A RT I C L E S

192 VOLUME 22 NUMBER 2 FEBRUARY 2004 NATURE BIOTECHNOLOGY

Skin, the largest organ of the human body, offers a painless and com-pliant interface for systemic drug administration1. As compared withinjections and oral delivery routes, transdermal drug deliveryincreases patient compliance, avoids metabolism by the liver, and pro-vides sustained and controlled delivery over long time periods. Andyet, after nearly four decades of extensive study, the success of thistechnology remains limited, with only a few transdermal productsavailable in the market, all of which are based on low molecularweight lipophilic drugs2. Development of transdermal products formacromolecules is hindered primarily by low skin permeability.Evolved to impede the flux of toxins into the body, skin naturallyoffers a very low permeability to the movement of foreign moleculesacross it3. A unique hierarchical structure of lipid-rich matrix withembedded corneocytes in the upper strata (15 µm) of skin, stratumcorneum (SC), is responsible for this barrier3. Overcoming this bar-rier safely and reversibly is a fundamental problem in the field oftransdermal delivery.

Several technological advances have been made in the past coupleof decades to overcome this barrier: iontophoresis4, sonophoresis5

and chemical penetration enhancers6,7, to name a few. CPEs provideseveral advantages such as design flexibility with formulation chem-istry, patch application over a large area (>10 cm2) and the absence ofexternal physical delivery mechanisms. Several different classes ofCPEs, including surfactants8, fatty acids and fatty esters9, have beenstudied for permeation enhancement. However, only a few induce atherapeutic enhancement of drug transport. This problem is aggra-vated for high molecular weight drugs, which are especially difficultto deliver. In addition, potent enhancers are also potent irritants tothe skin at concentrations necessary to induce sufficient penetrationenhancement10. Thus, improved enhancement with single enhancers

inevitably leads to a compromise on safety issues. Attempts have beenmade to synthesize novel CPEs, such as Azone11,12; however, achiev-ing sufficient potency without irritancy has proved challenging.

Potent CPEs usually enhance skin permeability by altering the stra-tum corneum structure13,14. Because the stratum corneum consists ofnonviable, keratinized cells, disruption of its structure does notinduce irritation. However, CPEs are usually not selective towardsstratum corneum lipids, and eventually they affect the viable epider-mal cells, thereby inducing irritation by interstitial release ofcytokines and by triggering other inflammatory responses15.

Our tests of 32 individual enhancers, chosen from a list of >250older and newer CPEs, did not find any that achieved therapeutic lev-els of macromolecular skin permeability without irritation. However,these CPEs might still be used as building blocks to construct combi-nations of CPEs that are more potent. Here we describe the search fora new family of potent and safe permeation enhancers that we desig-nate synergistic combinations of penetration enhancers (SCOPE).SCOPE formulations are rare mixtures of known CPEs that exhibithigh potency upon contact with the stratum corneum but a relativelack of irritation in the epidermis because of the differential retentionof components in the stratum corneum. SCOPE formulations are dis-tinct from empirically formulated enhancer combinations reportedin the literature16,17, which are usually designed from a limited num-ber of experiments and based on additive rather than synergisticeffects. We describe examples of SCOPE formulations for transder-mal drug delivery and a method for discovering them. NLS:S20 andSLA:PP, two leading SCOPE formulations discovered via thismethodology, increased the flux of macromolecules such as LHRHand heparin in vitro by 50- to 100- fold. The SLA:PP formulation alsodelivered leuprolide acetate in hairless rats without skin irritation.

Department of Chemical Engineering, University of California, Santa Barbara, California 93106, USA. Correspondence should be addressed to S.M.(samir@engineering.ucsb.edu).

Published online 4 January 2004; doi:10.1038/nbt928

Discovery of transdermal penetration enhancers byhigh-throughput screeningPankaj Karande, Amit Jain & Samir Mitragotri

Although transdermal drug delivery is more attractive than injection, it has not been applied to macromolecules because of low skin permeability. Here we describe particular mixtures of penetration enhancers that increase skin permeability tomacromolecules (∼ 1–10 kDa) by up to ∼ 100-fold without inducing skin irritation. The discovery of these mixtures was enabled by an experimental tool, in vitro skin impedance guided high-throughput (INSIGHT) screening, which is >100-fold more efficient than current tools. In vitro experiments demonstrated that the mixtures delivered macromolecular drugs, includingheparin, leutinizing hormone releasing hormone (LHRH) and oligonulceotides, across the skin. In vivo experiments on hairlessrats with leuprolide acetate confirmed the potency and safety of one such mixture, sodium laureth sulfate (SLA) and phenylpiperazine (PP). These studies show the feasibility of using penetration enhancers for systemic delivery of macromolecules froma transdermal patch.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy

A RT I C L E S

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 2 FEBRUARY 2004 193

RESULTSDiscovery process of transdermal formulationsSCOPE formulations were discovered in a large library of mixturesgenerated using known CPEs. The library was first screened to deter-mine formulation potency by the INSIGHT method. This method is>100-fold more efficient than other methods (Franz diffusion cells,(FDC)18), thus allowing exploration of experimental space that is wellbeyond the scope of traditional tools19. In contrast to the method ofref. 19, which relies on measurements of the penetration of radiola-belled solutes into the skin, INSIGHT uses measurements of skin con-ductivity, thereby allowing a substantial increase in experimentalthroughput. The leading hits from INSIGHT were then screened forirritation and for the delivery of macromolecules. The schematic of thediscovery process is shown in Figure 1.

Enhancer libraryA library of SCOPE candidates was created using 32 chemically distinctCPEs chosen from an even larger pool of about 100 chemicals that havebeen used for transdermal drug delivery. These CPEs represent eightdistinct categories of penetration enhancers: (i) anionic surfactants,(ii) cationic surfactants, (iii) zwitterionic surfactants, (iv) nonionicsurfactants, (v) fatty acids, (vi) fatty esters, (vii) azone and azone-likecompounds and (viii) others. Enhancers from some of these categories(for example, anionic and cationic surfactants) are traditionallyexcluded from consideration for transdermal applications becausethey exhibit irritancy at concentrations required for achieving substan-tial flux enhancements (>1% (wt/vol))20. However, these surfactantscan form SCOPE mixtures at low concentrations. Accordingly, weincluded anionic and cationic surfactants in the experimental design.Irritation potential (IP) of SCOPE formulations was evaluated onlyafter screening for potency. Many CPEs used in this study are alreadybeing used in approved or investigational dermatological and cosmeticproducts.

Random pairing of CPEs from various categories led to 496 binaryCPE pairs. For each pair, 44 distinct chemical compositions were created with the concentration of each CPE ranging from 0–2%(wt/vol), yielding a library of ∼ 25,000 candidate SCOPE formulations.

Here we report the results obtained by screen-ing about 20% of this library (5,040 formula-tions), the largest cohesive screening studyreported in the transdermal literature. Eachformulation was tested at least four times inover 20,000 experiments. Using the tradi-tional tools for formulation screening(FDCs), it would have taken over seven yearsto do this many experiments (about tenexperiments a day). With INSIGHT screen-ing, the same task was accomplished in abouttwo months with a screening rate of500–1,000 experiments per day.

Results from INSIGHT screeningEnhancement potentials of formulationsmeasured by INSIGHT screening from>20,000 experiments are reported in Figure2a. The formulations spanned a wide range ofenhancement potentials measured in terms ofconductivity enhancement ratios (ER). Someof the leading hits from INSIGHT screening(that is, formulations with high ER) were fur-ther screened for irritation using in vitro cul-

tures of human keratinocytes (EpiDerm), which has recently emergedas an excellent method for assessing IP21. IP was assessed by measuringthe viability of epidermal cells using the Methyl Thiazol Tetrazolium(MTT) uptake assay and the release of interleukin-1α. IP assessed byboth methods agreed with each other. IP values were used as a prelimi-nary assessment of irritancy. Safety of lead formulations was assessedin vivo in rats. ER and IP values for the leading hits from INSIGHTscreening (green circles) are shown in Figure 2b. For reference, IP andER values for a number of individual CPEs that were used to designSCOPE formulations are also shown in Figure 2b (red circles).

ER-IP plots were divided into four quadrants. Quadrant I corre-sponds to safe but weak enhancers, quadrant II corresponds to weakand irritating enhancers, quadrant III corresponds to potent and irri-tating enhancers and quadrant IV corresponds to potent and safeenhancers. Most single CPEs are located in quadrants I or II (red cir-cles). Furthermore, for single enhancers, IP increases proportionallywith ER, thus confirming the argument that it is very difficult if notimpossible to design a potent and safe formulation for transdermaldelivery based on a single enhancer. However, a substantial number ofleading hits from INSIGHT screening (green circles) are located inquadrant IV. Four hits were found to lie in quadrants II or III and wereeliminated from subsequent development.

We observed that leading hits from INSIGHT screening generallyresulted in enhancers that are safer and more effective. Two formula-tions were particularly outstanding (0.5% (wt/vol) SLA:PP, weightfraction of SLA, 0.7 and 1% (wt/vol) N-lauroyl sarcosine:sorbi-tan monolaurate (NLS:S20), weight fraction of NLS, 0.6, shown inFigure 2b by arrows 1 and 2, respectively). Both formulations exhibitedIP < 10 and were selected for further consideration because a numberof enhancers that exhibited IP ∼ 10 in our experiments (for example,0.5% (wt/vol) SLA, 0.5% (wt/vol) NLS and 1.5% (wt/vol) S20) havebeen considered suitable for topical applications22–24.

Validation of the hits from INSIGHT screening and safety studiesusing FDCs is reported in Figure 2c. Skin permeabilities to inulin fromtwo leading hits were measured, and it was confirmed that form-ulations discovered by INSIGHT screening increase permeability to inulin by a factor of 30–80. Inulin was chosen as a validating marker

Successful transdermal formulation

Candidate enhancer pool

a b

Bioavailabilityand safety

Flexenhancement

Irritation

Discovery

0.02%

0.5%

2%

100%

Figure 1 The process of discovery of transdermal formulations. (a) A library of enhancer mixturescreated from a pool of existing enhancers is screened using INSIGHT to identify potential SCOPEformulations. The leading hits from INSIGHT screening are evaluated for IP. SCOPE formulations withhigh potency and low IP are evaluated in vitro for flux enhancement using candidate drugs. Uponconfirming transdermal flux enhancement, the formulation is tested in vivo for bioavailability andsafety. (b) Pyramid of the formulation discovery process. Of all the formulations studied using INSIGHTscreening, 98% were eliminated from further consideration based on potency. 75% of the remaining2% formulations (that is, 99.5 % of the original library) were eliminated after irritation studies. Theremaining 0.5% formulations were tested in vitro for flux enhancement and 0.02% formulations (1 in∼ 5,000) were tested for bioavailability.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy

A RT I C L E S

194 VOLUME 22 NUMBER 2 FEBRUARY 2004 NATURE BIOTECHNOLOGY

because it is a poorly permeable hydrophilic macromolecule and pro-vides a standard high enough to select formulations for transdermaldelivery of macromolecules. Inulin permeability enhancementobtained by tape stripping the stratum corneum is also shown inFigure 2c. This number simply serves as an approximate upper limit ontransdermal transport enhancement. SCOPE formulations induced anenhancement approaching that induced by tape stripping, yet withouta high IP.

Two SCOPE formulations stood out from a large list of >5,000 initialcandidates: (i) SLA:PP (weight fractions, 0.7:0.3; total concentration of0.5% (wt/vol)); and (ii) NLS:S20 (weight fractions, 0.6:0.4; total con-centration of 1% (wt/vol)). The efficiency of these two formulationsfor enhancing skin permeability without irritation can be understoodfrom Figures 3a and 3b, which respectively show INSIGHT screeningresults for all tested combinations of SLA:PP and NLS:S20, as two-dimensional phase maps. Each phase map represents the ER of 44 for-mulations from a binary pair as a function of total enhancerconcentration and enhancer composition. Regions of the map arecolor coded, with red corresponding to highest enhancements and bluecorresponding to lowest. The location of thetwo SCOPE formulations in respective phasemaps is indicated by arrows. Interestingly,these formulations correspond to ‘hot spots’in the phase map that arise from the synergis-tic effect of individual CPEs.

The uniqueness of both SCOPE formula-tions in increasing potency without increasingirritation can also be seen in Figure 4. In thisfigure the placement of SCOPE formulationsand their individual enhancers is shown onthe ER-IP map. In both cases, the potency ofthe enhancers was increased owing to theircombination without aggravating irritation.In the case of NLS:S20, the IP was substantiallyreduced compared with that of individualenhancers. The reduced irritation of enhancer

mixtures is consistent with earlier reports25,26. The ability of CPEs toinduce skin permeation without irritation, quantified by the ratioER/IP, was enhanced 3- to 12-fold for the SCOPE formulations shownin Figure 4 compared with their respective individual components.

In vitro and in vivo validationThe ability of SCOPE formulations to deliver macromolecules acrossthe skin is shown in Figure 5a. The figure shows the dependence of skinpermeability to hydrophilic solutes (water-octanol partition coefficient< 0.1) on solute molecular weight. Permeability of untreated skin tohydrophilic solutes (open circles) decreases rapidly with increasingmolecular weight. In the presence of SCOPE formulations, the perme-abilities are increased up to 100-fold from the baseline for several ther-apeutic hydrophilic drugs including LHRH (1.2 kDa), low molecularweight heparin (LMWH, ∼ 10 kDa), oligonucleotide (ODN, 15 kDa),and methotrexate (460 Da) (open squares and closed circles) and twodummy drugs, inulin (5 kDa) and mannitol (182 Da).

To confirm the potency and safety of leading INSIGHT hits, we car-ried out in vivo experiments using hairless rats. We used a 9 cm2 trans-

a b c

Figure 2 Results from INSIGHT screening. (a) Summary of all INSIGHT screening experiments done in this study. The figure shows conductivityenhancements measured in ∼ 24,000 experiments. The colors correspond to the scale shown in Figure 3. Formulations exhibiting high ER were selected forfurther evaluation. (b) A plot of the IP versus ER for selected enhancers (red circle, individual enhancers; green circle, enhancer mixtures). The ER-IP graphis divided into four quadrants. Quadrant I corresponds to weak and safe enhancers. Quadrant II corresponds to weak and highly irritating enhancers.Quadrant III corresponds to strong and highly irritating enhancers. Quadrant IV corresponds to strong and safe enhancers. (c) Enhancement of skinpermeability to inulin from two SCOPE formulations (SLA:PP and NLS:S20) in FDCs. Enhancements are calculated with respect to skin permeability toinulin from PBS in all three cases.

Potency scale

a b

Composition(wt fraction of thefirst component)

Tota

l con

cent

ratio

n(%

w/w

)

6050403020102.8

Figure 3 Potency phase maps of two SCOPE formulations. Two examples of potency maps of binaryenhancer mixtures. (a) Combination of SLA and PP, which is one of the lead formulations. (b) Synergybetween NLS and S20.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy

A RT I C L E S

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 2 FEBRUARY 2004 195

dermal patch containing 2 mg/ml leuprolide acetate in SLA:PP in 1:1phosphate buffered saline (PBS):ethanol. SLA:PP was chosen as thepreferred combination because it induced an ∼ 80-fold enhancement ofinulin in vitro at a total concentration (SLA:PP) of only 0.5% (wt/vol)and passed the IP screen. Plasma concentrations of leuprolide weremeasured using enzyme-linked immunosorbent assay (ELISA).Therapeutically substantial plasma leuprolide concentrations wereachieved (Fig. 5b, closed circles). Plasma concentrations of leuprolideincreased over 24 h. No substantial plasma concentrations wereobserved in controls (Fig. 5b, open circles). The skin appeared normalthroughout the experiment and no erythema was observed at any time.Histological studies showed that the stratum corneum of the skinexposed to the formulation appeared normal, and no structural differ-ences were observed in the skin compared with controls. We did notobserve any signs of inflammation or detect the presence of inflamma-tory cells in the skin. Skin conductance was measured throughout theexperiment to assess skin permeabilization and recovery. These meas-urements showed that skin conductance increased by about 20-foldduring the 24 h the formulation was in contact with the skin; about80% of the skin conductance enhancement recovered within 12 h afterremoval of the formulation.

DISCUSSIONUsing INSIGHT screening of a binary enhancer library, we have identi-fied several mixtures of CPEs exhibiting ER > 60. These values com-pare well with experiments using skin samples in which thepermeation barrier was eliminated by tape-stripping, which yielded ERvalues in the range of 60–70. Another reference to calibrate the ER val-ues reported in Figure 2a was obtained by evaluating the ER ratio ofone of the most commonly used chemical enhancers, oleic acid (OA)(1.5% (wt/vol) in 1:1 PBS:ethanol), which gave an ER value of only 10.Compared with this reference, several formulations reported in Figure2a offer superior enhancement. All formulations exhibiting ER > 60were binary mixtures. Of all binary pairs studied, only a few were rep-resented in the most potent group of formulations (ER > 60), with no

more than two compositions for any binary pair exhibiting such highpotency. The low population density of formulations exhibiting ER >60 emphasizes the utility of INSIGHT screening in identifying success-ful formulations from a very large pool of randomly chosen enhancers.

The leading hits from INSIGHT screening corresponded to hotspots in potency maps (Fig. 3a,b). Hot spots are essential for SCOPEformulations because they offer the possibility of exposing the stratumcorneum to a potent formulation while exposing the epidermis to a rel-atively weak formulation. Hot spots for both binary pairs are rathersmall, thereby increasing the likelihood of enhancing potency withoutirritation. However, the narrow size of the hot spot also decreases thelikelihood of discovery. The mechanistic origins of such hot spots areunclear. They may originate from the molecular interactions of theenhancers in the donor solutions or with the stratum corneum lipids.The synergistic effect of enhancers was quantified by defining a param-eter, S, which quantitatively describes the potency of a binary formula-tion compared to the weighted mathematical average of the individualpotencies at the same concentration (see Methods). Based on the S-value, formulations can be classified into three categories: positivelysynergistic (S > 1), negatively synergistic (S < 1) and nonsynergistic (S= 1). Both SCOPE formulations exhibited S values greater than 1(SLA:PP, 3.5 and NLS:S20, 2.25). The highest S value found in thisstudy was 9 (benzyl dimethyl dodecyl ammonium chloride:lauric acid(BDAC:LA)).

The top hit from INSIGHT screening (SLA:PP) exhibited substantialenhancement of skin permeability (Fig. 5a). Permeation acrossuntreated skin depends strongly on solute molecular weight, indicatingthe importance of steric hindrance of solute diffusion. Skin permeabil-ity is especially low for large hydrophilic molecules (MW > 500 Da).The dependence of skin permeability on solute molecular weight isdramatically decreased in the presence of SCOPE formulations, almostapproaching that in an isotropic and structurally, almost approachingthat in an isotropic and structurally homogeneous medium, therebyindicating the disruption of the diffusion barrier. The enhancementsreported in Figure 5a are therapeutically substantial. For example, atherapeutic dose of LHRH or its synthetic analog (∼ 1 mg/day27) can bedelivered from a 10 cm2 patch containing 8 mg/ml LHRH. Similarly, adose of LMWH of about 1 mg/kg/day28 can be delivered from a 50 cm2

patch containing 100 mg/ml LMWH. In vivo studies done usingleuprolide acetate as a model drug and hairless rats as an animal modelconfirmed the efficacy of SLA:PP in enhancing macromolecular trans-dermal delivery (Fig. 5b).

Initial studies indicated that SCOPE formulations containingSLA:PP are safe for transdermal applications. Both SLA and PP have ahistory of use in topical and other medical products. Specifically, SLA isused as an inactive ingredient in several topical formulations approvedby the US Food and Drug Administration at concentrations usuallyhigher than 0.5% (wt/vol). Derivatives of PP are also used in approveddermatological and medical products (http://www.accessdata.fda.gov/scripts/cder/iig/index.cfm).

We have described a family of enhancers (SCOPE) for transdermaldrug delivery and a method for discovering them. These rare formula-tions, whose discovery was beyond the capabilities of available tools(FDCs), was enabled by the high screening rate of the INSIGHTmethod. Two SCOPE formulations (SLA:PP and NSL:S20) were par-ticularly potent in enhancing skin permeability to macromolecules.Even more potent formulations may be discovered by further screen-ing. It may also be possible to increase potency by combining CPEs internary or quaternary formulations. Finally, INSIGHT screening datashould be useful for advancing our understanding of the mechanismsof enhancer activity.

Figure 4 ER-IP relationships of two SCOPE formulations. ER and IP of two leading SCOPE formulations compared to that of their correspondingindividual enhancers. SLA:PP (total concentration of 0.5% (wt/vol), SLAweight fraction of 0.7); SLA, 0.5% (wt/vol); PP, 0.5% (wt/vol). NLS:S20(total concentration of 1% (wt/vol), NLS weight fraction of 0.6); NLS, 1% (wt/vol); S20, 1% (wt/vol).

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy

A RT I C L E S

196 VOLUME 22 NUMBER 2 FEBRUARY 2004 NATURE BIOTECHNOLOGY

METHODSLibrary generation. The formulation library consisted of 32 CPEs, which wereclassified into eight categories: (i) anionic surfactants (sodium lauryl sulfate,NLS, sodium octyl sulfate (SOS), SLA); (ii) cationic surfactants (cetyl trimethylammonium bromide, dodecyl pyridinium chloride, BDAC, octyl trimethylammonium bromide); (iii), zwitterionic surfactants (hexadecyl trimethylammoniopropane sulfonate, oleyl betaine, cocamidopropyl hydroxysultaine,cocamidopropyl betaine); (iv) nonionic surfactants (polyoxyethylene sorbitanmonolaurate (TWEEN20), sorbitan monolaurate (S20), polyethyleneglycoldodecyl ether, Triton X-100); (v) fatty acids (OA, linoleic acid, LA, linolenicacid); (vi) fatty esters (tetracaine, isopropyl myristate, sodium oleate, methyllaurate); (vii) azone/azone-like compounds (N-decyl-2-pyrrolidone, dodecylamine, PP, nicotine sulfate); and (viii) others (menthol, methyl pyrolidone,cineole, limonene). To limit the size of the library, CPEs were divided into fourblocks such that each group had one representation from each class ofenhancers. Enhancers within each block were paired to generate 28 binary com-binations (a total of 112 combinations over the entire set under study). For eachpair of enhancers four different total concentrations of 0.5, 1, 1.5 and 2%(wt/vol) were selected. At each total concentration the weight fraction of oneenhancer was varied from 0 to 1 in steps of 0.1. Thus for each enhancer pair, 44test formulations were generated. All formulations were prepared in a 1:1PBS:ethanol solution. The role of ethanol in the base solvent is to solubilizecomplex molecular moieties that may be formed in enhancer combinations.Ethanol may potentially be replaced by other organic solvents miscible withPBS.

INSIGHT screening. The potency of transdermal formulations is convention-ally screened using FDCs, which typically allow on the order of 10 experiments aday. INSIGHT screening, on the other hand is designed to do 500–1,000 experi-ments a day with less than one-tenth skin usage per experiment, thus making itfeasible to screen SCOPE candidates in a practical time19. This tool was devel-oped and used in this study.

The high efficiency of INSIGHT screening originates from efficient skinusage and from the use of skin impedance as a determinant of the efficacy of theformulations. Transepidermal current is mediated by the movement of charge-carrying ions and is thus related to the permeability of skin to these ions. Formal

relationships relating skin impedance to drug per-meability have been developed using Nernst-Planckflux equations and the Nernst-Einstein relations forideal solutions29–31. For screening purposes it is suf-ficient to establish that skin conductivity (reciprocalof impedance) is directly related to skin permeabil-ity. This correlation was confirmed using inulin as amodel permeant (data not shown).

Porcine skin was used as a model for skin in allthe experiments. Hair was clipped to ensure that thestratum corneum was not damaged. Skin was har-vested from the lateral abdominal region ofYorkshire pigs. Adipose tissue was removed andfull-thickness skin was sectioned into 4˝ × 4˝ pieces.The skin was stored at –70 °C until the experi-ments32. The INSIGHT screening apparatus con-sists of a Teflon plate that serves as the donor and apolycarbonate plate that serves as the receiver, each12.7 mm (a half-inch) thick19. The donor contains asquare matrix of 100 wells (each 3 mm in diameter)that serve as individual donor compartments. Asymmetric matrix of 100 wells in the polycarbonateplate serves as individual receivers. The receiverwells were filled with PBS to keep the skin hydratedduring the experiment (24 h). Skin, thawed at 25 °C,was placed between the two plates with the stratumcorneum facing the donor plate. Donor and receiverplates were clamped together using four screws. Theskin was incubated with 85 µl of each test formula-tion in the donor wells for 24 h with each formula-tion being repeated in four wells. Skin impedance in

each well was recorded using two electrodes; a common electrode in the dermisand a second electrode placed sequentially into each donor compartment. AnAC signal, 100 mV RMS at 100 Hz, was applied across the skin with a waveformgenerator (Agilent 33120A). Conductivity measurements were done using amultimeter (Fluke 189) with a resolution of 0.01 µA. Data acquisition parame-ters (frequency and amplitude of electric potential and number of measure-ments) were selected to provide maximum resolution and reproducibility in thedata. Current measurements were done at two time points, 0 h (I0) and 24 h(I24). The ER for each formulation was then calculated by taking the ratio ofskin conductivities at 24 h and 0 h. Correlation between skin ER measured byINSIGHT screening with those using standard diffusion cells was also con-firmed for selected formulations (r2 > 0.9).

Synergy values, S, were calculated using the following equation:

ER X,YA+B

S = —————————X.ERY

A + (1 – X).ERYB

where ERX,YA+B is the enhancement ratio obtained with the formulation contain-

ing A and B at a point {X (weight fraction of A), Y (total concentration of A +B)} on the map, and ERY

A and ERYB are the enhancement ratios obtained with

pure components A and B, respectively, at the same total concentration.

FDC validation. We did permeation experiments using 3H-labeled inulin,methotrexate, LHRH, ODN and heparin as model permeants. All radiochemi-cals except for ODN were obtained from American Radiolabeled Chemicals.Radiolabeled ODN was provided by ISIS Pharmaceuticals. FDCs (16 mm diam-eter, 12 ml receiver volume) were used to assess the transport enhancements oftest formulations. Simultaneous conductivity measurements were done in theFDC to validate the results from INSIGHT. A small stir bar and an Ag/AgCl diskelectrode (E242, InVivo Metric) were added to the receiver chamber. We tookadequate measures to prevent any inclusion of air in the receiver chamber. Theconductivity measurement assembly used was the same as that used in the caseof the INSIGHT screening assembly except that an Ag/AgCl electrode was usedin the receiver compartment instead of inside the skin. The electric resistance ofthe electrodes used in both the systems was verified to be similar. The receiverchamber was filled with PBS. Thawed pigskin was mounted on the diffusion cell

a b

Figure 5 In vitro and in vivo assessment of the efficacy of SLA:PP SCOPE formulation. (a) Dependenceon molecular weight of skin permeability to hydrophilic solutes. Open circles show permeability ofuntreated skin reported in the literature for a variety of hydrophilic solutes33. Open squares show skin permeability from leading SCOPE formulations (SLA:PP and NSL:S20). Closed circles showpermeabilities measured from leading scope formulations (SLA:PP and NLS:S20) that have beencorrected to take into account the amount of drug deposited in the skin. For ODN, the majority of the drug crossing the stratum corneum was trapped in the skin. Hence, only the permeability valuecalculated based on the amount deposited in the skin is reported. Permeability of untreated skin shows a strong decrease with increasing molecular weight. In contrast, permeabilities from SCOPEformulations show a relatively weak size dependence. (b) Plasma concentrations of leuprolide after theplacement of a leuprolide patch on the skin (n = 3, 4). Control experiments were done using leuprolidedissolved in PBS containing 1.8% (wt/vol) hyaluronic acid.

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy

A RT I C L E S

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 2 FEBRUARY 2004 197

using a clamp with the stratum corneum side facing the donor. Radiolabeledsolutes were directly added to the formulation at a concentration of 10 µCi/ml.The formulation was then added in the donor wells for a period of 96 h duringwhich the receiver was sampled periodically. Concentration of radiolabeledsolute was measured using a scintillation counter (Packard Tri-Carb 2100 TR).An independent study verified that all detected radioactivity came from themodel solute and not from tritiated water that may have resulted from tritiumexchange. Specifically, receiver samples were desiccated and analyzed forradioactivity and no substantial difference was observed between native anddesiccated receiver samples. Skin permeability was calculated using the standardequations18. Enhancement of permeability was calculated by dividing perme-ability obtained with SCOPE formulation with that obtained from 1:1PBS:ethanol or with PBS alone (untreated) depending on the objective.

IP assessment. IP of test formulations was estimated using Epiderm (MatTek), acell culture of normal epidermal keratinocytes derived from humans. Epidermcultures were stored and handled according to the standard protocol MTT-ET-50 supplied by MatTek. To study the effect of the test formulations on cell viabil-ity, cell cultures were exposed to 10 µl of the test formulation for 4 h. Each testformulation was analyzed in duplicate. At the end of 4 h the assay medium wasremoved and stored at –70 °C for an interleukin-1α assay. The cell cultures werethen handled as per the suggested protocol. The optical absorbance data fromthe extracted samples were then used to calculate the percentage cell viability asrecommended in the protocol. We define IP based on cell viability as follows:

(% cell viability with the formulation

)IP = 100 1 – ——————————————–

maximum % cell viability

1% (wt/vol) Triton X-100 was used as the positive control and 1:1 PBS:ethanolwas used as the negative control. PBS:ethanol by itself does not yield a positivescore and allows us to rank the ‘hits’ from high-throughput screening in terms oftheir IP. One must keep in mind, however, that this assay provides a quick pre-liminary estimate on the irritancy of enhancer mixtures and allows us to rankthe mixtures for irritation. Any final predicament on irritation behavior is sub-ject to in vivo studies done over extended exposure times or multiple exposures.

In vivo leuprolide delivery. All animal experiments were done according toinstitutionally approved protocols at UCSB. In vivo experiments were doneusing hairless rats (250–280 gm, Charles River Labs) as an animal model.Animals were anesthetized using isofluorane (1.25–3% isoflorane in oxygen).We placed 1 gm of leuprolide gel (2 mg/ml leuprolide in 1:1 PBS:ethanol alongwith 0.35 % (wt/vol) SLA and 0.15% (wt/vol) PP and 1.8% (wt/vol) hyaluronicacid or 1.5% (wt/vol) hydroxypropyl cellulose MF) on a skin area of 9 cm2. Athin polymer sheet was placed on the gel and the edges were sealed with acyanoacrylate adhesive. The animals were allowed to recover from anesthesiaafter 2 h. Blood samples were collected from the jugular vein over a period of 24h and plasma concentration of leuprolide was measured using ELISA (S-1159(EIAS-7680r) Bachem Bioscience). Throughout the experiment the skin wasobserved for erythema. At the end of the experiment, the skin was excised andfixed in 10% (vol/vol) formalin. The skin was sectioned and stained with hema-toxylin and eosin (Mass Histology services). Control experiments were doneusing leuprolide dissolved in PBS containing 1.8% (wt/vol) hyaluronic acid.PBS was used as a control formulation because it is a true indicator of baselineskin permeability.

ACKNOWLEDGMENTSThe authors acknowledge the technical assistance of Maricella Castaneda,Jeffery O’Neil and Drew Lassen. The authors also thank ISIS pharmaceuticals forproviding oligonucleotides and Chemron for providing betaines. This research was funded in part by Cottage hospital research program, Santa Barbara,California, USA.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnologywebsite for details).

Received 23 September; accepted 13 November 2003Published online at http://www.nature.com/naturebiotechnology/

1. Zaffaroni, A. Overview and evolution of therapeutic systems. Ann. NY Acad. Sci. 618,405–421 (1991).

2. Bos, J.D. & Meinardi, M.M. The 500 dalton rule for the skin penetration of chemicalcompounds and drugs. Exp. Dermatol. 9, 165–169 (2000).

3. Wertz, P.W. & Downing, D.T. in Transdermal Drug Delivery: Developmental Issues andResearch Initiatives (eds. Hadgraft, J. & Guy, R.H.) 1–17 (Marcel Dekker, New York,Basel, Hong Kong, 1989).

4. Guy, R. et al. Iontophoresis: electrorepulsion and electroosmosis. J. Control. Release64, 129–132 (2000).

5. Mitragotri, S., Blankschtein, D. & Langer, R. Ultrasound-mediated transdermal pro-tein delivery. Science 269, 850–853 (1995).

6. Finnin, B.C. & Morgan, T.M. Transdermal penetration enhancers: applications, limita-tions and potential. J. Pharm. Sci. 88, 955–958 (1999).

7. Williams, A.C. & Barry, B.W. Skin absorption enhancers. Crit. Rev. Ther. Drug CarrierSyst. 9, 305–353 (1992).

8. French, E., Potton, C. & Walters, K. in Pharmaceutical Skin PenetrationEnhancement (eds. Walters, K. & Hadgraft, J.) 113–144 (Marcel Dekker, New York,Basel, Hong Kong, 1993).

9. Kanikkannan, N., Kanimalla, K., Lamba, S.S. & Singh, M. Structure-activity relation-ship of chemical penetration enhancers in transdermal drug delivery. Curr. Med.Chem. 7, 593–608 (2000).

10. Lashmar, U.T., Hadgraft, J. & Thomas, N. Topical application of penetrationenhancers to the skin of nude mice—a histopatholgical study. J. Pharm. Pharmacol.41, 118–122 (1989).

11. Akimoto, T. & Nagase, Y. Novel transdermal drug penetration enhancer: synthesis andenhancing effect of alkyldisiloxane compounds containing glucopyranosyl group. J. Control. Release 88, 243–252 (2003).

12. Takanashi, Y., Higashiyama, K., Komiya, H., Takayama, K. & Nagai, T. Thiomentholderivatives as novel percutaneous absorption enhancers. Drug Dev. Ind. Pharm. 25,89–94 (1999).

13. Suhonen, M., Bouwstra, J. & Urtti, A. Chemical enhancement of percutaneousabsorption in relation to stratum corneum structural alterations. J. Control. Release59, 149–161 (1999).

14. Mitragotri, S. Effect of bilayer disruption on transdermal transport of low-molecularweight hydrophobic solutes. Pharm. Res. 18, 1022–1028 (2001).

15. Robinson, M.K. & Perkins, M.A. A strategy for skin irritation testing. Am. J. ContactDermat. 13, 21–19 (2002).

16. Mitragotri, S. Synergistic effect of enhancers for transdermal drug delivery. Pharm.Res. 17, 1354–1359 (2000).

17. Funke, A.P. et al. Transdermal delivery of highly lipophilic drugs: in vitro fluxes ofantiestrogens, permeation enhancers and solvents from liquid formulations. Pharm.Res. 19, 661–668 (2002).

18. Bronaugh, R.L. in Percutaneous Absorption: Mechanisms-Methodology-Drug Delivery(eds. Bronaugh, R.L. & Maibach, H.I.) (Marcel Dekker, New York, Basel, Hong Kong,1989).

19. Karande, P. & Mitragotri, S. High-throughput screening of transdermal formulations.Pharm. Res. 19, 655–660 (2001).

20. Black, G. in Pharmaceutical Skin Penetration Enhancement. (eds. Walters, K. &Hadgraft, J.) 145–174 (Marcel Dekker, New York, Basel, Hong Kong, 1993).

21. Fentem, J.H. & Botham, P.A. ECVAM’s activities in validating alternative tests for skincorrosion and irritation. Altern. Lab. Anim. 30, 61–67 (2002).

22. Lanigan, R.S. Final report on the safety assessment of cocoyl sarcosine, lauroyl sar-cosine, myristoyl sarcosine, oleoyl sarcosine, stearoyl sarcosine, sodium cocoyl sar-cosinate, sodium lauroyl sarcosinate, sodium myristoyl sarcosinate, ammoniumcocoyl sarcosinate and ammonium lauroyl sarcosinate. Int. J. Toxicol. 20, 1–14(2001).

23. Loffler, H. & Happle, R. Profile of irritant patch testing with detergents: sodium laurylsulfate, sodium laureth sulfate and alkyl polyglucoside. Contact Dermatitis 48,26–32 (2003).

24. Berardesca, E. et al. Ranking of surfactant skin irritancy in vivo in man using the plas-tic occlusion stress test (POST). Contact Dermatitis 23, 1–15 (1990).

25. Hall-Manning, J., Holland, H., Basketter, A. & Barratt, D. Skin irritation potential ofmixed surfactant systems in a human 4-hour covered patch test. Allergologie 18, 465(1995).

26. Fentem, J. et al. A prevalidation study on in vitro tests for acute skin irritation: resultsand evaluation by the Management Team. Toxicology In Vitro 15, 57–93 (2001).

27. Periti, P., Mazzei, T. & Mini, E. Clinical pharmacokinetics of depot leuprorelin. Clin.Pharmacokinet. 41, 485–504 (2002).

28. Frydman, A. Low-molecular weight heparins: an overview of their pharmacodynamics,pharmacokinetics and metabolism in humans. Haemostasis 26, 24–38 (1996).

29. Peck, K., Ghanem, A. & Higuchi, W. Hindered diffusion of polar molecules throughand effective pore radii estimates of intact and ethanol treated human epidermalmembrane. Pharm. Res. 11, 1306–1314 (1994).

30. Tang, H., Mitragotri, S., Blankschtein, D. & Langer, R. Theoretical description oftransdermal transport of hydrophilic permeants: application to low-frequencysonophoresis. J. Pharm. Sci. 90, 543–566 (2001).

31. Tezel, A., Sens, A. & Mitragotri, S. A theoretical description of transdermal transportof hydrophilic solutes induced by low-frequency sonophoresis. J. Pharm. Sci. 92,381–393 (2003).

32. Mitragotri, S. et al. Determination of the threshold energy dose for ultra-sound-induced transdermal drug delivery. J. Control. Release 63, 41–52 (2000).

33. Mitragotri, S. Modeling skin permeability to hydrophilic and hydrophobic solutesbased on four permeation pathways. J. Control. Release 86, 69–92 (2003).

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy