International Journal of Pharmaceutics 289 (2005) 167–178
`
`Skin permeation of propranolol from polymeric film containing
`terpene enhancers for transdermal use
`
`Chomchan Amnuaikita,b, Itsue Ikeuchia, Ken-ichi Ogawaraa,
`Kazutaka Higakia, Toshikiro Kimuraa,∗
`
`a Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan
`b Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,
`Prince of Songkla University, Songkhla 90112, Thailand
`
`Received 18 September 2004; received in revised form 30 October 2004; accepted 7 November 2004
`
`Abstract
`
`To develop the suitable film formulations of propranolol hydrochloride (PPL) containing enhancers for transdermal use,
`polymeric film formulations were prepared by employing ethyl cellulose (EC) and polyvinyl pyrrolidone (PVP) as a film former,
`and dibutyl phthalate (DBP) as a plasticizer. Terpenes such as menthol and cineole, and propylene glycol (PG) were also employed
`as a chemical enhancer to improve the skin penetration of PPL. The film preparations were characterized in physical properties
`such as uniformity of drug content, thickness and moisture uptake capacity. Release and skin permeation kinetics of PPL from film
`preparations were examined in the in vitro studies using a Franz-type diffusion cell. The uniformity of drug content was evidenced
`by the low S.D. values for each film preparation. The moisture uptake capacity and drug release rate increased with the increase
`of PVP in each preparation. Enhancers examined in the present study also increased the moisture uptake capacity and release
`rate of PPL from the film preparations. Increasing the concentration of PPL from 1 to 2 mg/cm2 in the film enhanced the release
`rate of PPL, while no effect of enhancer concentrations on the release rate from the film preparations was observed. In vitro skin
`permeation study showed that cineole was the most promising enhancer among the enhancers examined in the present study and
`suggested that the suitable compositions of film preparation would be EC:PVP:PPL = 6:3:4 with 10% (w/w) cineole and 7:2:4 with
`10% (w/w) PG and cineole, which provided high skin permeation rates at 93.81± 11.56 and 54.51± 0.52 ␮g/cm2/h, respectively.
`© 2004 Elsevier B.V. All rights reserved.
`
`Keywords: Propranolol hydrochloride; Terpene; Transdermal absorption; Polymeric film; Cineole; Propylene glycol
`
`∗
`
`Corresponding author. Tel.: +81 86 251 7948;
`fax: +81 86 251 7926.
`E-mail address: kimura@pheasant.pharm.okayama-u.ac.jp
`(T. Kimura).
`
`Oral administration is one of the most convenient
`ways that are acceptable for patients, useful and suit-
`able for some drugs that are not subjected to intestinal
`
`1. Introduction
`
`0378-5173/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
`doi:10.1016/j.ijpharm.2004.11.007
`
`Page 1
`
`Mylan v. MonoSol
`IPR2017-00200
`MonoSol Ex. 2024
`
`

`

`168
`
`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`and/or hepatic first-pass metabolism (Kimura and Hi-
`gaki, 2002). However, there are several disadvantages
`that should be overcome for achieving the efficient drug
`therapy as follows: the intestinal and/or hepatic first-
`pass elimination, high variance in bioavailability due to
`variable condition of gastrointestinal tract, difficulty in
`long-term and rate-regulated absorption and impossi-
`bility of arbitrary drug input and its interruption (Higaki
`et al., 2003). Transdermal route is one of the potent
`alternative routes that can improve undesirable charac-
`teristics of oral administration. Particularly, as propra-
`nolol, a ␤-blocker, has a short biological half-life and
`is subjected to extensive hepatic first-pass metabolism
`(Walle et al., 1979; Sawamoto et al., 1997), propra-
`nolol must be a potential candidate for the transder-
`mal use. Recently, development of transdermal drug
`delivery systems (TDDS) has been focused on the for-
`mulation that can achieve the desirable constant rate
`of drug penetration into the systemic circulation, es-
`pecially by employing several polymers as matrices
`or membranes controlling the release of drugs (Kou,
`2000). On the other hand, the impermeability of human
`skin is still a fundamental problem to be overcome for
`the therapeutic use of TDDS (Barry, 2001a). Although
`many approaches have been proposed to overcome the
`stratum corneum, a main barrier for transdermal drug
`absorption (Higaki et al., 2003), chemical approaches
`such as a utilization of chemical enhancers might be
`only applicable to patch preparations. Among many
`enhancers examined, terpenes have been extensively
`investigated for their clinical use as an penetration en-
`hancer and suggested to increase drug diffusivity in
`the skin by disrupting the intercellular lipid packing
`in the horny layer (Vaddi et al., 2002; Higaki et al.,
`2003). Considering the balance between efficiency and
`toxicity, several terpenes may be promising chemical
`enhancers for clinical use (Kitahara et al., 1993; Higaki
`et al., 2003). In the present study, we tried to develop a
`suitable film preparation of propranolol hydrochloride
`(PPL) by employing ethyl cellulose (EC) and polyvinyl
`pyrrolidone (PVP) as a film former, and dibutyl phtha-
`late (DBP) as a plasticizer. Furthermore, in order to
`improve the penetration of PPL, terpenes such as men-
`thol and cineole, and propylene glycol (PG) were em-
`ployed as a chemical enhancer. Release and permeation
`profiles of PPL from film preparations were exam-
`ined in the in vitro studies using a Franz-type diffusion
`cell.
`
`2. Materials and methods
`
`2.1. Materials
`
`EC (with an ethoxy content 47.5–53.5% by weight
`and a viscosity of 9–11 cps in a 5% (w/w), 80:20
`◦
`toluene/ethanol solution at 25
`C, Tokyo Kasei Kogyo
`Co., Ltd., Tokyo, Japan), PVP K30, DBP, chloroform
`(HPLC grade) and cineole were obtained from Nacalai
`Tesque (Kyoto, Japan). PPL, PG and menthol were
`purchased from Sigma Chemical Co. (St. Louis, MO,
`USA). Other chemicals obtained commercially were of
`a reagent grade.
`
`2.2. Animals
`
`Male Wistar rats (Japan SLC, Hamamatsu, Japan),
`◦
`maintained at 25
`C and 55% humidity were allowed
`free access to standard laboratory chow (Clea Japan,
`Tokyo) and water prior to the experiments. Rats weigh-
`ing 150–200 g were randomly assigned to each experi-
`mental group. Our investigations were performed after
`approval from the local ethical committee at Okayama
`University and in accordance with ‘Interdisciplinary
`Principles and Guidelines of the Use of Animals in Re-
`search’.
`
`2.3. Preparation of film formulations containing
`PPL
`
`Films composed of different ratios of EC, PVP, en-
`hancers and PPL were prepared by a method reported
`previously (Kurosaki et al., 1988). All the ingredients
`were weighed in requisite ratio and they were then dis-
`solved in 25 ml of chloroform. DBP was incorporated
`at a concentration of 30% (w/w) of dry weight of poly-
`mers as a plasticizer. An enhancer was dissolved at a
`concentration of 5% or 10% (w/w) of total dry weight
`of EC, PVP and DBP. The resultant chloroform solu-
`tions were poured into a Teflon tray, and were dried at
`◦
`45
`C for 12 h.
`
`2.4. Film thickness
`
`The thickness of films was measured at three dif-
`ferent places using a micrometer (Mitutoyo Co., Kana-
`gawa, Japan) and mean values were calculated.
`
`Page 2
`
`

`

`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`169
`
`2.5. Determination of drug content in the film
`
`The uniformity of drug distribution was evaluated
`by determining drug content at different places of the
`film by a spectrophotometric method (United States
`Pharmacopeia, 1995). A known weight of film was
`dissolved and diluted subsequently with chloroform,
`and the concentration of PPL was spectrophotomet-
`rically measured at 290 nm (Shimadzu UV-260, Shi-
`madzu, Kyoto) against the blank chloroform solution
`containing the same amount of polymer and plasticizer
`without drug.
`
`2.6. Moisture uptake study
`After films, of which the size is 1 cm× 1 cm in a
`square, were put in a desiccator with silica gel for 24 h
`and weighed (Ws), the films were transferred to another
`desiccator containing saturated NaCl solution (rela-
`◦
`tive humidity 75%) at 25
`C. After equilibrium was
`attained, the films were taken out and weighed (Wm).
`Moisture uptake capacity was calculated according to
`the following equation:
`
`Moisture uptake capacity (%) = Wm − Ws
`
`× 100
`
`Ws
`
`2.7. In vitro drug release study
`
`The release of drug from film preparations was ex-
`amined using a modified Franz-type diffusion cell. The
`films cut in a circle shape were put on a glass filter paper
`placed on the receptor cell, of which the effective area
`for diffusion was 3.14 cm2. The receptor compartment
`was filled with 18 ml of isotonic phosphate buffer so-
`lution (PBS). The diffusion cell was thermoregulated
`◦
`with a water jacket at 37
`C and the receptor compart-
`ment was stirred with a magnetic stirrer. Samples (2 ml)
`were withdrawn at 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h. An
`equal volume of fresh PBS was immediately added to
`the receptor cell after each sampling. The concentra-
`tion of PPL was spectrophotometrically determined at
`289 nm (Shimadzu UV-260).
`
`2.8. In vitro skin permeation study
`
`Abdominal hair was removed using 7% thioglycolic
`acid gel 2 days before performing the isolation of rat
`
`abdominal skin (Higaki et al., 2002). The shaved ab-
`dominal skin was carefully excised from Wistar rats
`as described previously and the subcutaneous tissue
`and adipose tissue were carefully removed (Yagi et al.,
`1998). The obtained skin preparations were mounted in
`a Franz diffusion cell. The film preparation was placed
`on the skin and fixed and covered by the upper com-
`partment of a Frantz-type cell. Experimental condition
`of diffusion cell and sampling procedure were the same
`as in the case of drug release study. Concentration of
`PPL in PBS of the receptor compartment was deter-
`mined by HPLC system, which consists of a model
`LC-6A HPLC pump (Shimadzu) and a UV detector
`(SPD-6A, Shimadzu) set at 289 nm. Analytical column
`was Inertsil ODS-3 (5C18, 250 mm× 4.6 mm i.d., GL
`Sciences, Tokyo). The mobile phase (CH3CN:20 mM
`NH4Cl:0.05% phosphoric acid = 1:1:1 (v/v)) was de-
`livered at 1 ml/min. The coefficient of variation (CV)
`for standard curves ranged from 0.06 to 18.7% and
`the squared correlation coefficient was over 0.9981.
`The cumulative amount of drug permeated was plotted
`against time. The flux values were calculated from the
`linear portions of the plots.
`
`2.9. Statistical analysis
`Results are expressed as the mean± S.D. of at
`least three experiments. Analysis of variance (ANOVA)
`was used to test the statistical significance of differ-
`ences among groups. Statistical significance in the dif-
`ferences of the means was determined by Dunnet’s
`method or Student’s t-test.
`
`3. Results
`
`Polymeric film formulations containing various
`loaded with 1 mg/cm2 PPL,
`ratios of EC:PVP,
`were prepared and their physicochemical properties
`such as uniformity of drug content, thickness and
`moisture uptake capacity were examined (Table 1).
`Estimation of drug content at different places on each
`film indicated that PPL was distributed uniformly
`throughout the films. There was no significant effect
`of film ingredients on the thickness of films. On
`the other hand,
`the increase in the ratio of PVP
`significantly enhanced the moisture uptake, which was
`confirmed by the significant relationship between the
`
`Page 3
`
`

`

`170
`
`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`Table 1
`Physicochemical properties of film formulations of PPL
`
`EC:PVP:PPL
`
`9:0:2
`8:1:2
`7:2:2
`6:3:2
`5:4:2
`
`9:0:2
`8:1:2
`7:2:2
`6:3:2
`5:4:2
`
`9:0:2
`8:1:2
`7:2:2
`6:3:2
`5:4:2
`
`9:0:2
`8:1:2
`7:2:2
`6:3:2
`5:4:2
`
`9:0:2
`8:1:2
`7:2:2
`6:3:2
`5:4:2
`
`r2a
`
`0.9469
`
`(p < 0.01)
`
`0.9499
`
`(p < 0.005)
`
`0.9661
`
`(p < 0.005)
`
`0.9095
`
`(p < 0.02)
`
`0.8591
`
`(p < 0.05)
`
`0.9210
`
`Enhancers
`
`No enhancer
`
`Propylene glycol
`
`Menthol
`
`Cineole
`
`Propylene glycol and menthol
`
`Propylene glycol and cineole
`
`Moisture uptake capacity (%)
`Thickness (mm)
`Drug content (%)
`1.8± 0.0
`0.060± 0.010
`87.7 ± 0.5
`2.1± 0.0
`0.050± 0.010
`97.8 ± 2.2
`3.7± 0.0
`0.060± 0.010
`102.5 ± 0.7
`4.5± 0.0
`0.060± 0.010
`93.1 ± 0.7
`6.6± 0.1
`0.057± 0.015
`102.8 ± 0.2
`1.8± 0.1
`0.057± 0.006
`83.1 ± 0.3
`2.9± 0.0
`0.050± 0.010
`91.6 ± 1.2
`5.9± 0.1
`0.057± 0.012
`102.7 ± 0.5
`6.2± 0.1
`0.057± 0.006
`95.3 ± 1.5
`8.0± 0.1
`0.063± 0.006
`89.7 ± 0.5
`2.2± 0.1
`0.060± 0.017
`85.5 ± 1.0
`2.8± 0.1
`0.060± 0.010
`90.9 ± 0.6
`5.4± 0.0
`0.050± 0.010
`101.4 ± 1.3
`7.2± 0.0
`0.063± 0.012
`92.3 ± 0.7
`8.1± 0.1
`0.050± 0.010
`92.2 ± 0.3
`2.4± 0.0
`0.057± 0.006
`90.1 ± 1.5
`3.2± 0.1
`0.060± 0.010
`95.8 ± 0.7
`6.9± 0.1
`0.060± 0.010
`94.5 ± 0.8
`7.4± 0.1
`0.050± 0.017
`98.5 ± 2.6
`8.4± 0.1
`0.060± 0.010
`101.6 ± 1.3
`2.1± 0.1
`0.060± 0.010
`91.4 ± 1.1
`3.0± 0.0
`0.053± 0.012
`102.9 ± 0.0
`7.1± 0.1
`0.060± 0.010
`99.5 ± 1.3
`7.1± 0.1
`0.060± 0.010
`101.9 ± 1.3
`7.9± 0.1
`0.050± 0.010
`97.7 ± 1.0
`3.1± 0.1
`0.063± 0.012
`97.4 ± 2.5
`9:0:2
`4.0± 0.1
`0.057± 0.006
`91.7 ± 1.2
`8:1:2
`7.0± 0.1
`0.050± 0.010
`93.5 ± 0.9
`7:2:2
`7.6± 0.1
`0.053± 0.006
`94.3 ± 0.1
`6:3:2
`8.2± 0.0
`0.053± 0.006
`96.5 ± 0.7
`5:4:2
`(p < 0.01)
`Results are expressed as the mean± S.D. of three experiments. EC, PVP and PPL mean ethyl cellulose, polyvinyl pyrrolidone and propranolol
`hydrochloride, respectively. Loaded amount of PPL in each film was 1 mg. Concentration of each enhancer was 5% (w/w).
`a A square of correlation coefficient between the moisture uptake % and the ratio of PVP in each film.
`
`Table 2
`Higuchi’s rate constant of PPL (1 mg/cm2) for film formulations calculated by following Higuchi’s model
`
`EC:PVP:PPL
`
`PG + cineole
`PG + menthol
`Cineole
`Menthol
`PG
`No enhancer
`51.0 ± 3.2b
`35.8 ± 1.0
`49.6 ± 8.5b
`59.0 ± 6.3b
`54.3 ± 6.8b
`50.8 ± 5.6b
`9:0:2
`80.1 ± 6.1b
`53.0 ± 2.7
`84.3 ± 17.4b
`61.6 ± 2.8
`69.7 ± 8.1
`73.6 ± 11.8
`8:1:2
`167.4 ± 13.4a,b
`61.0 ± 6.0
`105.2 ± 6.3a
`93.0 ± 14.8a
`207.1 ± 13.8a,b
`91.4 ± 42.6
`7:2:2
`159.5 ± 23.2a
`292.3 ± 22.3a,b
`297.7 ± 18.5a,b
`245.9 ± 13.8a,b
`243.8 ± 21.9a,b
`341.2 ± 7.9a,b
`6:3:2
`325.7 ± 29.9a,b
`332.7 ± 19.5a,b
`314.1 ± 54.6a,b
`288.7 ± 18.4a,b
`417.0 ± 32.1a,b
`212.8 ± 20.9a
`5:4:2
`Results are expressed as the mean± S.D. of three experiments. EC, PVP, PPL and PG mean ethyl cellulose, polyvinyl pyrrolidone, propranolol
`hydrochloride and propylene glycol, respectively. Unit of Higuchi’s rate constant of PPL is ␮g/cm2/h1/2. Concentration of each enhancer was
`5% (w/w).
`a p < 0.05 when compared with ratio of EC:PVP:PPL 9:0:2 as the control in no enhancer and each enhancer.
`b p < 0.05 when compared with no enhancer as the control in corresponding ratio of EC:PVP:PPL.
`
`Page 4
`
`

`

`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`171
`
`moisture uptake and the ratio of PVP in films. Each
`enhancer (5% w/w) increased the moisture uptake
`capacity, but cineol and the combination of PG with
`cineole tended to give higher capacity than other film
`preparations.
`Release of PPL from film preparations was exam-
`ined in an in vitro study using a Frantz-type diffusion
`cell (Fig. 1 and Table 2). As the regression analysis
`of obtained results for three kinetic models such as
`zero order, first order and Higuchi’s model showed that
`Higuchi’s model gave the highest value of r2 with sig-
`nificant difference (p < 0.05), Higuchi’s model, where
`the cumulative amount of released drug per unit area
`is proportional to the square root of time, is the most
`suitable model to describe the release kinetics of PPL
`from the film preparations examined in the present
`study. Higuchi’s rate constants calculated are summa-
`rized in Table 2. Fig. 1 shows the release profile of
`PPL from film preparations containing no enhancer, 5%
`(w/w) cineole or 5% (w/w) PG and cineole as a typical
`example. The release rate of PPL from film prepara-
`tions tended to increase as PVP fraction in the film in-
`creased (Fig. 1 and Table 2). Furthermore, the addition
`of an enhancer or enhancers also promoted the release
`of drug from the film preparations more (Fig. 1 and
`Table 2).
`In vitro skin permeation studies were performed to
`evaluate transdermal absorption of PPL from these film
`preparations. Fig. 2 depicts the permeation profile of
`PPL from film preparations containing 5% (w/w) cine-
`ole, which provided the highest permeation rate among
`enhancers examined in the present study. Table 3 shows
`the permeation rates of PPL for all the film prepara-
`tions. Results show that there is an optimal ratio of
`film formers for each enhancer to show the highest per-
`meation rate of PPL. The film (EC:PVP:PPL = 6:3:2)
`containing 5% (w/w) cineole gave the highest perme-
`ation rate among the film preparations containing 5%
`(w/w) enhancer or enhancers.
`To improve the skin permeation of PPL from film
`preparations further,
`the loading concentrations of
`PPL and enhancers were increased up to 2 mg/cm2
`and 10%, respectively. The ratio of film formers that
`gave the highest permeation rate of PPL for each en-
`hancer was selected based on the results shown in
`Table 3. Because of recrystallization, 2 mg/cm2 was
`almost a maximal dosing concentration of PPL in the
`film preparations. Physicochemical properties for these
`
`Fig. 1. Effect of ratio of EC and PVP on release profile of PPL
`from film preparations without any enhancer (A), film preparations
`containing 5% (w/w) cineole (B) and film preparations containing 5%
`(w/w) PG + cineole (C). PPL was contained in the films at 1 mg/cm2.
`Cumulative PPL amount released was plotted against the square root
`of time, because Higuchi’s model was found to be the suitable model
`for describing the release profile of PPL. Results are expressed as the
`mean with the bars showing S.D. values of three and more different
`experiments. Keys: EC:PVP = 9:0 (䊉), 8:1 ((cid:7)), 7:2 ((cid:1)), 6:3 ((cid:8)) and
`5:4 (♦).
`
`Page 5
`
`

`

`172
`
`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`Fig. 4(A) shows the in vitro skin permeation profiles
`for film preparations containing 2 mg/cm2 PPL and 5%
`(w/w) enhancers. Fig. 4(B) shows the effect of 10%
`(w/w) enhancer on the skin permeation of PPL. The per-
`meation rates for all the preparations are summarized in
`Table 5. Skin permeation of PPL was significantly en-
`hanced by increasing the loading concentration of PPL
`in every preparation except for one containing PG. The
`highest permeation rate was observed in the film con-
`taining cineole, which was followed by the film con-
`taining PG and cineole. Increasing the concentration of
`enhancers significantly enhanced the skin permeation
`of PPL only for the two preparations containing cineole
`or PG and cineole, and the film preparation containing
`10% (w/w) cineole provided the highest permeation
`rate of PPL among the preparations examined in the
`present study, around 8.5-fold of the film preparation
`(EC:PVP:PPL = 5:4:4) without any enhancer.
`
`4. Discussion
`
`TDDS is one of the promising alternatives to oral
`dosage forms especially for drugs that are subjected to
`the first-pass elimination such as PPL. To optimize the
`release of drug from the TDDS as close to a desired
`profile as possible for the long-time period of opera-
`tion, much attention has recently been focused on the
`development of a film preparation composed of several
`polymers (Jain et al., 1996; Rama Rao et al., 2000; Shin
`et al., 2002). In the present study, we tried to prepare
`polymeric film formulations of PPL that would make
`it possible for PPL to penetrate the skin at a high and
`constant rate by employing several terpenes such as
`menthol and cineole.
`
`Fig. 2. Effect of ratio of EC and PVP on skin permeation of PPL
`through rat skin from films containing 5% (w/w) cineole. PPL was
`contained in the films at 1 mg/cm2. Results are expressed as the mean
`with the bars showing S.D. values of three different experiments.
`Keys: EC:PVP = 9:0 (䊉), 8:1 ((cid:7)), 7:2 ((cid:1)), 6:3 ((cid:8)) and 5:4 (♦).
`
`film preparations are summarized in Table 4. Load-
`ing of 2 mg/cm2 PPL tended to thicken films and sig-
`nificantly enhanced the capacity of moisture uptake.
`The increase of enhancer concentration in the film
`tended to increase the moisture uptake capacity more.
`Fig. 3A shows the effect of PPL concentration on the
`release kinetics, indicating that Higuchi’s rate con-
`stant of PPL was increased around three times from
`212.79± 20.91 to 709.81± 53.79 ␮g/cm2/h1/2, as the
`concentration of PPL increased twice. On the other
`hand, there was no effect of cineole concentration on
`the release profile of PPL (Fig. 3B). Higuchi’s rate
`constants of PPL for the preparations containing cine-
`ole are as follows: the film containing 5% (w/w) cine-
`ole, 693.38± 132.74 ␮g/cm2/h1/2; 10% (w/w) cineole,
`672.02± 27.17 ␮g/cm2/h1/2.
`
`Table 3
`Skin permeation rate of PPL from film preparations containing 1 mg/cm2 PPL in an in-vitro transport study
`
`EC:PVP:PPL
`
`PG + cineole
`PG + menthol
`Cineole
`Menthol
`PG
`No enhancer
`12.9 ± 3.5b
`6.9 ± 1.4
`4.3± 1.8
`3.2 ± 0.3
`3.2 ± 0.9
`5.7 ± 0.2
`9:0:2
`6.3 ± 0.5a
`7.7 ± 0.8
`5.1± 0.9
`6.8 ± 1.8
`8.8 ± 1.9b
`6.7 ± 1.4
`8:1:2
`10.0 ± 1.0b
`8.3 ± 1.5
`3.6± 0.3
`21.1 ± 0.7a,b
`16.6 ± 4.7a,b
`15.4 ± 4.2a,b
`7:2:2
`13.5 ± 2.1a,b
`6.2± 1.9
`27.4 ± 3.9a,b
`6.8 ± 0.3a
`12.1 ± 5.5a
`6.8 ± 1.9
`6:3:2
`16.6 ± 2.4b
`9.8 ± 3.0
`6.3± 1.9
`9.8 ± 1.9a
`8.3 ± 2.6
`9.9 ± 3.0
`5:4:2
`Results are expressed as the mean± S.D. of three experiments. EC, PVP, PPL and PG mean ethyl cellulose, polyvinyl pyrrolidone, propranolol
`hydrochloride and propylene glycol, respectively. Unit of permeation rate of PPL is ␮g/cm2/h. Concentration of each enhancer was 5% (w/w).
`a p < 0.05 when compared with ratio of EC:PVP:PPL 9:0:2 as the control in no enhancer and each enhancer.
`b p < 0.05 when compared with no enhancer as the control in corresponding ratio of EC:PVP:PPL.
`
`Page 6
`
`

`

`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`173
`
`Table 4
`Physical properties of film formulations containing 2 mg/cm2 PPL
`
`Enhancers (%)
`
`No enhancer
`
`PG
`
`5
`10
`
`Menthol
`5
`10
`
`Cineole
`5
`10
`
`PG + menthol
`5
`10
`
`EC:PVP:PPL
`
`5:4:4
`
`6:3:4
`6:3:4
`
`5:4:4
`5:4:4
`
`6:3:4
`6:3:4
`
`7:2:4
`7:2:4
`
`Drug content (%)
`99.6± 0.7
`
`Thickness (mm)
`0.070± 0.010
`
`Moisture uptake capacity (%)
`8.9± 0.3a
`
`96.4± 1.4
`98.7± 0.4
`
`100.9± 0.6
`96.7± 1.9
`
`99.8± 0.8
`100.2± 0.9
`
`96.3± 2.4
`97.9± 1.7
`
`0.073± 0.006
`0.080± 0.010
`
`0.070± 0.010
`0.080± 0.010
`
`0.063± 0.006
`0.073± 0.006
`
`0.077± 0.006
`0.073± 0.012
`
`9.5± 0.4b
`13.8± 0.8c
`
`10.7± 1.5
`13.4± 0.5
`
`15.1± 0.9b
`16.8± 0.3
`
`11.1± 0.6b
`14.1± 0.8c
`
`PG + cineole
`10.4± 0.6b
`0.080± 0.010
`99.3± 0.6
`5
`7:2:4
`14.0± 0.8c
`0.073± 0.012
`99.8± 1.1
`10
`7:2:4
`Results are expressed as the mean± S.D. of three experiments. EC, PVP, PPL and PG mean ethyl cellulose, polyvinyl pyrrolidone, propranolol
`hydrochloride and propylene glycol, respectively. Loaded amount of PPL in each film was 2 mg.
`a p < 0.05 when compared with the same ratio of film former containing 1 mg/cm2 PPL.
`b p < 0.05 when compared with the same concentration of enhancer containing 1 mg/cm2 PPL.
`c p < 0.05 when compared with the same enhancer containing 5% (w/w) of enhancer.
`
`The moisture uptake capacity increased with the in-
`crease of the PVP ratio in the film (Table 1), which
`can be supported by the previous reports (Rama Rao
`et al., 2000; Arora and Mukherjee, 2002). PVP might
`
`enhance the absorption of water vapor by converting
`the crystalline drug into amorphous state and making
`the molecules spaced further apart than in a crystal
`(Rama Rao et al., 2000). Each enhancer also increased
`
`Fig. 3. Effect of PPL concentration (A) or cineole concentration (B) on release profile of PPL from film formulations. (A) PPL was contained at
`1 mg/cm2 ((cid:7)) or 2 mg/cm2 (䊉). Ratio of film formers was EC:PVP = 5:4. (B) Cineole was contained at 5% (w/w) ((cid:8)) or 10% (w/w) ((cid:3)) in the
`films containing 2 mg/cm2 of PPL. Ratio of film formers was EC:PVP = 6:3. Cumulative PPL amount released was plotted against the square
`root of time, because Higuchi’s model was found to be the suitable model for describing the release profile of PPL. Results are expressed as the
`mean with the bars showing S.D. values of three different experiments.
`
`Page 7
`
`

`

`174
`
`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`Fig. 4. Effect of enhancer concentration on permeation of PPL through rat skin from films. Each enhancer was contained at 5% (w/w) (A) or
`10% (w/w) (B). Concentration of PPL was 2 mg/cm2 for every preparation. Results are expressed as the mean with the bars showing S.D. values
`of three different experiments. Keys: no enhancer (䊉); PG ((cid:1)); menthol ((cid:7)); cineole ((cid:8)); PG + menthol (♦) and PG + cineole ((cid:4)).
`
`the moisture uptake capacity (Table 1), for which the
`reason remains to be clarified. In the case of PG, how-
`ever, it can absorb moisture from environment into the
`film where PG is contained because of its humectant
`ability (Barry, 1983).
`The analysis of drug release profiles showed that
`Higuchi’s model was the most suitable for describing
`the release kinetics of PPL from the films prepared in
`the present study (Figs. 1 and 3, Table 2), which means
`that the release of PPL from the film preparations is
`regulated by the diffusion of PPL within a matrix sys-
`tem (Siepmann and Peppas, 2001). The increase of
`
`Higuchi’s rate constant with the increase of PVP con-
`tent in the film (Fig. 1 and Table 1) can be explained by
`the leaching of PVP and pore formation (Rama Rao et
`al., 2000, 2003). Each enhancer and the combination
`of two enhancers also tended to promote the release of
`PPL from the film preparations (Fig. 1 and Table 2).
`Although the mechanisms for this effect remain to be
`clarified, the greater amount of water absorbed into the
`film by an enhancer or enhancers (Table 1) would con-
`tribute to the more rapid release of PPL from the films.
`Although the release rate of PPL did not increase in the
`film containing 10% (w/w) cineole compared with the
`
`Table 5
`Skin permeation rate of PPL from film preparations containing 2 mg/cm2 PPL and 5% or 10% (w/w) enhancers in an in vitro transport study
`Permeation rate (␮g/cm2/h)
`
`EC:PVP:PPL
`
`Enhancers
`
`Ratio to 1 mg/cm2-PPL Film
`
`10% (w/w) Enhancer
`5% (w/w) Enhancer
`Mean± S.D.
`Mean± S.D.
`11.3± 1.6a
`No enhancer
`5:4:4
`–
`1.79
`–
`20.6± 5.3
`16.1± 1.2
`PG
`6:3:4
`1.19
`1.28
`24.0± 5.0b
`28.2± 1.7b
`Menthol
`5:4:4
`1.45
`1.18
`49.3± 5.5b
`93.8± 11.6b,c
`Cineole
`6:3:4
`1.80
`1.90
`24.2± 1.7b
`30.6± 4.0b
`PG + menthol
`7:2:4
`1.46
`1.27
`36.1± 3.0b
`54.5± 0.5b,c
`PG + cineole
`7:2:4
`1.71
`1.51
`Results are expressed as the mean± S.D. of three experiments. EC, PVP, PPL and PG mean ethyl cellulose, polyvinyl pyrrolidone, propranolol
`hydrochloride and propylene glycol, respectively.
`a p < 0.05 when compared with the same ratio of film former containing 1 mg/cm2 PPL.
`b p < 0.05 when compared with no enhancer as the control.
`c p < 0.05 when compared with the same enhancer containing 5% (w/w) of enhancer.
`
`Ratio to 5%-enhancer film
`
`Page 8
`
`

`

`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`175
`
`film containing 5% (w/w) cineole, this finding might be
`explained by no change of the moisture uptake capac-
`ity (Table 4). Increasing the concentration of PPL two
`times resulted in over three times greater rate of release
`from the films (Fig. 3(A)), but this finding agrees with
`another report (Rama Rao et al., 2000), where it was
`suggested that increasing the amount of drug dispersed
`in the film would increase the porosity, leading to the
`greater release rate than expected based on the increase
`in drug concentration.
`Skin permeation studies showed that the film prepa-
`rations containing cineole or menthol could enhance
`the skin penetration of PPL (Figs. 3 and 4). Especially,
`the film preparation containing 10% (w/w) cineole
`(EC:PVP:PPL = 6:3:4) or 10% (w/w) cineole and PG
`(EC:PVP:PPL = 7:2:4) provided the high permeation
`rate, 93.81 and 54.51 ␮g/cm2/h, respectively (Table 5).
`These values are 3–6-fold of the films prepared by
`Rama Rao et al. (2003), suggesting that these film
`preparations can provide the good and long-lasting
`therapeutic effect. It is well known that the enhancing
`effect of chemical enhancers could be dependent on
`the physicochemical properties of drugs and the com-
`bination with vehicle or ingredients in preparations
`(Higaki et al., 2003), but terpenes can enhance both
`hydrophilic drugs including propranolol and lipophilic
`drugs such as testosterone (Williams and Barry,
`1991a, 1991b; Kunta et al., 1997; Kaplun-Frischoff
`and Touitou, 1997; Vaddi et al., 2002). Especially,
`cineole and menthol improved the skin permeation of
`hydrophilic drugs better than other terpenes (Jain et al.,
`2002; Narishetty and Panchagnula, 2004). A proposed
`mechanism for terpenes to improve the skin perme-
`ation of drugs is mainly the increase in drug diffusivity
`in the skin (Williams and Barry, 1991a; Cornwell and
`Barry, 1994; Zhao and Singh, 1998; Vaddi et al., 2002)
`by modifying the intercellular packing, disrupting
`highly ordered structure of lipids (Barry, 1991). It has
`also been suggested that the molecular mechanism
`is attributed to the preferential hydrogen bonding
`of oxygen-containing monoterpenes with ceramide
`head groups thereby breaking the lateral/transverse
`hydrogen bond network of lipid bilayer (Jain et al.,
`2002; Narishetty and Panchagnula, 2004). Because of
`the short lag time (around 2 h) for the film preparation
`without any enhancer and the shape of cumulative
`transport profile, we could not find the significant
`change in the lag time for the films containing
`
`terpenes (data not shown). However, the transported
`amount of PPL clearly increased at early time periods,
`suggesting the increase in diffusivity of PPL in the
`skin.
`Skin permeation studies also showed that there was
`an optimal ratio of film formers that gave the highest
`permeation rate of PPL for each enhancer (Table 3). The
`preparation giving the highest penetration rate was not
`necessarily coincided with the one giving the highest
`release rate of PPL (Table 2), meaning that the skin
`penetration of PPL is not regulated by drug release ki-
`netics, but by the effect of enhancers on the skin per-
`meation of PPL. Several factors such as the release
`kinetics of enhancers from the film and the interaction
`between enhancers and other ingredients in the film
`would influence the enhancing activity, and the effects
`of these factors might be changeable dependent on the
`ratio of film formers and enhancers. Cal et al. (2001)
`have investigated percutaneous penetration of five ter-
`penes from the matrix type transdermal patches through
`the skin, and have indicated that eucalyptol (cineole)
`is one of the terpenes with the highest skin penetra-
`tion rate. Narishetty and Panchagnula (2004) suggested
`that cineole would interact with lipid components of the
`stratum corneum more easily, because the boiling point
`of cineole was lower than other terpenes and the low
`boiling point is an indication of weak cohesiveness or
`self-association. These might explain the reason why
`cineole showed the highest enhancing effect on the skin
`permeation of PPL in the present study. In the case of
`the film preparations containing PG or cineole, the skin
`flux of PPL tended to be improved with the increase of
`PVP (Table 3), which might be attributed to an antinu-
`cleating effect of PVP that can convert the crystalline
`drug into amorphous state on the skin surface (Rama
`Rao et al., 2003). Compounds in amorphous state gen-
`erally posses a high energy state with improved solu-
`bility and the enhancement of solubility of drug close
`to the skin surface increases thermodynamic activity
`that facilitates the permeation rate of drug through the
`skin (Rama Rao et al., 2003).
`The effect of permeation enhancers often depends
`on their applied concentrations (Jain et al., 1996;
`Takayama et al., 1999; Narishetty and Panchagnula,
`2004), and this is also the case with the preparations
`examined in the present study (Table 5). Only cineole
`and the combination of PG with cineole significantly
`increased the enhancing effect as the applied concentra-
`
`Page 9
`
`

`

`176
`
`C. Amnuaikit et al. / International Journal of Pharmaceutics 289 (2005) 167–178
`
`tion increased, although the enhancing effect of other
`preparations was almost saturated.
`The enhancing effect of PG itself was not so remark-
`able for PPL (Fig. 4, Tables 3 and 5), although it was
`reported that PG itself enhanced the skin permeation
`of several drugs (Polano and Ponec, 1976; Wotton et
`al., 1985; Irwin et al., 1990) without any alteration in
`the skin structure (Fang et al., 2003). The increase in
`solubilizing ability of the aqueous site in the stratum
`corneum is considered to be a main mechanism for PG
`to improve the skin permeation of drugs (Barry, 1991).
`However, the enhancing activity of PG itself is quite
`controversial (Asbill et al., 2000; Higaki et al., 2003),
`because PG also has several characteristics that can
`decrease the skin permeation of drugs as follows: (1)
`PG, classified as a humectant (