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Biomedical subjects

R O Potts

Publications and source records attributed to R O Potts.

At least 19 recordsLinked to original sources
PubMed1992

Secondary structural analysis of two recombinant murine proteins, interleukins 1 alpha and 1 beta: is infrared spectroscopy sufficient to assign structure?

The secondary structure for two murine recombinant proteins, interleukins 1 alpha and 1 beta (rmIL-1 alpha and -1 beta), has been analyzed by Fourier transform infrared (IR) spectroscopy and then compared to results obtained by X-ray diffraction, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. The IR results obtained here for rmIL-1 alpha and -1 beta suggested that their secondary structures consisted predominantly of beta-sheets or strands. However, the analysis also revealed a significant absorption band near 1656 cm-1, which is typically assigned to alpha-helical or random structures. When these same murine polypeptides were analyzed by CD, no evidence of alpha-helical structures was observed. Further, published X-ray diffraction and NMR studies characterizing the human forms of IL-1 alpha and -1 beta indicate the absence of alpha-helices and that the human proteins are composed mainly of beta-strands (i.e., greater than 55%), with approximately 24% of the amino acids involved in large loops connecting the strands. The murine IL-1 proteins, when compared to their respective human counterparts, each show greater than 80% sequence homology. Given this fact, the CD analyses, and the result that this IR band amounted to 21% of the overall integrated area, the absorption peak at 1656 cm-1 was attributed to the presence of large loops rather than to alpha-helical or random structures. Such a structural assignment appears reasonable and is totally consistent with the established existence of large loops in the human forms as well as in other proteins found to fold similarly (viz., human bFGF).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Predicting skin permeability.

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 physiocochemical 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 greater than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1992

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 nondosed 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗
PubMed1992

Physical methods for studying stratum corneum lipids.

The skin's barrier to water loss and drug transport is closely related to the biophysical properties of the stratum corneum lipids. A number of in vitro and in vivo methods have been used to evaluate the physical properties of stratum corneum lipids. Two particularly useful techniques, infrared spectroscopy and differential scanning calorimetry, have provided a wealth of information about stratum corneum structure and function. The results of these studies demonstrate that the alteration of stratum corneum lipid packing results in increased skin permeability. Furthermore, the magnitude of increased permeability can be predicted from these physical properties. Therefore, these results provide both insight into the mechanism of transport through the stratum corneum, as well as a useful paradigm for selection of agents that reversibly alter the permeability of the skin.

Animals↗
PubMed1991

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Barrier function of human keratinocyte cultures grown at the air-liquid interface.

Stratum corneum (SC), the outermost and least permeable layer of skin, is the major barrier to passive transepidermal water loss. In the research described in this paper, we have used human keratinocyte cultures, grown at the air-liquid (A/L) interface, to examine the relationship between epidermal differentiation (including SC formation) and barrier function. Histologically, the A/L culture showed several markers of complete differentiation, including the presence of well-organized and defined epidermal cell layers, keratohyalin granules, and a multilayered SC. The permeability of tritiated water through epidermal cultures, which had grown for 3 weeks at the A/L interface, was measured with a microdiffusion apparatus. The results of these experiments demonstrated that: a) the human keratinocyte cultures developed a substantial barrier (i.e., a multilayered SC) to water diffusion across the entire surface. If the relative humidity of the culturing environment was lowered from 100% to around 75%, the barrier was significantly improved; b) the differentiation promoter, 1.25-dihydroxy-vitamin-D3, increased the number of SC layers and reduced water permeation through the culture; c) the nature of the keratinocyte support matrix could be altered to improve the morphology as well as the barrier function of the epidermal cultures. Overall, the observations are consistent with the relationship that is believed to exist between SC intercellular lipid content and percutaneous penetration. Confirmation of this hypothesis will further the considerable potential of human keratinocyte A/L cultures as a valuable and relevant model in which to study drug absorption and metabolism.

Air↗
PubMed1991

The influence of stratum corneum morphology on water permeability.

The stratum corneum (SC) provides the barrier to water loss for the skin of mammals. A significant body of evidence now exists suggesting that extracellular SC lipids are primarily responsible for this barrier. We have measured the permeability (P) and lag-time (Tlag) for water vapor transport through the SC and found that P is about 1000 times less than the values obtained for most other lipid membranes. In addition, we have measured the water partitioning into the lipid microdomain of the SC using a differential scanning calorimetry technique. These combined data provide an estimate of the diffusion coefficient (D) and diffusion pathlength (delta). The results show that the intrinsic diffusion of water is comparable to values obtained with other lipid membranes. The value obtained for delta, however, is fiftyfold greater than the sample thickness. These results are interpreted in terms of the unique morphology of the SC, where lipids form an extracellular continuum that is highly tortuous. Thus, the exceedingly low permeability of the SC may be due, in large part, to its unique morphology.

Animals↗
PubMed1990

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 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.

Administration, Cutaneous↗
PubMed1990

Percutaneous penetration enhancement in vivo measured by attenuated total reflectance infrared spectroscopy.

A novel application of attenuated total reflectance IR spectroscopy (ATR-IR) was used to monitor the outer several microns of the stratum corneum (SC) and, thereby, demonstrate enhanced percutaneous absorption in vivo in man. 4-Cyanophenol (CP) as a model permeant yielded a unique IR signal, distinct from those of the stratum corneum and the vehicle components. CP was administered for 1, 2, or 3 hr as a 10% (w/v) solution either in propylene glycol or in propylene glycol containing 5% (v/v) oleic acid. The absorbance at 2230 cm-1, which corresponded to C identical to N bond stretching, diminished significantly faster when CP was codelivered with oleic acid. An IR absorbance due primarily to propylene glycol at 1040 cm-1 (C-O stretching) also disappeared more quickly following application of the enhancer-containing solution. In addition, only the formulations with oleic acid induced a higher wavenumber shift in the frequency of the asymmetric C-H bond stretching absorbance. This change indicates increased lipid-chain disorder, the mechanism by which oleic acid is believed to cause enhanced drug transport across the stratum corneum. Therefore, ATR-IR permits one to examine noninvasively the kinetics, extent, and mechanism of percutaneous penetration enhancement in vivo in human subjects.

Adult↗
PubMed1990

Lipid biophysics of water loss through the skin.

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.

Animals↗
PubMed1990

Examination of stratum corneum barrier function in vivo by infrared spectroscopy.

It is generally accepted that the stratum corneum (SC) is the least permeable layer of the epidermis. Histologically, though, the SC is a non-uniform, inhomogeneous membrane, and the question "Is barrier function distributed uniformly across the SC thickness?" has been posed. To address this issue, human ventral forearm SC has been studied in vivo by attenuated-total-reflectance Fourier-transform infrared spectroscopy during the course of sequential tape-stripping. Because the intercellular lipids of the SC and the degree of hydration of the membrane have been shown to be crucial determinants of barrier function, attention has been focused on the spectral features, which report specifically on these parameters. The degree of disorder of the SC intercellular lipids has been found to decrease over the outer cell layers (up to three tape-strips) and then to remain essentially constant. The amount of lipids decreases similarly such that a 60% reduction (relative to the "no-strip" baseline) is observed after about four tape-strips. A plausible explanation for these measurements is that the lipids near the surface are a mixture of (a) "true" intercellular lipid (which is expected to be highly ordered), and (b) sebaceous lipid (which contains much greater amounts of low-melting components, such as fatty acids). The sequential infrared (IR) spectra provide at least circumstantial evidence to support this hypothesis. As expected, the IR spectra show that SC hydration increases from the surface towards the SC-stratum granulosum interface. Taken together, the results imply that the SC is indeed non-uniform. The properties of the outer layers (those removed by the first 3-4 tape-strips) change significantly with increasing depth.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Cells↗
PubMed1989

Transdermal drug transport and metabolism. I. Comparison of in vitro and in vivo results.

Using excised human skin and tissue grafted to athymic mice, the in vitro and in vivo delivery and metabolism of a salicylate diester were compared. Concentration profiles of this drug and its metabolites were obtained for the outer several hundred microns of the skin. These results show significant differences in the extent of enzymatic cleavage and distribution of metabolites between in vitro and in vivo studies. Furthermore, these data suggest that in vitro results may overestimate metabolism because of increased enzymatic activity and/or decreased capillary removal.

Administration, Cutaneous↗
PubMed1989

Transdermal drug transport and metabolism. II. The role of competing kinetic events.

The steady-state flux and skin tissue distribution of a topically applied diester of salicylic acid was measured in vitro in the presence and absence of an esterase inhibitor. When compared with data obtained previously under in vivo conditions, the results presented here provide insight into the role of competing diffusional and hydrolytic events in the delivery and distribution of topically applied drugs. Furthermore, these results, when combined with a theoretical analysis of topical drug delivery and metabolism, suggest that the differences in delivery and drug distribution seen under in vitro conditions are related to both altered drug removal and altered hydrolysis rates.

Administration, Cutaneous↗