`
`i;
`7'. g“
`ELSEVIER
`
`Journal of Controlled Release 51 (1998) 1937199
`
`
`
`journal of
`controlled
`release
`
`In vitro percutaneous absorption enhancement of a lipophilic drug
`tamoxifen by terpenes
`
`Shen Gao, Jagdish Singh*
`Department of Pharrr'aceutical Sciences, College of Pharrr'acy, North Dakota Sate University, Fargo, ND 58105, USA
`
`Received 16 March 1997; received in revised form 7 August 1997; accepted 14 August 1997
`
`
`
`Abstract
`
`Tamoxifen is a highly lipophilic drug that is widely used in breast malignancies and also as a prophylactic therapy in
`women at high risk for the development of this disease. Recently, the terpenes have been reported to show an enhancement
`effect on percutaneous drug absorption. The effect of terpenes (e.g. carvone, 1,8—cineole, menthol, and thymol) was studied
`on the in Vitro percutaneous absorption of tamoxifen through porcine epidermis. The above terpenes (5% w/ V)
`in
`combination with 50% ethanol significantly (P<0.01) increased the permeability coefficient of tamoxifen in comparison to
`the control (50% ethanol). The solubility of tamoxifen was determined in the control and enhancer solutions to correct the
`permeability enhancement by way of fractional solubility adjustment. Binding of tamoxifen to powdered stratum corneum
`from control and enhancer solutions was also determined. Binding studies reveal that the enhancement in the permeability
`coefficient of tamoxifen by menthol and thymol is due, at least in part, to improvement in the partitioning of the drug to the
`stratum corneum. In conclusion, terpenes in combination with ethanol can be used to enhance the percutaneous absorption of
`the highly lipophilic drug tamoxifen. © 1998 Elsevier Science B.V
`
`Keywords: Percutaneous absorption; Permeability coefficient; Tamoxifen; Penetration enhancer; Terpenes
`
`1. Introduction
`
`Various studies have demonstrated that the trans-
`
`dermal pathway may be a suitable alternative to the
`oral route in the administration of drugs with sys-
`temic activity. The advantages of transdermal drug
`delivery systems are well documented [1]. The
`primary barrier
`to transdermal diffusion is
`the
`stratum corneum,
`the thin outermost
`layer of the
`skin, which is comprised of a regular array of
`protein-rich cells that are embedded in a multilamel-
`lar lipid domain. The larnellar packing of stratum
`
`>x<Corresponding author: Tel.: +1 701 2317943; fax: +1 701
`2317606; e-mail: jsingh@plains.nodak.edu
`
`corneum intercellular lipids is established and several
`experiments have directly implicated these lipoidal
`domains as the integral components of the transport
`barrier which must be breached if drugs are to be
`administered at an appropriate rate. More recently it
`was found that diffusion barrier
`reduction may
`conveniently and elegantly be achieved by the use of
`chemical [2—4] and physical [5—9] enhancers.
`Terpenes are a series of naturally occurring com-
`pounds which consist of isoprene (C5H8) units.
`Terpenes are constituents of essential oils, which are
`the volatile and fragrant substances found mainly in
`flavorings, perfumes, and medicines. Recently,
`the
`terpenes were reported to show an enhancement
`effect on percutaneous drug absorption [10—12]. 1,8-
`
`0168-3659/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.
`PII SOI68-3659(97)00168-5
`
`AstraZeneca Exhibit 2100 p. 1
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`

`194
`
`S Gao, J. Sngh / Journal of Controlled Release 51 (1998) 1937199
`
`Cineole has been used to promote the percutanous
`absorption of several lipophilic drugs through hair-
`less mouse skin [13]. Patents exist for the use of
`1-carvone and eugenol as skin penetration enhancers
`[14]. Terpenes containing 50% ethanol increased the
`total flux of nicotine through the hairless-mouse skin
`[15].
`Some monocyclic monoterpenes
`such as
`limonene and menthol enhanced the transport of
`indomethacin [16] and diazepam [12], respectively,
`through the rat skin. Thymol is incorporated in some
`lotions, creams, topical mixtures, and mouth washes.
`Currently, natural products are receiving consider-
`able interest
`in the pharmaceutical
`industry, and
`terpenes may provide a series of relatively safe,
`clinically acceptable accelerants for lipophilic and
`hydrophilic drugs.
`Tamoxifen is a highly lipophilic drug [17]. It is a
`widely used adjuvant therapy following surgery for
`breast malignancies in postmenopausal women. This
`agent
`is also indicated for treatment of estrogen
`receptor-positive tumors in the premenopausal popu-
`lation [18]. Studies are also in progress to evaluate
`tamoxifen as a prophylactic therapy in women at
`high risk for the development of this disease [19,20].
`Tamoxifen undergoes extensive hepatic metabolism
`after oral administration in humans. The usual oral
`
`dose of tamoxifen is 10 mg twice daily. The steady-
`state plasma concentration of 77—274 ng ml’1 has
`been reported for tamoxifen [21]. The chemical
`stmcture of tamoxifen is given in Fig. 1.
`The basic data for in vitro human percutaneous
`absorption, with which animal models are compared,
`were obtained from Feldmann and Maibach [22,23].
`The histological characteristics of pig and human
`
`Tamoxifen
`
`1. Structural formula of tamoxifen, a lipophillc drug of
`Fig.
`molecular weight 563.65.
`
`\
`Carvone
`
`1,8-Cineole
`
`OH
`
`OH
`
`Menthol
`
`Thymol
`
`formula of terpenes used in the in vitro
`2. Structural
`Fig.
`percutaneous absorption of tamoxifen.
`
`skin have been reported to be comparable with
`similarities existing for epidermal
`thickness and
`composition [24,25], dermal stmcture [26],
`lipid
`content [27,28] and general morphology [25,29]. The
`ranking of skin permeability of different species in
`vitro has been determined by several
`investigators
`[30—32]. Increasing evidence supports the contention
`that
`in vitro permeability studies can accurately
`predict in vivo absorption [33]. Skin from the pig
`generally approximates the permeability of human
`skin [34,35]. Thus,
`the percutaneous absorption of
`tamoxifen through pig skin can well be used to
`predict the percutaneous absorption in humans.
`In this
`study, we selected four simple cyclic
`terpenes (e.g. carvone, 1,8-cineole, menthol, and
`thymol) from the chemical classes of ketones, ox-
`ides, and alcohols (Fig. 2) to investigate their effects
`on the in vitro percutaneous absorption of tamoxifen
`through porcine epidermis.
`
`2. Materials and methods
`
`2.1. Materials
`
`[3H]Tamoxifen (specific activity 85.0 Ci mmol’l)
`was obtained from Amersham Life Sci. (Cleveland,
`OH, USA). Carvone was purchased from Aldrich
`Chemical Company Inc.
`(Milwaukee, WI, USA).
`1,8-Cineole, menthol, thymol and ammonium acetate
`(HPLC grade) were purchased from Sigma Chemical
`Co. (St. Louis, MO, USA). Ethanol was purchased
`from CMS (Houston, TX, USA). Acetonitrile (HPLC
`grade) and Microsorb-MVTM C18 HPLC column
`
`AstraZeneca Exhibit 2100 p. 2
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`

`

`S Gao, J. Sngh / Journal of Controlled Release 51 (1998) 1934199
`
`195
`
`were obtained from EM Science (Gibbstown, NJ,
`USA) and Rainin Instrument Co.
`(Wobum, MA,
`USA), respectively. All other chemicals and reagents
`used were of analytical grade.
`
`2.2. Preparation of epidernis
`
`Porcine ears were obtained from a local slaughter
`house. Epidermal membranes were prepared by heat
`separation technique [36,37]. The whole skin was
`soaked in water at 60°C for 45 s, followed by careful
`removal of the epidermis. The epidermis was washed
`with water and used in the in vitro percutaneous
`absorption studies.
`
`2.3. Sblubility of tamoxifen
`
`Solubility of tamoxifen was determined by agitat-
`ing excess solute in the control or enhancer solutions
`for 24 h at 37°C, and then determimng the amount of
`tamoxifen in saturated solution following the HPLC
`method described by Lim et al.
`[38] with slight
`modification. A Hewlett-Packard series 1050 liquid
`chromatograph
`(Hewlett-Packard GmbH, Wald-
`bronnz, Germany) was used for tamoxifen analysis.
`A Microsorb-MVTM C18 column (5 um, 250><4.6
`mm) was used. The mobile phase consisted of a
`mixture of 0.5 M ammonium acetate (pH 6.4)—
`acetomtrile (50:50). The flow rate was 2 ml/min. A
`20-ul sample size was injected. Tamoxifen was
`detected using a variable wavelength UV detector at
`238 nm. Table 1 depicts the solubility of tamoxifen
`in control and enhancer solutions.
`
`2.4. Binding to powdered stratum corneum
`
`The binding behavior of tamoxifen in control and
`enhancer systems to powdered stratum corneum was
`
`Table 1
`Solubility of tamoxifen in control and enhancer solutions
`Enhancer solutions Solubility (ug/ ml)
`
`
`
`Control (50% ethanol)
`5% carvone/50% ethanol
`5% cineole/50% ethanol
`5% menthol/50% ethanol
`
`9.19
`55.52
`13.03
`1.01
`
`3.68
`5% thymol/50% ethanol
`
`Water 0.04
`
`determined following the method of Wester et al.
`[39]. Stratum corneum was pulverized in a mortar
`and pestle contaimng dry ice. Particles of stratum
`corneum that were passed through a 48-mesh sieve
`but retained by an 80-mesh sieve were used. In a
`centrifuge tube, 10 mg of powdered stratum corneum
`was mixed with 1 ml of either control or enhancer
`
`solution contaimng 0.2 uCi of [3H]tamoxifen by
`vortexing. After 10 h of contact time, the mixture
`was separated by centrifugation and the supemate
`removed. The stratum corneum pellet was again
`resuspended in 1 ml of the control or enhancer
`solution and immediately centrifuged to remove
`material adsorbed on the surface. The amount of
`
`radioactivity was determined in the supemates by
`liquid scintillation counting. The amount of tamox-
`ifen that bound to the stratum corneum was obtained
`
`by substracting the amount of tamoxifen recovered in
`supernates from the amount of tamoxifen originally
`added (0.2 [LCD to the control or enhancer solution.
`
`2.5.
`
`In vitro percutaneous absorption
`
`Franz diffusion cells were used in the in vitro
`
`percutaneous absorption studies. The epidermis was
`sandwiched between the cells with the stratum
`
`corneum facing the donor compartment. The maxi-
`mum capacity of each of the donor and receiver
`compartments was 2 and 5 ml,
`respectively. The
`surface area of epidermis exposed to the solution was
`0.785 cm2. The donor compartment contained 1 ml
`of tamoxifen solution (0.2 uCi of tamoxifen con-
`tained in 1 ml enhancer solution), and the receiver
`compartment contained 5 ml of phosphate-buffered
`saline, pH 7.4. Thus,
`the donor concentrations of
`tamoxifen used was 2.35><10’3 nmol ml’l. The
`
`donor compartment was capped with a glass cap that
`snuggly fits to the neck of donor compartment to
`prevent evaporation of the solvents. We used 50%
`ethanol in water in donor solution to solubilize the
`
`5% of terpenes. The cells were maintained at
`37 i0.5°C by a PMC Dataplate® stirring digital dry
`block heater (Crown Bioscientific Inc., Somerville,
`NJ, USA). The content of the receiver compartment
`was stirred with the help of a magnetic bar at 100
`rpm. At specified intervals, 0.5-ml samples were
`withdrawn from the receiver compartment, and an
`equivalent amount of phosphate-buffered saline (0.5
`
`AstraZeneca Exhibit 2100 p. 3
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`

`

`196
`
`S Gao; J. Sngh / Journal of Controlled Release 51 (1998) 1937199
`
`3. Results and discussion
`
`The effect of terpenes (e.g. carvone, 1,8-cineole,
`menthol, and thymol) on the in vitro percutaneous
`absorption profiles of tamoxifen through porcine
`epidermis is shown in Fig. 3. Five percent terpenes
`in combination with 50% ethanol in water increased
`
`the in vitro transport of tamoxifen as compared to the
`control (50% ethanol
`in water). The permeability
`coefficient and enhancement factors of tamoxifen
`
`through the epidermis are shown in Table 2. The
`permeability coefficient of tamoxifen in the presence
`of terpenes was significantly greater (P<0.01) than
`the control. However, the permeability coefficients of
`tamoxifen were not significantly different (P>0.05)
`among the terpene-treated groups.
`The drug flux through the skin should be directly
`proportional to drug activity in the vehicle, provided
`the membrane is unaltered.
`In all vehicles that
`
`contain saturated solutions of the drug, the thermo-
`dynamic activity of the drug is also maximal. We can
`also note that the chemical potential and the thermo-
`dynamic activity must remain the same in all satu-
`rated vehicles. In this study, we used unsaturated
`solutions of terpenes in 50% ethanol;
`therefore,
`relative activities and chemical potential of terpenes
`would vary. Table 2 shows the corrected enhance-
`ment of tamoxifen by terpenes in comparison to the
`control. The results show that carvone is a more
`
`effective terpene in enhancing the permeability of
`
`ml) was added to maintain the constant volume.
`Control experiments were also performed using 50%
`ethanol
`in water without terpenes. All the experi-
`ments were run for 10 h.
`
`The samples were assayed for tamoxifen contents
`by liquid scintillation counting. Each sample was
`mixed with
`10 ml
`of
`scintillation
`cocktail
`
`International
`(ECONOSAFE®, Research Products
`Corp., Mount Prospect, IL, USA), and counted in a
`liquid scintillation counter (Packard, Tri Carb® 2100
`TR, Downers Grove, IL, USA). The instrument was
`programmed to give counts for 10 min. The results
`were expressed as the mean iSD. of three experi-
`ments.
`
`2.6. Data analysis and statistics
`
`The tamoxifen concentration was corrected for
`
`sampling effects according to the equation described
`by Hayton and Chen [40]:
`
`Cl :CnNT/VT evstla/CM)
`
`(1)
`
`where C; is the corrected concentration of the nth
`sample, Cn the measured concentration of tamoxifen
`in the nth sample, Ch,1 the measured concentration
`of the tamoxifen in the (n71)th sample, VT the total
`volume of the receiver fluid, and VS the volume of
`the sample drawn.
`The cumulative amount of tamoxifen permeated
`per unit skin surface area was plotted against time,
`and slope of the linear portion of the plot was
`estimated as steady-state flux (J55). The permeability
`coefficient (Kp) was calculated as [41]:
`
`Kp : JSS /Cv
`
`where C is the total donor concentration of the
`tamoxifen.
`
`We also corrected the Kp from fractional solubility
`adjustment as [42]:
`
`Corrected Kp : Jss/(CV/Cs)
`
`Where Cs is the saturated solubility of tamoxifen in
`control/ enhancer solutions.
`
`Statistical comparisons were made using analysis
`of variance procedure
`(ANOVA)
`and Duncan’s
`multiple range test with the help of an SAS program.
`The level of significance was taken as P<0.05.
`
`
`
`
`
`
`
`TamoxifenFermented(11molcm‘z)x105
`
`Nu-
`
`NO
`
`_. u:
`
`._. O
`
`LII
`
`O
`
`0.0
`
`2.0
`
`4.0
`
`6.0
`
`8.0
`
`10.0
`
`Time (h)
`
`Fig. 3. Effect of terpenes on the in vitro percutaneous absorption
`of tamoxifen through porcine epidermis. Each data point is the
`
`meaniSD. of three determinations. Key:
`(O) control; (I)
`
`
`
`carvone; (
`) menthol; (A) cineole; (A) thymol.
`
`AstraZeneca Exhibit 2100 p. 4
`
`

`

`S Gao, J. Sngh / Journal of Controlled Release 51 (1998) 1937199
`
`197
`
`Table 2
`Effect of terpenes on the permeability coefficient, and enhancement factors (Ef) of tamoxifen through porcine epidermis
` Treatment Permeability coefficient (cm/ h) (meaniS.D.)><103 E: Corrected E:
`
`
`Control
`0.27i0.02
`7
`
`
`
`244.13
`40.41
`10.91i0.72 (P<0.01)
`Carvone
`51.35
`36.22
`9.78i0.69 (P<0.01)
`Cineole
`3.98
`36.22
`9.78i0.69 (P<0.01)
`Menthol
`16.75
`42.52
`11.48i1.25 (P<0.01)
`Thymol
`
`Water 0.01 0.42i0.12 (P>0.05) 1.56
`
`
`Control is 50% ethanol in water.
`
`E::Kp with enhancer/Kp with control.
`Corrected E::corrected Kp with enhancer/ corrected Kp with control.
`
`tamoxifen in comparison with control. On the basis
`of corrected enhancement factors, the rank and order
`of
`the effectiveness of
`terpenes
`are carvone>
`cineole>thymol>menthol
`Fifty percent ethanol in water was used as control
`to study the effect of 5% terpenes on the in vitro
`percutaneous absorption of tamoxifen. The effect of
`50% ethanol was also investigated on the permeabili-
`ty of tamoxifen with respect to water alone. The in
`vitro percutaneous absorption profiles are given in
`Fig. 4. The transport of tamoxifen was less with 50%
`ethanol in water than with water alone. Tamoxifen,
`being highly
`lipophilic,
`should be
`transported
`through the non-polar pathway (i.e.
`intercellular
`lipids of the stratum comeum). When ethanol was
`used beyond 50%, the solute and concomitant etha-
`nol
`fluxes
`started to decrease, probably due to
`ethanol’s dehydrating effect on the skin tissue [43].
`
`\l
`
`Table 3 shows the binding of tamoxifen to stratum
`comeum from water and 50% ethanol. The partition-
`ing of tamoxifen to stratum comeum is greater from
`water than 50% ethanol. The above findings explain
`the greater permeability coefficient of tamoxifen
`from water than 50% ethanol. An another explana-
`tion for the above findings can be offered based on
`the thermodynamic activity of tamoxifen in water
`and 50% ethanol. The flux is actually proportional to
`a gradient of thermodynamic activity rather than
`concentration. The drug activity will change in
`different solvents at a definite concentration. The
`
`solubility of tamoxifen is less in water than in 50%
`ethanol (Table 1). At a constant drug concentration,
`drug activity will be reduced as solubility in a
`solvent increased. The solvent where the drug is least
`soluble should provide the highest drug permeation,
`provided solvent does not alter the membrane. This
`explains the greater tamoxifen permeability in water
`than in 50% ethanol.
`
`Penetration enhancers improve drug permeation by
`interacting with the stratum comeum. The lipid
`
`Table 3
`
`
`
`Partition coefficient of tamoxifen in powdered stratum comeum/
`enhancer solutions
`Enhancer solutions Partition coef‘ficienta (X 102)
`Control (50% ethanol)
`1.35i0.01
`5% carvone/50% ethanol
`1.35i0.01
`5% cineole/50% ethanol
`1.34i0.03
`5% menthol/50% ethanol
`1.51i0.02
`
`1.65i0.01
`5% thymol/50% ethanol
`
`Water 1.61i0.03
`
`aPartition coefficient:concentration of tamoxifen in 1000 mg of
`powdered stratum comeum/ concentration of tamoxifen in 1000
`mg of control or enhancer solution.
`
`AstraZeneca Exhibit 2100 p. 5
`
`l Tamoxifen
`
`permeated(nmolcm")x10"
`
`0
`0.0
`
`111‘
`4.0
`
`2.0
`
`
`til__4_._.i_._.
`6.0
`8.0
`
`.
`
`1
`10.0
`
`Time (h)
`
`Fig. 4. Effect of 50% ethanol on the in vitro percutaneous
`absorption of tamoxifen through porcine epidermis. Each data
`point is the meaniS.D. of three determinations. Key: (O) water;
`(0) 50% ethanol in water.
`
`

`

`198
`
`S Gao, J. Sngh / Journal of Controlled Release 51 (1998) 1934199
`
`protein partitioning theory was introduced by Barry
`[44] to describe the modes of action of penetration
`enhancers. According to the theory, enhancers would
`act by one or more of three main mechanisms:
`(a)
`dismption of the highly ordered stmcture of stratum
`comeum lipids,
`(b)
`interaction with intracellular
`protein, and (c)
`improvement
`in partitioning of a
`drug, coenhancer, or cosolvent
`into the stratum
`comeum. For terpenes, the enhancer—protein inter-
`action might play a relatively small role because they
`are lipophilic compounds [37]. In the same study
`[37], it was suggested that the terpenes acted, at least
`in part, by modifying intercellular lipids and dismpt-
`ing their highly ordered structure to increase drug
`diffusivity. The above also suggested that partition
`phenomena might be more important in the accelera-
`tion of highly lipophilic drugs. Cyclic ether-type
`terpenes such as ascaridole increased the partitiomng
`of highly lipophilic estradiol [11]. It was reported
`that terpenes increased the percutaneous absorption
`of lipophilic indomethacin by affecting the barrier
`property of the skin [16,45].
`Synergistic effects with ethanol were investigated
`by Obata et a1.
`[46] for some terpenes. At lower
`concentrations of ethanol, the enhancement effects of
`relatively hydrophilic terpenes such as 1,8-cineole
`and menthol were much greater than those of the
`lipophilic terpenes. Kobayashi et a1.
`[47] reported
`that an aqueous vehicle contaimng menthol and
`ethanol showed a marked enhancement effect not
`
`only on water-soluble drugs, but also on lipophilic
`drugs. It has been generally accepted that solvents
`such as ethanol or propylene glycol used with
`enhancers accumulate in the tissue and increase the
`
`partitioning of drugs due to large affimties of drugs
`for the solvents [44].
`Table 3 shows that thymol and menthol increase
`the partitioning of tamoxifen in stratum comeum to a
`greater extent than the control. Carvone and cineole
`do not affect the partitiomng of tamoxifen into the
`stratum comeum in comparison with the control.
`Therefore enhancement in the permeability coeffi-
`cient by carvone and cineole is possibly due to
`dismption of the stratum comeum lipids and not due
`to improvement in partitiomng of tamoxifen into the
`stratum comeum. However, enhancement
`in the
`permeability of tamoxifen by menthol and thymol
`may be attributed, at least in part, to improvement in
`
`the partitioning of the drug in the stratum comeum.
`In this study,
`terpenes (e.g. carvone, 1,8-cineole,
`menthol, and thymol)
`in combination with 50%
`ethanol significantly enhanced (P<0.01) the per-
`meability coefficient of tamoxifen possibly dismpt-
`ing highly ordered intercellular lipids structure and/
`or improving the partitiomng of tamoxifen in the
`stratum comeum.
`
`Acknowledgements
`
`We acknowledge the financial support from NSF
`through ND/EPSCoR grant # OSR-9452892.
`
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`
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`AstraZeneca Exhibit 2100 p. 7
`
`