-
International Journal of Pharmaceutics 215 (2001) 229240
The effects of terpene enhancers on the percutaneouspermeation
of drugs with different lipophilicities
Ayman F. El-Kattan a,b, Charles S. Asbill a,c, Nanhye Kim
a,d,Bozena B. Michniak a,e,*
a College of Pharmacy, Uni6ersity of South Carolina, Columbia,
SC 29208, USAb Pfizer, Ann Arbor, MI, USA
c McWhorter School of Pharmacy, Samford Uni6ersity, Birmingham,
AL, USAd Watson Pharmaceuticals, Salt Lake City, UT, 84108, USA
e New Jersey Center for Biomaterials, Laboratory for Drug
Deli6ery Center, 111 Lock Street, Newark, NJ 07103, USA
Received 26 July 2000; received in revised form 7 December 2000;
accepted 11 December 2000
Abstract
Four model drugs were selected based on their lipophilicity
denoted as log P (nicardipine hydrochloride log P0.9990.1,
hydrocortisone log P 1.4390.47, carbamazepine log P 2.6790.38, and
tamoxifen log P 7.8790.75).The enhancing activities of four terpene
enhancers (fenchone log P 2.1390.30, thymol log P 3.2890.20,
D-limonenelog P 4.5890.23, and nerolidol log P 5.3690.38) were
tested in vitro across full thickness hairless mouse skin witheach
of the evaluated drugs formulated in hydroxypropyl cellulose gel
formulations. The relationships betweenlipophilicity (log P) of the
terpene enhancers and model drugs and efficacy (represented by the
enhancement ratio offlux ERflux) of the drugs when coadministered
with the enhancers were examined using linear regression.
Terpeneenhancers had significant effect on the percutaneous
permeation of the model drugs. Nerolidol (highest
lipophilicity)provided the highest increase in the flux of the
evaluated model drugs. The flux of nicardipine hydrochloride
increasedby approximately 135-fold, hydrocortisone by 33-fold,
carbamazepine 8-fold, and tamoxifen 2-fold. The lowestincrease in
the flux was observed with fenchone. Linear relationships were
generated between the ERflux of nicardipinehydrochloride,
hydrocortisone, carbamazepine, and tamoxifen and the log P of the
terpene enhancers [r0.951,(P0.049), r0.977, (P0.023), r0.942,
(P0.057), and r0.874, (P0.126), respectively]. Furthermore, thefour
terpene enhancers produced linear relationships, indicating that
they were more effective at enhancing thepenetration of hydrophilic
drugs rather than lipophilic drugs r 0.824 (P0.176) for fenchone, r
0.891(P0.109) for thymol, r 0.846 (P0.154) for limonene, and r
0.769 (P0.232) for nerolidol. 2001Published by Elsevier Science
B.V.
www.elsevier.com:locate:ijpharm
1. Introduction
Skin, the largest human body organ, envelopsand protects the
body from the external environ-
* Corresponding author. Tel.: 1-973-9729720; fax:
1-973-3535336.
E-mail address: [email protected] (B.B. Michniak).
0378-5173:01:$ - see front matter 2001 Published by Elsevier
Science B.V.
PII: S0 378 -5173 (00 )00699 -2
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240230
ment while helping maintain the integrity andappropriate
function of the complex inner bodyorgans. The utilization of the
transdermal drugdelivery is associated with various advantages
thathave been extensively discussed in the literature.This route
allows for controlled release of thedrug at rates approaching
zero-order simulatingthose provided by IV infusion. It also
delivers themedication in a continuous input, which is impor-tant
for drugs that have short biological half-livesand low therapeutic
indices. Once the drug isabsorbed through the skin, the first-pass
intestinaland hepatic metabolism is bypassed. As a conse-quence of
all these advantages, patient complianceis improved.
Despite the obvious advantages of transdermaldrug delivery, this
route presents unique chal-lenges. The greatest obstacle is the
stratumcorneum, the outermost layer of the skin, which isconsidered
to form the primary rate-limiting bar-rier to the permeation of the
drugs across the skin(Zettersten et al., 1997). It consists of
dead, flat-tened cells, filled with keratin, that are embeddedin a
lipid matrix (Bouwstra et al., 1999; Coderchet al., 1999). Lipids
in the intercellular spaces aremade of fatty acids, ceramides, and
cholesterol(esters) and are arranged in bilayer structures,which
run mainly parallel to the skin surface(Elias, 1996a,b). The
stratum corneum has beendescribed as hydrophilic protein bricks in
a hy-drophobic mortar (Heisig et al., 1996). The searchfor
solutions to this problem led investigators toemploy several
enhancement techniques such asiontophoresis, electroporation (Banga
et al.,1999), or the application of drug eutectic
mixtures(Kaplun-Frischoff and Touitou, 1997; Stott et al.,1998).
However, the most widely implementedtechnique is the use of
chemical penetration en-hancers, which reversibly perturb the
permeabilitybarrier of the stratum corneum. Examples includemany
compounds such as Azone and its ana-logues (Michniak, 1993;
Michniak et al., 1993,1994a,b), fatty acids (Aungst, 1989),
alcohols(Takahashi et al., 1991), pyrrolidones (Southwelland Barry,
1983), sulfoxides (Choi et al., 1991),and terpenes (Williams and
Barry, 1990; Cornwelland Barry, 1991; El-Kattan et al., 2000a,b).
How-ever, the skin has a special role as a major barrier
in protecting a living body from cutaneous expo-sure to toxic
chemicals, hence the safety of perme-ation enhancers is of primary
consideration.Terpenes are of low cutaneous irritancy, possessgood
toxicological profile, provide excellent en-hancement ability, and
appear to be promisingcandidates for pharmaceutical formulations
(Gaoand Singh, 1998). A variety of terpenes have beenshown to
increase the percutaneous absorption ofboth hydrophilic (Zhao and
Singh, 1999) andlipophilic drugs (Gao and Singh, 1998).
Differen-tial scanning calorimetery (DSC) studies showedthat cyclic
monoterpenes decreased the transitiontemperature associated with
the stratum corneumlipids (Yamane et al., 1995). The DSC
resultssupport the speculation that terpene enhancersmainly
increase drug diffusivity in the skin bydisrupting the highly
ordered intercellular lipidstructure of the stratum corneum.
Furthermore,this interaction with the stratum corneum lipids
isreversible.
In this study, hydroxypropyl cellulose (HPC),which is a
hydrophobic nonionic polymer, wasincorporated in the gel
formulations. HPC hasseveral useful properties that render it
widely usedin topical and transdermal formulations. It is usedas an
emulsifier, suspending, and stabilizing agentfor topical
preparations (Wu et al., 1998). Fur-thermore, it can inhibit the
formation of sedi-ments (Wade and Weller, 1995).
The purpose of the present study was to investi-gate if a
correlation exists between the efficacy ofthe terpene enhancers
with different lipophilicity(denoted as log P) (fenchone log P
2.1390.30,thymol log P 3.2890.20, D-limonene log P4.5890.23, and
nerolidol log P 5.3690.38) andthe log P of the model drugs
(nicardipine hydro-chloride log P 0.9990.1, hydrocortisone log
P1.4390.47, carbamazepine log P 2.6790.38, andtamoxifen log P
7.8790.75).
2. Methods
2.1. Materials
Fenchone, thymol, D-limonene, and nerolidolwere all purchased
from Aldrich Chemical Co.
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240 231
(Milwaukee, WI). Nicardipine hydrochloride, hy-drocortisone,
carbamazepine, tamoxifen, potas-sium phosphate monobasic, and
triethanolaminewere supplied by Sigma Chemical Co. (St. Louis,MO).
Acetonitrile, methanol, and water used wereof HPLC grade and
supplied by EM Science(New Briggs, NJ). Ethyl alcohol (USP) was
ob-tained from Aaper Alcohol and Chemical Co.(Shelbyville, KY). HPC
and glycerol were pro-vided by Spectrum Chemical ManufacturingCorp.
(Gardena, CA). Female hairless mice strainSKH1 (h:h), 68 weeks old
were obtained fromCharles River Lab., Inc., (Wilmington, MA).
Allreagents were of analytical grades and used with-out further
purification.
2.2. Preparation of gel formulations
HPC gels were prepared as described recentlyby El-Kattan et al.
(El-Kattan et al., 2000a,b).The HPC gel composition is shown in
Table 1.Briefly, a Sted Fast stirrer was used to mix etha-nol with
glycerol and distilled water. Then HPCpowder was added to the
obtained solution withcontinuous mixing until the gel was formed.
Thetested drug followed by a terpene enhancer wereadded to the gel
and mixed. The gels were leftovernight at ambient temperature. It
is worthnoting that tamoxifen, carbamazepine, and hydro-cortisone
were not completely soluble in the pre-pared gel formulations.
Whereas, nicardipinehydrochloride was completely soluble in the
gelformulations. Furthermore, all the evaluated ter-pene enhancers,
but limonene were completelysoluble in the gel formulations.
2.3. In 6itro skin permeability studies
Franz diffusion cells (effective diffusion area0.64 cm2,
receptor volume 5.1 ml) were used toevaluate the in vitro drug
percutaneous perme-ation from HPC gel. The receptor compartmentwas
filled with isotonic phosphate buffer (pH 7.2)and 0.1% formaldehyde
(37%) as a preservative.The temperature was maintained at
3790.5Cand the receptor was constantly stirred at 600rpm. Excised
female full-thickness hairless mouseskin (strain SKH1 h:h, 8 weeks
old, Charles RiverLaboratories, Wilmington, MA) was mounted
be-tween the donor and receptor compartments. Ap-proximately 300 mg
of HPC gel was placed oneach skin. All cell donors were occluded
with aParafilm. A 500-ml sample of the receptor wastaken at
predetermined time intervals over 24 hand replenished immediately
with equal volume ofthe diffusion buffer. Samples were frozen at
70C prior to HPLC analysis. After 24 h ofsampling, the skins were
removed from the cellsand washed briefly in methanol (25 ml) for 15
s(Michniak et al., 1994a,b). Following drying atroom temperature
for 10 min, each skin was cutup and then homogenized in 4-ml
methanol. Thesamples were then centrifuged and the superna-tant
layer was taken and stored at 70C beforeHPLC analysis.
Control gel formulations for each model drugwere developed.
Control formulations consistedof all the formulation components
except theterpene enhancer. All experiments were n5.
2.4. HPLC analysis of model drugs
All drugs were analyzed using a reverse phaseC18 column
(MICROSORB-MV, 15 cm, 5 mm)and at a flow rate 1 ml:min. HPLC
analysis wasperformed using Hewlett Packard 1100 with anautosampler
equipped with a quaternary pump, avariable-wavelength detector.
Nicardipine hydro-chloride was detected at 240 nm with a
mobilephase composition of 60:40 acetonitrile:0.02 Mpotassium
phosphate monobasic and injectionvolume of 20 ml (Kobayashi et al.,
1993). Hydro-cortisone was detected at 242 nm with a mobilephase
composition of 40:60 acetonitrile:water and
Table 1Composition (% w:w) of drug HPC gel formulations a
% w:wComponent
2Drug*2Terpene2HPC
Water 2810Glycerol
Ethanol 56
a * nicardipine hydrochloride, hydrocortisone, carba-mazepine,
and tamoxifen.
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240232
Table 2Effect of terpene enhancers on the percutaneous
parameters of nicardipine hydrochloride formulated in HPC gels
a
ERflux Q24 (mg:cm2)Terpene SC (mg:g)Flux (mg:cm2 per h)
1.0Control 45952.591.0 43059400Fenchone 44.895.0 17.9 793919 13
35991023Thymol 45.596.2 18.2 811962 98039845
60.0 33049350150.0920.0 21 48394532Limonene337.2931.2Nerolidol
134.8 61059590 18 04693425
a Mean9S.D., n5. ERflux, Enhancement ratio of nicardipine HCl
flux; Q24, cumulative amount of nicardipine HCl in thereceptor
after 24 h; SC, skin content of nicardipine HCl after 24 h.
injection volume of 40 ml (Michniak, 1993). Car-bamazepine was
detected at 210 nm with a mobilephase composition of
acetonitrile:water (30:70)and injection volume of 20 ml (Psillakis
et al.,1999). Tamoxifen was detected at 275 nm with amobile phase
composition of wa-ter:triethanolamine:methanol and injection
vol-ume of 100 ml (70:1.8:928) (Herrlinger et al.,1992).
2.5. Data analysis
The in vitro skin permeation data obtained wasgraphically
plotted as the cumulative correctedamount of drug penetrated into
the receptorphase as a function of time. The permeationprofiles
provided the following parameters: Theslope of the straight line
portions of this plot (atsteady state) yielded the values of flux
(mg:cm2 perh) and the cumulative corrected receptor concen-trations
at 24 h Q24 (mg:cm2). Skin content valueswere expressed as mg of
drug per gram of hy-drated full-thickness mouse skin.
Log P values of terpenes enhancers and drugswere determined
using ACD software program(Advanced Chemistry Incorporated,
Ontario,Canada).
Enhancement ratios for flux (ERflux) were calcu-lated using the
following equation
ERflux
Model drug flux with terpene in gelModel drug flux without
terpene in gel (control)
(1)
Controls were assigned the value of 1.00 andthe data was
presented as mean9S.D. of five
experiments. Statistical analyses were performedusing one-way
analysis of variance (one wayANOVA). Correlation analyses were
performedby the least squares linear regression method.Correlation
coefficients were examined for signifi-cance (PB0.05) by Students t
test.
3. Results
3.1. Percutaneous absorption of nicardipinehydrochloride in
6itro
The effects of terpene enhancers on the percuta-neous permeation
parameters of nicardipine hy-drochloride (flux, ERflux, cumulative
amount ofnicardipine hydrochloride after 24 h (Q24), andskin
content) from HPC gel formulations areshown in Table 2. Controls
consisted of gel for-mulations with no terpene enhancers. The
valuesfor the percutaneous permeation parameters ofnicardipine
hydrochloride control gels are as fol-lows 2.591.0 mg:cm2 per h for
flux, 4595 mg:cm2 for Q24, and 43059400 mg:g for
nicardipinehydrochloride skin content. All the evaluated ter-pene
enhancers had significant effects on the per-cutaneous permeation
of nicardipinehydrochloride relative to the control.
Nerolidolprovided the best enhancement activity fornicardipine
hydrochloride. It increased nicardip-ine hydrochloride flux
134.8-fold relative to thecontrol (P0.0000042) followed by
limonenewith 60.0-fold (P0.00021), thymol 18.2 fold(P0.000244), and
fenchone 17.9-fold (P0.00013). The highest increase in the Q24 was
alsoprovided by nerolidol (61059590 mg:cm2) fol-
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240 233
lowed by limonene (33049350 mg:cm2), thymol(811962 mg:cm2), and
fenchone (793919 mg:cm2). It should be emphasized that the increase
inthe Q24 provided by nerolidol, limonene, thymol,and fenchone was
significantly higher than thatrecorded with the control (P4106,
4106, 0.00011, and 0.000063, respectively). Thehighest nicardipine
hydrochloride skin contentwas observed with limonene (21 48394532
mg:g)followed by nerolidol (18 04693425 mg:g), fen-chone (13
35991023 mg:g), and thymol (98039845 mg:g), which were
significantly higher thanthat provided by the control (P0.0027,
0.0025,0.0014, and 0.00090, respectively).
3.2. Percutaneous absorption of hydrocortisone in6itro
The effects of terpene enhancers on the percuta-neous permeation
parameters of hydrocortisone(flux, ERflux, cumulative amount of
hydrocorti-sone after 24 h (Q24), and skin content) from HPCgel
formulations are presented in Table 3. Controlvalues for
hydrocortisone were determined to be6.091.5 mg:cm2 per h for flux,
14596 mg:cm2 forQ24, and 80529290 mg:g for hydrocortisone
skincontent. All tested terpene enhancers had signifi-cant effects
on both hydrocortisone flux and Q24relative to the control. The
highest increase in theflux was observed with nerolidol, which
increasedthe flux by 32.7-fold relative to the control (P0.0011)
followed by limonene with 28.0-fold (P0.00071), thymol 10.5-fold
(P0.00088), andfenchone with 7.8 fold (P0.000169). Similar tothe
ERflux results, nerolidol provided the highestincrease in the Q24
(18379106 mg:cm2) that was
significantly higher than that observed with thecontrol (P8106)
followed by limonene(15699232 mg:cm2) (P0.00095) and
thymol(11589168 mg:cm2) (P0.00059). The lowest in-crease in the Q24
was recorded with fenchone(10179148 mg:cm2) yet it was
significantly higherthan that observed with the control
(P0.00094).Terpene enhancers did not increase the hydrocor-tisone
skin content values significantly relative tothe control. All the
terpene enhancers, with theexception of fenchone (10 61193139 mg:g)
hadlower skin content values relative to the control.
3.3. Percutaneous absorption of carbamazepine in6itro
The effects of terpene enhancers on the percuta-neous permeation
parameters of carbamazepine(flux, ERflux, cumulative amount of
carba-mazepine after 24 h (Q24), and skin content) fromHPC gel
formulations are presented in Table 4.The control percutaneous
permeation parametersfor carbamazepine were 24.694.5 mg:cm2 per
hfor flux, 6809123 mg:cm2 for Q24, and 38739644 mg:g for
carbamazepine skin content. It isinteresting to note that all the
terpene enhancers,with the exception of fenchone had major
effectson the carbamazepine flux and Q24 relative to thecontrol.
Nerolidol provided the highest increase inthe carbamazepine flux
relative to the control. Itincreased the flux by 7.5-fold
(P0.00041) fol-lowed by limonene with 6.6-fold (P0.00011),thymol
4.2-fold (P0.00077), and fenchone with1.5-fold (P0.049). Similar to
the ERflux results,nerolidol provided the highest increase in the
Q24(12839251 mg:cm2) relative to the control (P
Table 3Effect of terpene enhancers on the percutaneous
parameters of hydrocortisone formulated in HPC gels a
Flux (mg:cm2 per h) ERflux Q24 (mg:cm2) SC (mg:g)Terpene
14598Control 1.0 805292906.091.510 61193139101791487.8Fenchone
47.294.9
10.563.4910.6 664092015Thymol 1158916815699232168.9929.3Limonene
28.0 834291250
32.7Nerolidol 46039142118379106196.5939.0
a Mean9S.D., n5. ERflux, enhancement ratio of hydrocortisone
flux; Q24, cumulative amount of hydrocortisone in the receptorafter
24 h; SC, skin content of hydrocortisone after 24 h.
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215 (2001) 229240234
Table 4Effect of terpene enhancers on the percutaneous
parameters of carbamazepine formulated in HPC gels a
ERflux Q24 (mg:cm2)Terpene SC (mg:g)Flux (mg:cm2 per h)
1.0Control 680912324.694.5 38739644Fenchone 37.896.4 1.5 7739123
16869525
103.9913.2Thymol 4.2 994979 7840920126.6 1121948162.5916.97 16
74291531Limonene
Nerolidol 7.5185.1925.0 12839251 34 91099924
a Mean9S.D., n5. ERflux, enhancement ratio of carbamazepine
flux; Q24, cumulative amount of carbamazepine in the receptorafter
24 h; SC, skin content of carbamazepine after 24 h.
0.020) followed by limonene (112148 mg:cm2)(P0.0041) and thymol
(994979 mg:cm2) (P0.020). The lowest increase in the Q24 was
pro-vided by fenchone (7739123 mg:cm2), which wasnot significantly
different from the control (P0.41). It is interesting to note that
nerolidol,limonene, and thymol significantly increased
thecarbamazepine skin contents relative to the con-trol (P0.0058,
0.00016, and 0.032, respectively).However, the carbamazepine skin
contentrecorded with fenchone (16869525 mg:g) wassignificantly
lower than that provided by the con-trol (P0.0081).
3.4. Percutaneous absorption of tamoxifen in 6itro
The effects of terpene enhancers on the percuta-neous permeation
parameters of tamoxifen (flux,ERflux, cumulative amount of
tamoxifen after 24 h(Q24), and skin content) from HPC gel
formula-tions are shown in Table 5. Controls consisted ofgel
formulations with no terpene enhancer and thevalues for the
percutaneous permeation of tamox-ifen control gel were found to be
2.891.0 mg:cm2per h for flux, 52925 mg:cm2 for Q24, and358491642
mg:g for tamoxifen skin content. Un-like the results found with
other model drugs,terpene enhancers did not have major effects
onthe percutaneous permeation parameters of ta-moxifen relative to
the control. However,nerolidol provided the best enhancement
activityfor tamoxifen. It increased the flux 1.7-fold rela-tive to
the control followed by limonene with1.6-fold, and thymol 1.4-fold.
Unlike other terpe-nes, fenchone decreased the flux 0.6-fold
relativeto the control. Nerolidol also provided the highest
increase in the Q24. It increased Q24 to 82913mg:cm2 followed by
limonene (81925 mg:cm2),thymol (74955 mg:cm2), and fenchone
(57915mg:cm2). It should be emphasized that the ob-tained Q24
results with nerolidol, limonene, thy-mol, and fenchone were not
significantly higherthan that provided by the control (P0.12,
0.21,0.51, and 0.78, respectively). The highest tamox-ifen skin
content was observed with limonene(714091932 mg:g) followed by
thymol (711593600 mg:g), fenchone (577391000 mg:g), andnerolidol
(553291431 mg:g). It should be empha-sized that limonene, thymol,
fenchone, andnerolidol did not increase tamoxifen skin
contentssignificantly relative to the control (P0.065,0.196, 0.117,
and 0.208, respectively).
3.5. Correlation of terpene efficacy with log P6alues of model
drugs
In this study, the effect of terpene enhancers onthe
percutaneous permeation of model drugs withdifferent lipophilicity
as denoted by their log Pvalues were investigated. Correlation
coefficientswere established between the lipophilicties of themodel
drugs and the ERflux and between the ter-pene enhancers
lipophilicities and the ERflux.
Fig. 1 presents the relationships between thelipophilicities of
the model drugs (nicardipine hy-drochloride, hydrocortisone,
carbamazepine, andtamoxifen) and their ERflux with the terpene
en-hancers. The findings suggest that the increase inthe
lipophilicities of the model drug is associatedwith a decrease in
the percutaneous permeation ofthe drug. The relationships for the
four terpeneenhancers were found to be linear [r 0.824,
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240 235
(P0.176) for fenchone, r 0.891, (P0.109)for thymol, r 0.846,
(P0.154) for limonene,and r 0.769, (P0.232) for nerolidol].
Fig. 2 represents the relationships between thelipophilicities
of the terpene enhancers (fenchone,thymol, limonene, and nerolidol)
and the ERflux ofthe tested model drugs. It can be suggested
thatthe increase in the lipophilicities of the terpeneenhancers is
associated with an increase in thepercutaneous permeation of the
drug. The rela-tionships for the five model drugs were found tobe
linear [r0.951, (P0.049) for nicardipinehydrochloride, r0.977,
(P0.023) for hydro-cortisone, r0.942, (P0.057) for carba-mazepine,
and r0.874, (P0.126) fortamoxifen].
4. Discussion
Terpenes were reported to be effective penetra-tion enhancers
for both hydrophilic and lipophilicdrugs (Cornwell and Barry, 1991;
Hori et al.,1991; Gao and Singh, 1998; Zhao and Singh,1998). In
this study, the tested terpene enhancersprovided significant
enhancements for the fluxand cumulative amounts of the evaluated
modeldrugs at 24 h (Q24). Nerolidol in particular wasthe most
effective terpene enhancer in promotingthe permeation of all the
model drugs followed bylimonene and thymol. Fenchone provided
thelowest increase in the flux for all the tested modeldrugs. The
obtained results are similar to thosereported with other research
groups. In 1991,Cornwell et al. evaluated the effect of
terpeneenhancers on the percutaneous permeation of 5-
fluorouracil across the skin. They reported thatnerolidol
provided the highest enhancement forthe permeation of
5-fluorouracil (Cornwell andBarry, 1991). Furthermore, the high
enhancementactivity of nerolidol was reported by Arellano etal.,
who found that nerolidol was an effectiveenhancer for the
permeation of diclofenac sodiumacross the rat skin (Arellano et
al., 1996). Theeffective promoting activity of nerolidol was
at-tributed to its amphiphilic structure that was suit-able for
alignment within the lipid lamellae of thestratum corneum and
disrupting its highly orga-nized packing (Cornwell and Barry,
1994).
It is worth noting that limonene providedhigher enhancing
activity for the permeation ofnicardipine hydrochloride
(hydrophilic calciumchannel blocker log P 0.9990.1) and
hydro-cortisone (a polar steroid with a log P of 1.4390.25)
relative to fenchone and thymol. Thesefindings conflict with the
results observed by otherauthors. It has been recognized that
hydrophilicterpenes capable of hydrogen binding (such asfenchone
and thymol) are more active towardspromoting the permeation of
hydrophilic drugs,whereas, hydrocarbon terpenes (such as
limonene)provide higher enhancing activity for lipophilicdrugs
(Hori et al., 1991; Moghimi et al., 1997). In1992, Katayama et al.
studied the effect of L-men-thol on the skin permeability of
mannitol, corti-sone or indomethacin using hairless mouse skin
invitro (Katayama et al., 1992). They observed thatwhen the donor
solution contained L-menthol inan aqueous ethanol vehicle at pH 7.4
the perme-ability coefficients of mannitol and indomethacinwere
increased by 100-fold relative to thatrecorded with the control (an
aqueous vehicle)
Table 5Effect of terpene enhancers on the percutaneous
parameters of tamoxifen formulated in HPC gels a
Flux (mg:cm2 per h) ERflux Q24 (mg:cm2) SC (mg:g)Terpene
35849164252925Control 2.891.0 1.057915 5773910000.6Fenchone
1.890.8749551.43.991.5Thymol 711593600
7140919351.64.591.3Limonene 819251.7Nerolidol
553291431829134.890.7
a Mean9S.D., n5. ERflux, enhancement ratio of tamoxifen flux;
Q24, cumulative amount of tamoxifen in the receptor after 24h; SC,
skin content of tamoxifen after 24 h.
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215 (2001) 229240236
Fig. 1. The relationship between the log P of model drugs and
the ERflux of the evaluated model drugs (nicardipine
hydrochloride,hydrocortisone, carbamazepine, and tamoxifen), using
fenchone, thymol, limonene, and nerolidol as penetration
enhancers.
and increased those of cortisone by about 10-fold.However,
L-menthol did not increase the perme-ation of indomethacin at pH
3.0, since the major-ity of the species would be in the unionized
form.
The effects of terpenes on the permeation ofpropranolol
hydrochloride (hydrophilic drug) anddiazepam (lipophilic drug) were
evaluated by Horiet al. The purely hydrocarbon terpenes
promotedboth propranolol and diazepam permeation (Horiet al.,
1991). Whereas, the terpenes with hydro-gen-bonding ability only
enhanced the flux ofpropranolol.
The data in our study can be explained by thefollowing
information. The higher enhancementactivity of limonene relative to
thymol and fen-chone can be attributed to its higher thermody-namic
activity in the gel since limonene was notcompletely soluble in the
gel at 2% concentration.On the contrary, thymol and fenchone were
foundto be completely soluble in the gel at the evalu-ated
concentration. Similar results were also re-ported by Obata et al.,
who attributed the higherenhancing activity of the permeation of
diclofenacby limonene to its higher thermodynamic activity
in the 40% ethanol-buffer solution at the evalu-ated
concentration relative to L-menthol (Obata etal., 1993).
The lipophilicity of the permeant, as well as theenhancer
molecule is thought to play an impor-tant role in determining the
enhancers promotingactivity on the permeation of the drug across
theskin (Okabe et al., 1990; Hori et al., 1991; Lee etal., 1993,
1994; Phillips and Michniak, 1995; Bor-ras-Blasco et al., 1997;
Godwin and Michniak,1999; El-Kattan et al., 2000a,b; Sung et al.,
2000).
In the present study, terpene enhancers in com-bination with the
gel solvent mixtures exhibitedhigh enhancement for flux and Q24 of
the testedmodel drugs. The enhancers produced the highestactivity
for the most hydrophilic drug i.e.nicardipine hydrochloride
followed by hydrocorti-sone, and carbamazepine. The lowest activity
ofthe enhancers was recorded with the mostlipophilic compound i.e.
tamoxifen. Terpene en-hancers did not increase tamoxifen flux and
Q24significantly relative to the control (Table 5). Theresults were
anticipated, since other studies havedemonstrated the significant
effect of model drug
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240 237
lipophilicity on the penetration enhancing proper-ties of
terpenes (Kitagawa et al., 1998). Guy et al.reported that compounds
with log P of more than2 may have potential problems in achieving
steadyplasma concentrations in reasonable time spansdue to the fact
that the drug is delayed in thestratum corneum where a reservoir
may be estab-lished (Guy and Hadgraft, 1989). Kitagawa et
al.evaluated the effect of L-menthol in 15% ethanolon the
permeation of paraben analogues throughexcised guinea pig dorsal
skin. They found thatthe permeability coefficients of the parabens
cor-related with their lipophilicity (log P). Addition of1%
L-menthol in 15% ethanol increased the per-meability coefficient of
methyl paraben 16-fold,whereas this enhancer decreased that of
butylparaben to about one fifth of the control value(Kitagawa et
al., 1997).
In our study, significant linear correlations wereestablished
between the ERflux and the log P ofthe tested model drugs for the
four tested terpeneenhancers [r 0.824, (P0.176) for fenchone,r
0.891, (P0.109) for thymol, r 0.846,(P0.154) for limonene, and r
0.769, (P0.232) for nerolidol (Fig. 1)].
In the present investigation, the lipophilicitydenoted as log P
of the terpene enhancers greatlyinfluenced the enhancement ratios
of the evalu-ated model drugs at log P ranges of 2.135.36and strong
correlation coefficients were estab-lished between the ERflux of
nicardipine hydro-chloride, hydrocortisone, carbamazepine,
andtamoxifen and the log P of the terpene enhancers[r0.951,
(P0.049), r0.977, (P0.023), r0.942, (P0.057), and r0.874, (P0.126),
re-spectively, (Fig. 2)]. These results are in a goodagreement with
earlier findings reported byWilliams et al., who found linear
relationshipsbetween the lipophilicity of the epoxide and ke-tone
terpenes and the enhancement ratio for 5-fluorouracil permeation
(Williams and Barry,1991). The mechanism of permeation enhance-ment
of the terpene enhancers has been evaluatedusing differential
scanning calorimetery, Fouriertransform infrared, and X-ray
diffraction. Thesestudies suggested that terpenes enhance the
per-meation of the drug across the skin mainly bydisrupting the
highly ordered intercellular packingof the stratum corneum lipids
and increasing drugdiffusivity (Williams and Barry, 1991; Cornwell
et
Fig. 2. The relationship between the log P of the terpene
enhancers and the ERflux of the evaluated model drugs
(nicardipinehydrochloride, hydrocortisone, carbamazepine, and
tamoxifen), using female hairless mouse skin.
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A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240238
al., 1994). Taking these finding into consideration,it can be
speculated that the increase in thelipophilicity of the terpenes
may increase theirability to disrupt the highly hydrophobic
lipidlamella of the stratum corneum.
5. Conclusions
In conclusion, terpene enhancer lipophilicityhad a significant
effect on the percutaneous per-meation of the model drugs tested.
Increasing thelog P of the terpene enhancers was associatedwith an
increase in drug permeation. Nerolidolwas the most effective
terpene enhancer for thepermeation of the drugs followed by
limoneneand thymol. Fenchone was the least effective ter-pene
enhancer. However, the higher enhancingactivity of limonene
relative to fenchone and thy-mol was unexpected and attributed to
its higherthermodynamic activity in the gel. Unlike fen-chone and
thymol, limonene was not completelysoluble in the gel at 2%
concentration. This maysuggest the importance of the gel
formulationvehicle compositions on the enhancing activity ofthe
terpene enhancers. Furthermore, model druglipophilicity had a
significant impact on the ter-pene enhancers promoting activity.
Increasing thelipophilicity of the model, drug was associatedwith a
decrease in the enhancement activity of theterpenes. Terpene
enhancers provided the highestenhancement activity for nicardipine
hydrochlo-ride, which was the most hydrophilic compound.The lowest
enhancement activity was provided fortamoxifen, which was the most
lipophilic com-pound. It should be stressed that the
obtainedresults were in agreement with earlier findings ofKitagawa
et al., who reported that the addition of1% L-menthol in 15%
ethanol increased the per-meability coefficient of methyl paraben
16-fold,whereas this enhancer decreased that of butylparaben to
about one fifth of the control value(Kitagawa et al., 1997).
Further work needs to be conducted using ad-ditional drugs and
other series of enhancercompounds.
References
Arellano, A., Santoyo, S., Martin, C., Ygartua, P.,
1996.Enhancing effect of terpenes on the in vitro
percutaneousabsorption of diclofenac sodium. Int. J. Pharm. 130,
141145.
Aungst, B.J., 1989. Structure:effect studies of fatty acid
iso-mers as skin penetration enhancers and skin irritants. J.Pharm.
Sci. 6, 244.
Banga, A.K., Bose, S., Ghosh, T.K., 1999. Iontophoresis
andelectroporation: comparisons and contrasts. Int. J. Pharm.179,
119.
Borras-Blasco, J., Lopez, A., Morant, M., Diez-Sales,
O.,Herraez-Dominguez, M., 1997. Influence of sodium laurylsulfate
on in vitro percutaneous absorption of compoundswith different
lipophilicity. Eur. J. Pharm. Sci. 5, 1522.
Bouwstra, J.A., Dubbelaar, F.E., Gooris, G.S., Weerheim,A.M.,
Ponec, M., 1999. The role of ceramide compositionin the lipid
organisation of the skin barrier. Biochim.Biophys. Acta 1419,
127136.
Choi, H.K., Amidon, G.L., Flynn, G.L., 1991. Some
generalinfluences of n-decylmethyl sulfoxide on the permeation
ofdrugs across hairless mouse skin. J. Invest. Dermatol.
96,822826.
Coderch, L., de Pera, M., Perez-Cullell, N., Estelrich, J., de
laMaza, A., Parra, J.L., 1999. The effect of liposomes onskin
barrier structure. Skin Pharmacol. Appl. Skin Physiol.12,
235246.
Cornwell, P., Barry, B., 1991. Sesquiterpene components
ofvolatile oils as skin penetration enhancers for the hy-drophilic
permeant 5-flurouracil. J. Pharm. Pharmacol. 46,261269.
Cornwell, P.A., Barry, B.W., 1994. Sesquiterpene componentsof
volatile oils as skin penetration enhancers for the hy-drophilic
permeant 5-fluorouracil. J. Pharm. Pharmacol.46, 261269.
Cornwell, P.A., Barry, B.W., Stoddart, C.P., Bouwstra,
J.A.,1994. Wide-angle X-ray diffraction of human stratumcorneum:
effects of hydration and terpene enhancer treat-ment. J. Pharm.
Pharmacol. 46, 938950.
Elias, P.M., 1996a. Stratum corneum architecture,
metabolicactivity and interactivity with subjacent cell layers.
Exp.Dermatol. 5, 191201.
Elias, P.M., 1996b. The stratum corneum revisited. J. Derma-tol.
23, 756758.
El-Kattan, A.F., Asbill, C.S., Michniak, B.B., 2000a. Theeffect
of terpene enhancer lipophilicity on the percutaneouspermeation of
hydrocortisone formulated in HPMC gelsystems. Int. J. Pharm. 198,
179189.
El-Kattan, A.F., Asbill, C.S., Michniak, B.B., 2000. The
effectof terpene enhancer lipophilicity on the percutaneous
per-meation of hydrocortisone formulated in HPMC gel sys-tems.
Inter. J. Pharm., in press.
Gao, S., Singh, J., 1998. In vitro percutaneous
absorptionenhancement of a lipophilic drug tamoxifen by terpenes.
J.Contr. Rel. 51, 193199.
-
A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240 239
Godwin, D.A., Michniak, B.B., 1999. Influence of
druglipophilicity on terpenes as transdermal penetration
en-hancers. Drug Dev. Ind. Pharm. 25, 905915.
Guy, R., Hadgraft, J., 1989. Selection of candidates
fortransdermal drug delivery. In: Hadgraft, J., Guy, R.(Eds.),
Transdermal Drug Delivery. Marcel Dekker, NewYork, pp. 5982.
Heisig, M., Lieckfeldt, R., Wittum, G., Mazurkevich, G.,Lee, G.,
1996. Non steady-state descriptions of drug per-meation through
stratum corneum. I. The biphasic brick-and-mortar model. Pharm.
Res. 13, 421426.
Herrlinger, C., Braunfels, M., Fink, E., Kinzig, M., Metz,R.,
Sorgel, F., Vergin, H., 1992. Pharmacokinetics andbioavailability
of tamoxifen in healthy volunteers. Int. J.Clin. Pharmacol. Ther.
Toxicol. 30, 487489.
Hori, M., Satoh, S., Maibach, H.I., Guy, R.H., 1991.
En-hancement of propranolol hydrochloride and diazepamskin
absorption in vitro: effect of enhancer lipophilicity.J. Pharm.
Sci. 80, 3235.
Kaplun-Frischoff, Y., Touitou, E., 1997. Testosterone
skinpermeation enhancement by menthol through formationof eutectic
with drug and interaction with skin lipids. J.Pharm. Sci. 86,
13941399.
Katayama, K., Takahashi, O., Matsui, R., Morigaki, S.,Aiba, T.,
Kakemi, M., Koizumi, T., 1992. Effect of L-menthol on the
permeation of indomethacin, mannitoland cortisone through excised
hairless mouse skin.Chem. Pharm. Bull. (Tokyo) 40, 30973099.
Kitagawa, S., Hosokai, A., Aseda, Y., Yamamoto, N.,Aneko, Y.,
Matsuoka, E., 1998. Permeability of benzoicacid derivatives in
excised guinea pig dorsal skin and theeffect of L-menthol. Int. J.
Pharm. 161, 115122.
Kitagawa, S., Li, H., Sato, S., 1997. Skin permeation ofparabens
in excised guinea pig dorsal skin, its modifica-tion by penetration
enhancers and their relationship withn-octanol:water partition
coefficients. Chem. Pharm.Bull. (Tokyo) 45, 13541357.
Kobayashi, D., Matsuzawa, T., Sugibayashi, K., Morimoto,Y.,
Kobayashi, M., Kimura, M., 1993. Feasibility of useof several
cardiovascular agents in transdermal therapeu-tic systems with
L-menthol-ethanol system on hairless ratand human skin. Biol.
Pharm. Bull. 16, 254258.
Lee, C.K., Uchida, T., Kitagawa, K., Yagi, A., Kim, N.S.,Goto,
S., 1993. Effect of hydrophilic and lipophilic vehi-cles on skin
permeation of tegafur, alclofenac andibuprofen with or without
permeation enhancers. Biol.Pharm. Bull. 16, 12641269.
Lee, C.K., Uchida, T., Kitagawa, K., Yagi, A., Kim, N.S.,Goto,
S., 1994. Skin permeability of various drugs withdifferent
lipophilicity. J. Pharm. Sci. 83, 562565.
Michniak, B., Player, M., Chapman, J., Sowell, J., 1993. Invitro
evaluation of a series of azone analogs as dermalpenetration
enhancers. Int. J. Pharm. 91, 8593.
Michniak, B., Player, M., Fuhrman, L., Christensen, C.,Chapman,
J., Sowell, J., 1993. In vitro evaluation of aseries of azone
analogs as dermal penetration enhancers.II. (Thio) amides. Int. J.
Pharm. 94, 203210.
Michniak, B., Player, M.R., Chapman, J.M., Sowell, J.W.,1994a.
Azone analogues as penetration enhancers: effectof different
vehicles on hydrocortisone acetate skin per-meation and retention.
J. Contr. Rel. 32, 147154.
Michniak, B.B., Player, M.R., Chapman, J.M., Sowell, J.W.,1993b.
Optimization of dermal delivery using azoneanalogs as enhancers.
Proc. 12th Pharm. Technol. Conf.2, 158173.
Moghimi, H., Williams, A., Barry, B., 1997. A lamellar ma-trix
model for stratum corneum intercellular lipids. V.Effects of
terpene penetration enhancers on the structureand thermal behavior
of the matrix. Int. J. Pharm. 146,4154.
Obata, Y., Takayama, K., Maitani, Y., Machida, Y., Nagai,T.,
1993. Effect of pretreatment of skin with cyclicmonoterpenes on
permeation of diclofenac in hairless rat.Biol. Pharm. Bull. 16,
312314.
Okabe, H., Obata, Y., Takayama, K., Nagai, T., 1990.
Per-cutaneous absorption enhancing effect and skin irritationof
monocyclic monoterpenes. Drug Design Del. 6, 229.
Phillips, C.A., Michniak, B.B., 1995. Transdermal delivery
ofdrugs with differing lipophilicities using azone analogs asdermal
penetration enhancers. J. Pharm. Sci. 84, 14271433.
Psillakis, T., Tsatsakis, A.M., Christodoulou,
P.,Michalodimitrakis, M., Paritsis, N., Helidonis, E.,
1999.Carbamazepine levels in head hair of patients underlong-term
treatment: a method to evaluate the history ofdrug use. J. Clin.
Pharmacol. 39, 5567.
Southwell, D., Barry, B., 1983. Penetration enhancers forhuman
skin: mode of action of 2-pyrrolidone anddimethylformamide on
partition and diffusion of modelcompounds water, alcohols, and
caffeine. J. Invest. Der-matol. 66, 243252.
Stott, P.W., Williams, A.C., Barry, B.W., 1998.
Transdermaldelivery from eutectic systems: enhanced permeation of
amodel drug, ibuprofen. J. Contr. Rel. 50, 297308.
Sung, K.C., Fang, J., Yoa-Pu Hu, O., 2000. Delivery ofnalbuphine
and its prodrugs across skin by passive diffu-sion and
iontophoresis. J. Contr. Rel. 67, 18.
Takahashi, K., Tamagawa, S., Katagi, T., Yoshitomi, H.,Kamada,
A., Rytting, J.H., Nishihata, T., Mizuno, N.,1991. In vitro
transport of sodium diclofenac across ratabdominal skin: effect of
selection of oleaginous compo-nent and the addition of alcohols to
the vehicle. Chem.Pharm. Bull. (Tokyo) 39, 154158.
Wade, A., Weller, P., 1995. Handbook of
PharmaceuticalExcipients, Second edn. American Pharmaceutical
Associ-ation, Washington DC.
Williams, A., Barry, B., 1990. Differential scanning
calorime-tery does not predict the activity of terpene
penetrationenhancers in human skin. J. Pharm. Pharmacol.
42,156P.
Williams, A.C., Barry, B.W., 1991. Terpenes and the
lipid-protein-partitioning theory of skin penetration enhance-ment.
Pharm. Res. 8, 1724.
-
A.F. El-Kattan et al. : International Journal of Pharmaceutics
215 (2001) 229240240
Wu, P., Huang, Y., Fang, J., Tsai, Y., 1998.
Percutaneousabsorption of captopril from hydrophilic cellulose
deriva-tives through excised rabbit skin and human skin. DrugDev.
Indust. Pharm. 24, 179182.
Yamane, M.A., Williams, A.C., Barry, B.W., 1995.
Terpenepenetration enhancers in propylene glycol:water co-sol-vent
systems: effectiveness and mechanism of action. J.Pharm. Pharmacol.
47, 978989.
Zettersten, E.M., Ghadially, K.R., Feingold, D., Crumrine,A.,
Elias, P.M., 1997. Optimal ratios of topical stratum
corneum lipids improve barrier recovery in chrono-logically aged
skin. J. Am. Acad. Dermatol. 37, 403408.
Zhao, K., Singh, J., 1998. Mechanisms of percutaneous
ab-sorption of tamoxifen by terpenes: eugenol, D-limoneneand
menthone. J. Contr. Rel. 55, 253260.
Zhao, K., Singh, J., 1999. In vitro percutaneous
absorptionenhancement of propranolol hydrochloride throughporcine
epidermis by terpenes:ethanol. J. Contr. Rel. 62,359366.
.