Synthesis and properties of two new membrane-impermeant high-molecular-weight carbonic anhydrase inhibitors

Article abstract of DOI:10.1006/bioo.1996.0024

The synthesis and inhibitory properties and stability of two nontoxic high-molecular-weight carbonic anhydrase inhibitors (F 3500 and POBUMS) are reported. F 3500 was prepared by the covalent linkage of aminobenzolamide, a potent carbonic anhydrase (CA) inhibitor, to polyoxyethylene bisacetic acid (MW 3350). Linkage of the same inhibitor to polybutadiene maleic acid copolymer (MW 20,000) gave POBUMS. They contained on a mole percentage basis 0.11-0.28% unreacted aminobenzolamide as contaminant. F 3500 and POBUMS were approximately equipotent as carbonic anhydrase inhibitors on a weight basis with K(i) at 37°C for CA II and CA IV of approximately 0.5 and 10 μg/ml, respectively. They showed a maximum release of 28% aminobenzolamide when heated at 70°C for 3 days at pH 10.5 and were completely stable toward enzymatic hydrolysis (protease and peptidase at 25°C). They were not membrane permeable as judged by their inability to bind to intracellular CA II in intact red cells, nor were they actively uptaken in rat kidney slices. Rats injected with F 3500 (200 mg/kg) showed no toxicity and excreted 93% of the polymer unchanged in the urine in 3 h. The two polymers should prove useful for in vivo and in vitro studies of selective CA IV inhibition in membranes.

Full text of DOI:10.1006/bioo.1996.0024

BIOORGANIC CHEMISTRY 24, 262–272 (1996)
ARTICLE NO. 0024
Synthesis and Properties of Two New Membrane-Impermeant
High-Molecular-Weight Carbonic Anhydrase Inhibitors
1
C
URTIS W. CONROY, GEORGE C. WYNNS
,
AND
THOMAS H. MAREN
Department of Pharmacology, University of Florida College of Medicine, P.O. Box 100267,
Gainesville, Florida 32610–0267
Received March 3, 1996
The synthesis and inhibitory properties and stability of two nontoxic high-molecular-weight
carbonic anhydrase inhibitors (F 3500 and POBUMS) are reported. F 3500 was prepared by
the covalent linkage of aminobenzolamide, a potent carbonic anhydrase (CA) inhibitor, to
polyoxyethylene bisacetic acid (MW 3350). Linkage of the same inhibitor to polybutadiene
maleic acid copolymer (MW 20,000) gave POBUMS. They contained on a mole percentage
basis 0.11–0.28% unreacted aminobenzolamide as contaminant. F 3500 and POBUMS were
approximately equipotent as carbonic anhydrase inhibitors on a weight basis with Ki at 37ЊC
for CA II and CA IV of approximately 0.5 and 10 Ȑg/ml, respectively. They showed a
maximum release of 28% aminobenzolamide when heated at 70ЊC for 3 days at pH 10.5 and
were completely stable toward enzymatic hydrolysis (protease and peptidase at 25ЊC). They
were not membrane permeable as judged by their inability to bind to intracellular CA II in
intact red cells, nor were they actively uptaken in rat kidney slices. Rats injected with F 3500
(200 mg/kg) showed no toxicity and excreted 93% of the polymer unchanged in the urine in
3 h. The two polymers should prove useful for in vivo and in vitro studies of selective CA
IV inhibition in membranes.
1996 Academic Press, Inc.
INTRODUCTION
Secretory tissues, e.g., ciliary process (eye), choroid plexus (brain), parietal cell
(stomach), and kidney tubule (1, 2), contain two forms of the zinc metalloenzyme
carbonic anhydrase (CA II and IV).2 Both forms effect the facile interconversion
of CO2 and HCOϪ3 at physiological pH, and it is the latter ion, bicarbonate, that
serves as counterion to Naϩ in fluid production. In addition to this role, CA isoen-
zymes are also responsible for proton secretion (kidney, stomach) and in bicarbonate
reabsorption (kidney). The cytosolic form CA II is slightly more efficient in the
Ϫ
catalysis of CO2 hydration, CO2 ϩ H2O S HCO3 ϩ Hϩ, compared with the
membrane-bound form CA IV (3) and, because of intracellular localization, is
present in much higher concentration. Inhibition with potent and permeant sulfon-
amide carbonic anhydrase inhibitors (CAIs) invariably leads to an approximate
40% reduction in fluid production (4) due to reduced bicarbonate formation from
1 To whom correspondence should be addressed.
2 Abbreviations used: CA, carbonic anhydrase; CAI, carbonic anhydrase inhibitor; STZ, succinyl
thiadiazolesulfonamide; EDC, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide; HOBT, hydroxybenzo-
triazole; DCC, dicyclohexylcarbodiimide; PAH, p-aminohippurate; PEG, polyethylene glycol.
262
0045-2068/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

TWO HIGH-MW CARBONIC ANHYDRASE INHIBITORS
263
CO2. Their effect on the kidney is to interfere with bicarbonate reabsorption in
the proximal segment of the tubule, resulting in a bicarbonate-rich (100 m ) urine.
M
This process is dependent both on brush border CA IV (bicarbonate reabsorption)
and on cytosolic CA II as a source of Hϩ (proximal acidification) (5).
All high-MW CAIs produced to date have consisted of heterocyclic sulfonamides
[prontosil, succinyl thiadiazolesulfonamide (STZ), and aminobenzolamide (p-
aminobenzenesulfonamido-1,3,4-thiadiazole-5-sulfonamide)] covalently bonded to
derivatized dextrans of MW 5000–1,000,000 (6–8). They have been employed in
perfusion studies in the isolated kidney tubule in the attempt to assess the relative
roles of CA II and IV in bicarbonate reabsorption. The tendency to induce anaphy-
laxis in the living animal has restricted their use, however. Karlmark et al. (6)
studied the effect of aminobenzolamide bonded to bromcyanide-activated dextran
(MW 17,000), on acidification in the isolated tubule, but the failure to appreciate
the effect of increasing Ki with temperature (9) leading to reduced potency likely
led to use of too little compound.
In two subsequent studies (7, 10) in which STZ was bonded to aminoethyl-derived
dextrans, MW 5000 and 100,000, a high degree of sulfonamide linkage was achieved,
up to eight substitutions per molecule, despite the fact that the stoichiometry of
drug binding to enzyme is 1 : 1. A later study using a series of weakly inhibitory
prontosil dextrans (8), (MW 5000–1,000,000) yielded variable results in the washout
of H14CO3Ϫ from the isolated rabbit hindlimb. The Ki values for the various inhibi-
tors, however, were determined for inhibition of cytosolic CA II despite the fact
that CA IV is the isoenzyme being inhibited by these compounds. Since inhibition
by sulfonamides is weaker for CA IV by 7 to 33-fold compared with CA II (3), it
is likely that too little compound was used. Additionally, it was determined (8) that
the STZ dextrans of Tinker et al. (7) liberated appreciable quantities of free drug
in solution.
We now report synthesis of two new high-MW CAIs based on nontoxic polymers,
polybutadiene maleic acid (MW 20,000) and polyethylene glycol bisacetic acid (MW
3350), covalently bonded to aminobenzolamide 4 (Fig. 1), using the carbodiimide
linking reaction. The first polymer has not previously been used for drug linkage.
The second has been extensively employed for this purpose (11) and yields products
that are water soluble, nontoxic, and generally well suited for in vivo studies and
in this case of bicarbonate reabsorption in the kidney and now for the selective
inhibition of CA IV in vivo.
MATERIALS AND METHODS
PEG dicarboxylate (polyoxyethylene glycol bisacetic acid, (Sigma, St. Louis, MO)
and polybutadiene maleic acid copolymer (Polysciences, Warrington, PA) were
used without further purification. CA II was obtained from hemolyzed dog red
cells and CA IV from bovine kidney microsomes by ultracentrifugation (12). 1-
Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide
(DCC), hydroxybenzotriazole (HOBT), acetazolamide, trichloroacetic acid, ammo-
nium sulfamate, sodium nitrite, bovine liver acetone powder, and bovine pancreas

264
CONROY, WYNNS, AND MAREN
F
IG. 1. Synthesis of aminobenzolamide and structures of POBUMS and F 3500.
protease were obtained from Sigma and used without further purification. N-Acetyl-
sulfanilyl chloride (Aldrich) and N-(1-naphthyl)ethylenediamine dihydrochloride
(Kodak) were used without further purification.
Analytical Methods
The inhibitory potency (Ki) of POBUMS and F 3500 against CA II and CA IV
was determined at 0 and 23ЊC by a changing pH-indicator method (13) using barbital
buffer and bromthymol blue as indicator. Ki was related to the equilibrium constant

TWO HIGH-MW CARBONIC ANHYDRASE INHIBITORS
265
(Keq) by the relationship Keq ϭ 1/Ki. Arrhenius plots of ln Keq vs 1/T(ЊK) were
then used to obtain estimates of Ki at physiological temperature (37ЊC). Degrees
of fractional inhibition of CA isoenzymes (i) were calculated from the relationship
If
i ϭ
,
If ϩ Ki
where If is the unbound drug concentration in tissue or fluids.
The presence of unreacted aminobenzolamide in preparations of F 3500 and
POBUMS was determined by a modification of the Bratton–Marshall procedure
(14) in which the pH of the trichloroacetic acid/sodium citrate solution is raised to
pH 4 by addition of 6
N
NaOH. Results were expressed as mole percentages (mole
free arylamine/mole polymer) ϫ 100.
The degree of drug substitution on polymers was determined by hydrolysis of F
3500 and POBUMS in 1
N
HCl at 100ЊC for 30 min prior to assay for free arylamine
by the Bratton–Marshall method. This number was also confirmed based on the
uv absorption at 270–290 nm.
The stability of drug polymers toward hydrolysis was determined both enzymati-
cally (in vivo and in vitro) and nonenzymatically. In the latter procedure 10 m
M
solutions were heated at 70ЊC in pH 3.5, 7, and 10.5 phosphate buffer (50 m
M
)
for 72 h. These solutions were monitored for free aminobenzolamide using the
Bratton–Marshall method. The stability of POBUMS and F 3500 toward enzymatic
hydrolysis was tested in vitro by incubation of 5 mg polymer in 1 ml pH 7.5 phosphate
buffer at 25ЊC for 1 h with 50 enzyme units of bovine pancrease protease or with
50 mg bovine acetone powder followed by analysis for free arylamine. In the in
vivo procedure, rats were administered F 3500 (200 mg/kg), and the urine was
collected for 3 h, pooled, and tested for arylamine. Washed red cells collected at
3 h were tested in an analogous manner.
The membrane permeability of POBUMS and F 3500 was determined by incuba-
tion of drug polymers (150 Ȑ ) with intact rabbit red cells for periods up to 3 h
M
according to Maren et al. (15). Red cells were washed three times against saline,
lysed, and analyzed for drug. The active uptake of both polymers in kidney slices
was studied by the method of Cross and Taggert (16) at 25ЊC in Ringer’s solution
containing 10 m acetate in an O2 atmosphere and compared with that of benzolam-
M
ide, aminobenzolamide, and PAH.
Synthesis of Aminobenzolamide
Aminobenzolamide was synthesized by a variation of the method of Vaughan et
al. (17), as shown in Fig. 1. Acetazolamide 1 (2-acetylamino-1,3,4-thiadiazole-5-
sulfonamide (111 g 0.5 mol) was refluxed for approximately 1 h in 1 liter 0.1 N
HCl. Complete solution was effected in 45 min followed by an additional 15 min
at reflux. The solution was cooled and adjusted to pH 5 by the careful addition of
sodium carbonate. The precipitate that formed after standing overnight at 5ЊC was
washed with 100 ml water. Recrystallization from boiling water gave 90 g (80%
yield) of 2-amino-1,3,4-thiadiazole-5-sulfonamide 2.
Fifty-four grams (0.3 mol) of 2 was dissolved in 155 ml 2.5
N
NaOH and cooled

266
CONROY, WYNNS, AND MAREN
to 10ЊC in an ice bath. N-Acetylsulfanilyl chloride (14 g, 0.06 mol) and 30 ml 5
N
NaOH were added simultaneously as the temperature was maintained at 10ЊC.
These additions were repeated four more times and the solution was refrigerated
overnight. The solution was then adjusted to pH 4 with 6
The precipitate, p-acetylaminobenzenesulfonamido-1,3,4-thiadiazole-5-sulfon-
amide 3, was not purified but deacetylated by dissolving in 300 ml of 6 HCl and
N
HCl and chilled in ice.
N
refluxing for 45 min. The cooled solution was taken to pH 4 with 1 N NaOH and
the precipitate collected. The crude aminobenzolamide was recrystallized from 20%
EtOH/H2O, giving 110 g of pure 4 mp 247–248ЊC (dec.) in 65% yield.
Synthesis of POBUMS
Polybutadiene maleic acid (2.5 g, 40% solids in water, representing 1 g polymer
or 0.05 mmol) was dissolved in 30 ml 50% acetone/water. The pH was raised to
4.9 by addition of 1
N
NaOH. A 25-fold molar excess of aminobenzolamide (1.25
mmol, 418 mg) was dissolved in boiling ethanol/water and added to the stirred
solution above. The pH was readjusted to 4.9. A 10-fold molar excess of EDC (0.5
mmol, 96 mg) was dissolved in water and added slowly overnight while the pH was
maintained at 4.9. Additional EDC (96 mg) was added on Days 2 and 3. After 5
days of reaction the crude reaction product was placed in benzolated dialysis tubing
(MW cutoff, 3500) and dialyzed for 1 week, first against dilute dibasic phosphate
(to facilitate removal of unreacted drug), then against distilled water. The product
was finally dried in air to an amorphous yellow waxy solid.
Synthesis of F 3500
The synthesis of F 3500 was the same as used by Johansson for the synthesis of
(Et3NEtO)2 Ph-NH-azelate-poly(EtO), a polymer used in the affinity partitioning
of synaptic membrane domains (18). Polyoxyethylene bisacetic acid (1 g 0.3 mmol),
HOBT (0.08 g, 0.6 mmol), DCC (0.14 g, 0.7 mmol), and aminobenzolamide (0.20
g, 0.6 mmol) were dissolved in 10 ml pyridine. The flask was sealed and heated in
a water bath at 45ЊC for 2 days during which time a white precipitate presumed to
be the corresponding dicyclohexyl urea formed. The solution was evaporated in
vacuo to remove all traces of pyridine and the resultant waxy solid was extracted
with three 10-ml volumes of CH2Cl2. The CH2Cl2 was evaporated and the solid
taken up in 5 ml H2O, adjusted to pH 8.5 with 0.1
to remove all insoluble matter. This was then applied to a 50 ϫ 600-mm Sephadex
G-50 column and eluted with 25 m ammonium carbonate. Fractions corresponding
to OD Ͼ 0.2 at 280 n were pooled and dried in vacuo to yield F 3500 in 70%
yield as a white waxy solid.
N
NaOH, and carefully filtered
M
M
RESULTS
Table 1 shows the inhibitory potency of aminobenzolamide, POBUMS, and F
3500 against CA II and CA IV isoenzymes as a function of temperature. Inhibition
data obtained at 0ЊC for these compounds were used to construct standard curves

TWO HIGH-MW CARBONIC ANHYDRASE INHIBITORS
267
TABLE 1
Inhibitory Potency of Aminobenzolamide, F 3500, and POBUMS against
Carbonic Anhydrase Isoenzymes as a Function of Temperature
Ki[Ȑg/ml (ȐM)]
0ЊC
23ЊC
37ЊC
Aminobenzolamide
(MW 334)
CA II
CA IV
CA II
CA IV
CA II
CA IV
0.0005
(0.0015)
0.008
(0.023)
0.10
(0.029)
2.30
(0.69)
0.10
0.002
(0.006)
0.027
(0.082)
0.28
(0.083)
7.36
(2.20)
0.26
0.004
(0.012)
0.047
(0.140)
0.48
(0.144)
13.40
(4.0)
F 3500
(MW 3350)
POBUMS
(MW 20,000)
0.44
(0.022)
9.5
(0.005)
2.5
(0.013)
5.8
(0.125)
(0.290)
(0.475)
for use in assays of drug concentration in tissues, fluids, and stock solutions. In
combination with data obtained at 23ЊC, Ki values at 37ЊC were extrapolated using
the Arrhenius relationship to provide estimates for the concentration range that
should yield a physiological response for inhibition of membrane-bound CA IV.
Previous work suggests this occurs when i Ն 0.98 from which it may be concluded
If ϭ Ki ϫ 50 (3). From the results of Table 1 at 37ЊC it would appear that responses
attributable to the inhibition of CA IV in membranes may be anticipated at solution
concentrations of approximately 0.48 mg/ml (0.024 m
M
) for POBUMS and 0.67
mg/ml (0.2 m ) for F 3500, both concentrations representing Ki ϫ 50. It should
M
be noted that the unreacted polymers do not inhibit the CA isozymes at 1 mg/ml,
the maximum concentration employed.
Aminobenzolamide was less inhibitory toward CA IV compared with CA II by
some 15-fold as judged by Ki values at 0ЊC. This is well within the range of an average
of 17-fold observed for a diverse range of heterocyclic sulfonamides (3), and this ap-
pearsto beanintrinsic property of the CA IV isoenzyme. Thedegree ofdrugsubstitu-
tion on F 3500 and POBUMS determined by uv spectra and hydrolysis and the typical
extent of contamination by free arylamine in different batches are shown in Table 2.
The contamination of POBUMS and F 3500 with free or unbound drug is given as the
mole ratio of free arylamine as determined by the Bratton–Marshall method to the
moles of polymer, the result expressed as a percentage. The last column of Table 2
shows the concentration of free drug in fully inhibitory solutions (perfusates) as given
above. Based on the Ki at 37ЊC, the 0.22 m contaminant of aminobenzolamide in F
M
3500 gives fractional inhibition of CA II corresponding to i ϭ 0.948, and of CA IV,
i ϭ 0.611. This low level of inhibition would not elicit any physiological effect (4).
Thus, the component of unbound diffusible drug in F 3500 and POBUMS solutions

268
CONROY, WYNNS, AND MAREN
TABLE 2
Degree of Drug Binding in F 3500 and POBUMS and Free Drug Content of
Polymers and Perfusate Solutions
Mol unreacted
Mol aminobenzolamide
bounda/
aminobenzolamide/
mol polymer
(%)
Perfusion solutions
of polymerb
Free aminobenzolamide
[column (2) ϫ column (3)]
mol polymer
(Ȑ
M
)
(ȐM
)
(1)
(2)
(3)
(4)
F 3500
0.14
2.6
0.11 Ϯ 0.03
0.28 Ϯ 0.05
200
24
0.22
0.07
(n ϭ 5)c
POBUMS
(n ϭ 3)
a Determined from uv absorption and from hydrolysis and analysis for free arylamine. See Materials
and Methods.
b Ki determined at 37ЊC for inhibition of CA IV. Solution concentration represents Ki ϫ 50.
c n ϭ number of preparations.
is not high enough to inhibit either isoenzyme, even if 5-fold greater concentrations
(250 ϫ Ki) are employed.
For CA II inhibition, attachment of aminobenzolamide to polyoxyethylene results
in a 200-fold weakening of Ki as judged on a microgram-per-milliliter basis. As
Table 2 shows, approximately 1 in 10 molecules of F 3500 has covalently bonded
drug. This combined with a 10-fold difference in molecular weight suggests F 3500
should be 100-fold weaker on a weight basis than aminobenzolamide. Attachment of
aminobenzolamide to polymer, therefore, results in an intrinsic loss of sulfonamide
potency of about 2-fold. For POBUMS there are approximately 2.6 substitutions
per molecule of polybutadiene maleic acid. Only one of these sulfonamides may
reasonably be assumed to bind to carbonic anhydrase, however, given the large
size of inhibitor and enzyme, a fact largely overlooked in studies cited above where
as many 8 inhibitor molecules were bound per polymer molecule. POBUMS should,
therefore, be 60-fold weaker than aminobenzolamide (compared with the 200-fold
observed) as given by the MW ratios (20,000/334), suggesting a 3-fold weakening
of sulfonamide Ki. This may be compared with a 4-fold loss in prontosil potency
when bound to MW 130,000 dextran (8).
The relative inhibitory potencies of F 3500 and POBUMS toward CA II were
nearly the same (0.10 mg/ml) at 0ЊC. Since the molecular weight of POBUMS is
six times that of F 3500 but the degree of drug substitution seven times as great
(0.14 to 1 per molecule), the two inhibitors are approximately equivalent on a
weight basis. The degree of inhibitor substitution on both polymers was low, approxi-
mately 7% of free carboxy groups in F 3500 and 2.2% in POBUMS. The polymer
preparations were, nevertheless, sufficiently inhibitory toward CA IV that complete
inhibition of this isoenzyme could be achieved without concern for insolubility
or solution viscosity. This finding, however, illustrates the unreactive nature of
arylamines compared with aliphatic amines toward nucleophilic substitution in
the carbodiimide linking reaction and the necessity to employ conditions of high
temperature or excess of reagent in their synthesis.

TWO HIGH-MW CARBONIC ANHYDRASE INHIBITORS
269
TABLE 3
Penetrance of Aminobenzolamide, F 3500, and POBUMS into Rabbit Red Cellsa
Concentration (ȐM)
Time (h)
in red cells
% Saturation
Aminobenzolamide
F 3500
0.5
1.0
1.0
3.0
1.0
3.0
24
42
1.1
3.5
0.08
0.35
48
85
2.2
7.0
0.16
0.70
POBUMS
a Incubation procedure and drug analysis as given under Materials and Methods and
Ref. (15). Full saturation corresponds to approximately 50 Ȑ bound inhibitor.
M
Table 3 shows the red cell penetrance of F 3500 and POBUMS after incubation
in 150 Ȑ polymer solutions with rabbit red cells for 1 and 3 h. The degree of drug
M
binding to red cell CA II at 3 hr for F 3500, which appeared to be the more permeant
inhibitor, amounted to only 4% of the total compared with near-full saturation
when unbound aminobenzolamide was used. The possibility must be considered
that the 7 Ȑ measured did not represent the polymer but rather the free component
M
of unbound aminobenzolamide, which was present as a contaminant. If this were
the case, then this concentration expressed as free drug may be given by dividing
by the Ki ratio at 0ЊC against CA II (19.3-fold, Table 1) or 0.36 ȐM. Since the
original bathing solution of F 3500 was 150 Ȑ , this concentration represents 0.24%
M
of the total on a mole percentage basis, similar to the 0.11% cited in Table 2. Thus
all drug measured in washed red cells likely comes from free drug contaminant,
suggesting that both F 3500 and POBUMS have no intrinsic membrane permeability.
Permeant inhibitors such as methazolamide quickly saturate intracellular CA II
under low drug gradients and generate bound drug concentrations in washed rabbit
red cells of approximately 50 Ȑ . More highly ionized compounds such as acet-
M
azolamide require higher gradients and longer times up to 1 h. It should, however,
be noted that even completely ionized compounds such as benzolamide fully saturate
red cell CA at 1 h using similar drug gradients (200 Ȑ ) (15). Thus, low-MW CAIs
M
that are fully ionized are not substitutes for polymer-linked drugs in studies of
selective CA IV inhibition. They do traverse biological membranes either passively
or by active uptake and elicit physiological effects identical to those of permeant in-
hibitors.
The presence of charged groups (unreacted COOH) on F 3500 and POBUMS
offers the possibility of active uptake into cells. The uptake of F 3500, POBUMS,
aminobenzolamide, benzolamide, and PAH (as control) was therefore studied by
the classic method of Cross and Taggert in rat kidney slices (16). The experiments
were run in an O2 atmosphere in the presence of 10 m acetate to ensure maximal
M
uptake. Under these conditions Cross and Taggert found a slice-to-medium (S/M)
ratio for PAH of 13.5. After incubation for 1 h the following S/M ratios were
obtained: F 3500 (0.6), POBUMS (0.8), benzolamide (6.1), aminobenzolamide (2.4),
and PAH (10.5). The values obtained for benzolamide and aminobenzolamide are

270
CONROY, WYNNS, AND MAREN
TABLE 4
Susceptibility to Hydrolysis of
F 3500 and POBUMSa
% Breakdown
pH
F 3500
POBUMS
3.5
7.0
10.5
16.2
8.3
28.1
1.10
0.25
2.3
a (Mol hydrolyzed aminobenzolamide/mol bound
aminobenzolamide) ϫ 100. Conditions of hydrolysis:
50 m
M phosphate buffer, 70ЊC, 72 h.
well within the range observed for a wide variety of sulfonamides (19), whereas
those for the two polymers are indicative of no active uptake.
The susceptibility of the peptide linkage in F 3500 and POBUMS to spontaneous
and enzymatic hydrolysis was studied in several ways. Table 4 shows the release
of free aminobenzolamide from the two polymers when incubated at acid, neutral,
and basic pH at 70ЊC for 72 h. POBUMS was the more resistant polymer, showing
a maximum release of only 2% bound drug. By contrast the hydrolysis of F 3500
was at least 10 times as large, although the predicted release of drug from neutral
solution and physiological temperature on short time scales would be miniscule.
Thus both polymers must be judged as completely stable toward spontaneous
hydrolysis. The STZ dextrans, by comparison, were found to generate 10 Ȑ
M
free
STZ after 2 h at 37ЊC (8). Given the inhibitory potency of free STZ, this amount
is sufficient to give a response characteristic of a permeant inhibitor. Based on this
result, it seems likely that there is a fundamental difference in the stability of peptide-
linked drugs obtained from amino-derived polymers (e.g., aminoethyl-dextrans)
compared with those obtained from carboxy-derived polymers (polyoxyethylene
bisacetic acid).
The susceptibility of F 3500 and POBUMS to enzymatic hydrolysis was studied
in vitro at 25ЊC (pH 7.5) by incubation for 1 h of 5 mg of either polymer dissolved
in 1 ml pH 7.5 phosphate buffer with 50 units of bovine pancreas protease or 50
mg bovine liver acetone powder as a source of proteolytic enzymes. A 100-Ȑl aliquot
corresponding to 0.5 mg F 3500 (0.15 mmol, equivalent to 0.021 Ȑmol bound
aminobenzolamide) or 0.5 mg POBUMS (0.025 or 0.065 Ȑmol bound aminoben-
zolamide) was taken for analysis using the Bratton–Marshall method. In each
solution free arylamine concentration was below the limits of detection of the
method, and this corresponded to an absorbance Ͻ 0.005 or arylamine concentration
Ͻ 0.0002 Ȑmol. Thus, hydrolysis of the polymers should have been detected if only
0.95% of bound drug in F 3500 or 0.31% in POBUMS had hydrolyzed, and this
was not observed.
The urinary excretion of F 3500 was measured after intravenous administration
of 50 mg (200 mg/kg) or 15,000 nmol in the rat along with urinalysis for free

TWO HIGH-MW CARBONIC ANHYDRASE INHIBITORS
271
arylamine as well as red cell drug content. It should be noted this dose contained
19 nmol unbound aminobenzolamide as contaminant. In 3 h 46.6 mg of 50 mg
administered (93%) was recovered unchanged in the urine. Analysis for free arylam-
ine showed 0.008 Ȑmol/ml or 56 nmol unbound drug. This is the same order as in
the 19 nmol administered as contaminant. Analysis of washed red cells at 3 h
showed 1.35 Ȑ
M
F 3500 present or 0.07 Ȑ
M
if measured as aminobenzolamide in
the hydration assay. Assuming 8 ml packed red cells in a 240-g rat, this translates
to 0.56 nmol free aminobenzolamide. Thus no hydrolysis of F 3500 occurred in vivo
since essentially no drug was bound in red cells and little excess free drug appeared
in the urine. Both F 3500 and POBUMS, when injected at 200 mg/kg, showed no
evidence of toxicity over a 3-h interval. POBUMS did appear to interfere with the
ability of the blood to clot, possibly due to the chelation of Ca2 by free carboxyl
ϩ
groups on POBUMS.
DISCUSSION
F 3500 and POBUMS represent two new potent high-MW CAIs suitable for use
in in vivo and in vitro studies of membrane-bound CA IV activity. Compared with
similar previous compounds based on dextran, the present ones show a relatively
low degree of drug substitution yet retain high potency against CA isoenzymes.
The results of their chemical stability toward both enzymatic and nonenzymatic
hydrolysis suggest that these compounds are superior to those based on dextran
(with the exception of the prontosil dextrans) toward release of free drug. The
binding of inhibitor molecules to polymers in numbers in large excess of that given
by the 1:1 stoichiometry of the enzyme–inhibitor complex clearly may be related
to the release of free drug from these compounds.
The selective inhibition of membrane-bound CA with polymers of the type re-
ported here concerns the relationship between the inhibitory potency of the com-
pound at 37ЊC against CA IV and the minimum concentration of drug that elicits
a physiological effect. Previously, certain high-MW CAIs were used at concentra-
tions of 1500 (8) and 7400 (7) ϫ Ki, far in excess of the amount needed for
CA IV inhibition. In a recent report Matsui (20) found a maximal change in the
transmembrane potential of the isolated ciliary epithelial bilayer (150% increase)
when POBUMS was perfused at 900 mg/ml (95 ϫ Ki, i ϭ 0.989) and this result
was attributed to the selective inhibition of the membrane-bound enzyme. This is
in good agreement with previous work that shows that i approaching 0.98 should
mark the threshold for observing the full effect attributable to CA IV inhibition (3).
A second report (21) has now shown that intravenous administration of F 3500
(100 mg/kg) to rats results in a partial inhibition of HCOϪ3 reabsorption in the
kidney, resulting in a urine that contained 40 m
reabsorption, which occurs when CA II and IV are inhibited, results from adminis-
tration of 3 mg/kg aminobenzolamide and gives a value of 100 m
HCOϪ3 in the
M
HCOϪ3 . Full inhibition of tubular
M
urine. Thus it appears that infusion of F 3500 achieved inhibition of the brush border
enzyme (CA IV) without inhibition of the cytosolic enzyme (CA II), accounting for
the partial bicarbonate effect observed. This result contrasts with previous studies

272
CONROY, WYNNS, AND MAREN
based on certain inhibitors where either there was no effect on bicarbonate reabsorp-
tion (6) or an effect was obtained that was indistinguishable from that of permeant
inhibitors (7).
F 3500 was also used in ventricular cisternal perfusion in the rat to find if inhibition
at the membrane of the choroid plexus facing the cerebrospinal fluid was responsible
for the secretory process. Results, surprisingly, were negative, leading us to conclude
either that CA IV was not involved in the secretion or that F 3500 did not have
access to the membrane-bound enzyme. This new series of membrane-impermeant
CAIs, therefore, may represent potent, chemically stable, biocompatible inhibitors
superior to those based on dextran for studies of CA IV function in membranes.
ACKNOWLEDGMENTS
This research was supported by National Institutes of Health Grant EY-O2227, Merck Sharp &
Dohme, and the University of Florida Division of Sponsored Research.
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