Stereoselective hydrolysis and penetrati

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Stereoselective Hydrolysis and Penetration of Propranolol Prodrugs: In Vitro
Evaluation Using Hairless Mouse Skin
SHAMIM

AHMED,TERUKOIMAI',

AND

MASAKIOTAGlRl

Received October 24, 1994, from the Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kumamoto University,
5-1 Oe-honmachi, Kumamoto 862, Japan.
Accepted for publication March 22, 1995@.

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Abstract 0 Stereoselective hydrolysis of two ester prodrugs of propranolol, isovaleryl propranolol (IV-PL) and cyclopropanoyl propranolol (CPPL), was studied in Tris-HCI buffer (pH 7.4) containing 0.15 M KCI, skin
and liver homogenates, 5% plasma in Tris-HCI buffer, skin cytosol and
microsomes, and liver cytosol and microsomes. The hydrolysis rate
constants of (@-isomers of the prodrugs were 1.1-30.3 times greater
than those of the respective (9-isomers in tissue preparations. Skin
showed considerable metabolic activity and very high stereoselectivity
(RIS ratio: 7.3-30.3). The hydrolyzing capacities of buffer and different
tissue preparations per milligram of protein content were in the following
increasing order: buffer < skin homogenatec plasma c liver homogenate.
The studies with microsomes and cytosol indicated that the esterases,
which are responsible for the hydrolysis of prodrugs, were mainly present
in the cytosolic and microsomal fractions of skin and liver, respectively.
There was a good correlation between the octanol-buff er partition
coefficients of propranolol and its prodrugs and the skin partition coefficient.
In vitro stereoselective penetration of propranolol and the prodrugs through
full-thickness hairless mouse skin was evaluated with flow-through diffusion
cells. Although the concentration of propranolol was 14-22 times greater
than those of the prodrugs in the donor chamber, the steady-state flux of
propranolol isomers [10.72 and 10.64 pg/cm2.h for (4-and (9-isomers,
respectively] were similar to those of CP-PL [10.80 and 10.78 pg/cm2.h

for (4-and (9-isomers, respectively] and even lower than those of IVPL [14.51 and 14.33 pg/cm2.h for (@- and (9-isomers, respectively].
Moreover, the permeability coefficients of IV-PL t2.82 x
and 2.78 x
c d h for (4-and (Sj-isomers, respectively] and CP-PL (1.29 x
c d h for each isomer) were 14-30-fold greater than those of
propranolol isomers (0.09 x
cmlh for each isomer). The diffusion
coefficients of all the compounds were similar, but their solvent membrane
distribution coefficients differed greatly and proved that the higher
permeability coefficients of the prodrugs were due to the higher affinity
of the prodrugs for skin. Neither propranolol nor the prodrugs showed
stereoselective penetration. However, highly stereoselective hydrolysis
occurred during penetration of the prodrugs, and the RIS ratios of the
cumulative amount of delivered propranolol in 12 h were 11 and 13 for
IV-PL and CP-PL, respectively. A skin irritation test was performed in
Japanese white male rabbits and no irritation was observed. In conclusion,
the hairless mouse skin possesseshighly stereoselective esterase activity,
and IV-PL and CP-PL might be promising prodrugs for transdermal delivery
of higher amounts of drug from a much lower initial concentration
compared with propranolol.

tion of ester prodrugs. However, because the skin is not an
inert organ as commonly believed (it is actually a highly active
~ r g a nand
~,~
contains a multitude of different enzymes that
can metabolize a wide range of synthetic and naturally
occurring xenobioti~s~,~),
the prodrugs might be hydrolyzed
to propranolol during permeation through skin, which in turn
could affect the penetration of intact prodrug. Therefore, it
is necessary to study the hydrolysis of the prodrugs in skin.
Also, skin possesses many of the same enzymes as liver and
plasma, so it is also necessary to compare skin enzyme activity
with enzyme activity of liver and plasma to evaluate the
potential of the skin as a metabolizing organ. Propranolol
shows stereoselective activity; this is the S(-)-isomer is 100
times more active than the R(+)-isomer as P-bl~cker.~
Stereoselective hydrolysis and penetration may therefore play an
important role in the delivery of the active isomer of propranolol as well as its prodrugs. Unfortunately, t o date, the

stereoselective hydrolysis of drug in skin has been overlooked
and, although transdermal delivery has received considerable
attention in recent years, little attention has been paid to
transfer characteristics of individual enantiomers of chiral
species.lOJ1Moreover, regulatory authorities are demanding
more information concerning all aspects of the administration
of chiral drugs,12including those delivered transdermally. All
these reasons prompted us to conduct studies considering
stereoselectivity.
The purpose of this study was to evaluate the increase of
percutaneous penetration of propranolol by prodrug formation,
to evaluate the stereoselective hydrolyzing activity of skin
compared with that of liver and plasma, to determine the
location of the esterases, and to study skin irritation caused
by the drugs in rabbit.

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Experimental Section

Materials-Racemic propranolol hydrochloride was obtained from

Sigma Chemical Company (St. Louis, MO). Bovine serum albumin
(fraction V, Sigma Chemical Company) was purchased. All other
chemicals and reagents used were of analytical grade. The prodrugs
were synthesized from the racemic propranolol hydrochloride as the
hydrochloride salt (99.9% pure) according to a previously described
method.13 Propranolol hydrochloride was used in prodrug synthesis
to protect the amino group from amido formation with fatty acid.
Moreover, the esters of propranolol base are unstable because they
undergo intramolecular catalyzed hydrolysis and intramolecular O-N
acyl transfer reaction. So, in our study, propranolol and the prodrugs
were always used as the hydrochloride salts.
Propranolol, a nonselective P-adrenergic receptor antagoAnimals-Male hairless mice (8-9 weeks old; Kyudo, Fukuoka,
nist, has a very low and variable oral bioavailability because
Japan), weighing 28-32 g, and Japanese white male rabbits (average
of extensive stereoselective hepatic first-pass metabolism.1*2 weight, 2.5 kg; Kyudo, Fukuoka, Japan) were used for hydrolysis and
To avoid hepatic first-pass metabolism, the transdermal route
penetration experiments and the skin irritation test, respectively. The
can be used as an alternative route of drug a d m i n i s t r a t i ~ n . ~ ? animals
~
were kept at room temperature (25 f 1 "C) and given a

Propranolol hydrochloride, which is the commercially availcommercial diet and tap water ab libitum.
able form of propranolol, is hydrophilic in nature and its
Solubility Determination-A normal equilibrium solubility determination was undertaken in 0.01 M acetate buffer (pH 4, 37 "C).
absorption through skin is very poor. To increase its percuTo 1 mL of buffer, -30 mg of prodrug or 300 mg of propranolol was
taneous penetration, the drug was made lipophilic by formaadded. The samples were sonicated for 30 min a t room temperature,
then shaken mechanically in a temperature-controlled waterbath a t
37 "C for 6 h. The supernatant was then filtered through cotton and,
Abstract published in Advance ACS Abstracts, May 15, 1995.

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@

0 1995, American Chemical Society and
American Pharmaceutical Association

0022-3549/953184-0877$09.00/0

Journal of Pharmaceutical Sciences / 877

Vol. 84, No. 7, July 1995

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after dilution, the solubility of each isomer from the racemic mixture
was determined by HPLC.
Dissociation Constant (p&) Determination-The pKa values
for the prodrugs were determined by a normal potentiometric titration
(logarithmic) method a t 25 "C. The determination of pK, was made
with a Horiba F11 pH-meter equipped with a two decimal digit display
of pH and a glass-electrode combined with a silver-silver chloride
reference system. Solutions of propranolol and the prodrugs were
prepared in water (5.4 mM, 25 mL). The solutions were titrated with
1 M NaOH in 10-pL aliquots (using a 10-pL microsyringe). To
minimize degradation, the titrant was added rapidly and the overall
titration time was limited to 4 min.
M e a s u r e m e n t of Partition Coefficient-The partition coefficients of propranolol and the prodrugs were determined in l-octanol-pH 4 phosphate buffer @ = 0.155) system. The buffer solution
and 1-octanol were mutually saturated at 25 "C before use. The
concentration of each isomer of the drugs in pH 4 buffer was measured

by HPLC before and after shaking with a n equal volume of 1-octanol
for 1 h. The partition coefficients were determined as the ratios
between the concentrations measured in 1-octanol and the buffer.
Preparation of Skin Homogenate and Its Microsomal and
Cytosolic Fractions-All operations were carried out a t 0-4 "C.
After sacrificing the mouse by decapitation, cutaneous strips were
removed from the back and the abdomen, The fat and the muscular
tissues as well as capillaries adhering to the dermis were removed.
The skin was minced, mixed with five volumes of cold Tris-HC1 buffer
(pH 7.4) containing 0.15 M KC1, and subjected to four separate bursts
of a tissue homogenizer (Ultra Turrax Antrieb T25, Ika Labortechnik,
Germany), at 20 500 rpm. There was a pause of 1 min between each
burst to permit cooling of the tissue. The whole homogenate was
filtered with a funnel through cotton soaked in the buffer and
centrifuged at 10 000 x g for 20 min a t 0 "C to remove mitochondria
and nuclei. The 10 000 x g supernatant was further centrifuged at
100 000 x g for 1 h with a Hitachi ultracentrifuge. The resulting
supernatant and pellet contained cytosol and microsomes, respectively. The microsomes were resuspended in Tris-HC1 buffer (pH 7.4)
containing 0.15 M KCl by homogenization, and isolated after a second
centrifugation at 100 000 x g for 1 h. The 10 000 x g supernatant

and microsomal and cytosolic fractions were stored in aliquots a t -80
"C until used in the hydrolysis experiments.
Storage of liver preparations by freezing is a widely used technique,
and there are also some reports suggesting this method for skin
preparations.14J5 Experiments with fresh versus frozen skin preparations, used in the present studies, showed no alteration of esterase
activities due to freezing. Skin preparations were used within 1
month of storage at -80 "C, and the esterase activities underwent
no significant changes during this period.
Preparation of Liver Homogenate and Its Microsomal and
Cytosolic Fractions-The liver was removed from the animal and
throughly washed with 0.15 M KCl. The liver was minced, added to
three volumes of cold 0.15 M KC1, and homogenized with a PotterElvehjem homogenizer. The homogenate was centrifuged a t 10 000
x g for 20 min a t 0 "C. Cytosolic and microsomal fractions were
prepared according t o the procedure described for skin, using 0.15 M
KC1 instead of Tris-HC1 buffer (pH 7.4) containing 0.15 M KC1. The
10 000 x g supernatant and microsomal and cytosolic fractions were
stored in aliquots a t -80 "C until used.
Plasma-Hairless mice were sacrificed by decapitation, the blood
was collected in a heparinized tube and centrifuged a t 3000 rpm, and
the plasma was collected. Due to the paucity of plasma, it was diluted

to 5%with Tris-HC1 buffer (pH 7.4) containing 0.15 M KCl and used
immediately.
Hydrolysis of Prodrug in Skin Preparations-All kinetic
measurements were carried out a t 37 "C in a shaking thermostatic
waterbath. The 10 000 x g supernatant, cytosol, and microsomes,
after appropriate dilution with Tris-HC1 buffer (pH 7.4), were used.
The protein contents of the 10 000 x g supernatant from skin
(16.67%),of the cytosol, and the microsomes were 7.35,6.35, and 3.59
mg/mL, respectively, as determined by the method of Lowry e t a1.I6
and with bovine serum albumin a s the reference standard. The
reaction was initiated by addition of 4 yL of a stock solution of the
prodrug in dimethyl sulfoxide (DMSO; 0.05 M) with a microsyringe
to 4 mL of the skin preparation, which had been preincubated for 10
min. At appropriate intervals, 100-yL aliquots were withdrawn and
added to 300 ,uL of acetonitrile and 100pL of 0.01 M phosphoric acid
kept in an ice-water bath to deproteinize and to prevent further

hydrolysis. After centrifugation at 3000 rpm and 0 "C for 5 rnin the
clear supernatant was filtered through a poly(tetrafluoroethy1ene)
(PTFE) 0.5-pm pore size filter and stored at 0 "C until analysis by

HPLC. The adsorption of drug by PTFE was checked and found to
be nil. A 20-yL aliquot of the sample was loaded onto the column.
First-order rate constants for the hydrolysis were determined from
the slopes of linear plots of the logarithm of residual prodrug against
time. Heat treatments were performed by heating skin homogenates
a t 50 and 60 "C for 10 rnin before use.
Hydrolysis of Prodrug in Liver Preparations-The liver
hydrolysis study was performed as described for skin homogenate
using stock solutions that were diluted to 0.5% (protein content: 512
pglmL) with Tris-HC1 buffer (pH 7.4) containing 0.15 M KC1 just
before use. The same procedure was followed with liver cytosol and
microsomes, which were diluted before use to adjust to about the
protein content of the liver homogenate (548 and 514 pg/mL,
respectively) with Tris-HC1 buffer (pH 7.4) containing 0.15 M KCl.
Hydrolysis of Prodrug in Plasma-The reaction was initiated
as just described using 5.0%(v/v) plasma (protein content: 3.25 mgl
mL) in Tris-HC1 buffer (pH 7.4) containing 0.15 M KCl.
Hydrolysis of Prodrug in Buffer-The study on the hydrolysis
of the prodrugs was also performed in Tris-HC1 buffer (pH 7.4)
containing 0.15 M KC1 according to the procedures just described for
plasma, but without centrifugation.
Skin Partitioning Determination-Distribution of the compounds studied between hairless mouse skin and pH 4 phosphate
buffer was estimated by the methods of Scheuplein17 and Durrheim
and Flynn18 as the difference between the initial and equilibrium
aqueous phase concentration. The relationship used was
(1)

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where C, is the initial drug solution concentration, C, is the solution
concentration at equilibrium, W, is the tissue weight expressed in
grams, and V,, is the solution phase volume expressed in milliliters.
Therefore, the partition coefficients are expressed in cubic centimeters
per gram. Given the obvious heterogeneity (multiphase nature) of
the tissue, no attempt was made to factor in density to obtain unitless
values. The hairless mouse skin-pH 4 buffer partition coefkients
were evaluated by equilibrating propranolol and prodrug solutions
with a known mass of skin (-200 mg) immediately after skin removal
from the animal. The skin pieces were immersed in 4 mL of
phosphate buffer that contained 5 mM drug. The samples were
equilibrated in a mechanical waterbath-shaker a t 37 "C for 6 h, which
was a sufficient amount of time t o attain equilibrium. The initial
and equilibrium concentrations of each drug in the aqueous phase
were determined by HPLC.
In Vitro Penetration Study-The in vitro percutaneous penetration study was performed with a flow-through diffusion cell.19 The
receiving chamber had a volume of -4 mL and the area available for
diffusion was 1.02 cm2. The membrane used was a full-thickness skin
sample taken from the dorsal surface of the hairless mouse. Hairless
mice were sacrificed by snapping the spinal cord a t the neck. A
rectangular section of dorsal skin was excised from the animal with
surgical scissors. Adhering fat and other visceral debris were removed
from the undersurface with tweezers. The excised skin was immediately mounted between the half-cells, with the dermis side in
contact with the receptor fluid (0.01 M acetate buffer, pH 4). One
milliliter of drug suspension (drug amount: 1.2 times of the amount
required for saturation) in pH 4 acetate buffer was added to the donor
half-cell. Both half-cells were maintained a t 37 "C by a n external
circulating waterbath. The receptor chamber was perfused with
receptor fluid at a rate of 5 mUh with a pump (Roller Pump; Furue
Science Company Ltd., Japan). Fractions were collected a t definite
time intervals and assayed for the presence of propranolol and
prodrugs. To minimize problems with this tissue,20-22studies were
conducted for only 12 h.
D e t e r m i n a t i o n of Prodrug and Propranolol in Penetration
Study-Two milliliters of the collected sample was added to 1 mL of
0.1 M phosphate buffer (pH 4) that was saturated with NaCl.
Propranolol and intact prodrug were simultaneously extracted by 6
mL of ether. After shaking for 10 min, 5 mL of the organic phase
was evaporated to dryness under reduced pressure and dissolved in
either 100 ILLof mobile phase from reversed phase HPLC for the
determination of prodrugs or 100 pL of 2-propanol for the determi-

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878 /Journal of Pharmaceutical Sciences
Vol. 84, No. 7, July 1995

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nation of propranolol. Then, 20 pL of each of these solutions was
injected onto the HPLC system.
Chromatography-An Hitachi 655 A-11 liquid chromatograph
equipped with a fluorescence spectrophotometer (Hitachi F-1050,
Hitachi Company Ltd., Japan) was used at excitation (Aex) and
emission (Aem) wavelengths of 285 and 340 nm, respectively. Concentrations of prodrug and propranolol were determined with an
Ultron ES/OVM column (150 x 4.6 mm i.d., Shinwa Chemical
Industries, Japan) and a Chiralcel OD column (250 x 4.6 mm i.d.,
Daicel Chemical Industries Ltd., Japan), respectively. The eluent
used for prodrug determination consisted of 20 mM KH2P04 (pH
4.6): acetonitrile [80:20 and 83:17 (v/v) for IV-PL and CP-PL,
respectively], and the eluent used for propranolol determination
consisted of n-hexane:ethanoldiethylamine[85:15:0.6(&)I. Constant
flow rates of 0.5 m u m i n (ES/OVM column) and of 0.6 m u m i n (OD
column) were maintained with the columns at ambient temperature.
Samples of 20 pL were injected onto the chromatograph.
Skin Irritation Test-Primary skin irritations caused by 0.1, 1,
and 10% drug solutions dissolved i n PEG 400 and PEG 400:water
(3:1),respectively, were determined with Japanese white male rabbits.
The drug solutions (0.5 mL) were applied to the intact and abraded
skin areas of each of four rabbits (each group contained four rabbits).
Skin irritation was evaluated at 24,48,76,and 96 h after application
of drug by the method described by
Calculation of Permeation Parameters-The in vitro permeation parameters were calculated from the penetration data with the
following equations:

Table 1-Solubility, Partition Coefficients (PC) and Dissociation
Constants (pKJ of Propranolol and its Prodrugs
OR

I

S)CH$HCH~NHCH(CH-J)~

Propranolol / Prodrug

compound

R

Solubility, mg/mLa

PCb

pKac

Propranolol
IV-PL
CP-PL

H
COCHzCH(CH3)z
COCC~HS

115.87
5.15
8.35

2.4
88.9
14.4

9.44
8.59
8.72

Determined in pH 4 acetate buffer at 37 "C;the values represent the solubility
of each isomer from racemate. Done in octanol-pH 4 phosphate buffer =
0.155) at 25 "C; the values are same for both the isomers. CDetermined in
deionized water at 25 "C.

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J , = (K,DC)IG = KpC

(2)

z = SZl6D

(3)

where J, is the steady-state flux,K, denotes the solvent membrane
partition coefficient of drug, D is the diffusion coefficient, C is the
drug concentration in the donor chamber, 6 is the thickness of mouse
skin (0.07 cm), Kp denotes the permeability coefficient of drug, and 5
represents the lag time. The permeation parameters were finally
calculated with a nonlinear least square computer program (MULTI).%
Statistical Analysis-Results of hydrolysis and penetration experiments were expressed a s the mean i SD. The student's t test
was applied, where necessary, to evaluate significance of difference.

Results and Discussion
Physicochemical Properties-Physicochemical parameters, such as aqueous solubility,have been shown to influence
formulation variables, activity, and pharmacokinetic profiles.
Therefore, the physicochemical properties of propranolol and
the prodrugs are important, especially in determining the
loading and release properties of a transdermal therapeutic
device. The ester derivatives of propranolol used in this study
undergo chemical hydrolysis, particularly under alkaline
~0nditions.l~Therefore, they are too unstable for normal
equilibrium solubility determination in water. To overcome
this problem, the solubility of the prodrugs was determined
in pH 4 acetate buffer in which the prodrugs proved to be most
stable. Analysis by HPLC showed insignificant degradation
(