Inn-rampant? Jotrrrmi' of Pharmaceutics. "H ”9911 IST—lfiB
`!2-
`i991 Elserier Science Publishers BX. E1_1?fi-5IT3_/9If5[l3.5tl
`ADONL'S' 03185 I 301003354
`
`IJP 1.1148“
`
`15?
`
`The enhancement index concept applied to terpene penetration
`enhancers for human skin and model lipophilic (oestradiol)
`and hydrophilic (S-fluorouracil} drugs
`
`AC. Williams and B.W. Barryr
`Postgraduate Smditts- in Pfirurmrreeurik-ui' Ti’t'IIHHIrI’JKK'. The \‘t'hooi'rd'Pirrirmut'J'. ifurr'rrsi'rv of Bruifliird. Hm'rtffrmi E't'fi'J
`(Received 32 March I‘Nll
`[Accepted 12 April I99”
`
`Key words: Percutaneous absorption; Skin penetration enhancer; 'l‘erpene: Oestradioi: S—I’luurouracil;
`Enhancement index
`
`Summary
`
`.-’\ series of cyclic monotei'penes has been assessed as skin penetration enhancers towards a model lipnphilir drug. tie-.[radioL In
`vitro permeation experiments on human epidermal membranes showed that the lerpenes varied in their act ivities. hydrocarbon
`{e.g.. iimonene] and cyclic ether (my...
`l.l5-t:irle0le1 [emenes were effective accelerartls providing approximately 4-fold increases in
`
`the permeability coefficient of aqueous oestradiol. whereas alcohols te.g., caneol]. ketones (ere. menthonc} and epoxides le.g..
`
`
`
`pinene oxide] were Inel’ft
`Ive. The results of this study are compared with Ierpene. activil
`s towards a model hydrophilie drug.
`SAt'htorouracii. A novel concept. the enhancement index (H). is introduced to compare dilferenees in terpene acti\.ities towards the
`two permeartts; F.l provides information as to the partition coefficient and maximum achievable permeation enhancement for a
`drug. together with a measure or a penetration enhancur's activity towards that drug expressed as a percentage of the maximum
`effect. J'Itis approach permits usetul comparisons helwtcrl the m‘livilies oi \‘ilriulis crllraltcurg [awards different drugs.
`
`Introduction
`
`The rate determining step for transdermal de—
`livery of most drugs is provided by the stratum
`eorneum (Seheuplein,
`1965}.
`Its structure has
`been depicted in the brick and mortar model
`[Michaels et al._ l‘1'i'5; Elias.
`i981) in which anu-
`cleate keratinised cells are embedded in a lipid
`mortar. The stratum corneum lipids are arranged
`
`(hrrrwmnn'eur-i’: B.W. Harry. Postgraduate Studies in Pharma‘
`ceutit‘a] Technology. The School ol Pharmacy. University of
`Bradford Brittlfurd. BD? lDI’. U.K.
`
`in multiple bilayers providing alternate hydropho—
`bic and hydrophilic barriers. Drugs must diffuse
`through the intercellular lipid matrix. and to re—
`duce reversibly the resistance of
`this pathway
`researehers employ penetration enhancers (01' ac—
`celeranls]. These materials interact reversibly with
`stratum corneum constituents to disrupt the highly
`ordered structure and hence facilitate drug diffu-
`sion. Many established penetration enhancers are
`synthetic chemicals which are not yet approved
`by regulatory authorities for use with drugs. Re-
`cently. a Itt‘wci series of penetration enhancers.
`classed as lerpenes or terpenoids. has been de-
`scribed (Williams and Barry. 198.9. 1990]. These
`
`Noven Pharmaceuticals, Inc.
`EX2012
`
`0001
`
`Mylan Tech., Inc. v. Noven Pharma., Inc.
`|PR2018—00174
`
`

`

`158
`
`chemicals may provide a series of safe naturally
`occurring penetration enhancers whose toxicities
`
`isoiated from the oil (supplied by Field and Co.)
`by fractional distillation in vacuo (Finder. 1%”)
`
`are well documented (e.g., Opdyke. 19?4«l976),
`Several terpenes were shown to be effective ac-
`
`The chemical
`given in Fig. 1.
`
`formulae of these terpenes are
`
`eelerants for the hydrophilic cytotoxic drug. 23-Ho—
`orouracil (Williams and Barry, 1991). The present
`
`study extends the investigation to the effects of
`some terpencs on transdermal permeation of a
`model lipophilic drug, oestradiol (ES).
`Topical oestrogens are employed when en-
`
`dogenous hormones are lacking, such as in post~
`menopausal! women. A transdermal oestradiol
`patch, Estraderm TTS. has recently been devel—
`
`oped to treat menopausal symptoms. Clinical tri-
`
`als have indicated that transdermal delivery holds
`many advantages over oral oestrogen administra-
`tion.
`including reduced variation in serum hor-
`mone concentrations. a more normal oestrone:
`
`ocstradiol ratio and minimai pharmacological ef-
`
`fects on hepatic proteins (Powers et al.‘ 1085:
`Crust et £11.. 1989; Yum. 19891.
`
`Our report also compares the activities of ter-
`
`penc penetration enhancers towards the model
`hydrophobic drug {oestradiol} and the mode! hy-
`drophilic drug {S—fluorouracii). A novel concept.
`the enhancement index (El), is used to compare
`accelerant actions for the two drugs; it
`is hoped
`that such an approach may be of value with a
`
`wide variety of drugs and enhancers. A significant
`
`it allows an
`advantage of the method is that
`assessment of the maximum benefit which can be
`
`expected in chemically enhancing the skin perme-
`
`ation of a particular drug.
`
`Materials and Methods
`
`The terpenes used as received were a-pinene.
`
`terpinen»4-ol. carveol. carvone, pule“
`3-carcne,
`gone. menthone, a-pinene oxide. limonene oxide.
`cyclohexene oxide cyclopentene oxide and ?-
`oxabicycloi2.2.11heptane
`supplied by Aldrich
`Chemical Company, d-limonene and l,8~eineole
`
`a:-
`provided by Sigma Chemical Company.
`terpineol obtained from BDI-l Chemicals Limited
`and piperitone from Field and Co. Ascaridole,
`
`An assessment of the terpenc purities has been
`published (Williams and Barry. 1991): no single
`impurity was present in each terpenc at greater
`than 2%, and such traces were considered to he
`
`at sufficiently low thermodynamic activities that
`their effects on skin permeability would he negli—
`gihle compared with that of the main terpenc. As
`an initial assessment of accelerant activity. all
`
`terpenes were employed as neat liquids.
`The model
`lipophilic permeant was [2.4.63-
`3Hllhllloestradioi (NEN Research Products}.
`ra-
`
`diochemical purity 99174:. Unlabelled oesli‘adiol
`{Sigma Chemical Company} was used to prepare
`
`a saturated aqueous drug solution {0.11113 mg/mi
`at 311°C, Michaels et ai., 1915}.
`
`65
`
`Il
`
`1"'|..'|"‘.E
`
`22
`
`\
`
`dALimoner-e
`
`3'33’909
`
`a .Terpineot
`
`0 H
`
`0
`
`O
`
`D H
`
`\
`
`\
`
`Terpmen-d-ol
`
`Carvers
`
`Carvone
`
`Pulegane
`
`0
`
`Z
`
`O
`
`i 2"“0 (j
`
`P-peritone
`
`Msnthone
`
`Cyclohexerie
`oxide
`
`g
`
`F’inent-
`cut-:26:
`
`fi
`
`d 1
`_
`“at till c L
`
`.
`
`? Oxaoicydo
`l?- 2‘ 1]heptane
`
`O
`
`i\
`
`Limonene
`aside
`
`0
`
`/
`
`t
`
`1.8 C“. 1-)“!
`
`the main constituent of oil of chenopodium was
`
`Fig.
`
`l. The structural formulae ol' terpenc-s used in this slutty.
`
`0002
`
`

`

`159
`
`Preparation of human skin membranes
`Caucasian abdominal skin (male and female,
`
`swabbing. The stratum corneum membranes were
`floated on water before use to ensure full tissue
`
`age 17-89) was obtained postmortem. Excess fatty
`and connective tissues were removed and the
`
`hydration.
`A diagrammatical representation of the skin
`
`samples stored at — 20°C (Harrison et al., 1984).
`Full thickness membranes.
`Skin samples were
`trimmed of fatty material
`to provide tissue ap—
`
`membranes used in this study is shown in Fig. 2.
`
`Permeation experiments
`
`proximately 1 cm thick and essentially flat. The
`
`Experiments at 32: 1°C used an automated
`
`samples were clamped between stainless steel
`plates with a polythene sheet covering the stra-
`tum corneum and the membrane refrozen to ad-
`
`here the fatty layer to the metal. The upper plate
`
`diffusion apparatus with 24 stainless-steel diffu-
`sion cells (diffusional area 0.126 cm3) and 0.002%
`
`aqueous sodium azide as flow-through receptor
`solution (Akhter et al., 1984).
`
`and polythene sheet were removed and the stra-
`tum corneum surface of the tissue was thawed
`
`Fully hydrated epidermal membrane samples
`were mounted in the cells and treated with 150
`
`slightly before a membrane, approximately 430
`
`pm, was out using a Duplex Electro Dermatome
`7. providing a sample of full thickness skin com-
`
`prising stratum corneum, nucleate epidermis and
`some dermal tissue.
`
`Samples of
`Stripped faii thickness membranes.
`full thickness skin with the stratum corneum re-
`
`moved were prepared by tape stripping (Clipper
`
`tape}. Typically 25—30 strippings were required to
`remove the stratum corneum from full thickness
`
`skin membranes. Fully hydrated stratum corneum
`
`comprises approximately 30 am of the mem-
`brane, hence the resulting stripped full thickness
`
`tissue provided a sample approximately 400 um
`thick.
`
`Epidermai membranes. Epidermal mem-
`branes,
`incorporating the
`anucleate
`stratum
`
`tissue, were
`corneum and nucleate epidermal
`prepared by a heat separation technique (Klig-
`man and Christophers,
`1963). Skin samples
`trimmed of fatty tissue were immersed in water at
`
`60°C for 45 s, after which the epidermal mem-
`
`branes were teased off the underlying dermis.
`The membranes were floated on an aqueous solu»
`tion of 0.002% sodium azide for 36 h to ensure
`
`full hydration of the stratum corneum.
`Stratum comeam membranes.
`
`Stratum
`
`corneum samples were prepared from epidermal
`membranes (Kligman and Christophers, 1963);
`epidermal membranes were floated overnight on
`an aqueous solution of trypsin (0.000l% w/v}
`and sodium hydrogen carbonate (0.5% w/v) at
`37°C. The enzyme digests the nucleate epidermal
`tissue allowing the remnants to be removed by
`
`0003
`
`pl aliquots of saturated radiolabellcd ES. To
`ensure saturation of the donor solution a crystal
`
`of ES, with the same radioactivity as the drug
`solution, was placed in each donor compartment,
`and the donor drug solution was replenished ev-
`ery 8 h. Under these conditions. BS is at maximal
`
`
`
`FULL THICKNESS
`MEMBRANE
`
`STRIPPED FULL
`THICKNESS MEMBRANE
`
`
`
`MEMBRANE
`
`STRATUM COHNE UM
`
`EPIDEHMAL
`MEMBRANE
`
`Fig. 2, A diagrammatical representation of skin membranes
`employed in this study {not to scale). SC. stratum corneum: F..
`nucleate epidermis: D. dermis.
`
`

`

`loll
`
`thermodynamic activity throughout the diffusion
`
`membranes was as described previously (Williams
`
`experiment with negligible donor depletion. Sam~
`pics of the receptor solution (2 ml) were collected
`
`to which 5 ml OptiPhase
`every hour for 24 h.
`HiSafe
`ll
`scintillation fluid (Pharmacia) was
`
`added. and the radiolabelled drug determined by
`
`liquid scintillation counting (Packard Tri-Carb
`460}. The permeant solution was washed from the
`
`membrane with 0.002% aqueous sodium azidc
`
`and replaced with 150 pl of a tcrpene. After I2 h
`
`the tcrpcne was washed from the
`treatment,
`membrane and ES permeation was rcdctermincd
`
`as above. Linear regression analysis of the pseudo
`
`steady-state diffusion results after the lag time
`
`allows evaluation of the permeability coefficient
`
`(Kpl of the drug in the membrane before and
`after terpcne treatment. As a measure of the
`
`penetration enhancing activity of the terpenes.
`the enhancement ratio (ER) was caICulated as
`
`(Goodman and Barry. 1988):
`
`Kp after terpene treatment
`
`ER
`
`_ Kp before terpcne treatment
`
`(1)
`
`Values reported are mean ratios from 4—13 repli—
`cates.
`
`Permeation across the epidermis and dermis
`
`and clearance into the aqueous receptor fluid
`may provide the rate determining step in the
`
`permeation of very highly lipophilic drugs. The
`
`barrier to ES diffusion was therefore investigated
`
`using skin membranes composed of a variety of
`layers: stratum corneum alone. epidermis (includ-
`
`ing stratum corneum}. full thickness skin (stratum
`corneum, nucleate epidermis and some dermal
`
`tissue} and tape stripped full thickness skin (stra—
`tum corncum removed). Ocstradiol is not a very
`
`highly lipophilic drug (log P octanoI/water =
`
`small but significant aqueous
`2.29) and has a
`solubility (0.003 mg/ml at 30°C). Thus. removal
`of the drug into the flow through receptor fluid is
`
`unlikely to provide a significant resistance to drug
`permeation. but passage
`across epidermal/
`
`dermal tissue may.
`
`Partitioning experiments
`The method used to assess the effects of ter—
`
`penes on ES partitioning into stratum corneum
`
`and Barry. 1991). Fully hydrated stratum corneum
`samples were cquilibrated in a terpene for 12 h.
`The tissues were blotted dry and placed in a
`saturated radiolabellcd aqueous solution of ES
`
`for 4 h. The samples were blotted dry. solubilised
`and the drug determined by liquid scintillation
`counting. Triplicate partition coefficients (stra-
`
`tum corncum/watcrl were determined using tis-
`sue samples from three different human sources.
`
`Controls were stratum corneum samples un-
`
`treated with terpcne. The results were expressed
`as a partition ratio PR where:
`
`P
`
`
`partition coefficient after terpene treatment
`_
`_.
`._
`__
`.
`a _
`partition coefficient with untreated membrane
`
`l-J ._r
`
`Solubility studies
`
`The solubility of ES in the terpencs was deter-
`
`mined by the method of Williams and Barry
`(1991). Terpenes were saturated with radiola—
`
`belied crystals of ES and the saturated drug con-
`centration determined in triplicate by liquid scin-
`
`tillation counting.
`
`Results and Discussion
`
`The experimental design for permeation stud—
`ies of determining Kp.
`treating the membranes
`
`with a terpenc and then redetermining Kp. allows
`each piece of tissue to act as its own control.
`thereby reducing errors due to the biological vari-
`
`ability of human skin. The conditions for drug
`delivery were optimised by the use of saturated
`
`replenished every 8 h. which
`drug solutions.
`maintains the permeant at near maximal thermo—
`
`dynamic. activity. Typical permeation profiles un-
`der
`these conditions are in Fig. 3. From the
`
`diffusion experiments. the mean permeability co-
`efficient of aqueous ES through normal
`(un-
`
`treated) human epidermal membranes at 32°C is
`3.68 i 0.36X 10
`‘ cm/h (n = 144). This result
`shows good agreement with literature values of
`3.2 X 10‘" cm/h (Goodman and Barry, 1988} and
`5.2 X It]
`7‘ cm/h (Michaels et al..
`[975: Flynn
`and Stewart. 1988}.
`
`0004
`
`

`

`(a)
`
`tun“)
`cuuuuuNECPU/em?
`
`
`
`
`16
`
`18 20 '1"?
`
`24
`
`0
`
`2 $681012 14
`TIME
`(I!)
`
`(b)
`
`lfil
`
`employed in diffusion experiments. Relevant data
`are summarised in Table 1.
`
`These results illustrate no significant differ-
`
`ence in ES permeation through stratum corneum.
`
`thickness membrane with an
`epidermal or full
`aqueous receptor fluid (P=0.05). This clearly
`illustrates that intact stratum corneum is the main
`
`barrier to oestradiol permeation The resistance
`
`to drug permeation provided by various skin lay-
`ers may be calculated as the reciprocal of the
`
`drug permeability coefficient. It should be noted
`that
`the resistance of each membrane includes
`
`those contributions arising from donor and recep-
`
`tor stationary layers. Due to the natural variabil-
`
`ity of human skin. no significant difference exists
`between the resistances of stratum corneum, epi-
`dermal and full thickness membranes (P= 0.05).
`
`However,
`
`the stratum corneum provides signifi-
`
`cantly greater resistance to drug permeation than
`stripped full
`thickness skin (E/D in Table 1;
`P= 0.05). After removal of the stratum corneum
`
`the permeability coefficient of ES increases by a
`
`factor of 5 compared with full thickness skin with
`
`5.0
`as
`41.0
`3.5
`3.0
`2.5
`2.0
`1.5
`1.0
`0.5
`0.
`
`
`
`
`
`CUMULATWECPU/cmz(xto‘)
`
`n
`
`2
`
`4
`
`e
`
`a 10121;:513202224
`TIuE
`(I1:
`
`the stratum corneum intact. This result
`that
`the maximum enhancement effect
`
`implies
`that an
`
`Fig. 3. Typical permeation profiles of aqueous oestradiol
`through human epidermal membranes showing the effects of
`donor replenishment. a. Donor not
`replenished. h. Donor
`replenished. Every second experimental value plotted.
`
`For highly lipophilic drugs, the rate determin—
`
`accelerant such as a terpene may induce is a
`
`5-fold increase in permeability corresponding to
`full removal of the barrier resistance of the stra—
`
`tum corneum. Clearly, if the barrier nature of the
`
`stratum corneum is diminished by the use of
`penetration enhancers,
`then the resistance pro-
`
`ing step in transdermal permeation may reside in
`the partitioning process into the essentially aque-
`
`tissue will exert a
`vided by epidermal/dermal
`proportionally greater
`influence on oestradiol
`
`ous nucleate epidermis and dermis, and/or in
`clearance from the skin into the systemic circula-
`tion. To investigate the rate limiting step for ES
`
`permeation, a variety of skin membranes were
`
`permeation.
`
`Table 2 shows data on penetration enhancing
`
`activities of the terpencs. These results demon-
`strate that the hydrocarbon terpencs are acceler—
`
`TABLE ]
`
`The permeability coefficient (1(1)) of aqueous mistrudt'r)! permeating through t‘rm'rms foyt’n' of httrrtrm skirt. with 51':-
`
` Membrane Description Kp (cm/h. X103) Tissue resistance {h/cm}
`
`
`n
`
`3
`SC
`Stratum corneum
`4.47 i 0.77
`'
`224 i 39
`144
`SC/E
`Epidermal
`3.68 1r 0.36
`2?3 i 27
`o
`SC/E/D
`Full thickness
`2.45 i- 042
`408
`i 89
`
`Stripped full thickness 13.0 i 1.23 76.9 t 1813/13 8
`
`
`
`
`J
`
`SC. stratum corneum; E, epidermis; D, dermis; n. number of replicates.
`
`0005
`
`

`

`1h2
`
`TABLE 2
`
`'I‘erpene
`
`The J'ttt'wt permeabifity t'tltjflr‘tf‘rtrs (KM. Wt}! standard error of the mam. of aqueous om‘tmdiol throng/t epidt'rttwl' tttt'ttthrtmtir before
`and after treatment mm a rerpenc. with mean {and Sli'l mhrmrmnertr ratios (ER) and the number of motivate.»- in}
`
`Kp (em/h. x 10"}
`Initial (control)
`Treated
`
`
`FR
`
`u
`
`Hydrocarbons
`a—Pinene
`d-IJmonene
`ji—Carene
`
`Air'flhthlt
`
`rr—'l"erpineol
`Terpinen-4-ol
`Calvert]
`
`Katrina’s
`
`('farvonc
`Pulegone
`Piperitone
`Menthone
`
`(hides
`
`('yctohexene oxide
`a-Pinene oxide
`Limonene oxide
`Ascaridule
`2-Oxahicyclu-
`[2.2.1]heptane
`lfi—(‘ineole
`
`3.72 1: 11,61
`2.89 i 0.47
`1.57 4:11.32
`
`5.98 i 0.73
`2.52 i 0.49
`2.364 0.49
`
`4.09 4; 1.06
`3.01 _+ Lil
`3.54 31:03")
`3.22 r (1.71
`
`333 i 0.50
`2.68 i {1.31
`2.20 i “.64
`3.29 i 11.8?
`
`2.94 + 0.99
`3.26 1 1.24
`
`:1: {193
`10.1
`8.40 i {14}
`6.57 + ILhLI
`
`2.2—1 i {1.38
`I.b3 i 0.68
`1.06 + [1.31
`
`[1.41) :3; (H?
`{1.98 {r {1.36
`11.63 4: (1.18
`1.0] t1l.35
`
`4.68 i 0.84
`5.14 i— 1.2‘~J
`3.41% _+ 1.00
`15.8 -_1- 4.12
`
`12.7 i- 143
`15.1 1 3.75
`
`10951.89
`3.?5 i 1.33
`4.36 —_t_ 1.02
`
`0.33 :t 0.10
`“.45 + {1.1“
`”.42 4._— ll. 10
`
`I]. it) at (1.04
`£1.34 t {ms
`1|. I 7 1-11.02
`11.36 _+ 11.1?
`
`1.42 t 0.75
`1.011 10.8?
`1.6! 1-0.76
`4.75 + 1.3-1.
`
`4.93 4: 1.50
`4.4017115?
`
`12
`l2
`J
`
`h
`5
`U
`
`U
`13
`5
`7'
`
`h
`5
`5
`H
`
`-1
`?
`
`ants for the lipophiiic drug, providing enhance-
`ment ratios of between 3 and 4. The alcohol and
`
`ketone terpencs did not enhance oestradiol per-
`meation and may in fact hinder passage of the
`lipophilic drug. The oxide terpenes show varied
`accelerant activities; cyclohexene oxide. limonenc
`
`oxide and a-pinene oxide provide no significant
`increase
`in oestradiol permeation (P = {1.05}
`whereas
`ascaridole. 7-0xabicyclol22.fiheptane
`and 1.8-cineole all induce a 4—5-fold increase in
`
`diffusion of the lipophilic drug. 11 is interesting to
`note that the ineffective oxide terpenes all con-
`tain a 1,2-oxygen linkage and may broadly be
`classified as ep0xides. The effective oxide ter-
`pencs contain 1.4-(ascaridole. 7-0xahicyclol2.2.1]—
`heptane} or i.8-li.8-cineoie) oxygen linkages and
`may be classed as cyclic others. It has been sug-
`
`gested that structural conformations may he a
`
`factor in determining penetration enhancer activ—
`
`ities of terpenes (Williams and Barry. 1991).
`The mechanisms underlying terpcne penetra‘
`
`tion enhancement were investigated by partition~
`
`ing and solubility studies. Terpene effects on
`oestradiol partitioning into isolated fully hydrated
`stratum corneum membranes were assessed {Ta-
`
`ble 3). The centre! (non-terpene treated) parti-
`
`tion coefficient (P) was 14.5. providing a tog i’
`
`(stratum corncum/water) of 1.16. Literature P
`values include 46 for human stratum corneum
`
`(Scheuplein et a1.. 1969). 58.9 for male abdominal
`hairless mouse skin (Valia and Chicn. 1984} and
`
`80 for rat stratum corneum (chierre et al..
`
`1990). The partition coefficient (octanol/ water).
`which gives a rank order approximation to parti-
`tioning into the stratum corneum (Barry. 1983).
`for oestradiol is 195. Thus. the results from this
`
`0006
`
`

`

`TABLE 3
`
`The eflk’t‘ts of terpenes on the partitioning of oestradioi from aqueous solution into fiii‘h' hydrated s'trtttttm come-um membranes. and the
`.fl'ofttbiftfl’ of (Mrstradioi in the termites
`
`Terpene
`P t SE
`PR t SE
`Solubility
`
`{mg/ml)
`
`163
`
`0.003
`1.00
`[4.5 i 1.79
`Control (water)
`0.011
`0.99 10.15
`13.6 i 0.19
`a-Pinene
`0.025
`0.96 i 0.08
`13.5 1- 0.77
`d—Limonene
`0.046
`1.93 1- 0.17
`32.1 i 3.02
`3-Carene
`5.00
`3.29 1 1.21
`54.6 i 19.9
`o—Terpineol
`6.96
`2.04 :I-. 0.06
`29.5 i 3.56
`Terpinen—4-ol
`5.2?
`2101-046
`28.3 _+_- 3.95
`Can-co]
`12.80
`3.31 1— 025
`47.4 :t 5.94
`("an-one
`12.33
`4.56 i- 081
`61.7 i 3.06
`Pulegone
`13.06
`3.9-1 _+_ 0.27
`56.0 —1_— 5.51
`Piperitone
`5.60
`7.26 i- l.|5
`100
`i 14.1
`Menthone
`7.55
`1.63 i 0.18
`23.5 i 1.76
`Cyciohexene oxide
`6.83
`2,34 _-1- 0.60
`30.6 i 3.25
`a-Pinenc oxide
`7.36
`2.65 i 0.30
`37.? i 5.71
`Limonene oxide
`0.291
`4.30 ft 0.86
`71.8 +_ 14.9
`Ascatidole
`64.57
`1.57 i 0.27
`21.3 i 0.66
`T-Oxabicyclo[2.2.l Jhcptanc
`
`1.8-Cine01c 8.03 30.0 i 2.37 2.11 _+ 0.12
`
`
`
`P. partition coefficient. stratum corneum/ water. mean of 3 replicates. with SE. PR — partition ratio : P/control r P/14.5, mean of
`3 individual values. with SE.
`
`study and the literature confirm that the stratum
`
`corneum provides a more polar environment than
`octanol.
`
`Terpene treatment generally increases parti-
`tioning of the lipophilic drug into the stratum
`corneum, as illustrated by the partition ratio. P“.
`where:
`
`partition coefficient after terpene treatment
`PR_
`partition coefficient with untreated membrane
`
`(3)
`
`However. it is evident from Table 3 that treat-
`
`ment with d-limonene and a-pinene does not
`improve such partitioning whereas the other hy-
`drocarbon tested, 3-carene, provides a near dou-
`
`bling in partitioning. Improved partitioning w0uld
`be expected considering the solubility of BS in
`the terpenes; the drug is more soluble in all the
`tcrpenes than in water. and is more soluble in
`
`oxygen-containing terpenes than in the hydrocar-
`bons (Table 3). No mathematical relationship ex-
`ists between the partition ratios and drug solubili-
`ties. although a trend is apparent with the hydro-
`carbons having little or no effect on partitioning
`and providing a drug solubility of 0011—0046
`
`mg/ml compared with the ketones which im-
`prove partitioning approximately 4—f01d and pro-
`vide a greater drug solubility of 5.6—13 rag/ml.
`Terpene effects on the apparent lag time to
`pseudo steady-state diffusion have been used to
`
`gain an insight into the accelerant effects towards
`S-fluorouracil permeation (Williams and Barry,
`1991). However, oestradiol permeation through
`untreated epidermal membranes provides a rela—
`tively short lag time of 4.42 i 0.36 h (SE; :1 =93).
`
`Consequently, variations in the apparent lag times
`are not sufficient
`for a critical analysis of the
`
`terpene penetration en—
`modes of action of
`hancers. The inconsistent nature of the apparent
`lag time. due to the biological variability of hu-
`man skin.
`is well illustrated by the above value;
`the 93 replicate values provide a standard devia-
`tion of 19%. Additionally. the lag time above is
`considerably lower than literature values of 19 h
`
`(Mollgaard and Hoelgaard.
`(Scheuplein et al.. 1969).
`The oestradiol work has shown that hydrocar-
`bon and cyclic ether terpencs are effective accel-
`
`1983) and 103 h
`
`erants whereas alcohols. ketones and epoxides
`
`000?
`
`

`

`2 3D 6 7 a 91011121[L31¢15161?
`
`-FU .ES
`
`0!)
`
`l234567891011121514151617
`TERPENE
`III 5—FU - ES
`
`100
`
`90
`Q
`no
`I;
`a: m
`
`60
`50
`
`40
`so
`20
`to
`o
`
`1t)
`
`a
`6
`
`4
`
`2
`
`0
`
`~2
`-4
`
`E
`u
`
`ag
`
`g
`2
`w
`
`5a
`
`E
`
`t—
`Ez
`az
`1|!
`Lu
`E
`
`Fig. -l, a. The mean enhancement ratios of lerpenes towards
`5- tluorouracil and oeslradiol b. The enhancement
`indiees
`calculated from Eon '2'. l. a pinenc: .1w! Iimonene 3. 3-carcnc.
`4.
`rr- lerpineol: 5.
`terpincn-4-ol: h. carveol:
`7". cannot: 5.
`pulegone19. piperitone; 1t]. menthone: ll. cyclohexcne oxide:
`12.
`limooene oxide: 13. a—pinene oxide:
`l-‘l. cyclopentene
`oxide: 15. ascaritlole: lo. loxabicyclolllllhcptane: 17. LR
`t‘ineolc.
`
`respectively. h.
`
`f) and K refer to the thickness.
`
`diffusion coefficient and partition coefficient of
`the various
`layers, and stationary layers and
`
`clearance are neglected.
`Assuming that the nucleate epidermis and der-
`mis provide similar barrier properties per unit
`thickness. then:
`
`h
`h“.
`R = —u-‘—- + #—,
`DSt‘KSC‘
`01‘
`
`(s)
`
`=—
`where 2-17sz + fin, D = D... = DI) and K: K}.
`Kn- A problem with tape stripping studies arises
`from the mechanical weakness of epidermal
`
`I (14
`
`are
`
`not. Hydrocarbon terpenes,
`
`including
`
`increased percutaneous absorption of
`limoncne.
`indomethacin in rats (Nagai et al.. 1989: Okabe et
`al.. 1989). by direct effects on the barrier nature
`
`of skin. This implies that the hydrocarbons act by
`
`increasing diffusivity of the drug in the stratum
`
`Indomethacin (log P octanol/ water
`corneum.
`42?).
`like oestradiol (log P 2.39).
`is a lipophilic
`
`drug and the authors reported that oxygen-con-
`taining terpenes. such as carvone. ar-terpineol
`
`and l.8-cineole were ineffective. Our results par-
`
`tially support
`this view; hydrocarbon tcrpenes
`promoted ES permeation whereas alcohol and
`ketone terpenes did not. HOWever. this study also
`demonstrated that
`the
`cyclic ether
`terpenes
`(asearidole. ?-oxabicyclo[2.2.l]heptane and 1.8-
`cineole} promoted ES permeation across human
`
`epidermal membranes. to a similar extent as the
`
`hydrocarbons. Another study employing lipophilie
`
`drugs and terpene penetration enhancers found
`
`that tcrpenes containing polar groups did not act
`as enhancers {Hori et al.. 1989}.
`
`A comparison of the terpcne activities towards
`the polar drug S-fluorouraeil (Williams and Barry.
`
`is given in Fig. 4a. The
`l99l) and oestradiol
`terpenes apparently are more active towards 5-
`fluorouracil
`(S—FU)
`than towards ES. The en-
`
`hancement ratios of lfi—cineole. an effective ac—
`
`celerant
`
`for both drugs. are 94.5 and 4.40 for
`
`S-FU and ES respectively. However. a compari-
`son between these results may be misleading.
`
`particularly in respect of the apparent low value
`for ES. as the scope for enhancement of these
`
`drugs varies considerably. The enhancement ratio
`for S-FU provided by tape stripping the stratum
`corneum from full thickness human skin is 1045;
`
`the corresponding value for ES is only 5.3. We
`can deal with this consideration by treating the
`skin as a laminate with barriers in series.
`
`The resistance (R) of
`
`intact.
`
`full
`
`thickness
`
`human skin to drug diffusion may be described
`mathematically by (Barry. 1983):
`
`
`R 2 J's
`DSFKSt'
`
`f”-
`DF.K1.
`
`...i) _
`DIJKI)
`
`where the subscripts SC.
`
`13 and D refer to the
`
`stratum corneum, nucleate epidermis and dermis
`
`0008
`
`

`

`[65
`
`membranes;
`
`it
`
`is not practical
`
`to tape-strip the
`
`systems. assuming the epidermis to be a porous
`
`stratum corneum from epidermal membranes.
`Thus, the maximum enhancement ratios for the
`
`hydrogel.
`To describe the activities of penetration en-
`
`drugs obtained with the barrier layer removed
`
`were determined using stripped full thickness skin
`samples, approximately 4011 p.111 thick. These ER
`values are then compared with data obtained
`
`using epidermal membranes, approximately 80
`um thick of which approximately 30 pm is fully
`hydrated stratum corneum and 50 pm nucleate
`epidermis (Marks. 1981; Sato et al., 1991). Since
`the nucleate epidermal tissue represents approxi-
`mately one eighth of the stripped full
`thickness
`skin, then for epidermal membranes used in dif-
`
`fusion studies Eqn 5 may be rewritten as:
`
`hancers in a more informative way. we propose
`that an enhancement index (El) may be useful.
`The maximum achievable enhancement is depen-
`dent,
`in part, on the partition coefficient of the
`model permeant. The log P (octanol/water} of
`the drug is thus provided as a superscript to the
`enhancement index, and the maximum enhance-
`
`ment
`
`ratio provided by stripping the stratum
`
`corneum from nucleate epidermal membranes,
`corrected as discussed above,
`is given in a sub-
`
`script. These two values provide information
`which places into context
`the enhancement ef-
`fect. The enhancement index is then calculated as
`
`it sc
`R = ——- +
`
`
`it
`
`('5)
`
`the fraction of the maximum achievable enhance-
`ment ratio induced by accelerant treatment. ex-
`
`With the stratum corneum intact, drug perme—
`ation across the bulk of the skin (nucleate epider-
`mis and dermis)
`is
`rapid for hydrophilic and
`moderately Iipophilic molecules compared with
`passage across the stratum corneum. However.
`
`when the barrier layer is removed. permeation
`across the nucleate epidermal and dermal tissue
`
`becomes rate limiting. Assuming that the epider-
`mis and dermis provide similar
`resistances to
`
`drug permeation per unit thickness, a correction
`may be made for the differences in skin sample
`
`thickness between the two experimental proto-
`cols;
`the permeability coefficients of the drugs
`will be approximately 8 times greater through 50
`pm of nucleate epidermis than through 4110 p.111
`epidermal/dermal
`tissue. Thus,
`the maximum
`achievable enhancement ratios through nucleate
`epidermal membranes can be corrected to ap-
`
`proximately 8400 for S-FU and 42 for E5. Clearly
`these two values are approximations, but they are
`more appropriate than the maximum enhance-
`
`ment ratios taken simply from the stripped full
`thickness tissue studies. Literature reports of
`
`penetration enhancers seldom provide data for
`drug permeation through epidermal
`tissue with
`the stratum corneum removed. In such cases an
`
`estimate of drug permeability coefficients may be
`
`calculated from the drug diffusivity in aqueous
`
`0009
`
`pressed as a percentage. However, our definition
`of the enhancement ratio. which is the ratio of
`
`treat-
`drug Kp after to that before accelerant
`ment. dictates that an enhancement ratio of 1.0
`
`indicates that an accelerant has no penetration
`enhancing activity. To correct
`for this,
`the en-
`hancement index may be defined as:
`
`_ MINING I,
`I:‘Iminirttum Lil-{i =
`
`(Enhancement ratio after
`
`accelerant treatment}—l
`.
`_
`(MaXImum enhancement ratio,
`stratum corneum removed)- -
`l
`
`X 1””
`
`(7)
`
`The enhancement indices of the terpenes as
`calculated towards the model drugs are listed in
`Table 4. These values show that,
`for example,
`while the enhancement ratio for 1,8-cineole to-
`
`wards S-FU is large,
`
`in terms of the maximum
`
`the terpene activity is
`achievable enhancement
`low, Elflajlflq= 1.1%. Contrariwise,
`for oestra-
`diol, although the ER is apparenriy low, E1343” =
`8.3%, i.e., the terpene shows 8 times more activ—
`
`ity towards the lipOphilic drug as compared with
`the polar drug based on assessments of the maxi-
`mum achievable effect. Table 4 illustrates that
`
`the hydrocarbon and oxide terpenes are more
`active,
`in terms of the maximum possible en-
`hancement,
`towards ES than towards S-FU.
`whereas the alcohol and ketone terpenes are
`
`

`

`lhh
`
`TABLE 4
`
`(ER) and iridit‘cs Hill of some
`ratios
`The enhancement
`monoterpencs,
`towards tl' mode! point penetmm. 5-flrmroiimci!
`Hog? = — 0.89} and a mode! firmphilic penchant. wstrudioi
`Hot: P 2 2.3}. E] t'alues quoted to 2 significant figures
`
`
`Terpene
`
`i-Fluorouracil
`
`Oeslradiol
`
`ER
`E1 tint."
`ER
`El a:
`(”at 1
`[’I‘i-l
`
`
`o—Pincnc
`Lintonene
`3-Carene
`
`a—Terpincol
`Terpinen—4—ol
`Carvcol
`Can'one
`
`Pulegone
`Piperitone
`Menthone
`
`C‘yclohexene oxide
`n—Pinene oxide
`Lirnnnene oxide
`
`Cyclopentenc oxide
`Ascaridole
`
`|.2
`ll
`3.2
`
`9.4
`10.4
`20.“
`Ill
`
`3|.2
`27.?
`37.9
`
`2.4
`ll?
`1H)
`
`30.9
`82.5
`
`0.0024
`0.1113
`{1.026
`
`(1.10
`0.1 1
`(1.23
`0.]?-
`
`11.24
`0.32
`[1.44
`
`[1.017
`{1.15
`£1.12
`
`0.35
`[1.07
`
`3.09
`3.75
`4.36
`
`0.33 ‘l
`0.45 ‘l
`0.42 “
`{1. If} "
`
`0.34 "
`ll. [7" “
`{1.36 "‘
`
`1.42
`1.9”
`|.nl
`
`-—
`4.75
`
`5.|
`(1.?
`8.2
`
`— Ln
`— 1.3
`— L4
`__ 3.3
`
`— Lo
`— 2.1]
`— 1.6
`
`1.“
`2.2
`l5
`
`—
`9.1
`
`7-Oxabicyclo—
`[2.2.1]hcplane
`Lit—Cirieole
`
`9J1
`4.93
`[.1
`91.7
`
`
`
`4.40[.194.5 8.3
`
`" Enhancement ratio chs than [.01).
`
`P.
`
`log partition coefficient of the permeunt: M. maximum
`enhancement with stratum corneum removed.
`
`more active towards the polar drug. For ES. the
`hydrocarbon and cyclic ether terpenes produce
`between 5 and 10% of the maximum enhance-
`
`i.e.. up to 10% of the stratum corneum
`ment,
`resistance is removed. a value at which the resis-
`
`tance of other skin iayers or drug clearance into
`
`the receptor fluid may become significant. For
`S-FU. the hydrocarbons remove less than 0.1% of
`the barrier resistance and even the relatively ef-
`
`fective cyclic ether terpenes remove only 1% of
`the stratum corneum resistance.
`‘Accelerants’
`
`ratios of less than 1.0
`providing enhancement
`inhibit drug permeation. Such agents provide a
`
`negative enhancement index.
`The use of enhancement indiees demenstrates
`
`that comparisons between values obtained from
`permeation studies may at first sight be mislead-
`ing and could be viewed more profitably with
`respect
`to the maximum permeation rate a
`
`0010
`
`molecule may achieve through skin after all the
`stratum corneum resistance is removed. This is
`
`illustrated in Fig. 4 which compares the enhance—
`ment ratios and enhancement indices of the ter-
`
`pcnes towards the two drugs. An apparently ef-
`fective penetration enhancer. increasing