Fourier transform infrartd (FTIR) spectroscopy is an established tool for the structural character- ization of proteins. However, many potential pitfalls exist for the unwary investigator. In this review we critically assess the application of FIlR spectroscopy to the determination of protein structure by (1) outlining the principles underlying protein secondary structure determination by FZZR spectroscopy. (2) highhghting the situations in which FZZR spectroscopy should be considered the technique of choice, (3) discussing the manner in which experiments should be conducted to derive as much physiologically relevant information as possible, and (4) outlining current methods for the determination of secondary structure from infrared spectm of proteins,

The Use and Misuse of FTIR Spectroscopy in the Determination of Protein Structure

Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment.

Skin penetration enhancers.

Published permeability coefficient (Kp) data for the transport of a large group of compounds through mammalian epidermis were analyzed by a simple model based upon permeant size [molecular volume (MV) or molecular weight (MW)] and octanol/water partition coefficient (Koct). The analysis presented is a facile means to predict the percutaneous flux of pharmacological and toxic compounds solely on the basis of their physicochemical properties. Furthermore, the derived parameters of the model have assignable biophysical significance, and they provide insight into the mechanism of molecular transport through the stratum corneum (SC). For the very diverse group of chemicals considered, the results demonstrate that SC intercellular lipid properties alone are sufficient to account for the dependence of Kp upon MV (or MW) and Koct. It is found that the existence of an “aqueous-polar (pore) pathway” across the SC is not necessary to explain the Kp values of small, polar nonelectrolytes. Rather, their small size, and consequently high diffusivity, accounts for their apparently larger-than-expected Kp. Finally, despite the size and breadth of the data set (more than 90 compounds with MW ranging from 18 to >750, and log Koct ranging from −3 to + 6), the postulated upper limiting value of Kp for permeants of very high lipophilicity cannot be determined. However, the analysis is able to define the physicochemical characteristics of molecules which should exhibit these maximal Kp values. Overall, then, we present a facile interpretation of a considerable body of skin permeability measurements that (a) very adequately describes the dependence of Kp upon permeant size and lipophilicity, (b) generates parameters of considerable physicochemical and mechanistic relevance, and (c) implies that the SC lipids alone can fully characterize the barrier properties of mammalian skin.

Predicting skin permeability.

The past twenty five years have seen an explosion in the creation and discovery of new medicinal agents. Related innovations in drug delivery systems have not only enabled the successful implementation of many of these novel pharmaceuticals, but have also permitted the development of new medical treatments with existing drugs. The creation of transdermal delivery systems has been one of the most important of these innovations, offering a number of advantages over the oral route. In this article, we discuss the already significant impact this field has made on the administration of various pharmaceuticals; explore limitations of the current technology; and discuss methods under exploration for overcoming these limitations and the challenges ahead.

Current status and future potential of transdermal drug delivery

The influence of stratum corneum morphology on water permeability

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 (T
m) in addition to increasing the conformational freedom or flexibility of the endogenous lipid alkyl chains above their T
m. At temperatures lower than T
m, 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 T
m 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.

Evidence that Oleic Acid Exists in a Separate Phase Within Stratum Corneum Lipids

The regulation of water loss through the skin is a poorly understood but crucial process in maintaining terrestrial life-forms. In mammalian skin, the outermost layer, called the stratum corneum (SC), is rate-limiting to water loss. We have evaluated temperature-dependent changes in water vapor permeability and infrared spectra of porcine SC. In particular, we have analyzed the infrared absorption peaks due to the extracellular lipids of the SC. These results show a remarkable correlation between water permeability and the frequency of the C-H stretching vibrations over a broad range of temperature. Since the spectral changes reflect an increased number of alkyl gauche conformers, these results suggest that water permeability is dependent upon the hydrocarbon-chain disorder of SC lipids.

https://www.pnas.org/content/pnas/87/10/3871.full.pdf

Lipid biophysics of water loss through the skin

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 Tm's 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 delta Tm, 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.

Michael L. Francoeur

Papers

The influence of stratum corneum morphology on water permeability

Evidence that Oleic Acid Exists in a Separate Phase Within Stratum Corneum Lipids

Lipid biophysics of water loss through the skin

Oleic acid: its effects on stratum corneum in relation to (trans)dermal drug delivery.

Characterization of low-temperature (i.e., <65°C) lipid transitions in human stratum corneum