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

SHAMIM AHMED, TERUKO IMAI', AND MASAKI OTAGlRl

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@.

Abstract 0 Stereoselective hydrolysis of two ester prodrugs of propra- nolol, isovaleryl propranolol (IV-PL) and cyclopropanoyl propranolol (CP- PL), 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 homogenate c 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 IV- PL [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 possesses highly 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.

Propranolol, a nonselective P-adrenergic receptor antago- nist, has a very low and variable oral bioavailability because of extensive stereoselective hepatic first-pass metabolism.1*2 To avoid hepatic first-pass metabolism, the transdermal route can be used as an alternative route of drug adminis t ra t i~n.~?~ Propranolol hydrochloride, which is the commercially avail- able form of propranolol, is hydrophilic in nature and its absorption through skin is very poor. To increase its percu- taneous penetration, the drug was made lipophilic by forma-

@ Abstract published in Advance ACS Abstracts, May 15, 1995.

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 n ~ , ~ and 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.~ Ste- reoselective hydrolysis and penetration may therefore play an important role in the delivery of the active isomer of propra- nolol as well as its prodrugs. Unfortunately, to 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.lOJ1 Moreover, regulatory authorities are demanding more information concerning all aspects of the administration of chiral drugs,12 including 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.

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.

Animals-Male hairless mice (8-9 weeks old; Kyudo, Fukuoka, Japan), weighing 28-32 g, and Japanese white male rabbits (average weight, 2.5 kg; Kyudo, Fukuoka, Japan) were used for hydrolysis and penetration experiments and the skin irritation test, respectively. The animals were kept at room temperature (25 f 1 "C) and given a commercial diet and tap water ab libitum.

Solubility Determination-A normal equilibrium solubility de- termination was undertaken in 0.01 M acetate buffer (pH 4, 37 "C). To 1 mL of buffer, -30 mg of prodrug or 300 mg of propranolol was added. The samples were sonicated for 30 min a t room temperature, then shaken mechanically in a temperature-controlled waterbath at 37 "C for 6 h. The supernatant was then filtered through cotton and,

0 1995, American Chemical Society and American Pharmaceutical Association

0022-3549/953 184-0877$09.00/0 Journal of Pharmaceutical Sciences / 877 Vol. 84, No. 7, July 1995

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 at 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.

Measurement of Partition Coefficient-The partition coef- ficients of propranolol and the prodrugs were determined in l-oc- tanol-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 an 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 at 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, respec- tively. 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 prepara- tions, 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 Potter- Elvehjem 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 to 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 et a1.I6 and with bovine serum albumin as 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 100 pL 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 com- pounds 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)

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 to 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 penetra- tion 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 im- mediately 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 an 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,20-22 studies were conducted for only 12 h.

Determinat ion 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 ILL of mobile phase from reversed phase HPLC for the determination of prodrugs or 100 pL of 2-propanol for the determi-

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

nation of propranolol. Then, 20 pL of each of these solutions was injected onto the HPLC system.

Chromatography-An Hitachi 655 A-1 1 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. Con- centrations 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 mumin (ES/OVM column) and of 0.6 mumin (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 in 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 perme- ation parameters were calculated from the penetration data with the following equations:

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 ex- periments were expressed as the mean i SD. The student's t test was applied, where necessary, to evaluate significance of difference.

Results and Discussion Physicochemical Properties-Physicochemical param-

eters, 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 (<0.05%) of the esters during the solubility determination. As shown in Table 1, the solubility of the esters decreased many- fold compared with propranolol, and IV-PL was the least soluble. There was no difference between the solubility of (R)- and (5')-isomers when solubility was determined from a racemic mixture. The actual value of the solubility of the racemic mixture is twice the value mentioned in Table 1. The partition coefficient values of the prodrugs in octanol-pH 4 buffer increased markedly compared wtih that of propranolol and complied with the solubility in respective order. Partition coefficients of (R)- and (S)-isomers were always the same. Lipophilicity is very important for transdermal penetration because the stratum corneum, the major barrier of drug penetration, is lipid in nature and generally favors the penetration of lipophilic drugs. The data shown in Table 1

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

OR I

S)CH$HCH~NHCH(CH-J)~ a- Propranolol / Prodrug

compound R Solubility, mg/mLa PCb pKac

Propranolol H 115.87 2.4 9.44 IV-PL COCHzCH(CH3)z 5.15 88.9 8.59 CP-PL COCC~HS 8.35 14.4 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.

provide a usable estimate of the pK, for propranolol of 9.44, which compares favorably with the values of 9.20 and 9.51 reported p r e v i o u ~ l y . ~ ~ , ~ ~ The pK, values of the two prodrugs are slightly decreased compared with that of propranolol, and this type of decrease in p& value was previously reported with some other 0-acyl prodrugs of p r o p r a n o l ~ l . ~ ~ , ~ ~ A small change in pK, values indicates a slight effect on the dissociation of drug in skin because all compounds, being salts of amines, will be primarily in their ionized forms due to their higher pK, values (8.59-9.44). Furthermore, the protonation that may occur on contact with the acidic environment (-pH 5.5) of the skin surface will be of the same magnitude for all the three drugs.

Hydrolysis in Buffer and Tissue Preparations-The hydrolysis of two prodrugs in buffer and different tissue preparations exhibited typical first-order kinetics, and the hydrolysis rate constants of the prodrugs are shown in Table 2. On the basis of protein content (mg), the hydrolysis rate was greatest in liver homogenate followed by plasma and skin homogenate, and the lowest rate was observed in the buffer. No stereoselectivity was observed in the buffer; this result is in accord with an earlier report on hydrolysis of 0-acetyl propranolol in buffer.2s But, the hydrolysis rate of (R)-isomer was faster (1.21 to 16.67 times) than that of the respective @)-isomer in tissue preparations; this type of stereoselectivity was also observed in dog plasma and 1 i ~ e r . l ~ ~ ~ ~ It is evident that the highest stereoselectivity was observed in skin homo- genate (RJS ratios: 9.82 and 16.67 for IV-PL and CP-PL, respectively); this high stereoselectivity is mainly due to the high resistance of the (5')-isomers of the prodrugs to hydrolysis in skin homogenate. Stereoselectivity of CP-PL in skin homogenate was greater than that of IV-PL. Moreover, the hydrolysis rates of CP-PL in skin homogenate were faster than those of IV-PL. Apparently, a relatively high amount of esterases that are responsible for the hydrolysis of CP-PL is located in hairless mouse skin. In plasma, the hydrolyzing activity was similar for both the prodrugs. However, in liver, IV-PL was preferentially hydrolyzed compared with CP-PL. These data suggest that either the hydrolytic enzymes are different in various tissue preparations or the same enzymes behave differently with the prodrugs in various tissue prepa- rations, which might be due to their different concentration and proportion in each tissue. As already mentioned, the (S)- isomer is pharmacologically 100 times more potent as a B-adrenoreceptor blocker than the (R)-isomer, so the bioavail- ability of the (S)-isomer is important. Interestingly the (S)- isomers were relatively more stable than the (R)-isomers of the prodrugs, suggesting that (R)-propranolol converted from (R)-prodrug will be preferentially metabolized in liver and other metabolizing organs. Therefore, (S)-propranolol be-

Journal of Pharmaceutical Sciences / 879 Vol. 84, No. 7, July 1995

Table 2-Stereoselective Hydrolysis Rate Constants of Propranolol Prodrugs in Buffer and Different Tissue Preparations of Hairless Mouse at 37 "C, and Comparison of the Rate Constant in Skin Homogenate with Liver Homogenate and Plasma per Protein Weight (A) and per Tissue Weight (6)

Rate Constant x 1 O3 Ratio, %

Buffera Plasmab Liver Homogenateb Skin Homogenateb Skin/Liver Skin/Plasma (PH 7.41, (5%), (1 0 OOOxg), (10 OOOxg),

prodrug (min-l) min-lmg-l min-l.mg-' min-l.mg-' (A) (6) (A) (6)

IV-PL (4 0.46 k 0.02 47.05 f 4.96 155.98k 19.11 1.67 k 0.26 1.07 0.46 3.55 2.41 IV-PL (SJ 0.46 f 0.05 31.79 k 5.75 78.50 k 14.21 0.17 5 0.05 0.22 0.09 0.53 0.36 ais 1 .oo 1.48 1.99 9.82

CP-PL (s) 0.58 k 0.04 12.89 f 0.13 34.99 k 8.03 0.31 k 0.06 0.89 0.38 2.40 1.63 RIS 0.98 3.54 1.21 16.67

NOTE: Each value is the mean f SD ( n = 4). a Tris-HCI buffer (pH 7.4) containing 0.15 M KCI. In tissue preparations, hydrolysis rate constant was determined

CP-PL (R) 0.58 f 0.04 45.57 f 0.26 42.45 f 5.65 5.20 _+ 0.71 12.25 5.27 11.41 7.74

per milligram of protein. Protein content: plasma (5%), 3.25 mg/mL; liver homogenate, 512 ,ug/mL; skin homogenate, 7.35 mg1mL.

Table 3-Stereoselective Hydrolysis Rate Constants (per Protein Weight) of Two Prodrugs in Microsomes and Cytosols of Liver and Skin of Hairless Mouse at 37 "C

Rate Constant x lo3, min-'.mg-'

Liver Skin

Compound Microsomes Cytosol Microsomes Cytosol

IV-PL (RJ 771.58 f 109.82 19.23 f 1.55 0.76 f 0.01 3.93 k 0.13 IV-PL (SJ 357.43 f 32.30 7.49 f 1.52 0.10 k 0.01 0.26 f 0.04

CP-PL (R) 360.88 i 31.80 9.38 f 0.59 2.93 f 0.12 7.88 f 0.48 CP-PL (SJ 290.02 f 49.84 8.51 f 0.78 0.40 f 0.06 0.26 k 0.00

(44s) 2.16 2.57 7.60 15.12

(R)/(SJ 1.24 1.10 7.33 30.31

NOTE: Each value is the mean k SD (n = 3). Protein content: liver microsomes and cytosol, 514 and 548 pg/mL, respectively; skin microsomes and cytosol, 3.59 and 6.35 mg/mL, respectively.

comes more effective at avoiding metabolism, which can further help in improving the bioavailability of propranolol.

Skin possesses many of the same enzymes as liver and plasma, so it would be interesting to compare their relative activity. The activity of skin, per protein (column A in Table 2) and tissue weight (column B in Table 2) as compared with that of liver and plasma are shown in Table 2. It is evident from the data in the table that, considering per protein weight, skin possesses low metabolic activity, especially for the (5')- isomers of the prodrugs, and the comparative enzyme activity of skin towards CP-PL is greater. The metabolizing activity of skin compared with that of liver was similar to that published in previous report^.^^^^ Considering per tissue weight, cutaneous activity seems to be very low compared with that of hepatic activity, but these data may not be representa- tive of the true picture because, during homogenate prepara- tion, we used full-thickness mouse skin but the enzymes are present mainly in the epidermis layer,31 which is only -4% of total mouse skin.32 So, the real activities range from 2 to 130% of those in the liver. Moreover, the hydrolyzing activity ratio of skidplasma was greater than that of skidliver considering both per protein and tissue weight. All these results suggest that skin possesses considerable metabolic activity.

Location of Enzymes-To find out the location of en- zymes, experiments to determine hydrolysis of prodrugs in the microsomal and cytosolic fractions of skin and liver were performed. Stereoselective hydrolysis was observed in all the fractions, and the (R)-isomers were more susceptible to hydrolysis (Table 3). In skin, the cytosol was more enzymati- cally active than the microsomes and the reverse was observed in liver cytosol and microsomes; these results are in agreement with previous reports with mouse and other animals.29s33-35 The enzymatic nature of the hydrolytic reaction is further substantiated by experiments in which the cutaneous 10 000

x g preparations had undergone a heat treatment prior to their addition to the incubation mixture. These results indicate (data not shown) that pretreatment at 50 "C has little effect on the biological activity. After treatment a t 60 "C, however, the activity is markedly lost. The increase from 50 to 60 "C thus provides a sudden and extensive loss of activity, a behavior compatible with enzyme denaturation. Further- more, the relative heat stability is suggestive of a soluble (cytosolic) enzyme.34 It is also clear from the data in Table 3 that the microsomal and cytosolic fractions of skin showed very high stereoselectivity (RIS ratio: 7.33-30.31) compared with that of liver fractions, indicating that the esterases of skin might be different from those of liver. In liver, both the cytosolic and microsomal fractions showed similar stereose- lectivity. Interestingly, however, the hydrolysis rates of (R)- isomers of both the prodrugs in skin cytosol were much higher than those in skin microsomes, whereas the hydrolysis of (S)- isomers was very slow and similar in both fractions of the skin. Therefore, the stereoselectivity of the cytosolic fraction of skin is significantly higher than its microsomal fraction. However, because the skin cytosol contains high amounts of esterases, the prodrugs might be easily hydrolyzed to propra- nolol during penetration.

Skin Partitioning-The partitioning of the prodrugs and propranolol a t 37 "C between hairless mouse skin and pH 4 buffer was measured. An important relationship between hydrophobicity as measured by the octanol-pH 4 buffer partition coefficient and the skin partition coefficient (R = 0.995) is shown in Figure 1. The relationship with the hydrophobicity is in agreement with the expectation that the more hydrophobic the derivative is, the easier it should be for the drug to bind to or t o partition into the skin. As already mentioned, a t pH 4, prodrugs and propranolol will be prima- rily in their ionized forms, and the positive charge on the amine might impart a similar degree of polarity for all the three drugs. Apparently, this could not overrule hydrophobic- ity in partitioning, as is evident from the highly linear relationship seen in Figure 1.

Percutaneous Penetration-The best way to determine the penetration potential of a compound is to perform the actual study in humans. But studies with living human subjects are costly and time consuming because of the neces- sary safeguards, including approved clinical protocols.1* On the other hand, in vitro studies by S t ~ u g h t o n ~ ~ using human and hairless mouse skin, which is both economic and readily available, showed remarkable similarities in absorption be- tween the skin of the two species for many drugs. Little is known about skin esterases and there are few reports in which hairless mouse skin was used as a model for the study of esterase activity in transdermal de1ive1-y.~~ In Figure 2, the cumulative penetrated amount of propranolol and the pro- drugs (sum of intact prodrug and delivered propranolol)

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

0.6

8 0.4 5 n

Ia *

5 0.2 2 9 0.0

(I)

m

-0.2 ' ' 1 I

0.0 0.5 1 .o 1.5 2.0

log PCOctanol/pH 4 buffer

Figure 1 -Plot of skin partition coefficient against hydrophobicity for propranolol and its prodrugs.

-

-

-

.

N- 120 r

0 2 4 6 8 1 0 1 2

Time (h)

Figure 2-Stereoselective penetration of propranolol and the prodrugs (total amount; both intact prodrug and its delivered propranolol) across full-thickness hairless mouse skin at 37 "C. The donor concentrations were maintained at the aqueous saturation concentrations of the drugs; that is, 115.87, 8.35, and 5.15 mg/mL for each isomer of propranolol, CP-PL, and IV-PL, respectively. Each value is the mean k SD (n = 3). Key: (0) IV-PL (R); (0) CP-PL (R); (A) propranolol (R); (0) IV-PL (9; (m) CP-PL (9; (A) propranolol (9.

through hairless mouse skin was plotted against time, and the resulting curves indicate that both propranolol and the prodrugs have similar penetration. No stereoselectivity was observed during penetration of propranolol or the prodrugs. Although Miyazaki et al.l0 reported stereoselective penetration of propranolol through rat skin, Heard et al." proved that human skin did not cause stereoselective penetration of propranolol, a result which further supports the suitability of hairless mouse as a model for human skin for in vitro study. The permeation parameters are depicted in Table 4. The highest flux value was obtained with IV-PL, whereas CP-PL and propranolol showed similar flux values. It is interesting to note that although a 14-22 times higher concentration of propranolol than prodrugs was used in the donor compart- ment, the flux of propanolol was still lower or almost equal to those of the prodrugs. The permeability coefficients of the prodrugs are enhanced 14-fold (CP-PL) and 30-fold (IV-PL) compared with propranolol. The permeability coefficient is independent of donor concentration, unlike flux, so this parameter is important for the comparison of permeation among drugs. Therefore, the higher permeability coefficients

Table 4-Steadystate Flux (&), Permeability Coefficient (Kp), Solvent Membrane Distribution Coefficient (K,,,), Diffusion Coefficient (a), and Lag Time (t) of the Isomers of Propranolol and the Prodrugs through Full-Thickness Hairless Mouse Skin

Compound J$, (ugkm2.h Kpx lo4, cm/h k, D x lo4, cmYh 5, h

Propranolol (4 10.72 k 0.55 0.93 k 0.05 0.05 f 0.01 1.45 k 0.32 5.81 k 1 .I9 Propranolol (s) 10.64 i 0.65 0.92 A 0.06 0.05 f 0.01 1.46 k 0.30 5.74 f 1.08 IV-PL(4 14.51 i4.25 28.17f8.25 1.53k0.71 1.36A0.23 6.12k1.06 IV-PL (s) 14.33 k 4.93 27.82 i 9.57 1.52 k 0.93 1.42 k 0.36 6.01 i 1.63 CP-PL(FI) 10.8Ok2.13 12.93k2.55 0.55k0.23 1.78k0.55 4.98k1.85 CP-PL(S) 10.78k2.59 12.91 f3.10 0.58+0.24 1.8OkO.54 4.88i1.72

NOTE: Values are the mean k SD (n = 3).

observed for the prodrugs could support their superiority over propranolol for transdermal delivery. However, the differ- ences in permeability coefficient depend on different physi- cochemical characteristics of the compounds; for examples, as shown in eq 2, the permeability coefficient depends on solvent membrane distribution coefficient, diffusion coefficient, and membrane thickness. Membrane thickness is almost constant because the skin of hairless mouse was always used. The diffusion coefficient should not differ much because it depends on the structure, molecular weight, and properties of solute, which are closer among the drugs. Therefore, the perme- ability coefficients of propranolol and the prodrugs should vary due to their variation in solvent membrane distribution coefficient values. This conclusion was supported by the values of D and K, mentioned in Table 4; that is, the affinity to the skin in relation to the buffer was greatest for IV-PL followed by CP-PL and then propranolol, and this order was maintained in the K, as well as Kp values of the three compounds. The skin-buffer partition coefficient was also linearly correlated with K , [K, = 0.49(skin PC) - 0.31; r = 0.9931. These results show that by doing only this simple experiment we can choose better prodrugs of propranolol for transdermal delivery in near future. The lag time was lowest with CP-PL, and IV-PL and propranolol showed comparable values. However, in all the permeation parameters, there were no stereoselective differences between the isomers. Finally, from the flux and the permeability coefficient values it can be concluded that both the prodrugs are promising, especially if the goal is to get greater penetration with a lower amount of drug. Greater penetration with a lower concentra- tion of drug is always preferable and it is also necessary to minimize skin irritation, which generally increases with an increase of drug concentration in the transdermal device.

Although no stereoselectivity was observed in the pen- etrated amount of total prodrug (both as intact and delivered propranolol), there was stereoselectivity in the contents of both intact prodrug and its delivered propranolol in the receptor fluid. Although the (S)-isomers of intact prodrugs were always greater than (R)-isomers, the stereoselective differ- ences were very small due to low hydrolysis (5-10% of penetrated amount of total drug) of the prodrugs during penetration. One possible reason for low hydrolysis was washout of enzymes during penetration, but no esterase leaching was found when the esterase activity of washed buffer was checked every 2 h up to 12 h by a separate experiment (data not shown). The low hydrolysis was perhaps because the prodrugs contacted only a very small area of skin (only 1.02 cm2) and therefore a lower amount of esterases for hydrolysis during penetration. However, when the hydrolyzed propranolol from the isomers of the prodrugs were compared, significant difference was observed. The propranolol delivered from IV-PL and CP-PL are shown in Figures 3A and 3B, respectively. The amount of (R)-isomers of propranolol de- livered from both the prodrugs were remarkably greater than the amount of (S)-isomers. The difference is statistically significant from 4 h, and the RIS ratios of cumulative amount

Journal of Pharmaceutical Sciences / 881 Vol. 84, No. 7, Ju/y 1995

A Conclusions

d 2 4 6

1 I I WL 8 10

1 12

2 4 6 8 1 0 1 2

Time (h) Figure 3-Stereoselective hydrolysis of IV-PL (A) and CP-PL (B) during penetration through hairless mouse skin. Open and closed bars represent (R)-and (5)-propranolol, respectively. Each value is the mean ? SD (n = 3); (*) p c 0.05 compared with propranolol (5) .

of delivered propranolol from the prodrugs at 12 h were 11 and 13 for IV-PL and CP-PL, respectively. The chemical hydrolysis of the prodrugs in pH 4 buffer was nonstereose- lective and insignificant (<0.1%) during the period of penetra- tion study. Therefore, the highly stereoselective hydrolysis of the prodrugs during skin penetration was enzymatic. As we have already mentioned, the skin possesses stereoselective metabolic activity which is very high in the cytosol. Therefore, the prodrugs were partially hydrolyzed during skin penetra- tion, resulting in streoselectivity of intact prodrug as well as delivered propranolol in the receptor fluid. Finally, it should be noted that neither propranolol nor the prodrugs showed stereoselective penetration but both were stereoselectively hydrolyzed by the esterases of skin during penetration.

Skin Irritation-Skin irritation by drugs is a commonly encountered problem in transdermal delivery and limits the use of the transdermal route in drug d e l i ~ e r y . ~ ~ . ~ ~ The rabbit skin irritation test of propranolol and prodrugs used in this study has a maximum possible score of 4. Primary irritation responses of propranolol and prodrugs were 0 in all the cases, indicating no irritation. Although it is possible that propra- nolol, as well as other ,!?-blocking agents, could be skin sensitizers in rabbits and other species,40 no evidence of propranolol or prodrug skin sensitization was observed in this particular strain of rabbits.

We have demonstrated the high stereoselective hydrolytic activity of skin, especially in the cytosolic fraction, using two ester prodrugs as substrates. This activity is very important with regard to transdermal drug delivery but has been overlooked until now. The permeability coefficients were increased many-folds by prodrug formation, and the prodrugs showed fluxes equal to or greater than that propranolol from concentrations lower than that of propranolol. Stereoselec- tivity of intact prodrug was indicated by the observed concur- rent stereoselective cutaneous hydrolysis of the prodrugs. The prodrugs also did not cause any skin irritation in rabbit. These results indicate that IV-PL and CP-PL might be promising prodrugs for transdermal delivery of propranolol, but further studies are needed to confirm this.

References and Notes 1. Paterson, J . W.; Conolly, M. E.; Dollery, C. T.; Hayes, A,; Cooper,

R. G. Pharmacol. Clin. 1970.2. 127-133. 2. Walle, T.; Walle, U. K.; Wilson: M. J.; Fagan, T. C.; Graffney,

T. E. Br. J . Clin. Pharmacol. 1984, 18, 741-747. 3. Oeiso. T.: Shintani. M. J . Pharm. Sci. 1990. 79. 1065-1071. 4. Cgien, Y.' W. In Peptide and Protein Drug Delivery; Lee, V. H.

L., Ed.; Marcel Dekker: New York, 1990; pp 667-690. 5. Pannatier, A.; Jenner, P.; Testa, B.; Etter,-J. C. Drug Metab.

Rev. 1978,8, 319-324. 6. Bickers, D. R.; Kappas, A. In Extratherapeutic Metabolism of

Drugs and other Foreign Compounds; Gram, T. E., Eds.; Spectrum: New York, 1980; pp 295-318.

7. Rongone, E. L. In Adu. Med. Toxicology; pp 93-137. Vol. 4, Wiley: New York, 1977.

8. Noonan, P. K.; Wester, R. C. In Percutaneous Absorption Mechanism-Methodology-Drug delivery; Bronaugh, R. L.; Mai- bach, H. I., Eds.; Marcel Dekker: New York, 1985; pp 65-85.

9. Barrett, A. M.; Cullum, V. A. Br. J. Pharmacol. 1968, 34, 43- 45.

10. Miyazaki, K.; Kaiho, F.; Inagaki, A.; Dohi, M.; Hazemoto, N.; Haga, M.; Hara, H.; Kato, Y. Chem. Pharm. Bull. 1992, 40, 1075-1076.

11. Heard, C. M.; Watkinson, A. C.; Brain, K. R.; Hadgraft, J . Znt. J . Pharm. 1993, 90, R5-R8.

12. Bridges, J . W. Recent Advances in Chiral Seoaration: Plenum: Lonaon,' 1991; pp 1-3.

13. Shameem, M.; Imai, T.; Otagiri, M. J . Pharm. Pharmacol. 1993,

14. Yang, S. ,K.; Hsieh, Y. Y.; Chang, W. C.; Huang, J. D. Drug 45, 246-252.

Metab. DZSPOS. 1992. 20. 719-725. 15. McCrackei, N. W.;'Blain, P. G.; Williams, F. M. Biochem.

16. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J . Pharmacol. 1993,45, 31-36.

Biol. Chem. 1951.193. 265-275. 17. Scheuplein, R. J. J. Invest. Dermatol. 1965,45, 334-346. 18. Durrheim, H.; Flynn, G. L.; Higuchi, W. I.; Behl, C. R. J . Pharm.

19. Gummer, C. L.; Him, R. S.; Maibach, H. I. Znt. J . Pharm. 1987,

20. Bond, J. R.; Barry, B. W. J . Invest. Dermatol. 1988, 90, 486-

21. Bond, J. R.; Barry, B. W. J . Znuest. Dermatol. 1988, 90, 810-

22. Hinz, R. S.; Hodson, C. D.; Lorence, C. R.; Guy, R. H. J . Invest.

23. Draize, J. H. Assoc. Food Drug Off. 1959, 46-47. 24. Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. J .

25. Schoenwald, R. D.; Huang, H. S. J . Pharm. Sci. 1983,72,1266-

26. Irwin, W. I.; Belaid, K. A. Drug. Dev. Ind. Pharm. 1987, 13,

27. Buur, A.; Bundgaard, H.; Lee, V. H. L. Int. J . Pharm. 1988,42,

Sci. 1980, 69, 781-786.

40,101-104.

489.

813.

Dermatol. 1989, 93, 87-91.

Pharmabio-Dyn. 1981, 4, 879-885.

1272.

2033 - 2045.

51-60. 28. Takahashi, K.; Tamagawa, S.; Haginaka, J.; Yasuda, H.; Katagi,

29. Shameem, M.; Imai, T.; Yoshigae, Y.; Sparreboom, A.; Otagiri, T.; Mizuno, N. J . Pharm. Sci. 1992,81, 226-227.

M. J . Pharm. Sci. 1994.83, 1754-1757. 30. Pohl, R.; Philpot, R.; Fouts, J. Drug. Metab. Dispos. 1978, 4,

31. Wester, R. C.; Noonan, P. K. Znt. J . Pharm. 1980, 7, 99-110. 442-450.

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

32. Bronaugh, R. L.; Stewart, R. F.; Congdon, E. R. Toxicol. Appl. Pharmacol. 1980, 62, 481-488.

33. Wiegrebe, W.; Retrow, A. Arzneim.-Forsch. 1984, 34, 48-51. 34. Pannatier, A.; Testa, B.: Etter, J. C. Znt. J. Pharm. 1981,8,167-

37. Yu, C. D.; Gordon, N. A,; Fox, L.; Higuchi, W. I.; Ho, N. F. H. J .

38. Lammintausta, K.; Maibach, H. I.; Wilson, D. Contact Dermatitis Pharm. Sci. 1980, 69, 775-780.

1988.19.84-90. 174. 39. Finne, U'.; Urtti, A. Int. J . Pharm. 1992, 84, 217-222.

40. Cargill, R.; Engle, K.; Rork, G.; Caldwell, L. J. Pharm. Res. 1986, 35. Junge, W.; Krisch, K. Crit. Rev. Toxicol. 1975, 3, 371-434. 36. Stoughton, R. B. In Animal Models in Dermatology; Maibach, 3, 225-229.

H. I., Eds. Churchill Livingstone: New York, 1975; pp 121- 132. JS940615S

Journal of Pharmaceutical Sciences / 883 Vol. 84, No. 7, July 1995