocular hypotensive activity.2 This article explores and extends these observations with several structurally different compounds.
Theoretical and practical implications of these findings are discussed. Preliminary accounts of this work
were presented in abstract form at ARVO meetings89
along with attempts to rationalize the findings in terms
of ionic trapping mechanisms.10
METHODS
From University of Florida Health Science Center, Department of Pharmacology
and Therapeutics, Gainesville Florida.
Supported by National Institutes of Health grant *EYO2227.
Submitted for publication: August 10, 1992; accepted December 8, 1992.
Proprietary interest category: N.
Reprint requests: William F. Brechue, University of Florida Health Science Center,
Department of Pharmacology, P.O. Box 100267, Gainesville, FL 32610.
Adult male rabbits weighing 2—3 kg, either ocular normotensive New Zealand albino (18.6 ±2.1 mmHg) or
ocular hypertensive Dutch Belted pigmented (30.5
± 2.5 mm Hg) were used. The experimental procedures conform to the Association for Research in Vi-
Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
2582
sion and Ophthalmology Resolution on the use of animals. The rabbits were kept in individual cages with
food and water provided ad libitum. The animals were
maintained on a 12-hr: 12-hr light/dark cycle in a temperature controlled room (21-26°C). More details on
the handling, care, and housing of animals have been
given previously.11
Drug Administration
All drugs were topically applied. MK-927 was administered as a 2% (53 mM) or 0.5% (13.3 mM) solution.
The nascent pH level of these solutions was 4.5-4.8.
L662,583 was always used as a 2% solution (48 mM).
Its nascent pH was 4.4. Both of these compounds were
obtained from Merck Sharp & Dohme Research Laboratories (West Point, PA). AHR16329 was given as a
2% (63 mM) or 0.5% (9 mM) solution. Its nascent pH
level was 4.6-4.8. This compound was obtained from
the A. H. Robins Company. All solutions were prepared and applied in 0.5% hydroxyethylcellulose. To
alter ionization of the drug, solutions were titrated
with 1 N sodium hydroxide to the desired pH. Solutions of 0.5% MK-927 and AHR-16329 were used to
allow study over the entire pH profile of the drug. The
upper limit of water solubility for a 2% solution is
about pH 5.5; lower limit for 2% solution is about
pH 9.
Using a syringe, a 50-/il drop was applied to the
upper quadrant of the eye and allowed toflowover the
cornea. The contralateral eye served as a control during pressure studies. We have determined that hydroxyethylcellulose vehicle alone does not affect
IOP.2 The physicochemical properties of these drugs
TABLE l.
are given in Table 1 and the structures are given in
Figure 1.
Physiologic Measures
Tonometric IOP. IOP was measured using a Digilab
model 30R pneumatonometer (Bio-Rad, Cambridge,
MA).212 The pressure readings were matched with
two-point standard pressure measurements at least
twice each day using a Digilab Calibration Verifier. All
IOP measurements were made by the same investigator with the same tonometer.
One drop of 0.5% proparacaine (Alcaine, Alcon
Inc., Humaco, PR) diluted 1:2 with saline, was instilled
in each eye immediately before each set of pressure
determinations. The animals were familiarized with
the experimental procedure by handling and routine
pressure measurements taken before the experiments.
Rabbits were not sedated or otherwise restrained during IOP measurements except for the hand of the investigator placed lightly on the back and shoulders of
the rabbit. IOP was measured three times at each time
interval and the means reported. IOP measurements
were taken immediately before drug administration,
after which IOP was determined at 30 min and then
hourly until the drug effect had dissipated. In all IOP
studies, rabbits that showed a consistent difference in
IOP between eyes during baseline measurements or
any sign of eye irritation were excluded from the
study, ^ 3%.
Manometric IOP. To determine onset of pressure
reduction during the first hour after treatment, IOP
was measured continuously using manometric techniques.1213 Rabbits were anesthetized with sodium
Physicochemical Properties of Topical and Systemic Sulfonamides
Cornea
K, vs. CA II
(nM)
CHCl3
Buffer
PK
NH+
SO2NH~
OH~
MK-927*
4
5.8
8.3
L-662,583
0.3
5.7
8.4
10.8
AHR-16329f
7
6.0
8.9
—
10
1
—
—
7.4
8.0
—
—
Methazolamide|
EthoxzolamideJ
pH 7.4
pH5
pH6
pH7
pH8
pH9
0.3
0.8
0.1
0.2
0.5
pH 4.6
pH7.0
pH9.0
pH 4.5
pH7.4
pH 9.0
pH7.2
pH 7.2
0.01
0.50
0.02
0.013
0.60
0.50
0.06
Solubility
(mM)
25
* From reference 2.
f From reference 5.
X From reference 1.
Data on L-662,583 are new.
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60
20
13
20
50
>50
>50
>50
70
3
7
5
0.04
In vitro
(X103-hr-\ )
In vivo
(X103-hr-')
0.4
—
3
—
—
—
—
—
2.7
3.2
—
4
6
5
—
—
2.4
7
1
8.7
6.3
—
8
330
2
40
2583
Drug Ionization Alters CAI Pressure Lowering
Drug Distribution in Eye
SO 2 HN 2
SO,NH 2
CH 2 N ( C H J I J H C I
L662.583
O-C2H4-O-SO2NH2
HC1
AKR-16,329
FIGURE 1. Structures of the three compounds investigated in
this study.
pentobarbital (30 mg/kg intravenous) with additional
30-60-mg doses titrated as needed. Animals were intubated by tracheotomy and ventilated on a positive
displacement ventilator. End tidal carbon dioxide levels were maintained at 4.2 ± 0.4% throughout the experiment. Body temperature was maintained at 39
± 0.5°C with a heating pad. The right femoral artery
and vein were isolated and cannulated. The arterial
line was used to monitor systemic arterial pressure and
to sample blood. The venous line was used to inject
anesthesia.
Animals were prone with their heads supported
upright by a stereotaxic device such that the head was
at about the normal standing height, approximating
the position during tonometric IOP studies. Needles
(25-gauge) were introduced into the anterior chamber
of the eye through the cornea. A 4 cm, PE 50 polyethylene catheter connected the needle to an RP-1500
pressure transducer (Narco-Bio Systems, Houston,
TX). Output was recorded on a DMP-4B Narco physiograph (Narco-Bio Systems, Houston, TX). Calibrations were performed daily with a mercury manometer. Animals were killed with a pentobarbital overdose
followed by supersaturated potassium chloride solution.
Baseline arterial pressure, IOP, and arterial blood
gases were monitored for 30-45 min after surgical
preparation and insertion of eye needles and before
starting experiments to ensure steady-state levels in
the animals.
The distribution of MK-927 was studied 30 min and 2
hr after topical administration of 1 drop 0.5% MK-927
in 1% hydroxyethylcellulose at pH 4.7, 7.0, and 8.9.
Rabbits were killed by intraperitoneal injection of 50
mg/kg pentobarbital followed by centripetal pooling
of blood away from the head. Each eye was washed for
5 sec under a stream of water. The aqueous humor was
sampled, and the corneas were excised and then
briefly rinsed again before removal of the ciliary process. These were nearly blood-free and were excised
from well-blotted uveas by scraping with a flat scalpel
blade. The tissue was carefully blotted to remove excess liquid before weighing. Tissue samples (5-60 mg)
were homogenized in 0.2 ml distilled water and briefly
boiled before assay for drug concentration.2 These experiments were carried out by Dr. Curtis Conroy.
Calculations
For all IOP experiments drug was administered to
only one eye leaving the contralateral eye as an untreated control. The ocular hypotensive activity is expressed as the average difference in IOP between the
treated (T) and control (C) eyes ( I O P ^ Q , mmHg), thus
minimizing the diurnal, seasonal, and interindividual
variations commonly observed in the rabbit.11 Change
in IOPfr.Q versus time curves were plotted for each
animal after tonometric studies. The area between the
IOP = 0 line and the IOP/time curve was integrated to
yield the area under the line (AUL) or the integrated
time effect.
Statistics. All data are expressed as mean ± SE.
Data were analyzed using a one-tailed t test. Original
alpha levels were set at 0.05 with the per comparison
error rate corrected by the modified Bonferroni technique 14
RESULTS
Figure 2 shows the effect of ionization on the ocular
hypotensive activity of 2% MK-927. At the low and
high pH, the most ionized forms of the drug (^ 90%),
pressure lowering is the same both in peak IOP reduction (3.5 mm Hg) and area under the no-effect line
(AUL, 11 mm Hg/hr). When the drug is applied in the
less ionized form (72%, pH 5.4), peak IOP reduction is
3.1 mmHg, which is not statistically different from the
other pH solutions. However, by 2 hr the pressure
lowering is significantly less than after application of
the more ionized forms resulting in an attenuated duration of activity, seen as a significantly lower AUL, 6.1
mmHg/hr. To study this phenomenon more completely, we used a 0.5% solution that would confer
water solubility over a wide pH range. The results are
shown in Figure 3. Again, at the ionization extremes,
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Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
AUL-6.1 mmHg-hr A
AUL-10.6mmHg-hj
AUL-25.9 mmHg • hr
-1-
AUL-11.1 mmHg-hr
-2-
A
-32
3
4
HOURS
5
6
-4-I
AUL-13.7 mmHg • hr
0
1
2
3
HOURS
4
5
6
AUL-14.9mmHg-hr
FIGURE 2. Effect of ionization on the ocular hypotensive activity of 2% MK-927 in albino rabbits. Values are mean ± SE.
Mean starting pressures were 18.6 ± 2.4 mmHg.
pH
4.6, 94% ionized; A—A pH 5.4, 72% ionized; • — • pH 9.1,
86% ionized. Area under the no effect line (AUL) is given, n
= 8 for each pH experiment.
B
91% (pH 4.8) and 84% (pH 9), the IOP lowering is the
same with respect to peak reduction (2.5 and 2.7 mm
Hg, respectively) and AUL (6.7 and 6.6 mm Hg/hr,
respectively). Intermediate levels of ionization (56% at
pH 5.7 and 33% at pH 8) resulted in slightly less peak
IOP reduction and AUL. The most significant finding
was that at pH 7, the least ionized (11%) and most lipid
soluble form of the drug, peak IOP reduction (0.8 mm
Hg) and AUL (1.2 mm Hg/hr) were far lower than
those that occurred with the more ionized drug. Ocular pressure activity was essentially and surprisingly
eliminated. Figure 4 shows the same general phenomenon for MK-927 in Dutch Belted pigmented rabbits.
AUL-6.61 mmH(rhr
0
1
2
3
4
HOURS
FIGURE 3. Effect of ionization on the ocular hypotensive activity of 0.5% MK-927 in albino rabbits across the pH range.
Values are mean ± S.E. Mean starting pressures were 18.5
±1.6 mm Hg. Solution pH, percentage of ionization and
AUL are given, n = 8 for each pH experiment.
3
HOURS
FIGURE 4. Effect of ionization on the ocular hypotensive activity of MK927 in ocular hypertensive pigmented rabbits.
(A) 2% solution. Mean starting pressures were 31 ± 3.4
pH 5.4, 72% ionmmHg. •—• pH 4.5, 95% ionized;
ized. (B) 0.5% solution. Mean starting pressures were 30
pH 7.0, 11%
± 2.1 mmHg. •—• pH 4.8, 91% ionized,
ionized; A—A pH 9.1, 86% ionized. AUL is given, n = 8 for
each individual pH experiment. Values are mean ± SE.
The upper curve shows the 2% experiment and the
lower curve the 0.5% experiment. These animals were
naturally ocular hypertensive, and therefore pressure
lowering and AUL were significantly greater than in
the albino rabbits. However, the same effect of ionization is seen, decreasing ionization reduces the pressure lowering ability of MK-927.
Figure 5 shows the results of manometric pressure
studies performed in the albino rabbit. These experiments show clearly that the initial pressure lowering
(1.4 mm Hg at 20 min) is independent of ionization,
but between 30 and 60 min the ocular hypotensive
activity is significantly greater with the low pH, high
ionization drug.
Figures 6 and 7 show the same ionization phenomenon with other topical CA inhibitors with ampholyte
characteristics. L- 662,583 has a very different ring
system from MK-927 (Fig. I).4 AHR-16329 is an organic sulfamate and is also structurally quite different
from both other compounds (Fig. I). 515
Table 2 gives tissue distribution data of MK927.10
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Drug Ionization Alters CAI Pressure Lowering
2585
0-
-1-
o»
I.2-I
CL
o
•3-
-4-J
0
1
-4-J
20
10
30
50
40
60
MINUTES
FIGURE 5. Effect of ionization on the ocular hypotensive activity of 0.5% MK-927 in albino rabbits. Pressure was determined manometrically. Values are mean ± SE. Mean starting
pressures were 19.1 ± 1.8 mm Hg. • — • pH 4.8, 91% ionized; • — • pH 7, 11% ionized, n = 8 for each pH experiment.
The more ionized form of the applied drug (pH 4.7
and 8.9) results in significantly higher drug levels in
the cornea, anterior aqueous humor, and ciliary process at 30 min compared to those after application of
the less ionized form. These differences in cornea and
ciliary process persisted through 2 hr. Differences in
the aqueous humor were small throughout. Expanded
data along with their theoretical basis are being prepared for publication.
2
3
HOURS
4
5
6
FIGURE 7. Effect of ionization on the ocular hypotensive activity of 2% AHR-16329 in albino rabbits. Values are means
± SE. Mean starting pressures were 19.1 ± 2.6 mm Hg.
• — • pH 4.1, 98% ionized; • — • pH 5.3, 83% ionized.
AUL is given, n = 8 for each pH experiment.
DISCUSSION
Our main finding is that in this series of drugs the
ionized forms are more active than unionized. The
three compounds used are structurally different, but
all contain a sulfonamide group that confers inhibitory
activity against CA. Another major similarity is that
each compound is an ampholyte consisting of an ionizing group with pK of 5.5-6 and an acidic sulfonamide
group with pK of 8.3-8.9. Their ampholytic characteristic confers relatively good water and lipid solubility.2'45 Each compound has a constellation of physicochemical properties that allows good corneal and tissue permeability and significant reduction of IOP.1'5
TABLE 2.
Effect of Ionization on Distribution
of MK-927 in Ocular Tissues and Fluids
(/umol/kg)
0
1
2
3
4
5
6
HOURS
FIGURE 6. Effect of ionization on the ocular hypotensive activity of 2% L662.583 in albino rabbits across the pH range.
Values are means ± . Mean starting pressures were 18.9
±2.6 mm Hg. Solution pH, percentage of ionization and
AUL are given, n = 8 for each pH experiment.
Cornea
pH4.7
pH 7.0
pH8.9
Anterior Aqueous
pH4.7
pH 7.0
pH8.9
Ciliary Process
pH4.7
pH7.0
pH8.9
30 min
2hr
10.4 ± 1.3
2.5 ±0.4
9.9 ± 1.1
5.0 ± 0.6
2.5 ± 0.2
3.5 ±0.4
2.5 ±0.4
1.5 ±0.3
3.8 ±0.4
1.6 ±0.1
1.1 ±0.1
1.0 ±0.1
7.2 ± 0.8
3.0 ±0.8
6.5 ± 1.4
2.7 ±0.1
1.4 ±0.1
2.3 ±0.2
From reference 10 and amended by Dr. Curlis Conroy. (To be
published.)
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Investigative Ophthalmology & Visual Science, July 1993, Vol. 34, No. 8
We have shown that these ampholytes have another property that, in addition to good corneal and
non-corneal permeability, appears to be a necessity for
effective long-term lowering of IOP: namely the ability
to sequester in the cornea and form a drug delivery
depot.213 Analysis of corneal drug levels and decay
from the cornea and other ocular tissue and fluids
suggests that the cornea controls the process. The cornea continually supplies drug to the ciliary process,
replacing delivery from plasma, as for systemic CA inhibitors.13
Each of the three compounds have similar in vivo
corneal permeability comparable to that of methazolamide but significantly less than for ethoxzolamide (see
Table 1). Methazolamide and analogs and ethoxzolamide in solution do not lower IOP by the topical route;
it is significant, however, that there is activity with
some of the homologs and ethoxzolamide when administered as a suspension in which case there must be
some depot effect.1216 After topical application of
methazolamide or ethoxzolamide, in solution, high
corneal and aqueous humor concentrations are
reached early but decline quickly.117 Giving ethoxzolamide as a 2% suspension, where modest ocular hypotensive activity is seen, increases the corneal/aqueous
humor ratio from 1.1 to 4.12 Trifluormethazolamide
also penetrates the cornea and reduces IOP.1 This
fluorination of methazolamide increased the cornea/
aqueous humor ratio of drug from 0.67 to 1.2117 as a
result of increased ionization and corneal sequestration.
Each of the compounds studied here result in cornea/aqueous humor ratios of 2.5-5 when the drug is
administered in the ionized form5 (and unpublished
data). Other ocular hypotensive agents have similar
ratios, epinephrine, 4,18 and timolol, 1019 notably,
these are also bases.
The theoretical effect of increasing ionization of
CA inhibitors would be to greatly decrease in vivo permeability. However, previous studies have shown that
acidic ionized compounds adequately penetrate the
cornea at about 1/4 the rate of the unionized species.20 We now show that in the case of MK-927 corneal permeability is independent of ionization of the
base (Table I).2 The same pattern is seen for
AHR16329 whereas for L-662,583 it appears that the
unionized species is somewhat more permeable (Table 1).
It is emphasized that formation of the anionic species does not in itself confer activity in all structural
series. We have shown clearly that methazolamide and
its more active analogs do not lower pressure when
administered as their anionic solutions at pH 8-9. U 6
The same is true of acetazolamide and ethoxzolamide.1 What appears necessary is the anionic species
(at high pH) in an ampholyte setting, in the current
case an unionized base. Conversely, at low pH levels
corneal sequestration and IOP lowering may be dictated also by ionization of the base while the acid appears neutral.
The effects of ionization on IOP lowering suggests
for several reasons that it affects the ability of the
drugs to form a corneal depot, rather than affecting
permeability. First, in vivo corneal permeability is independent of ionization (Table 1). Second, the similarity
in IOP reduction at the different pH levels before 30
min (Fig. 5) is probably related to noncorneal absorption of drug to the ciliary processes. We have shown
that MK-927 appears in the anterior uvea before detection in aqueous humor and is associated with a significant decrease in IOP.13 The differences in IOP response after 30 min reflects the rapid decline of drug
from the eye because of rapid corneal clearance. From
Table 2 it can be seen that decreasing ionization results in significantly lower corneal, aqueous humor,
and ciliary process drug levels at all times; the data
show that the cornea/aqueous humor ratio decreases
twofold to threefold with decreasing ionization. The
corneal depot effect results in a higher concentration
of drug in the ciliary processes with the more ionized
drug (pH 4.7; 8.9) compared to the unionized drug
(pH 7).
The significance of these findings may relate to
clinical practice. Significant problems encountered
when placing many drugs on the cornea involve discomfort, irritability, tearing, and redness. Therefore, a
goal in developing topical drugs relates to corneal
comfort and the attempt to apply drugs as close to
physiologic pH as possible. Most drugs are bases and
given in acid solution; epinephrine, dipivefrine, pilocarpine, and MK-927 sting at pH < 6. Because the
topical CA inhibitors are relatively inactive at neutral
pH and are maximal at pH 9, usage at this pH level
may eliminate many of the corneal discomfort problems without sacrificing pressure lowering activity.
In conclusion, ampholyte carbonic anhydrase inhibitors when applied topically to the eye, significantly
reduce IOP because of a series of physicochemical
properties that result in good corneal and noncorneal
permeability and the ability to sequester in the cornea.
Decreasing the percentage of ionization of the applied
drug does not alter in vivo corneal or noncorneal permeability or early IOP reduction (< 30 min). Decreasing ionization attenuates the ability of these drugs to
sequester in the cornea resulting in lower drug levels
in the ciliary process after 30 min. Thus neutral pH
greatly limits their effectiveness in lowering IOP. This
is analogous to methazolamide, which has good corneal permeability and solubility but does not significantly reduce IOP after topical application because of
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2587
Drug Ionization Alters CAI Pressure Lowering
lack of corneal sequestration. The mechanisms or anatomic location of this corneal depot remain to be investigated.
9.
Key Words
ampholytes, intraocular pressure, ionization, AHR-16329,
L662.583, MK-927
10.
Acknowledgment
The authors thank David Godman for animal experiments
and Joyce Hearn for editing and managerial assistance.
Drug analyses were carried out by Dr. Curtis Conroy.
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