Terpenes increase the partitioning and molecular dynamics of an amphipathic spin label in stratum corneum membranes

In this work, the interaction of the skin penetration enhancers dl-menthol, -terpineol, 1,8-cineole and (+)-limonene with the uppermost skin layer, the stratum corneum and with multilamellar vesicles from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is investigated by electron paramagnetic resonance (EPR) spectroscopy of the small spin label 2,2,6,6,-tetramethylpiperedine-1-oxyl (TEMPO), which partitions the aqueous and hydrocarbon phases. The EPR spectrum allows for the determination of the actual partition coefficient and the rotational diffusion rates of the spin probe in the two environments. The enthalpy changes, ΔH°, to transfer the spin probe from the aqueous to the hydrocarbon phase, as well as the activation energies associated to its rotational motion, were considerably smaller for stratum corneum, indicating less pronounced thermal reorganization. For DPPC, the terpenes increased both the partition coefficient and the rotational diffusion rate of the spin label in the membrane, except in the liquid–crystalline phase, while these increases in stratum corneum were observed in the entire temperature range measured with the exception of the rotational motion parameter for dl-menthol and -terpineol at temperatures below their melting point (32–41 °C). It is suggested that the terpenes effectively acting as spacers in the membrane fluidize the lipids and cause ruptures in the hydrogen-bonded network of the polar interface.     

Interaction of Antidepressant Drug,Clomipramine, with Model and Biological Stratum Corneum Membrane as Studied by Electron Paramagnetic Resonance

The interactions of tricyclic antidepressant drug, clomipramine (CLO), with pig ear stratum corneum (SC) and model membranes were investigated by electron paramagnetic resonance (EPR) spin labeling to get some insight into the possible application of this drug in transdermal delivery. The changes in membrane characteristics caused by CLO in the regions that are close to the water–lipid interfaces and the central parts of the membranes were searched. The experimental results were supported by computer simulation of EPR spectra, which showed heterogeneity of the membranes composed of regions with different fluidity characteristics. CLO was effective in both parts of the layers, indicating intercalation of the drug into model membranes as well as into the pig ear SC. Introduction of various molar ratios of CLO caused a decrease in the order parameter and an increase in the rotational diffusion of nitroxide moiety in different membrane regions as well as an increase in the polarity of spin probe environment. It also changed the number of resolved spectral components, which reflects the heterogeneity of the membrane. The fluidizing effect of CLO on pig ear SC throughout the whole membrane layers indicates that CLO penetrates into the SC, which is important for its transdermal delivery.     

Comparative studies on the effects of water, ethanol and water/ethanol mixtures on chemical partitioning into porcine stratum corneum and silastic membrane.

The effects of water and ethanol vehicles on stratum corneum and silastic membrane partitioning of 11 industrial and agricultural compounds were studied to aid in characterizing and assessing risk from skin exposure. Zero percent, 50% and 100% aqueous ethanol solutions were used as solvents for (14)C labeled phenol, 4-nitrophenol, pentachlorophenol, dimethyl parathion, parathion, chloropyrifos, fenthion, triazine, atrazine, simazine and propazine. Compound partitioning between the solvents and porcine stratum corneum/silastic membrane were estimated. Stratum corneum was exposed to aqueous ethanol ranging from 0% to 100% v/v ethanol in 20% increments and Fourier transform infrared spectroscopy (FT-IR) was used to obtain an index of lipid disorder. Gravimetry and FT-IR were used to demonstrate lipid extraction in aqueous ethanol solutions. Partitioning patterns in silastic membranes resembled those in stratum corneum and were correlated with octanol/water partitioning. Partitioning was highest in water and was higher from 50% ethanol than from 100% ethanol, except for parathion, 4-nitrophenol, atrazine and propazine. Correlation existed between molecular weight and partitioning in water, but not in ethanol and ethanol/water mixtures. Lipid order, as reflected in FT-IR spectra, was not altered. These studies suggest that stratum corneum partitioning of the compounds tested is primarily determined by relative compound solubility between the stratum corneum lipids and the donor solvent. Linear relationships existed between octanol/water partitioning and stratum corneum partitioning. Partitioning was also correlated with molecular weight in water solvent systems, but not in ethanol and ethanol/water mixtures. Ethanol and ethanol/water mixtures altered the stratum corneum through lipid extraction, rather than through disruption of lipid order.     

Effect of surfactants on human stratum corneum: electron paramagnetic resonance study

Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes are useful for studying biological membranes and chemical-membrane interactions. Recently, we established a stripping method to remove stratum corneum (SC) for this purpose. To assess this stripping method with EPR and correlate with standard methods, we quantified the irritant effects of three types of surfactants by measurements of visual score and transepidermal water loss (TEWL), SC hydration and chromametry and studied EPR spectra measurements of surfactant-treated cadaver SC (C-SC) and stripped off SC (S-SC) on patch tested sites. 5-Doxyl stearic acid was the spin label. The order parameter S obtained from the spectra of S-SC correlated with those of C-SC and TEWL values. The results suggest that this method is capable of evaluating the fluidity of SC and correlates with the above bioengineering parameters.     

Interaction of chlorpromazine with biological membranes. A photochemical study using spin labels.

Fatty acid spin labels have been included into erythrocyte ghosts and synaptic plasma membranes in order to study the interaction of phenothiazine derivatives (particularly chlorpromazine— CPZ) with these membranes. The following results have been obtained. (1) Weak modifications of the spin label spectroscopic response are observed only on the label of the polar part of the membrane and with CPZ concentrations higher than 5 × 10^?4 M. (2) Under ultraviolet irradiation (λ = 310 nm) phenothiazine derivatives reduce fatty acids spin labels. Measurement of the reduction kinetic constants of two different types of spin labels give information about the location of the drugs inside the membranes. The photochemical interaction is influenced by the membrane proteins. These results suggest that, in the pharmacologically active concentration range, chlorpromazine seems to localize at the interface between the phospholipids and the proteins of the membranes.     

Influence on intestinal mucous permeation of paclitaxel of absorption enhancers and dosage forms based on electron spin resonance spectroscopy

The aim of this paper is to investigate the permeation mechanism of the hydrophobic drug, paclitaxel in intestinal membranes of mice in relation to enhancers and preparation factors. The alteration fluidity of lipid and protein in mucous membrane were determined using electron spin resonance (ESR) when the membrane was treated with several enhancers including Pluronic F68, polyethylene glycol (PEG), Brij78 and lecithin. At the same time, the enhanced permeation of paclitaxel across the intestinal intercellular membrane of stratum corneum was studied for three formulations: inclusion complex, microemulsion and injection. The results showed that use of paclitaxel-hydroxypropyl-beta-cyclodextrin inclusion complexation and of paclitaxel microemulsion as vehicle and PEG 1500 as enhancer could significantly increase the permeation kinetics of paclitaxel in a fluid diffusion study. The effect on absorption characteristics of enhancing permeation of this hydrophobic drug in the intestinal mucosa was considered in the light of the change in membrane fluid.     

The influence of ethanol on permeation behaviour of the porous pathway in the stratum corneum

Relationship between in vitro percutaneous penetration of baclofen, a model zwitterion, and ethanol concentration in the vehicle was studied. In comparison with water, 95% ethanol increases the permeability coefficient of baclofen by a factor of 10, which suggests rapid and maximal pore formation in the stratum corneum. A slow time-dependent increase of penetration rate was observed for lower ethanol concentrations. For ethanol concentrations in the range 0–70%, solubility of baclofen in the vehicle is a dominant factor influencing the penetration rate. The reasons for an unexpectedly small effect of 70° ethanol on the process of pore formation in the stratum corneum are discussed.     

Parallel artificial membrane permeability assay: a new membrane for the fast prediction of passive human skin permeability

This work was devoted to the search for new artificial membranes allowing a rapid evaluation of passive human skin permeation of compounds with a parallel artificial membrane permeability assay (PAMPA). Effective permeability coefficients (Pe) determined for a set of compounds using the PAMPA technique with isopropyl myristate (IPM) and silicone oil, alone or in mixture, were compared to the corresponding human skin permeability coefficient values (Kp). A good correlation between Pe and Kp was found for compounds tested through a membrane consisting of 70% silicone and 30% IPM. Moreover, positive correlation between the membrane retention of compounds and stratum corneum/water partition coefficients (PSC) was established. These results showed that this new artificial membrane, defined as PAMPA-skin, is able to mimic the main barrier properties of human stratum corneum and can be used for the fast prediction of passive human skin permeability coefficients.     

Assessment of fluidity of different invasomes by electron spin resonance and differential scanning calorimetry

The aim of this study was to investigate the influence of membrane-softening components (terpenes/terpene mixtures, ethanol) on fluidity of phospholipid membranes in invasomes, which contain besides phosphatidylcholine and water, also ethanol and terpenes. Also mTHPC was incorporated into invasomes in order to study its molecular interaction with phospholipids in vesicular membranes. Fluidity of bilayers was investigated by electron spin resonance (ESR) using spin labels 5- and 16-doxyl stearic acid and by differential scanning calorimetry (DSC). Addition of 1% of a single terpene/terpene mixture led to significant fluidity increase around the C16 atom of phospholipid acyl chains comprising the vesicles. However, it was not possible to differentiate between the influences of single terpenes or terpene mixtures. Incorporation of mTHPC into the bilayer of vesicles decreased fluidity near the C16 atom of acyl chains, indicating its localization in the inner hydrophobic zone of bilayers. These results are in agreement with DSC measurements, which showed that terpenes increased fluidity of bilayers, while mTHPC decreased fluidity. Thus, invasomes represent vesicles with very high membrane fluidity. However, no direct correlation between fluidity of invasomes and their penetration enhancing ability was found, indicating that besides fluidity also other phenomena might be responsible for improved skin delivery of mTHPC.     

Percutaneous Absorption Enhancement of an Ionic Molecule by Ethanol–Water Systems in Human Skin

Ethanol–water systems enhance permeation of ionic solutes through human stratum corneum. Optimum enhancement of salicylate ion permeation has been observed with ethanol volume fractions near 0.63. The mechanism of action of ethanol–water systems enhancing skin permeation was investigated by in vitro skin permeation studies combined with Fourier transform infrared spectroscopy experiments. The increased skin permeation of the ionic permeant by the ethanol–water systems may be associated with alterations involving the polar pathway. Polar pathway alterations may occur in either or both the lipid polar head and proteinaceous regions of the stratum corneum. Ion-pair formation may also contribute to increased permeation. However, the decreased permeation of salicylate ion observed at higher volume fractions of ethanol may be attributed to decreased uptake of permeant into the stratum corneum.     

Mechanism of skin penetration-enhancing effect by laurocapram.

In order to clarify the mechanism of action of laurocapram (Azone) on the skin permeation of drugs, the following experiments were done. First, the effect of Azone on the skin components was compared with that of other penetration enhancers. Azone markedly fluidized liposomal lipids (as a model lipid system) compared with other enhancers. Ethanol extracted large amounts of the stratum corneum lipids, whereas Azone did not. These results suggest that the effect of Azone on the lipids in the stratum corneum is not the same as that of ethanol. In addition, ethanol increased the amount of free sulfhydryl (SH) group of keratin in the stratum corneum, whereas Azone did not directly affect the stratum corneum protein. Azone increased water content in the stratum corneum, as measured by skin conductance. This effect might be a reason for the action of Azone. For further understanding, the enhancing effects of Azone on the skin permeation of several model compounds (alcohols, sugars, and inorganic ions) were compared with the effects of pretreatment with distilled water, which was thought to increase water-holding capacity, and pretreatment with ethanol, which was thought to affect the lipids and protein in the skin barrier (i.e., stratum corneum). Pretreatment with water or ethanol enhanced skin permeation of hydrophilic compounds, whereas they decreased that of octanol, a hydrophobic compound. The tendency of Azone to increase or decrease the skin permeation rate of most compounds was similar to that of pretreatment with water or ethanol. However, the effect of Azone on the skin permeation of inorganic ions was relatively low, whereas that of pretreatment with water or ethanol was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sesquiterpene Components of Volatile Oils as Skin Penetration Enhancers for the Hydrophilic Permeant 5-Fluorouracil

Abstract— Twelve sesquiterpene compounds, derived from natural volatile oils, were investigated as putative skin penetration enhancers for human skin. Pretreatment of epidermal membranes with sesquiterpene oils, or solid sesquiterpenes saturated in dimethyl isosorbide, increased the rate of absorption of the model hydrophilic permeant, 5-fluorouracil (5-FU). Enhancers with polar functional groups were generally more potent than pure hydrocarbons. Furthermore, enhancers with the least bunched structures were the most active. The largest effect was observed following pretreatment with nerolidol, which increased pseudo-steady-state 5-FU flux over 20-fold. Molecular modelling suggested that terpenes with structures suitable for alignment within lipid lamellae were the most potent enhancers. Sesquiterpene enhancers had long durations of action implying that they did not wash out of the skin easily. This study attempted to improve enhancer clearance by replacing the aqueous donor and receptor phases by ethanol: water (1:1) solutions. Ethanol increased the permeability coefficient for 5-FU 13-fold, demonstrating that, in aqueous solution, it is a moderately potent penetration enhancer. Sesquiterpene and ethanol enhancement effects were approximately additive. Sesquiterpene effects were almost fully maintained for at least 4·5 days following pretreatment, illustrating poor reversibility. Stratum corneum/water drug partitioning studies suggested that an important mechanism of action of the enhancers was to increase the apparent drug diffusivity in the stratum corneum. Increases in drug partitioning into the entire stratum corneum following enhancer pretreatment were relatively small. Diffusivity increases were directly related to overall rises in permeability. This study has shown that sesquiterpene compounds, which are of low toxicity and cutaneous irritancy, can promote 5-FU absorption across human skin. Sesquiterpene compounds, therefore, show promise as clinically-acceptable skin penetration enhancers.     

Measuring the Diffusional Pathlength and Area Within Membranes of Excised Human Stratum Corneum

By using lipid-free membranes it was possible to measure the extended diffusional pathlength (L*) and reduced diffusional area (A*) within samples of human stratum corneum. The value of 883 obtained for the factor L*A/LA* (A = external membrane area and L = membrane thickness) compared well with previously published theoretical values. It was also consistent with measurements of lag-time and permeability made on both lipid-free and intact stratum corneum. The geometrical barrier formed by the corneocytes was found to be quantitatively of equal importance to the extra-cellular lipid barrier for maintaining the overall barrier function of the stratum corneum.     

Promoting mechanism of menthol derivative, 1-O-ethyl-3-buthylcyclohexanol, on the percutaneous absorption of ketoprofen

Menthol derivatives were synthesized and evaluated for their promoting activity on the percutaneous absorption of ketoprofen and skin irritation in vivo, choosing O-ethylmenthol (MET) as the mother compound. The compound having a C-3 positionned n-butyl group (1-O-ethyl-3-n-buthylcyclohexanol, OEBC) indicated the most promoting activity and caused relatively little skin irritation. In order to understand enhancement mechanism of OEBC an in vitro permeation study of ketoprofen was performed. The time course of the cumulative amounts of drug permeated through the rat skin exhibited a linear relation after an initial lag time. This was analyzed in membrane diffusion model and the diffusion and partition parameters of ketoprofen were estimated. Both parameters were remarkably enhanced when a hydrogel containing a small quantity of OEBC (0.5%) was applied. Furthermore, to clarify the site of action of OEBC, we also investigated in vitro permeation study of ketoprofen employing different skins of state, reversed skin and stratum corneum stripped skin. When OEBC was added to the hydrogels which were applied to the reversed and stripped skins, almost no changes of the flux were observed compared with the control (without OEBC). These results suggested that the site of action of OEBC was stratum corneum. Morphological changes of the stratum corneum surface were microscopically observed with 0-2% OEBC. The spaces between the stratum corneum cells treated with 0.5-2% OEBC became extended and the shape of each cell became clear. This may suggest that the site of action of OEBC was the intercellular of stratum corneum. Furthermore, an electron spin resonance study was performed to investigate the effect of OEBC on the intercellular lipid bilayer fluidity of the stratum corneum and the rotational correlation times were calculated. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were used as the spin label. In use of OEBC, the fluidity of TEMPO labeled the stratum corneum lipid increased as the addition of OEBC. The results suggested that OEBC promote the penetration of drugs by enhancing fluidity of the local lipid bilayers around TEMPO.     

Amphiphilic transdermal permeation enhancers: structure-activity relationships

Transdermal drug delivery offers numerous advantages over conventional routes of administration; however, poor permeation of most drugs across the skin barrier constitutes a serious limitation of this methodology. One of the approaches used to enlarge the number of transdermally-applicable drugs uses permeation enhancers. These compounds promote drug permeation through the skin by a reversible decrease of the barrier resistance. Enhancers can act on the stratum corneum intracellular keratin, influence desmosomes, modify the intercellular lipid domains or alter the solvent nature of the stratum corneum. Even though, hundreds of substances have been identified as permeation enhancers to date, yet our understanding of the structure-activity relationships is limited. In general, enhancers can be divided into two large groups: small polar solvents, e.g. ethanol, propylene glycol, dimethylsulfoxide and amphiphilic compounds containing a polar head and a hydrophobic chain, e.g. fatty acids and alcohols, 1-dodecylazepan-2-one (Azone), 2-nonyl-1,3-dioxolane (SEPA 009), and dodecyl-2-dimethylaminopropanoate (DDAIP). In this review we have focused on structure-activity relationships of amphiphilic permeation enhancers, including the properties of the hydrophobic chains, e.g. length, unsaturation, and branching, as well as the polar heads characteristics, e.g. hydrogen bonding ability, lipophilicity, and size. We present over 180 examples of enhancers with different polar head to illustrate the structural requirements and the possible role of the polar head. We have given an overview of the methods used for investigation of the mechanisms of permeation enhancement, namely differential scanning calorimetry (DSC), infrared (IR) and Raman spectroscopy, X-ray diffraction and future perspectives in this field. Furthermore, biodegradability and chirality of the enhancers are discussed.     

Permeability of benzoic acid derivatives in excised guinea pig dorsal skin and effects of l-menthol

Penetration through excised guinea pig dorsal skin was examined for nine mono-substituents of benzoic acid derivatives. Permeability coefficients of the derivatives correlated well with their n-octanol/water partition coefficients. Since the regression coefficient was similar to the value obtained in human skin, it is suggested that the lipid lamellae of guinea pig skin resembled that of human skin in lipophilicity. Addition of penetration enhancer, 1% l-menthol in 15% ethanol markedly increased the flux and permeability coefficients of relatively hydrophilic derivatives and decreased the dependency of the permeability coefficients on the partition coefficients. Electron spin resonance analysis using 5-doxylstearic acid revealed the presence of a strongly immobilized component of the spin label in the skin and its disappearance in the presence of 1% l-menthol in 15% ethanol. These results suggest that the rigid lamellar structure of the stratum corneum was disrupted by l-menthol with ethanol, and caused the enhancement of penetration of relatively hydrophilic benzoic acid derivatives.     

Sensitivity of adipocyte basal and insulin-stimulated hexose transport to the membrane lipid structure

A series of anesthetic alcohols inhibited basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose transport in adipocytes over total alcohol concentration ranges that cause local anesthesia of rat sciatic nerve. The relative potencies of the inhibition caused by the alcohols increased in the following order: methanol less than ethanol less than propanol less than butanol less than benzyl alcohol less than hexanol less than octanol. The inhibition was reversible and correlated well with the known partitioning of the alcohols into lipids of biological membranes. Adipocyte membranes were labeled with the 5-nitroxide stearate spin probe to investigate the effects of the alcohols on the dynamic structure of membrane lipids of the adipocyte. The alcohols increased the membrane "fluidity", and the relative concentration dependence of the effects closely paralleled that noted from methanol to octanol in transport studies. Alcohols from methanol to hexanol caused inhibition of hexose transport at molar potencies comparable to that observed for membrane disordering. This suggests that hydrophobic regions of the transporter and its lipid environment are perturbed by a comparable mechanism for each alcohol. The cholesterol-complexing polyene antibiotic filipin inhibited hexose transport and influenced the mobility of lipid domains sampled with the nitroxide cholestane, cholesterol-like spin probe. The data are consistent with the concept that the membrane structural/functional effects are mediated by formation of 1:1 cholesterol:filipin complexes. Alcohols and filipin inhibited inherent transporter activity and perturbed the membrane lipid structure without dramatically diminishing transport stimulation by insulin above basal. The specific organization of membrane lipids (particularly cholesterol) may provide an essential environment for optimal transport system activity.     

Effect of ethanol/propylene glycol on the in vitro percutaneous absorption of aspirin, biophysical changes and macroscopic barrier properties of the skin.

The effect of the solvent systems ethanol (EtOH), propylene glycol (PG) and combinations thereof was examined on the in vitro percutaneous absorption of the antithrombotic, aspirin, through porcine epidermis. Biophysical changes in the stratum corneum lipids were studied through the use of Fourier transform infrared (FTIR) spectroscopy. Macroscopic barrier properties of the epidermis were examined through the use of in vitro transepidermal water loss (TEWL). The flux of aspirin increased with increasing concentrations of EtOH in the solvent systems. The maximum flux of aspirin was achieved by 80% EtOH in combination with 20% PG beyond which (i.e. 100% EtOH) there was no increase in the flux. FTIR spectroscopic study was enacted in order to determine the biophysical properties of the stratum corneum when the solvents were applied. The FTIR spectra of the stratum corneum treated with 80% EtOH/20% PG showed a maximum decrease in absorbance for the asymmetric and symmetric C&z. sbnd;H peaks, which suggests a greater loss of the lipids in the stratum corneum layers. In vitro TEWL studies allowed an investigation into the macroscopic barrier integrity properties of the stratum corneum. The TEWL results indicated that each of the solvent systems significantly enhanced (P<0.05) in vitro TEWL in comparison to the control. In conclusion, 80% EtOH/20% PG enhanced the percutaneous absorption of aspirin by perturbing the macroscopic barrier integrity of the stratum corneum and through a loss of stratum corneum lipids. Copyright     

Hydrophobic membrane interaction of etidocaine,bupivacaine and 2-chloroprocaine

Summary It has been suggested that local anesthetics may block sodium conductance through nervous membranes also by hydrophobic interaction, e.g., by expanding the membrane. Decreased anisotropy (fluidization) and depressed phase transition temperatures have been shown by relatively high local anesthetic concentrations. We studied the dose dependence of the effect of three clinically used local anesthetics, with different lipid solubility, on lipid fluidity parameters of four different model membranes. With stearic acid spin labels in dipalmitoyl lecithin vesicles etidocaine (1–5 mM) had the clearest fluidizing effect followed by bupivacaine (5 mM); 2-chloroprocaine was without effect on lipid fluidity. In synaptic plasma membranes a fluidizing effect near the hydrophilic part of the lipid bilayer was similar with etidocaine and bupivacaine (5–10 mM); 2-chloroprocaine had no effect. Bupivacaine at 125 and 250 M had a small ordering effect, which was not seen at a more hydrophobic site of the membrane. Etidocaine had the strongest fluidizing effect at the latter site of the synaptic plasma membranes. In erythrocyte ghost membranes, probed by stearic acid spin labels near the hydrophilic end, none of local anesthetics affected fluidity at 24° C, while at 37° C etidocaine (1–5 mM) and bupivacaine (5 mM) had a fluidizing effect. Dimyristoyl lecithin vesicles were probed by cis-and trans-parinaric acid. Etidocaine and bupivacaine (5–10 mM) clearly depressed the phase transition temperature evaluated from fluorescence intensity scans. The effect was most marked with bupivacaine (1–10 mM) when dis-parinaric acid was used. While isolated mammalian nerves are blocked by local anesthetic concentrations below 100 M, this study shows that the clinically used local anesthetics increase fluidity and depress phase transition temperature only at 10–100 times higher concentrations at physiological pH. This kind of hydrophobic membrane interaction may not be important for the nerve blocking effect.     

Terpenes and the Lipid–Protein–Partitioning Theory of Skin Penetration Enhancement

A series of terpenes has been assessed as skin penetration enhancers towards the model polar penetrant 5-fluorouracil (5-FU). Cyclic terpenes were selected from the chemical classes of hydrocarbons (e.g., -pinene), alcohols (e.g., -terpineol), ketones (e.g., carvone), and oxides (e.g., 1,8-cineole, ascaridole). Permeation experiments were performed on excised human epidermal membranes and the terpenes varied in their activities; -pinene only doubled the permeability coefficient of aqueous 5-FU, whereas 1,8-cineole caused a near 95-fold increase. Essential oils, e.g., chenopodium (70% ascaridole), were less effective than the corresponding isolated terpenes. 5-FU is less soluble in the terpenes than in water, and the terpenes did not exert their action by increasing partitioning of the drug into the membranes as illustrated by stratum corneum:water partitioning studies. The penetration enhancers increased drug diffusivity through the membranes, an effect which correlated empirically with the enhancer activities. The principal mode of action of these accelerants may be described by the lipid–protein–partitioning theory; the terpenes interacted with intercellular stratum corneum lipids to increase diffusivity, and the accelerant effects were not due to partitioning phenomena. Keratin interaction was assumed negligible.