Design, in Vitro Stability, and Ocular Hypotensive Activity of t-Butalone Chemical Delivery Systems

Article abstract of DOI:10.1002/jps.1055

The objective of this work was to synthesize two bioreversible diacyl derivatives of t-butalone (chemical delivery systems), determine their chemical and in vitro stability, and investigate their potential use as topical antiglaucoma agents. The stability of these compounds was determined in isotonic phosphate buffers (pH range 5-8) and in selected biological media, including human whole blood, rabbit and rat blood, and the anterior segment tissues of rabbit. The ocular hypotensive activity of these compounds in unrestrained, normotensive albino rabbits was determined with a pneumatonometer. The two compounds were stable at lower pH. The stability decreased as the pH increased, suggesting their lability to base-catalyzed hydrolysis. These compounds exhibited significant differences in the hydrolytic rates in the whole blood among species examined (rat > rabbit > human). The observed rates of disappearance in different ocular tissues were indicative of relative enzyme activity in these media (iris-ciliary body > cornea > aqueous humor). The two compounds exhibited a significant ocular hypotensive activity (P < 0.01) at 2 percent dose level. The peak activity was found between 2 and 4 h, and the activity was maintained for 4.5 to 7 h. The dipivalyl derivative of t-butalone exhibited more pronounced decrease in intraocular pressure than that of diisovaleryl derivative. The present study suggests the possible use of diacyl derivatives of t-butalone as ocular hypotensive agents.

Full text of DOI:10.1002/jps.1055

Design, In Vitro Stability, and Ocular Hypotensive Activity of
t-Butalone Chemical Delivery Systems
INDRA K. REDDY,1* SIVA R. VAITHIYALINGAM,1 MANSOOR A. KHAN,1 NICHOLAS S. BODOR2
1Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo,
Texas 79106
2Center for Drug Discovery, J. Hillis Miller Health Center, College of Pharmacy, University of Florida, Gainesville,
Florida 32610
Received 21 August 2000; accepted 16 January 2001
ABSTRACT: The objective of this work was to synthesize two bioreversible diacyl derivatives of t-
butalone (chemical delivery systems), determine their chemical and in vitro stability, and
investigate their potential use as topical antiglaucoma agents. The stability of these compounds
was determined in isotonic phosphate buffers (pH range 5±8) and in selected biological media,
including human whole blood, rabbit and rat blood, and the anterior segment tissues of rabbit. The
ocular hypotensive activity of these compounds in unrestrained, normotensive albino rabbits was
determined with a pneumatonometer. The two compounds were stable at lower pH. The stability
decreased as the pH increased, suggesting their lability to base-catalyzed hydrolysis. These
compounds exhibited signi®cant differences in the hydrolytic rates in the whole blood among
species examined (rat > rabbit > human). The observed rates of disappearance in different ocular
tissues were indicative of relative enzyme activity in these media (iris-ciliary body > cornea >
aqueous humor). The two compounds exhibited a signi®cant ocular hypotensive activity (P < 0.01)
at 2% dose level. The peak activity was found between 2 and 4 h, and the activity was maintained
for 4.5 to 7 h. The dipivalyl derivative of t-butalone exhibited more pronounced decrease in
intraocular pressure than that of diisovaleryl derivative. The present study suggests the possible
use of diacyl derivatives of t-butalone as ocular hypotensive agents. ß 2001 Wiley-Liss, Inc. and the
American Pharmaceutical Association J Pharm Sci 90:1026±1033, 2001
Keywords: chemical delivery systems; stability; t-butalone esters; antiglaucoma agents
INTRODUCTION
isms of the eye, such as instilled volume drainage
and tear turnover, the biological (corneal) barrier,
and the inherent physicochemical insuf®ciencies
of the drug molecule itself.1,2
Design and development of topical ocular delivery
systems is one of the most interesting and
challenging areas of current research. Medica-
tions designed for ophthalmic use are currently
undergoing a process of optimization because of
inherent problems associated with absorption
ef®ciencies of topically applied drugs. The poor
ocular availability of topical ophthalmic agents is
attributed to the physiological protective mechan-
In recent years, novel metabolism-based drug
delivery concepts and strategies were proposed to
design and develop safe, speci®c, and effective
ophthalmic agents.3±9 One of the popular con-
cepts is the site-speci®c chemical delivery system
(CDS) approach that is based on the predictable
multistep metabolic activation of bioreversible,
inactive agents at the site of action.3,5,10 The che-
mical delivery system concept was successfully
applied to design and develop site-speci®c, short-
acting mydriatic9,11±13 and anti-glaucoma
agents.14±16 The present work expands the ap-
plicability of the CDS concept to design biorever-
Correspondence to: I.K. Reddy (Telephone: 806-356-4000,
ext. 335; Fax: 806-356-4034;
E-mail: reddy@cortex.ama.ttuhsc.edu)
Journal of Pharmaceutical Sciences, Vol. 90, 1026±1033 (2001)
ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
1026
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001

CHEMICAL DELIVERY SYSTEMS OF t-BUTALONE
1027
concept in delivering t-butalone derivatives site-
speci®cally to iris-ciliary body tissues. The che-
mical and in vitro stability of novel bioreversible
derivatives were investigated in buffers (pH 5±8)
and in selected biological test media. The ocular
hypotensive activity was evaluated in the rabbit
model.
MATERIALS AND METHODS
All chemicals used were of reagent grade.
Reagents and deuterated solvents were obtained
from Aldrich Chemical Company unless other-
wise stated and were used without further puri-
®cation. Melting points were determined with a
Fisher Johns melting point apparatus and were
uncorrected. The proton nuclear magnetic reso-
nance (1H NMR) spectra were obtained with
Varian T-90 or VXR-300 spectrometers. Chemical
shifts are reported as parts per million (ppm)
units relative to tetramethylsilane (TMS) as
internal standard. Elemental analysis was per-
formed by Atlantic Microlab Inc. (Atlanta, GA).
Figure 1. Chemical structures of t-butaline (1), t-
butalone (2), and diacyl ketone derivatives (chemical
delivery systems; 8 and 9).
Synthesis
sible t-butalone derivatives to target t-butaline (1)
to iris-ciliary body tissues in the eye. According to
this concept, lipophilic ester derivatives (8, 9) of
the inactive chemical precursor of 1, the corre-
sponding ketone (2) is capable of site-speci®c
delivery of the active 1 (Figure 1), via a reduc-
tion-hydrolysis sequence, as shown in the case of
diacyl adrenalone derivatives.11
t-Butaline (1) is selective b2-adrenoreceptor
agonist and is used in the treatment of broncho-
constrictive disorders. Some selective b2-receptor
agonists, such as t-butaline, salbutamol, and
carbuterol, were found to lower intraocular
pressure (IOP) as effectively as isoproterenol in
rabbits.17 Their potential use as ocular hypoten-
sive agents is based on the suggestion that IOP
could be lowered effectively with minimal cardiac
stimulation, which is a b1 -adrenoreceptor effect.
However, by virtue of hydrophilic nature of 1,
topical administration of their aqueous solutions
at 2% dose level had little effect on IOP of
normotensive rabbits.17,18 This result may be
attributed to their poor permeability across
biphasic corneal membrane that is due to their
hydrophilic nature.
The synthetic scheme of diacyl derivatives of t-
butalone is depicted in Figure 2.
Synthesis of 30,50-Diisovaleryloxyacetophenone, 4
To a continuously stirred solution of 30,50-dihy-
droxyacetophenone, 3 (10 g) in pyridine (50 mL)
was added isovaleryl chloride (7.6 g) at ice-cooling
temperature. The reaction mixture was stirred
overnight at room temperature and then poured
into ice water (100 mL). The oil was extracted
twice with ethyl acetate (100 mL each time), and
the extract was washed ®rst with water followed
by 10% aqueous HCI and water. The extract was
dried over anhydrous Na2SO4, and the solvent
was removed by evaporation under vacuum to
give 4 (13 g, 65%) as crude oil, which was then
puri®ed by column chromatography on silica gel
1
(eluent CH2Cl2). H NMR (CDC13): 1.13 (12H, d,
J ˆ 9 Hz, CH3), 7.13 (1H, t, J ˆ 2 Hz, C4H), 7.53
0
0
(2H, d, J ˆ 2 Hz, C2 and C6 H).
Synthesis of 30,50-Dipivaloyloxyacetophenone, 5
In the present study, chemical modi®cations of
t-butaline are proposed to enhance its corneal
permeability and to assess the validity of CDS
To a continuously stirred solution of 30,50-dihy-
droxyacetophenone, 3 (4.2 g) in pyridine (20 mL)
was added pivaloyl chloride (4.6 g) at ice-cold
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001

1028
REDDY ET AL.
Figure 2. Synthetic reaction sequence of diacyl ketone derivatives (chemical delivery
systems) of t-butaline (8) and (9).
temperature. The reaction mixture was stirred for
5 h at room temperature and then poured into
ice-water (50 mL), which was later extracted
twice with ethyl acetate, taking 100 mL each
time. The extract was washed with water, dried
over anhydrous Na2SO4, and evaporated under
vacuum to give the residue. The crude products
were puri®ed by silica gel column chromatogra-
phy (eluent CH2Cl2) to obtain 5 (7.0 g, 77%) as
colorless oil. 1H NMR (CDC13): 1.4 (s, 18H, CH3 Â
6), 2.6 (3H, s, COCH3) 7.05 (1H, t, J ˆ 2 Hz, 40-H),
2.46 (2H, d, J ˆ 2 Hz, 20 and 60-H).
Synthesis of 2-Bromo-3,50-
dipivaloyloxyacetophenone, 7
To a continuously stirred solution of 5 (7.0 g) in
AcOH (70 mL) was added pyridinium bromide
perbromide (15.5 g). After stirring for about an
hour at room temperature, the mixture was
poured into ice water (200 mL) and extracted
with AcOEt (200 mL). The extract was washed
with water, dried over anhydrous Na2SO4, and
then evaporated in vacuum to give the crude 9,
which was then recrystallized from hexane to
obtain 7 (6.0 g, 67%) as colorless needles. The
melting point was 125±1278C. 1H NMR (CDC13):
1.40 (18H, s, CH3 Â 6), 4.40 (2H, s, COCH2Br),
7.03 (1H, J ˆ 2 Hz, 40-H), 7.43 (2H, d, J ˆ 2 H, 20
and 60-H).
Synthesis of 2-Bromo-30,50-
diisovaleryloxyacetophenone, 6
To a continuously stirred solution of 4 (13 g) in
AcOH (50 mL) was added pyridiniurn bromide
perbromide (15 g). The mixture was stirred for
about an hour at room temperature and then
poured into ice water (200 mL). After extraction
with ethylacetate (200 mL), the extract was
washed with water and aqueous NaHCO3, dried
over anhydrous Na2SO4, and then evaporated in
vacuum to give a crude oil (14.8 g). This material
was later puri®ed by column chromatography on
silica gel using CH2Cl2 as eluent to obtain 6 (11. 8
g, 74%) as oil. 1H NMR (CDCl3): 1.10 (12H, d, J ˆ 9
Hz, CH3 Â 4), 1.80±2.46 (2H, m, CH Â 2), 2.40 (4H,
s, CH2 Â 2), 4.40 (2H, s, CH2Br), 7.15 (1 H, t,
J ˆ 2 Hz, C4 H), 7.53 (211, d, J ˆ 2 Hz, C2 and
C6 H).
Synthesis of 2-tert-Butylamino-30,50-
diisovaleryloxyacetophenone, 8
To a continuously stirred solution of tertiary
butylamine (3.2 g) in CH2Cl2 (40 mL) was added
a solution of 6 (3.2 g in 10 mL of CH2Cl2) under
ice-cold conditions. After stirring for about an
hour, CH2Cl2 (40 mL) and 10% aqueous HCI
(50 mL) were added while stirring. The organic
layer was separated, dried over anhydrous
Na2SO4, and then evaporated under vacuum.
The residue thus obtained was recrystallized from
a mixture of CH2Cl2 and ether that contained few
drops of water to obtain hydrochloride salt of 8
(0.81 g, 24%) as colorless needles. The melting
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001

CHEMICAL DELIVERY SYSTEMS OF t-BUTALONE
1029
point was 186±1888C. 1H NMR (CDC13): 1.10
(12H, d, J ˆ 6 Hz, CH3  4), 1.50 (9H, s, CH3  3),
1.85±2.26 (2H, m, CH(CH3)2 Â 2), 2.46 (4H, s,
CH2 Â 2), 4.60 (2H, s, C CH2 N), 7.20 (2H, t,
J ˆ 1.5 Hz, C4 H), 7.50 (2H, d, J ˆ 1.5 Hz, C2
and C6 H), 9.50 (IH, bs, NH).
respectively. The calibration curves were con-
structed from linear plots of peak versus concen-
tration.
Stability in Aqueous Buffers
A stability study of 8 and 9 in isotonic phosphate
buffers (0.05 M), with pH values of 5.00, 5.4, 5.8,
6.44, 6.86, 7.40, and 8.00, was performed at 378C
to determine the hydrolytic stability of the
compounds under investigation as a function of
pH. Ten milliliters of buffer was equilibrated at
378C for 15 min prior to the introduction of
solutions of 8 or 9 in methanol to result an initial
concentration of 100 mM. The buffer was mixed by
vortex for 15 s. Twenty-microliter samples were
periodically withdrawn at set time intervals from
the media and injected directly into the HPLC
system. The disappearance of 8 or 9 was mon-
itored by measuring the peak heights. The
pseudo-®rst-order rate constant for disappear-
ance of compounds under investigation was deter-
mined by linear regression from the plot of natural
logarithm of peak height versus time. The pH
pro®les for each compound were generated
by plotting apparent ®rst-order rate constant
(log kapp) versus pH.
Synthesis of 2-tert-Butylamino-30,50-
dipivaloyloxyacetophenone, 9
To a continuously stirred solution of tertiary
butylamine (4.0 g) in CH2Cl2 (50 mL) was added
7 (4.0 g) under ice-cooling conditions. After
stirring for about an hour, CH2Cl2 (50 mL) and
10% aqueous HCI (50 mL) were added to the
mixture. The organic layer was separated, dried
over anhydrous Na2SO4, and evaporated under
vacuum to give 9. HCl was washed with Et2O
followed by recrystallization from CH2Cl2 Et2O
to give 13.HCl (1.3 g, 30%). The melting point was
1
1968C. H NMR (CDC13): 1.40 (18H, s, CH3 Â 6),
1.55 (9H, s, CH3  3), 4.50 (2H, d, J ˆ 1.5 Hz), 7.10
(1H, t, J ˆ 3 Hz), 7.56 (2H, d, J ˆ 3 Hz ), 9.50 (1H,
bs, NH).
Analytical Method
High-performance
liquid
chromatographic
(HPLC) analyses of CDS (8 and 9) as well as the
parent species (1) were performed with a Waters
Associates multicomponent system consisting of a
model 6000A double-piston reciprocating chroma-
tography pump; model U6K universal injector;
and model 440 absorbance detector, which can be
operated at two different wavelengths. A 25-cm
reversed-phase analytical C18 column (Water
Corp., Milford, MA) with mean particle diameter
of 5 mm was used at ambient temperature. A
guard column (Rainin Instrument Inc., Woburn,
MA) was connected to protect the analytical
column from contamination of ®ne particles of
sizes ꢀ 0.5 mm present in biological materials. The
guard column has a 3-cm cartridge (RP-18/18-
GU) (Brownee Labs) which had a 4.6-mm internal
diameter and a 10-mm sorbent packing. The
mobile phase consisted of 50% acetonitrile,
50% potassium dihydrogen phosphate monobasic
(0.05 M), and tetrabutylammonium phosphate
(0.005 M). The ¯ow rate was set at 2 mL/min.
With absorbance detection at 254 nm, the peak
heights of CDS and their corresponding parent
species were monitored. The retention times for 8,
9, and t-butaline were 4.7, 4.9, and 3.5 min,
In Vitro Stability in Biological Media
Whole Blood
The stability of 8 and 9 was determined in human
whole blood and in the whole blood of rabbits and
rats. Freshly drawn heparinized human blood,
obtained from the Civitan Regional Blood Center,
Inc. (Gainesville, FL) was stored in a refrigerator
and used within 72 h from the time it was
collected. Freshly prepared solutions of 8 or 9 in
methanol (50 mL, 50 mM) were mixed with 5 mL of
blood that had been previously equilibrated for
10 min at 378C in a waterbath. These prepara-
tions were then mixed thoroughly with a vortex
mixer for 15 s. At set time intervals, 0.4-mL
samples were withdrawn from the test medium
and mixed immediately with 0.8 mL of ice-cold
acetonitrile containing 10% dimethylsulfoxide
(DMSO) and mixed by vortex for 15 s. The
mixtures were centrifuged at 3000 g for 5 min.
Aliquots from the supernatant were ®ltered using
0.45-mm HV Millipore ®lters and analyzed by
injecting onto the HPLC column. The apparent
®rst-order rate constants for the hydrolysis of 8 or
9 were determined by monitoring the disappear-
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001

1030
REDDY ET AL.
ance of the respective compounds by linear
regression from the plot of natural logarithm of
the HPLC peak height versus time. Half-lives,
correlation coef®cients, and rate constants were
calculated for each compound studied.
The rabbit and rat blood was collected from
male New Zealand albino rabbits and Sprague-
Dawley rats, respectively. The blood was collected
into heparinized test tubes, and these heparinized
blood samples were pooled (from three animals in
each case) and refrigerated. Sampling and analy-
sis to determine hydrolytic stability of 8 and 9 in
rabbit and rat blood were done according to the
procedure already described.
in individual cages with free access to food and
water. The compounds (8 or 9) were administered
as 2.0% w/v solution (50 mL) in buffer, pH 7.4, or
saline into both eyes of a group of six rabbits. Prior
to the IOP measurements, corneal anesthesia
was provided by topical administration of one
drop of 0.5% proparacaine (Allergan) to minimize
discomfort from tonometry. Each measurement
lasted 5±10 s, and a precise reading was recorded
at set time points (0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h)
on a chart calibrated in mmHg. The tonometer
was calibrated at least twice a day, once at the
beginning of experiment and once in between,
using a 3-point calibration veri®er (Digilab). The
reported readings are the average values of six
experiments (ÆSEM). The signi®cance (P < 0.01)
of the change in IOP was determined using a
Student's t test. After allowing a washout period
of at least 8 days, the same group of rabbits was
administered 50 mL of saline or buffer without the
drug into both eyes and the readings served as
control.
Anterior Segment Tissues of Rabbits
Ocular tissues were obtained from male New
Zealand albino rabbits weighing 2±3 kg. Rabbits
were sacri®ced with lethal dose of pentobarbital
(100 mg/kg) administered intravenously (iv) into
the marginal ear vein. Each eye was enucleated
and rinsed in cold saline to remove any traces of
blood. Aqueous humor was then obtained by
making a single puncture at the limbus using 25
G Â 5/80 needle attached to a 3-cc syringe. Once
the aqueous humor was removed, corneal and iris-
ciliary body tissues were separated. Ten percent
homogenates of corneal and iris-ciliary body
tissues were prepared in isotonic phosphate
buffer, pH 7.4, using a tissuemiser (Tekmar
SDT, Cincinnati, OH) for 2 min. Aqueous humor
was used as such without further dilution. The
homogenates of corneal and iris-ciliary body
tissues and aqueous humor were taken into
stoppered tubes and incubated for 15 min at
378C. After equilibrium had been attained, 50 mL
of freshly prepared solutions of 8 or 9 in methanol
were added to each milliliter of the tissue
homogenate and aqueous humor. Fifty-microliter
samples were withdrawn at set time points, and
the enzymatic activity was destroyed by adding
200 mL of acetonitrile. The disappearance of CDS
was monitored by HPLC, and the kinetic para-
meters, such as half-life and rate constants, were
determined.
RESULTS AND DISCUSSION
Chemical Stability
The disappearance rates of 8 and 9 in aqueous
buffers with pH values in the range of 5±8 were
determined to assess their chemical stability. The
pH pro®les generated by plotting logarithmic
values of apparent ®rst-order rate constant, log
kapp (based on the disappearance of the com-
pounds under investigation from the buffer
media) versus pH are shown in Figure 3. The
two compounds were relatively stable at lower pH
values studied and the stability decreased as the
pH increased. The higher stability at low pH
was expected because the nucleophiles involved in
the hydrolysis of these ester compounds are
protonated, thereby decreasing the possibility of
nucleophilic attack and the resulting hydrolysis.
The dipivalyl ester derivative (9) was a relatively
more stable diisovaleryl ester derivative of t-
butalone (8) at all the pH values studied. This
result can be explained on the basis of steric
hindrance of the nucleophilic attack of hydroxyl
ions on the bulky tertiary butyl groups in the
hydrolysis, which is consistent with our earlier
®ndings with dipivalyl ester derivative of pheny-
lephrone.13 At physiological pH of 7.4, the half-
lives of 8 and 9 were 347Æ23 and 431Æ28 min,
respectively.
Ocular Hypotensive Activity in Rabbits
IOP was measured with a pneumatonometer
(Digilab model 30R), which utilizes a pneumatic
sensor. Adult male New Zealand albino (unrest-
rained and normotensive) rabbits, weighing 2.0±
3.0 kg, were used in this study. Rabbits were kept
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001

CHEMICAL DELIVERY SYSTEMS OF t-BUTALONE
1031
ciliary body > cornea > aqueous humor. Com-
pounds 8 and 9 were ꢁ2 times more stable in
corneal homogenates than in iris-ciliary body
homogenates. The iris-ciliary body tissues are
implicated as one of the major sites of drug
metabolism because they have the most well-
developed drug metabolizing systems found in the
eye, so the highest rates in these tissues were
expected. The half-lives of the compounds under
investigation in aqueous humor were ꢁ7±9 times
longer than those in iris-ciliary body tissue
homogenates.
Among the diesters tested, the diisovaleryl
ester derivatives (8) was relatively more labile
than dipivaloyl derivative (9) in all the biological
media. The ester linkages exhibited a steric order
of reactivity that had previously been reported for
esterase-catalyzed hydrolysis of phenyl acet-
ates,21 adrenalone esters,11 and phenylephrone
esters.13 Thus, the results suggest that it is
possible to prepare bioreversible derivatives with
different biological stability based on steric fac-
tors that allow the duration of action to be
controlled for the desired therapeutic need.
Figure 3. Experimentally determined pH±rate pro-
®les of diacyl ketone derivatives of t-butaline (8 and 9)
in pH range 5±8 at 378C (average value of three runs
was reported).
Stability in Biological Media
The in vitro stability of 8 and 9 was investigated
in selected biological media, which include the
whole blood from human, rabbit, and rat, and the
anterior segment tissues and ¯uids of rabbit,
which include 10% w/v corneal tissue homoge-
nates, 10% w/v iris-ciliary body tissue homoge-
nates, and aqueous humor. The half-lives based
on disappearance of CDS in the biological media
and in buffer solution (pH 7.4) are shown in
Table 1. In all the investigations, a signi®cant
difference in the disappearance rate was found
among species studied (rat > rabbit > human).
The rates of disappearance of all the CDS were
ꢁ2-fold faster in rat blood than in rabbit blood,
which were in turn ꢁ2- to 3-fold greater than in
human blood. The greater enzymatic activity in
smaller animals, such as rats and rabbits, is well
established.19,20
Intraocular Pressure
The effects of single dose of 8 and 9 on the IOP of
normotensive rabbits are depicted in Figure 4.
The compounds were tested at 2% dose level. The
change in IOP in mmHg [(treated value treated
baseline) (control value control baseline)] fol-
lowing topical administration of the CDS as a
function of time is shown. The two compounds
tested exhibited signi®cant (P < 0.01) IOP low-
ering activity. The activity (P < 0.001) peaked
between 3 to 4 h and it lasted for ꢁ6±7 hours.
Compound 9 was relatively more potent in low-
ering the IOP than 8 and had longer duration of
action (7 h compared with ꢁ 6 hours for that of 8).
The stability data of CDS in rabbit anterior
segment tissues, including corneal and iris-ciliary
body homogenates and aqueous humor, indicate
the following order of enzymatic activity: iris-
Table 1. The In Vitro Hydrolytic Half-Lives (min) of Diisovaleryl (8) and Dipivalyl (9) t-Butalone of in the
Biological Media and in the Buffer Solution (pH 7.4) at 378Ca
Biological Media
(8)
(9)
Human blood
Rabbit blood
Rat blood
Corneal tissue homogenate (10%)
Iris-ciliary body homogenate (10%)
Aqueous humor
5.42Æ0.4
2.41Æ0.2
1.27Æ0.1
3.78Æ0.4
2.23Æ0.1
20.38Æ1.8
347.0Æ23
13.80Æ1.5
6.0Æ0.9
3.27Æ0.2
8.58Æ0.9
4.66Æ0.3
39.29Æ2.9
431.0Æ28
Buffer (pH 7.4)
aEach half-life value presented in the table represents the mean of three experimentsÆSEM.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 8, AUGUST 2001

1032
REDDY ET AL.
hypotensive agents. Of the two compounds inves-
tigated, the dipivaloyl derivative of t-butalone
showed pronounced and longer duration of action.
The chemical and biological stability studies of
these compounds suggest that with the proper
selection of stearic bulk in the esteri®cation, the
duration of action can be manipulated.
ACKNOWLEDGMENTS
Figure 4. Effect of topical administration of a single
dose of 50 mL of a 2% solution of 8 and 9 in buffer or
saline on intraocular pressure (IOP) of normotensive
rabbit. Each value represents average (ÆSEM) of six
experiments.
Part of this work was done as a Doctoral
Dissertation at the University of Florida.
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The IOP-lowering activity of diacyl ketone
derivatives of t-butaline, a selective b2-receptor
agonists, is not unexpected. t-Butaline and other
selective b2-receptor agonists, such as salbutomol
and carbuterol, were shown to lower IOP of
normal rabbits.22 Furthermore, t-butaline was
equipotent to salbutamol and the effect was found
to be more than that of isoproterenol, a nonselec-
tive b-receptor agonist in the rabbit model.22 The
topical administration of aqueous solutions of t-
butaline in normal saline (at 2% dose level),
however, had little effect in lowering IOP of
normotensive rabbits.17,18 This result may be
explained on the basis of their inadequate
lipophilicity resulting in poor partitioning into
cornea. Because the cornea consists of lipophilic
epithelial and endothelial layers and a hydro-
philic middle region, the collagenous stroma,
biphasic solubility has been the major theory
describing the transcorneal drug permeation.23,24
Substitution of two hydroxyls on the rings of
compounds 3 by ester groups renders them more
lipophilic, thereby improving their penetration
into cornea across epithelium. After penetrating
the corneal membrane barrier, the effectiveness
of CDS becomes dependent on the ability of the
target tissue to regenerate the parent compounds,
1 from CDS (8 and 9, respectively) at an optimal
rate.15
9. Goskonda VR, Ghandehari H, Reddy IK. Novel site-
speci®c chemical delivery systems as a potential
mydriatic agent: Formation of phenyephrine in the
irisciliary body from phenylephrone CDS. J Pharm
Sci, in press.
CONCLUSIONS
The diacyl derivatives of t-butalone exibited
signi®cant IOP lowering activity in normotensive
rabbits, suggesting their use as potential ocular
10. Bodor NS. 1984. Novel approaches to the design of
safer drugs: Soft drugs and site-speci®c chemical
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