Michael L. Francoeur

Evidence that oleic acid exists in a separate phase within stratum corneum lipids

Oleic acid is known to be a penetration enhancer for polar to moderately polar molecules. A mechanism related to lipid phase separation has been previously proposed by this laboratory to explain the increases in skin transport. In the studies presented here, Fourier transform infrared spectroscopy (FT-IR) was utilized to investigate whether or not oleic acid exists in a separate phase within stratum corneum (SC) lipids. Per-deuterated oleic acid was employed allowing the conformational phase behavior of the exogenously added fatty acid and the endogenous SC lipids to be monitored independently of each other. The results indicated that oleic acid exerts a significant effect on the SC lipids, lowering the lipid transition temperature (Tm) in addition to increasing the conformational freedom or flexibility of the endogenous lipid alkyl chains above their Tm. At temperatures lower than Tm, however, oleic acid did not significantly change the chain disorder of the SC lipids. Similar results were obtained with lipids isolated from the SC by chloroform:methanol extraction. Oleic acid, itself, was almost fully disordered at temperatures both above and below the endogenous lipid Tm in the intact SC and extracted lipid samples. This finding suggested that oleic acid does exist as a liquid within the SC lipids. The coexistence of fluid oleic acid and ordered SC lipids, at physiological temperatures, is consistent with the previously proposed phase-separation transport mechanism for enhanced diffusion. In this mechanism, the enhanced transport of polar molecules across the SC can be explained by the formation of permeable interfacial defects within the SC lipid bilayers which effectively decrease either the diffusional path length or the resistance, without necessarily invoking the formation of frank pores.

Oleic Acid: Its Effects on Stratum Corneum in Relation to (Trans)Dermal Drug Delivery

Calorimetric studies with porcine stratum corneum (SC) have shown that the lipid phase transitions associated with the intercellular bilayers are markedly affected by treatment with oleic acid. Specifically, the transition temperatures (Tm) and cooperativity are reduced, whereas no effect was observed on the endotherm associated with keratin denaturation, suggesting that oleic acid primarily affects the SC lipids. The decrease in the lipid-associated Tms was further correlated with the amount of oleic acid taken up by the SC. Parallel experiments with silastic implied that the uptake is dependent on the thermodynamic activity of oleic acid in the vehicle itself. The in vitro transport of Piroxicam across human and hairless mouse skin (HMS) was significantly enhanced by oleic acid, as a function of the extent of oleic acid uptake, with an attendant change in Tm. These results emphasize the role of SC lipids in percutaneous absorption. Transport also depended on the donor concentration of ionized drug suggesting that the enhanced transport mechanism cannot be accounted for solely on the principles of the classical pH-partition hypothesis. Accordingly, a model of skin permeability enhancement involving solid-fluid phase separation within the SC lipids is proposed for oleic acid, consistent with the existing phospholipid literature. In conjunction with the use of oleic acid as an enhancer, very soluble hydrophilic salts were recognized as key factors in attaining maximum delivery. Oleic acid uptake, lipid ΔTm, and enhanced drug flux were all found to correlate, exhibiting a bell-shaped curve as a function of the ethanol vehicle concentration. Therefore, uptake and/or DSC experiments are useful for formulating enhanced topical delivery systems.

Polymorphism in stratum corneum lipids

Fourier transform infrared spectroscopy (FTIR) was employed to investigate the thermotropic phase behavior of stratum corneum lipid multilamellae. Stratum corneum (SC), the uppermost layer of mammalian skin, is unusual in many respects. It has been demonstrated that the lipids of the stratum corneum provide the primary electrical and transport resistance in the skin. These lipids are unusual in their composition, structure and localization; they contain only cholesterol, fatty acids and ceramides and they form broad, multi-lamellar sheets which are located extracellularly. The FTIR results from both the symmetric CH2 stretching and the CH2 scissoring vibrations suggest that the SC lipids exhibit polymorphic phase behavior below the main phase transition temperature. The multiple phases are most likely crystalline mixtures of different alkyl chain packings, along with solid-liquid phases. Similarities between the FTIR results reported here for SC lipids and those obtained for cholesterol-containing gel phase phospholipids suggest that the non-uniform distribution of cholesterol occurs in each system.

Enhanced percutaneous absorption via iontophoresis I. Evaluation of an in vitro system and transport of model compounds

Abstract In vitro methodology was developed to investigate the iontophoretic transport of select ionic and non-ionic compounds across hairless mouse skin. Using sodium benzoate and a constant current of 0.1 mA, it was observed that alterations in the diffusion cell configuration and/or return electrode placement relative to the membrane had little effect on the transport of benzoate ions, thus permitting the use of a simple experimental design. Increases in applied current (from 0.0 to 0.2 mA) produced a linear increase in observed benzoate flux. The steady-state flux was also slightly increased (apparently linearly) with greater donor concentrations, but was reduced when competitive ions (NaCl) were added to the donor chamber. Employing a direct current of 0.1 mA and identical solutions, the iontophoretic flux enhancement ratios (flux with current/flux without current) were calculated for benzoate (22.61) and the phenethylamine cation (43.32, using reversed electrode polarity). The flux of a non-ionic compound (benzyl alcohol) was not significantly altered during the application of a 0.1 mA direct current. Upon termination of the current in benzoate iontophoresis experiments (0.1 mA × 3 h), subsequent fluxes were observed to be quite inconsistent. Many yielded values fairly close to the average control (no current exposure) flux for the benzoate ion. However, several of the residual fluxes were nearly 10-fold higher than the control, suggesting compromised skin barrier integrity of a variable nature. This occasional alteration in membrane transport resistance was not, however, observed in experiments performed with benzyl alcohol. It is speculated that the diffusional path followed specifically by ionized species undergoes sporadic current-related changes, and that the flux of the uncharged benzyl alcohol is not affected by alterations in this path. These results suggest that iontophoresis may be a convenient means by which to achieve constant and readily controllable transdermal delivery, locally or to the systemic circulation, for ionized drug species (including peptides). Transport rates may be optimized by adjustment of donor ionic composition and utilization of current densities and patterns deemed physiologically appropriate.

Local Enhanced Topical Delivery (LETD) of Drugs: Does It Truly Exist?

There is considerable uncertainty over whether and to what extent topically applied drugs can be delivered directly to anatomical sites beneath the skin, without prior entry into the systemic blood circulation. The in vivo studies reported in this work were designed to assess whether local enhanced topical delivery (LETD) can be achieved with piroxicam, a nonsteroidal antiinflammatory drug. Equivalent doses of tritium-labeled drug were administered by the i.v. or topical routes to male rats. The topical plasma profile reveals a maximum concentration (Cpmax) at 12 hr, compared to a typical, multiexponential decline in plasma concentration after i.v. dosing. All four muscles from the topically dosed shoulder exhibit two distinct peaks, the first at 4 hr and a later one at 12 hr (which coincides with the topical Cpmax). The contralateral muscles from the non-dosed shoulder, in contrast, produce only a single peak at 12 hr after topical dosing. After the i.v. administration of piroxicam, the concentration-time profiles for each muscle closely parallel that seen for the i.v. plasma. Tissue-to-plasma ratios (T/P) show that the topical nondosed and the i.v. muscles are nearly constant over the entire time course of this study, indicating a pseudo-equilibrium between the plasma and those muscles. However, the early T/P ratios for the topically dosed muscles are markedly elevated and gradually decline to a constant value only after 12 hr, indicating that a similar pseudo-equilibrium is not established in this case. Thus, these results strongly imply that the topical administration of a drug can lead to LETD for tissues subjacent to the skin. Further, based on the elevated T/P ratios, these local enhanced drug levels cannot be solely attributed to entry from the systemic blood and suggest summarily that the cutaneous microvasculature is simply not an “infinite sink” for removal of all topically applied drugs.

Mechanism and enhancement of solute transport across the stratum corneum

Abstract This paper summarizes recent biophysical investigations of (a) stratum corneum (SC) barrier function, and (b) percutaneous penetration enhancement. Specifically, applications of differential scanning calorimetry (DSC) and infrared spectroscopy (IR) to probe the intercellular lipid domains of the SC are described. In vitro DSC experiments on isolated SC have determined the thermal melting behavior of the membrane and have indicated the presence of lipid phase transitions in the range 65–85 °C. Corresponding IR studies have confirmed this observation and, in conjunction with measurements of tritiated water permeability (Kp), have shown that solute flux increases with the number of gauche conformers along the lipid acyl hydrocarbon chains. There is an excellent correlation between Kp and the absorbance shift (to higher wavenumber) of the C-H antisymmetric stretching vibration associated with the SC lipids. Furthermore, it was found that certain putative penetration enhancers (e.g., cis-unsaturated fatty acids, such as oleic and vaccenic acids) induced similar shifts when applied to excised SC at ambient temperature. Concomitantly, the flux of another model solute (salicylic acid) was significantly enhanced by the treatment. The implied mechanism of penetration promotion (i.e., through the overall increased freedom of motion of the lipid acyl chains) was then examined in vivo, in humans, using attenuated total reflectance IR. Again, significant and sustained lipid disordering was induced by (in this case) oleic acid. In addition, through the assignment of unique absorbances, it was possible to obtain semi-quantitative measurements of (1) the enhancer in the upper layers of the SC, (2) the co-applied penetrant (4-cyanophenol, unequivocally identified by the intense C  N absorbance) and (3) the major vehicle component used (namely, propylene glycol, via the C-O stretching vibration). In this way, we have (i) documented the effect of the enhancer on the SC lipids, (ii) assessed the relative amount of the enhancer responsible for this action, and (iii) observed the kinetics of penetrant (solute) and solvent transport (in the presence and absence of enhancer) through the SC, from a single series of spectroscopic experiments in vivo in man. We suggest, therefore, that biophysical measurements of this type have considerable potential, often in clinically relevant situations, to reveal the crucial details of the mechanism and enhancement of solute transport across the SC.

Topical Penetration of Piroxicam Is Dependent on the Distribution of the Local Cutaneous Vasculature

The mechanism of the topical delivery of piroxicam, a nonsteroidal antiinflammatory drug, has been controversial as to whether systemic absorption is required for topical efficacy. This study, using in vivo pigs treated with topical 3H-piroxicam gel, was designed to assess the role of systemic absorption on its delivery to deep tissues. Further, the role of the structure of the cutaneous vasculature (e.g., direct cutaneous or musculocutaneous) was studied. Finally, piroxicam delivery was measured using in vitro diffusion cells with pig skin obtained from the same sites to determine inherent permeability independent of vascular anatomy. These studies showed that penetration of the radiolabel occurred in subcutaneous and muscle tissue only under the dosed sites and not at the remote sites, ruling out systemic absorption as a prerequisite for local delivery. Tissue penetration in vivo was enhanced at the musculocutaneous compared to the direct cutaneous sites. In contrast, in vitro flux was identical in skin harvested from the two vascular sites, suggesting that the vasculature plays a pivotal role in deep tissue penetration of piroxicam. In conclusion, local delivery of topical drugs occurs independent of systemic absorption and the nature of the cutaneous vasculature at different sites must be taken into consideration for optimal delivery.

Iontophoretic delivery of piroxicam across the skin in vitro

Iontophoresis of the non-steroidal anti-inflammatory drug, piroxicam, across hairless mouse skin in vitro has been investigated. The following drug delivery issues have been examined: (a) to what extent is the percutaneous absorption of the drug enhanced by iontophoresis; (b) how do current density, current duration, and drug formulation variables affect the efficiency of delivery; and (c) does the combination of iontophoresis with a chemical penetration enhancer (oleic acid) elicit additive or synergistic transport-promoting effects? Cathodal iontophoresis of negatively-charged piroxicam was carried out using three pH 7.4 formulations (a solution, a gel, and a gel containing 0.3% w/w oleic acid). From the solution, following a 6-h application period, iontophoresis at constant current densities in the range 0.07–0.50 mA/cm2 delivered 100–1000 times more drug than passive diffusion. Penetration of intact piroxicam was directly proportional to current density. At the highest current density, iontophoretic flow of other ions in the system (especially Na+ and Cl−) caused the piroxicam flux to peak and then gradually fall off with time. Short-duration current application (30 min) also increased the 6-h delivery of drug significantly (more than 10 times the no-current control at 0.07 mA/cm2,150-fold at 0.50 mA/cm2 ). Piroxicam transport from a simple gel was not as well correlated with current density as that from the solution. A lower drug concentration and altered ionic transport numbers are believed to be the reason for this behavior. Nevertheless, enhancement over passive diffusion (following iontophoresis for both 0.5 and 6 h) was substantial. When the gel containing oleic acid was used, drug delivery at the lowest current density was reduced relative to the gel with no enhancer. At higher current densities, on the other hand, piroxicam transport is augmented by oleic acid. This change in the relative delivery could be a consequence of oleate ion competition for transport dominating at low current density, with the enhancement effects taking over at higher current. This conclusion was consistent with pretreatment experiments in which the skin was exposed to oleic acid for 3 h prior to iontophoretic piroxicam delivery from the simple gel without oleic acid. The degree of drug delivery was significantly greater than the sum of those achieved by pretreatment alone and by iontophoresis alone.

Strategies to Enhance Permeability via Stratum Corneum Lipid Pathways

Publisher Summary Stratum corneum (SC) lipids, like those of other biomembranes, can be studied by a variety of biophysical techniques. Methods that have been used to investigate lipid membrane biophysics include X-ray diffraction, differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), electron spin resonance (ESR), and infrared (IR) spectroscopy. This chapter discusses the techniques of IR spectroscopy and DSC. These techniques have been particularly useful in establishing the phase behavior of a number of lipid membrane systems. The SC lipid pathway is important to the permeability of water and numerous drugs. Despite differences in lipid composition and distribution, the mechanisms of molecular permeation through the SC and other biomembranes are similar. The technique of Fourier transform IR (FTIR) spectroscopy has been extensively used to study the phase behavior of lipid membranes. The lipids of the SC can also be studied by FTIR techniques.

Routes of ionic permeability through mammalian skin

Abstract The skins highly resistive nature to the transport of ions and neutral compounds is primarily due to the lipids of the stratum corneum. The skin also contains a number of appendageal structures such as hair follicles and sweat ducts which can serve as shunt pathways for ion transport. Evidence is presented to suggest that ion transport can occur via the extracellular lipid domains of the stratum corneum through interfacial defects present at sites of lipid phase separation. Moreover, a similar mechanism of transport has been hypothesized for other lipid membranes.