How an increase in the carbon chain length of the ester moiety affects the stability of a homologous series of oxprenolol esters in the presence of biological enzymes

Article abstract of DOI:10.1021/js970280p

β-Blockers including timolol and propranolol are administered in eye- drops for the treatment of glaucoma. Due to high incidence of cardiovascular and respiratory side-effects, their therapeutic value is limited. As a result of poor ocular bioavailability, many ocular drugs are applied in high concentrations, which give rise to both ocular and systemic side-effects. Therefore, some methods have been employed to increase ocular bioavailability such as (a) the development of drug delivery devices designed to release drugs at controlled rates, (b) the use of various vehicles that retard precorneal drug loss, and (c) the conversion of drugs to biologically reversible derivatives (prodrugs) with increased corneal penetration properties, from which the active drugs are released by enzymatic hydrolysis. A series of structurally related oxprenolol esters were synthesized and investigated as potential prodrugs for improved ocular use. The stability of each ester was studied in phosphate buffer (pH 7.4), also in the presence of (a) 30% human plasm, (b) aqueous humor, and (c) corneal extract at pH 7.4 and at 37 °C. An account is given of how the stability of a homologous series of oxprenolol esters in the presence of biological enzymes is affected by an increase in the carbon chain length of the ester moiety.

Full text of DOI:10.1021/js970280p

How an Increase in the Carbon Chain Length of the Ester Moiety
Affects the Stability of a Homologous Series of Oxprenolol Esters in
the Presence of Biological Enzymes
,†
C. GERALDINE M. JORDAN*
Contribution from Department of Food Science, Faculty of Agriculture, University College, Belfield, Dublin 4, Ireland.
Received July 21, 1997.
Final revised manuscript received January 27, 1998.
Accepted for publication April 3, 1998.
epithelium, and iris-ciliary body of the pigmented rabbit.
A series of propranolol esters were synthesized5,11 and their
rates of hydrolysis were measured in 0.02 M phosphate
buffer (pH 7.4) in the presence of 80% human plasma.5
Propranolol and oxprenolol are used in the treatment of
glaucoma, and hence the ability of their respective esters
to regenerate the parent compound at a reasonable rate
in ocular tissue homogenates is an important factor when
considering propranolol and oxprenolol esters as promising
prodrugs for ocular delivery.
The susceptibility of the esters of propranolol and
oxprenolol to undergo conversion to their respective parent
compounds was studied in vitro. The degradation of each
ester was studied in phosphate buffer (pH 7.4), also in the
presence of (a) 30% human plasma, (b) aqueous humor, and
(c) corneal extract at pH 7.4 and at 37 °C.12 The reason
this homologous series of esters was synthesized is because
the parent compound oxprenolol, due to poor ocular bio-
availability, needs to be applied in high concentrations
thereby giving rise to both ocular and systemic side-effects.7
Therefore, these prodrugs were produced in the hope of
improving targeting to particular tissues, to decrease the
incidence of side-effects and hopefully to provide a stable
formulation.
Abstract 0 â-Blockers including timolol and propranolol are admin-
istered in eye-drops for the treatment of glaucoma. Due to high
incidence of cardiovascular and respiratory side-effects, their thera-
peutic value is limited. As a result of poor ocular bioavailability, many
ocular drugs are applied in high concentrations, which give rise to
both ocular and systemic side-effects. Therefore, some methods have
been employed to increase ocular bioavailability such as (a) the
development of drug delivery devices designed to release drugs at
controlled rates, (b) the use of various vehicles that retard precorneal
drug loss, and (c) the conversion of drugs to biologically reversible
derivatives (prodrugs) with increased corneal penetration properties,
from which the active drugs are released by enzymatic hydrolysis. A
series of structurally related oxprenolol esters were synthesized and
investigated as potential prodrugs for improved ocular use. The
stability of each ester was studied in phosphate buffer (pH 7.4), also
in the presence of (a) 30% human plasma, (b) aqueous humor, and
(c) corneal extract at pH 7.4 and at 37 °C. An account is given of
how the stability of a homologous series of oxprenolol esters in the
presence of biological enzymes is affected by an increase in the carbon
chain length of the ester moiety.
This paper gives an account of how the stability of a
homologous series of oxprenolol esters is affected by an
increase in the carbon chain length of the ester moiety in
the presence of biological enzymes.
1. Introduction
Esters are the best known prodrugs due to the predomi-
nance of carboxylic and hydroxyl substituents in drug
molecules along with the availability of enzymes in living
systems capable of hydrolyzing them. Therefore, consider-
able attention has been focused on the use of bioreversible
(prodrugs) in order to improve the delivery characteristics
of various drugs.1 A fundamental requisite for the useful-
ness of the prodrug approach is the ready availability of
chemical derivatives satisfying the prodrug requirements,
principally reconversion of the prodrug to the parent drug
in vivo.
In previous studies, esters of timolol have been developed
to potentially diminish the systemic absorption of topically
added timolol through increased corneal absorption. The
cardiovascular and respiratory side-effects2-4 are thereby
reduced. However, these esters are unstable in aqueous
solutions. In respect of this, a series of esters of both
propranolol5-7and timolol8-10 were synthesized and the
kinetics of degradation of the prodrugs in aqueous solution
studied.
2. Materials and Methods
2.1. Ch em ica lssSamples of oxprenolol hydrochloride were
obtained from both Ciba-Geigy and Sigma (UK). The acid
chlorides were obtained from Aldrich (UK). All solvents used were
either high-performance liquid chromatography (HPLC) grade or
distilled before use. Solid reagents were analytical or reagent
grade and were used as supplied or were recrystallized before use.
All solvents used in HPLC (i.e. acetonitrile, methanol, acetone,
and tetrahydrofuran) were HPLC grade. All buffer substances
were of reagent or analytical grade. The ionic strength of each
buffer solution was adjusted to 0.5 by adding a specific quantity
of analytical grade potassium chloride.
2.2. Syn th esis of Oxp r en olol Ester ssThe homologous series
of oxprenolol esters were prepared by heating oxprenolol hydro-
chloride (1 g) under reflux for a specified length of time with the
appropriate acid chloride. Excess acid chloride was removed under
vacuum. A solid product was obtained with the O-pivaloyl
derivative. An oily residue was obtained in all other cases. The
oil was crystallized by adding 5 mL of acetone followed by 30 mL
of petroleum ether. Continuous removal of the acetone and
petroleum ether under vacuum and repetition of the process
resulted in the formation of a milky precipitate which was
recrystallized from 2-propanol.
The degradation of esters of the â-adrenergic blocker
timolol was studied.8 They were all hydrolyzed to yield
timolol in quantitative amounts in buffer solutions, human
plasma, and homogenates of the conjunctiva, corneal
2.3. P r ep a r a t ion of Biologica l Sa m p less2.3.1. Human
PlasmasHuman plasma was obtained from the Blood Transfusion
Board, Dublin, and stored frozen.
2.3.2. Aqueous HumorsEyes were obtained from freshly killed
cattle in Red Meats, Dublin. The aqueous humor was extracted
from each eye using a 1 mL sterile disposable syringe fitted with
* Corresponding author.
† Present address: Department of Food & Environmental Science,
Dublin Institute of Technology, Cathal Brugha St., Dublin 1, Ireland.
Fax (01) 4024495.
880 / Journal of Pharmaceutical Sciences
S0022-3549(97)00280-3 CCC: $15.00
Published on Web 06/06/1998
© 1998, American Chemical Society and
Vol. 87, No. 7, July 1998
American Pharmaceutical Association

Figure 2sFirst-order plots for the degradation of O-propionyloxprenolol in
0.05 M phosphate buffer (pH 7.4) and also in the presence of (a) 30% human
plasma, (b) aqueous humor, and (c) corneal extract, at the same pH and at
37 °C.
Figure 1sFirst-order plots for the degradation of O-acetyloxprenolol in 0.05
M phosphate buffer (pH 7.4) and also in the presence of (a) 30% human
plasma, (b) aqueous humor, and (c) corneal extract, at the same pH and at
37 °C.
a brown 25 G 3/8 in. needle. Each eye contained approximately 1
mL of aqueous humor, and it was refrigerated in sterile vials at 4
°C until used.
2.3.3. Corneal ExtractsAn incision was made in the cornea
using a scalpel, and the cornea was dissected using a forceps and
scalpel. This transparent tissue was then chopped into small
pieces using a pair of scissors and was put into a 4 in. mortar
that contained approximately 3 g of hydrochloric acid washed sand
and approximately 5 mL of ice-cold 0.05 M phosphate buffer (pH
7.4). The contents were homogenized using a Heto motor-driven
tissue grinder. The ocular tissue homogenate was then poured
into centrifuge tubes and centrifuged at 12 000 rpm for ap-
proximately 45 min in an AGB bench centrifuge. The clear
supernatant was decanted off and stored in sterile vials at 4 °C.
The corneal extract and aqueous humor were only stored for a
maximum of 3 days at 4 °C. After that time fresh ocular tissue
was obtained.
2.4. Ap p a r a tu ssHPLC was carried out using
a system
consisting of a Waters 501 HPLC pump, a variable wavelength
detector attached to a Houston omniscribe recorder and a 20 µL
Rheodyne loop injection valve. The column used, 100 × 4.6 mm,
was packed with Spherisorb C-8 (5 µm particles). A precolumn,
50 × 4.6 mm, was similarly packed. A 10 µL sample was
introduced by means of a Hamilton syringe.
The pH value of each solution was determined using a Radi-
ometer M-26 pH meter fitted with a glass electrode (Radiometer
G-202B) and a calomel reference electrode (Radiometer K-401).
Reference buffers were Radiometer standard solutions (pH 4.00/
22 °C, pH 6.97/22 °C, and pH 8.86/22 °C). A Heto thermostat
water-bath with a Heto contact thermometer were used in all
experiments.
The corneal extract contents were homogenized using a Heto
motor-driven tissue grinder.
Figure 3sFirst-order plots for the degradation of O-butyryloxprenolol in 0.05
M phosphate buffer (pH 7.4) and also in the presence of (a) 30% human
plasma, (b) aqueous humor, and (c) corneal extract, at the same pH and at
37 °C.
20 min intervals a 10 µL sample was removed and immediately
chromatographed.
2.5. Sta bility Stu d iessA 20% stock solution of each ester was
prepared in HPLC grade, distilled water. To a 3 mL stock solution
of each ester, 4 mL of phosphate buffer (0.05 M, pH 7.4) was added,
and then 3 mL of aqueous humor or corneal extract was added.
Before immersing the solution in a Heto temperature-controlled
water-bath at 37 °C ( 0.2 °C, the mixing was agitated by hand
for approximately 2 min in order to ensure complete mixing. At
To a 3 mL stock solution of each ester, 4 mL of phosphate buffer
(0.05 M, pH 7.4) was added, and then 3 mL of human plasma was
Journal of Pharmaceutical Sciences / 881
Vol. 87, No. 7, July 1998

Figure 4sFirst-order plots for the degradation of O-valeryloxprenolol in 0.05
M phosphate buffer (pH 7.4) and also in the presence of (a) 30% human
plasma, (b) aqueous humor, and (c) corneal extract, at the same pH and at
37 °C.
Figure 5sFirst-order plots for the degradation of O-pivaloyloxprenolol in 0.05
M phosphate buffer (pH 7.4) and also in the presence of (a) 30% human
plasma, (b) aqueous humor, and (c) corneal extract, at the same pH and at
37 °C.
added. It was kept in a temperature-controlled Heto water bath
at 37 °C ( 0.2 °C. At 20 min intervals, the solution was agitated
quickly by hand and a 250 µL sample was withdrawn using a
micropipet and added to a 1000 µL of analytical grade ethanol in
order to deproteinize the plasma. After mixing and centrifugation
at 12 000 rpm for 2 min in an AGB bench centrifuge, 10 µL of the
clear supernatant liquid was removed using a Hamilton syringe
and immediately chromatographed.
A control was used in each experiment, and it consisted of ester
sample and 0.05 M phosphate buffer (pH 7.4) only. It was also
maintained at 37 °C ( 0.2 °C, and at 20 min intervals, a 10 µL
sample was withdrawn and immediately chromatographed.
Table 1sObserved Pseudo-First-Order Rate Constants (kobs) for the
Degradation of a Homologous Series of Oxprenolol Esters in 0.05 M
Phosphate Buffer (pH 7.4) and Also in the Presence of (a) 30%
Human Plasma, (b) Aqueous Humor, and (c) Corneal Extract, at the
Same pH and at 37 °C (µ ) 0.5)
kobs (min-1) × 10-2
buffer
(pH 7.4)
human plasma
(30%)
aqueous
humor
corneal
extract
ester
O-acetyl
7.5
6.6
3.6
3.2
0.03
16.0
10.7
5.3
4.2
0.26
11.1
4.7
3.7
3.5
0.10
7.1
6.1
5.3
4.2
0.18
O-propionyl
O-butyryl
O-valeryl
O-pivaloyl
3. Results and Discussion
Quantitation of the esters was determined by measuring
the peak heights in relation to those of standards chro-
matographed under identical conditions or by measuring
the peak areas. A plot of the log of residual concentration
(log % remaining) against time (min) was drawn for each
Table 2sThe Ratio of the Hydrolysis Rates in Different Media Relative
to That in Buffer (pH 7.4) and at 37 °C (µ ) 0.5)
human plasma
(30%)
aqueous
humor
corneal
extract
ester (Figures 1-5). Pseudo-first-order rate constants (kobs
)
ester
were calculated from the slopes of these linear plots using
eq 1
O-acetyl
2.13
1.62
1.47
1.31
8.66
1.48
0.71
1.02
1.09
3.33
0.95
0.93
1.47
1.31
6.0
O-propionyl
O-butyryl
O-valeryl
O-pivaloyl
slope ) -kobs/2.303
(1)
Those rate constants were also calculated for the overall
degradation of the esters in 0.05 M phosphate buffer only
(pH 7.4), and also in the presence of (a) human plasma,
(b) aqueous humor, and (c) corneal extract. Their half-lives
and shelf lives were calculated from the rate constants
using eqs 2 and 3, respectively
of the ester at a particular pH. These data are represented
in Tables 3 and 4, respectively. Fresh ocular tissue only
was used in these experiments, as only fresh extract
provided maximal esterase activity. Every effort was made
to use the ocular tissue on the day it was obtained, or if
this was not possible, ocular tissue that was stored at 4 °C
for no more than 3 days would be used. Ocular tissue that
had been stored for longer than 3 days at 4 °C was inactive
and could not be used in experiments. Preliminary experi-
ments carried out with inactive ocular tissue (boiled to over
t0.5 ) 0.693/kobs
t90 ) 0.105/kobs
(2)
(3)
where kobs ) the observed pseudo-first-order-rate constant
882 / Journal of Pharmaceutical Sciences
Vol. 87, No. 7, July 1998

Table 3sCalculated Half-Life (t0.5) Values for a Homologous Series of
Oxprenolol Esters in 0.05 M Phosphate Buffer (pH 7.4) and Also in
the Presence of (a) 30% Human Plasma, (b) Aqueous Humor, and (c)
Corneal Extract, at the Same pH and at 37 °C (µ ) 0.5)
Table 5sYields of Oxprenolol Formed (%) during the Degradation of
a Homologous Series of Oxprenolol Esters in 0.05 M Phosphate
Buffer (pH 7.4) and Also in the Presence of (a) 30% Human Plasma,
(b) Aqueous Humor, and (c) Corneal Extract, at the Same pH and at
37 °C after 120 min (µ ) 0.5)
t0.5 (min)
oxprenolol formed (%)
buffer
(pH 7.4)
human plasma
(30%)
aqueous
humor
corneal
extract
ester
buffer
(pH 7.4)
human plasma
(30%)
aqueous
humor
corneal
extract
ester
O-acetyl
9.1
10.4
19.1
21.1
2035.5
4.3
6.4
12.8
16.2
263.2
6.1
14.6
18.3
19.6
687.8
9.6
11.3
13.0
16.4
375.5
O-acetyl
96
95
98
97
98
95
90
91
92
98
99
98
96
97
95
92
94
95
97
96
O-propionyl
O-butyryl
O-valeryl
O-pivaloyl
O-propionyl
O-butyryl
O-valeryl
O-pivaloyl
Table 4sCalculated Shelf-Life (t90) Values for a Homologous Series of
Oxprenolol Esters in 0.05 M Phosphate Buffer (pH 7.4) and Also in
the Presence of (a) 30% Human Plasma, (b) Aqueous Humor, and (c)
Corneal Extract, at the Same pH and at 37 °C (µ ) 0.5)
Table 6sPartition Coefficients (log P), Apparent Partition Coefficients
(log P)app, and Capacity Factors (log k1) of a Homologous Series of
Oxprenolol Esters at 22 °C
t90 (min)
compounda
log P
log Papp (pH 7.4)b
log k1 (pH 7.4)c,d
buffer
human plasma
(30%)
aqueous
humor
corneal
extract
O-acetyl
3.82
3.76
3.95
4.0
2.52
2.58
3.29
3.57
2.78
0.43
0.58
0.74
0.90
0.95
ester
(pH 7.4)
O-propionyl
O-butyryl
O-valeryl
O-pivaloyl
O-acetyl
1.4
1.5
2.9
3.1
308.4
0.65
0.97
1.9
2.4
39.8
0.93
2.2
2.7
2.9
104.2
1.4
1.7
1.9
2.4
56.7
O-propionyl
O-butyryl
O-valeryl
O-pivaloyl
3.54
a All of the O-acyl esters synthesized were soluble in aqueous solvents,
i.e., water and buffers, and also in organic solvents such as methanol,
chloroform, ethanol, ethyl acetate, acetone and petroleum ether. However,
the N-acetyl and N-propionyl derivatives were also synthesized, and they were
completely insoluble in aqueous solvents but soluble in organic solvents. b pH
7.4, calculated from log P. c pH 7.4. d k1 ) capacity factor and is defined by
the following equation: k1 ) tr − to/to, where tr is the retention time of the
solute and to represents the elution time of the solvent.
40 °C and allowed to cool before use) produced results
similar to those obtained in buffer only.
The homologous series of oxprenolol esters degrade faster
in ocular tissue homogenate than in buffer. This is due to
the presence of many esterases in the ocular tissues to
catalyze the ocular hydrolysis, particularly acetylcholinest-
erase and the predominant butyrylcholinesterase enzymes.
These esterases succeed in exhibiting their catalytic effect
over the chemically stable O-pivaloyloxprenolol derivative,
thereby overcoming the steric hindrance induced by its
tertiary butyl ester group and hence speeding up its rate
of ocular hydrolysis. This same derivative of oxprenolol
exhibits a faster rate of hydrolysis in ocular tissue than in
buffer only. This phenomenon is probably due to the
overriding effect of the OCH2CHdCH2 group in the ortho
position of the benzene ring of oxprenolol, which suppresses
the resistance of this sterically hindered ester group to
enzymatic catalysis. Therefore, the ocular esterases present
are allowed to exert their catalytic effect, hence resulting
in an increased rate of ocular hydrolysis for this ester. For
the homologous series of oxprenolol esters, their rates of
ocular hydrolysis decrease as the chain length increases
(Table 1). This is in sharp contrast to research done on
adult albino rabbits.18 They found that the ocular hydroly-
sis of a series of R- and â-naphthyl esters increased as the
ester chain length increased. The ratio of the hydrolysis
rates in different media relative to that in buffer (pH 7.4)
and at 37 °C (µ ) 0.5) was determined (Table 2). From
the results obtained, one could speculate that the O-
propionyl, O-butyryl, and O-valeryl esters would reach the
intracular tissues most effectively because of their moder-
ate rates of hydrolysis. In contrast, the O-acetyl prodrug
is too unstable and the O-pivaloyl derivative too stable to
gain entry effectively. As the rate of hydrolysis of each
ester was not measured in rabbit ocular tissue, one might
expect the ocular hydrolysis rate to increase as the ester
chain length increased. These results would compare
favorably with the results of research done on adult albino
rabbits.18
increases. The lipophilic nature of the corneal epithelium
severely constrains the ability of topically applied oph-
thalmic drugs, the polar ones especially, to traverse this
barrier. For a drug to gain entry to the intraocular tissues,
it must be lipophilic enough to partition from the tears to
the corneal epithelium.19 Therefore, the basis for improve-
ment in ocular absorption by prodrugs is an increase in
the lipophilicity of the prodrugs relative to their compound,
which favors their uptake by, and diffusion across, the
lipophilic corneal epithelium. It should be anticipated that
corneal prodrug absorption may be altered when the
corneal epithelial barrier is breached. This is because the
prodrug would then be exposed to the corneal stroma which
differs from the epithelium in both lipophilicity and es-
terase activity.20 A number of explanations can be put
forward as to why the ocular hydrolysis of the homologous
series of oxprenolol esters decreases as the chain length
and their lipophilicity increases (Table 6), since the chemi-
cal nature of the parent compound can significantly influ-
ence the magnitude of the acylation rate of the enzyme and
in turn the hydrolytic rate of the associated ester prodrug.
Therefore, possibly the O-propionyl, O-butyryl, and O-
valeryl derivatives of oxprenolol are not as susceptible to
attack by the acetylcholinesterase and butyrylcholinest-
erase present in the ocular tissues, because of the effect
exerted by the ortho substituent on the benzene ring of
oxprenolol which slows down the interaction between the
esterases in the ocular tissue and the ester moiety, thereby
resulting in a reduced rate of ocular hydrolysis. The
amount of interference would increase as the chain length
of the ester increased.
Another possible explanation is that perhaps there were
not enough esterases present in the ocular tissue, e.g.
butyrylcholinesterase, to increase the ocular hydrolysis of
the prodrugs, and therefore chemical hydrolysis was the
dominant reaction taking place. Therefore, it could be
There are a number of reasons why the rate of ocular
hydrolysis increases as the lipophilicity of the ester prodrug
Journal of Pharmaceutical Sciences / 883
Vol. 87, No. 7, July 1998

proven that as fresh ocular tissue was used in those
experiments, there were enough esterases present to
increase the ocular hydrolysis of the oxprenolol prodrugs.
Finally, the most valid explanation for the reduced rate
of ocular hydrolysis of oxprenolol esters in bovine eye tissue
is due to the dependence of the rate of ester hydrolysis in
ocular tissue homogenates on ester chain length. Maxi-
mum hydrolytic rate was achieved with the valerate ester
when a series of 1- and 2-naphthyl esters were incubated
with the ocular tissue homogenates of albino rabbits.
However, the opposite trend was observed, when the
hydrolytic rates of 1- and 2-naphthyl acetate, propionate,
and valerate esters were measured in microsomal fractions
of the corneal epithelium and iris-ciliary body of bovine
eyes.16 Therefore, a similar trend is probably occurring
here, where the rate of ocular hydrolysis decreases as the
ester chain length and lipophilicity increases (Tables 1 and
6, respectively).
In contrast to a decrease in the rate of ocular hydrolysis
as the ester chain length increases for a homologous series
of oxprenolol esters, the opposite was observed for a similar
series of timolol esters. The ocular hydrolysis of the timolol
esters was measured in homogenates of the conjunctiva,
corneal epithelium, and iris-ciliary body of the pigmented
rabbit.8
Significant increases were observed in the enzymatic
ocular hydrolytic rate as the number of carbons in the alkyl
side chain increased and also with increasing lipophilicity
for a homologous series of timolol esters.8 Unlike the
O-pivaloyl derivatives of propranolol12 and oxprenolol,
O-pivaloyltimolol showed enhanced stability to hydrolysis
in ocular tissue homogenates, relative to the companion
esters. This high stability is most probably due to steric
hindrance to attack by acetylcholinesterase and butyryl-
cholinesterase present in these ocular tissues.21 The
timolol esters hydrolyzed readily in ocular tissue like those
of propranolol and oxprenolol, and they also displayed a
higher hydrolytic rate in corneal extract than in aqueous
humor.22 Intramolecular aminolysis did not take place in
ocular tissue homogenates or in buffer-only solutions for
timolol esters; this is probably due to the high stability of
the tertiary butylamino side-chain of the timolol molecule,
which inhibits formation on the N-acyltimolol derivative.
Therefore the percentage of timolol formed was the same
during ocular hydrolysis as during hydrolysis in buffer only,
because there was no N-acyl derivative formed to interfere
with the amount of parent compound formed.8
Esterases play a major role in the pharmacology of
prodrugs, which are enzymatically labile derivatives of
drugs designed to improve the pharmacokinetics of their
parent compounds.13 It is reasonable to expect that an
understanding of the esterase composition in various ocular
tissues would facilitate ester prodrug design. From a study
carried out using albino and pigmented rabbits,14 the
esterase activities in their corneas, irises, ciliary bodies,
and aqueous humor were delineated. It was found that at
least some of the already named esterases present in the
aqueous humor were different from those in the cornea,
iris, and ciliary body. It was also shown that the esterase
activity was highest in the iris-ciliary body followed by the
cornea and then the aqueous humor. Specifically, the
esterase activity in the cornea and aqueous humor was,
respectively, 50 and 2-5% of that seen in the iris-ciliary
body. The higher level of esterase activity in the iris and
ciliary may be due to the fact that these tissues are more
cellular than both the cornea and aqueous humor and
therefore more abundant in esterases. Even though the
cornea is not as enzymatically active as the iris and ciliary
body, it is still in a strategic position to determine the
amount of intact drug ultimately reaching the internal eye
Scheme 1sE and EH are the active and inactive forms of the enzyme; S,
is the substrate; ES and EHS are the active and inactive forms of the
enzyme−substrate complex; ES′ and EHS′ are the corresponding forms of
the acyl enzyme intermediate.
from topical application. It was found that in both breeds
of rabbits, esterase activity was the highest in the iris-
ciliary body followed by the cornea and then the aqueous
humor.15 However, on comparing results on the esterase
activity in the bovine eye with those obtained in the rabbit,
it is apparent that significant species differences in ocular
esterase activity exist.16 The bovine and rabbit eyes differ
in a number of ways. First, the esterase activity is 2-40
times higher in the rabbit than in bovine eye with the most
prominent difference in the corneal epithelium. Second,
whereas, in the rabbit the corneal epithelium possesses
twice as much esterase activity as the stroma, this rank
order is reversed in the bovine eye.17 It was also found
that the cornea was richer in esterases than the aqueous
humor. As there were no experiments carried out on rabbit
eye tissue, one can only speculate from the above informa-
tion that the rate of ocular hydrolysis would be greater in
rabbit eye tissue for all esters. This is because ocular
esterase activity is 2-40 times higher in rabbit eye tissue
than in bovine eye.
For the homologous series of oxprenolol esters, i.e., from
the O-acetyl to the O-valeryl derivatives, it was observed
that the rate of degradation decreased as the chain length
of the O-acyl group increased (Table 1). This occurred with
and without plasma. It is relevant in this case to explain
why it occurs in the presence of plasma. There are a
number of enzymes present in plasma which are capable
of hydrolyzing esters. It is possible to predict which
enzyme is involved in the in vivo hydrolysis of a prodrug
with an ester functional group as its reversible group18 by
carrying out experiments with plasma selectively enriched
with specific enzymes, and one might obtain a reasonably
good idea of which enzyme is responsible for the major
portion of the hydrolysis. Furthermore, data for the
variation of ester reactivity with structure is not available
for most of these enzymes. However, ester prodrug hy-
drolysis catalyzed by plasma enzymes is believed to follow
the path shown below23 (Scheme 1):
The rate-determining step in this type of reaction is
deacylation. Thus, the rate of deacylation is accelerated
by electron acceptors and by increasing hydrophobicity.24
It is decelerated in the case of the oxprenolol esters already
mentioned, because of the increasing chain length of the
ester moiety and also because of increasing lipophilicity
and the electron-releasing character of the O-acyl groups.
This gives rise to a decrease in the rate of hydrolysis as
the ester group becomes larger (Table 1).
Like O-pivaloylpropranolol,12 the O-pivaloyloxprenolol
derivative exhibits a very low rate constant at pH 7.4 with
or without plasma, in comparison with those of the other
oxprenolol esters. This is due to the steric hindrance
exhibited by the bulky tertiary butyl group of the O-acyl
moiety which makes this ester very stable. Due to their
increased rates of hydrolysis in plasma, their half-lives and
shelf lives are decreased (Tables 3 and 4). Enzymatic
hydrolysis of the oxprenolol esters is accompanied by
intramolecular aminolysis. The percentage of oxprenolol
formed during enzymatic hydrolysis of each ester is shown
in Table 5. A slight decrease in the formation of oxprenolol
884 / Journal of Pharmaceutical Sciences
Vol. 87, No. 7, July 1998

8. Bundgaard, H.; Buur, A.; Chang, S.-C.; Lee, V. H. L. Prodrugs
of Timolol for Improved Ocular Delivery: Synthesis, Hy-
drolysis Kinetics and Lipophilicity of Various Timolol Esters.
Int. J . Pharm. 1986, 33, 15-26.
is observed in some cases, and this is possibly due to an
increase in the formation of the N-acyl derivative.
9. Bundgaard, H.; Buur, A.; Chang, S.-C.; Lee, V. H. L. Timolol
Prodrugs: Synthesis, Stability and Lipophilicity of Various
Alkyl, Cycloalkyl and Aromatic Esters of Timolol. Int. J .
Pharm. 1988, 46, 77-88.
10. Chang, S.-C.; Bundgaard, H.; Buur, A.; Lee, V. H. L.
Improved Corneal Penetration by Prodrugs as a means to
Reduce Systemic Drug Load. Invest. Ophthalmol. Vis. Sci.
1987, 28, 487-491.
11. Quigley, J . M.; J ordan, C. G. M.; Timoney, R. F. The
Synthesis, Hydrolysis Kinetics and Lipophilicity of O-Acyl
Esters of Propranolol. Int. J . Pharm. 1994, 101, 145-163.
12. J ordan, C. G. M.; Davis, G. W.; Timoney, R. F. Studies
Showing the Effect of Enzymes on the Stability of Ester
Prodrugs of Propranolol and Oxprenolol in Biological Samples.
Int. J . Pharm. 1996, 141, 125-135.
13. Grass, G. M.; Robinson, J . R. Mechanisms of Corneal Drug
Penetration 1; in vivo and in vitro Kinetics. J . Pharm. Sci.
1988, 77, 3-14.
14. Lee, V. H. L. Esterases Activities in Adult Rabbit Eyes. J .
Pharm. Sci. 1983, 72, 239-244.
15. Chien, D. S.; Sasaki, H.; Bundgaard, H.; Buur, A.; Lee, V.
H. L. Role of Enzymatic Lability in the Corneal and Con-
junctival Penetration of Timolol Ester Prodrugs in the
Pigmented Rabbit. Pharm. Res. 1991, 8, 728-733.
16. Lee, V. H. L.; Iimoto, D. S.; Takemoto, K. A. Subcellular
Distribution of Esterases in the Bovine Eye. Curr. Eye Res.
1983, 2, 869-876.
4. Conclusion
One could conclude that the stability of the homologous
series of oxprenolol esters increased as the carbon chain
length of the ester moiety increased. This phenomenon
was observed in phosphate buffer (pH 7.4) only, also in the
presence of biological enzymes, thereby resulting in in-
creased half-lives and shelf lives. Enzymatic hydrolysis is
accompanied by intramolecular aminolysis in the case of
each ester. From the results of this study, the O-propionyl,
O-butyryl, and O-valeryl prodrugs are the most effective
because of their moderate hydrolysis rates and would most
probably reach the iris-ciliary body most effectively, whereas
the O-acetyl is too unstable and the O-pivaloyl is too stable.
The O-pivaloyloxprenolol derivative displays a very high
stability in the presence of biological enzymes due to the
steric hindrance exhibited by the bulky tertiary butyl group
of the O-acyl moiety which makes this ester very stable.
This conclusion about the stability of the esters was drawn
from physicochemical data such as the partition coefficients
(log p) and the capacity factors (log k1) (Table 6). As in
vivo studies were not carried out with these esters, it would
be very interesting to find out the best ways of administer-
ing them, to be able to comment on the relative ease of
stabilizing the formulations, and to compare the rates of
hydrolysis in the iris-ciliary body. This undoubtedly would
make very interesting future work.
17. Chien, D. S.; Bundgaard, H.; Lee, V. H. L. Influence of
Corneal Epithelial Integrity on the Penetration of Timolol
Prodrugs. J . Ocular Pharmacol. 1988, 4, 137-146.
18. Chang, S.-C.; Lee, V. H. L. Influence of Chain Length on the
In-Vitro Hydrolysis of Model Ester Prodrugs by Ocular
Esterases. Curr. Eye Res. 1983, 2, 651-656.
19. Bodar, N.; El-Koussi, A. Improved Delivery Through Biologi-
cal Membranes: LVI. Pharmacological Evaluation of Al-
prenoxime-a New Potential Antiglaucoma Agent. Pharm.
Res. 1991, 8, 1389-1395.
20. Lee, V. H. L.; Morimoto, K. W.; Stratford, R. E. Esterase
Distribution in the Rabbit Cornea and its Implications in
Ocular Drug Bioavailability. Biopharm. Drug Disp. 1982, 3,
291-300.
21. Lee, V. H. L.; Chang, S.-C.; Oshiro, C. M.; Smith, R. E. Ocular
Esterase Composition in Adult and Pigmented Rabbits:
Possible Implications in Ocular Pro-Drug Design and Evalu-
ation. Curr. Eye Res. 1985, 4, 1117-1125.
22. Chang, S.-C.; Bundgaard, H.; Buur, A.; Lee, V. H. L. Low
Dose O-butyryl Timolol Improves the Therapeutic Index of
Timolol in the Pigmented Rabbit. Invest. Ophthalmol. Vis.
Sci. 1988, 29, 626-629.
23. Charton, M. The Prediction of Chemical Lability through
Substituent Effects. Design of Biopharmaceutical Properties
through Prodrugs and Analogues; In Roche, E. B., Ed.;
American Pharmaceutical Association: Washington, DC,
1977; pp 228-280.
24. Huang, H.-S.; Schoenwald, R. D.; Lach, J . L. Corneal
Penetration Behaviour of â-Blocking Agents. II: Assessment
of Barrier Contributions. J . Pharm. Sci. 1983a , 72,
1272-1279.
References and Notes
1. Bundgaard, H., Design of Prodrugs: Bioreversible Deriva-
tives for Various Groups and Chemical Entities. In Design
of Prodrugs; Bundgaard, H., Ed.; Elsevier: Amsterdam,
1985; pp 1-92.
2. Velde, T. M.; Kaiser, F. E. Ophthalmic Timolol Treatment
Causing Altered Hypoglycemic Response in a Diabetic Pa-
tient. Arch. Intern. Med. 1983, 143, 1627.
3. Munroe, W. P.; Rindone, J . P.; Kershner, R. M. Systemic Side-
Effects Associated With the Ophthalmic Administration of
Timolol. Drug Intell. Clin. Pharm. 1985, 19, 85-89.
4. Nelson, W. L.; Fraunfelder, F. T.; Sills, J . M.; Arrowsmith,
J . B.; Kuritsky, J . N. Adverse Respiratory and Cardiovas-
cular Events Attributed to Timolol Ophthalmic Solution,
1978-1985. Am. J . Ophthalmol. 1986, 102, 606-611.
5. Buur, A.; Bundgaard, H.; Lee, V. H. L. Prodrugs of Propra-
nolol: Hydrolysis and Intramolecular Aminolysis of Various
Propranolol Esters and an Oxazolidin-2-one Derivative. Int.
J . Pharm. 1988, 42, 51-60.
6. Irwin, W. J .; Belaid, K. A. Drug-delivery by Ion-Exchange.
Hydrolysis and Rearrangement of Ester Pro-Drugs of Pro-
pranolol. Int. J . Pharm. 1988a , 46, 57-67.
7. J ordan, C. G. M.; Quigley, J . M.; Timoney, R. F. The
Synthesis, Hydrolysis Kinetics and Lipophilicity of O-Acyl
Esters of Oxprenolol. Int. J . Pharm. 1992, 84, 175-189.
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