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Penetration enhancers are chemicals that temporarily and reversibly diminish the barrier function of the outermost layer of skin, the stratum corneum, to facilitate drug delivery to and through the tissue. In the current study, the complex mechanisms by which 1,8-cineole, a potent terpene penetration enhancer, disrupts the stratum corneum barrier is investigated using post-mortem skin samples. In order to validate the use of excised tissue for these and related studies, a fibre optical probe coupled to an FT-Raman spectrometer compared spectroscopic information for human skin recorded from in vivo and in vitro sampling arrangements. Spectra from full-thickness (epidermis and dermis) post-mortem skin samples presented to the spectrometer with minimal sample preparation (cold acetone rinse) were compared with the in vivo system (the forearms of human volunteers). No significant differences in the Raman spectra between the in vivo and in vitro samples were observed, endorsing the use of post-mortem or surgical samples for this investigational work. Treating post-mortem samples with the penetration enhancer revealed some unexpected findings: while evidence for enhancer-induced disruption of the barrier lipid packing in the stratum corneum was detected in some samples, spectra from other samples revealed an increase in lipid order on treatment with the permeation promoter. These findings are consistent with phase-separation of the enhancer within the barrier lipid domains as opposed to homogeneous disruption of the lipid lamellae. Copyright (C) 2006 John Wiley & Sons, Ltd.

Incorporating edge activators (surfactants) into liposomes was shown previously to improve estradiol vesicular skin delivery; this phenomenon was concentration dependent with low or high concentrations being less effective. Replacing surfactants with limonene produced similar behaviour, but oleic acid effects were linear with concentration up to 16% (w/w), beyond which it was incompatible with the phospholipid. This present study thus employed high sensitivity differential scanning calorimetry to probe interactions of additives with dipalmitoylphosphatidylcholine (DPPC) membranes to explain such results. Cholesterol was included as an example of a membrane stabiliser that removed the DPPC pre-transition and produced vesicles with a higher transition temperature (T(m)). Surfactants also removed the lipid pre-transition but reduced T(m) and co-operativity of the main peak. At higher concentrations, surfactants also formed new species, possibly mixed micelles with a lower T(m). The formation of mixed micelles may explain reduced skin delivery from liposomes containing high concentrations of surfactants. Limonene did not remove the pre-transition but reduced T(m) and co-operativity of the main peak, apparently forming new species at high concentrations, again correlating with vesicular delivery of estradiol. Oleic acid obliterated the pre-transition. The T(m) and the co-operativity of the main peak were reduced with oleic acid concentrations up to 33.2mol%, above which there was no further change. At higher concentrations, phase separation was evident, confirming previous skin transport findings.

The role of hair follicles in transdermal delivery remains difficult to elucidate due partly to animal model complications. This paper explores a novel technique employing two human skin membranes to differentiate shunt route delivery from bulk transepidermal input. The method monitors penetration through epidermal membranes and compares this with delivery through a sandwich of stratum corneum and epidermis, with the corneum forming a top membrane. As orifices of shunts occupy only 0.1% of the area, there is negligible chance that shunts in the membranes will superimpose. The top layer blocks shunts available in the bottom layer. If shunts are important, delivery through sandwiches will be much reduced compared with that through epidermis, allowing for increased double membrane thickness. Experiments with penetrants under passive, iontophoretic and electroporation conditions illustrated the value of the method. A Monte Carlo simulation suggested that any failure of membrane adherence would not affect conclusions drawn.

5-Fluorouracil permeates the stratum corneum through the intercellular pathway. 5-Fluorouracil is hydrophilic and, therefore, its partitioning from the aqueous region into the hydrocarbon interior of stratum corneum lipids is expected to be an important stage of its permeation and a target for some permeation enhancers. It has also been reported that complexation plays a role in the enhancement effect of some accelerants. These mechanisms have been investigated. For partitioning-permeation studies, isooctane was chosen as a model of the hydrocarbon interior of stratum corneum lipid bilayers and the effects of 26 different terpene enhancers on the solubility of 5-fluorouracil in isooctane were measured. Results were then compared with the effects of the same enhancers on the permeation of 5-fluorouracil through the epidermis in man. The stoichiometry of interaction of cineole and limonene with 5-fluorouracil were also studied to reveal possible complex formation. Solubility studies revealed good correlation between solubility and enhancement ratios for the majority of terpenes, indicating that one mechanism by which terpenes increase permeation of the stratum corneum by 5-fluorouracil is by improvement of partitioning. Stoichiometry studies showed that cineole can form 1:1 or higher complexes with 5-fluorouracil. With limonene, only a weak 1:1 complex was indicated. Data obtained using epidermis from man show that the enhancement effect of cineole toward 5-fluorouracil is much higher than that of limonene. These data reveal that terpenes might increase the permeation of 5-fluorouracil through the stratum corneum as a result of complex formation and a form of facilitated transport.

Thermal transitions in desiccated stratum corneum membranes of neonatal rats, mice and rabbits and adult abdominal human skin were investigated using differential scanning calorimetry (DSC). Four endothermic transitions at 39–45 (T1), 55–58 (Tx), 68–74 (T2) and 77–86°C (T3) were observed. T1, T2 and T3 were attributed to phase changes in the intercellular lipid bilayers. Tx was a weak transition, the exact nature of which is unknown. A fifth transition at 48°C (Ty) was only observed with neonatal rabbit stratum corneum and was attributed to lipid melting. The DSC results indicated that the temperatures of lipid transitions of human and animal tissues were different although the patterns of their thermograms were similar. The temperatures of T2 and T3 of human stratum corneum were higher than those of the animals. DSC of neonatal rabbit stratum corneum showed a lipid transition not seen in stratum corneum of human or other animal species.

The molecular basis of lipid packing in human stratum corneum and a model phospholipid system has been studied as a function of temperature using Fourier Transform (FT) Raman spectroscopy. Thermally induced molecular rearrangements of the model lipid system, dipalmitoylphosphatidyl choline (DPPC), and stratum corneum were investigated using FT Raman spectroscopy coupled to a heating chamber. Spectra were recorded for a range of sample temperatures and the results for the two systems were compared, producing previously unreported information of the thermal behaviour for the different systems. Discrete thermal events were recorded for both systems by plotting band separation of the lipid v(CH2) symmetric and asymmetric stretching modes against temperature. The main thermal events observed for DPPC included a 'pre-melting' between 37 and 39 degrees C, the main transition observed between 41 and 42 degrees C, a 'post-transition' between 42 and 43 degrees C and three minor transitions at 58-60, 65-70 and 75-80 degrees C. No evidence was found for the pre-transition of DPPC, previously observed at 34-35 degrees C. The main transitions for human stratum corneum were observed at 35-45, 55, 72 and 83 degrees C, measured from lipid CH2 stretching and bending vibrations. The keratin thermal transition at about 100 degrees C exerted little effect on the lipid bands and no characterisable structural changes were reflected in the keratotic bands.

Raman spectroscopy has been used in a variety of biomedical applications including normal and diseased human tissues, single cells, implants and the presence of foreign inclusions following implantation and the interaction of certain chemical agents with tissues. This paper surveys recent advances in biomedical Raman spectroscopy. © 1997 by John Wiley & Sons, Ltd.

One approach to transdermal permeation enhancement investigated in this paper is to increase the thermodynamic activity of a drug in the transdermal delivery system to supersaturation levels. Saturated solubilities of the model drug oestradiol (OE) in different concentrations of propylene glycol/water (PG/W) co-solvent systems were determined. Utilising the saturated solubility curve, a series of OE supersaturated solutions at different multiples of saturation in PG/W systems was prepared using a mixed co-solvents method. Antinucleating polymers including hydroxypropylmethylcelluose (HPMC), hydroxypropylcelluose (HPC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP) and polyethylene glycols, 4000 and 8000 (PEG 4000 and PEG 8000), were used to stabilise the supersaturated solutions. All but the PEGs were able to retard the crystallisation of OE and polyvinylpyrrolidone (MW 40 000) was the most efficient. Mass transport characteristics of OE from saturated and supersaturated solutions through human skin and silastic membranes were then examined using an automated diffusion apparatus. OE flux was enhanced from saturated solutions with increased PG concentration. This increase was attributed to increased OE solubility in the membrane. Also, uptake of OE by human stratum corneum and silastic membrane from supersaturated solution was determined. The results were compared with those from saturated solutions having the same PG/W co-solvent compositions. Generally, both the uptake and flux of OE through both human skin and silastic membrane increased with increasing degree of saturation. Stratum corneum showed an 18.8 ± 4.88 times increase in uptake from a supersaturated solution of 18 times saturation. OE flux through human skin from a solution of 18 times saturation increased about 13-fold during the 12 h experiment when compared with the flux from saturated solution.

Recent investigations have shown that human skin permeabilities may not always follow a Gaussian-normal distribution as usually assumed. Permeability coefficients (Kp) for the model hydrophilic permeant, 5-fluorouracil (5-FU), and the model lipophilic permeant, estradiol (ES), were demonstrated to follow more closely log-normal distributions. The present study has evaluated the effect of terpene penetration enhancer treatment on the distribution of 5-FU and ES permeabilities in human skin. Control permeability coefficients (Kpe), enhanced permeability coefficients (Kpe) and enhancement ratios (ER) were tested for both Gaussian-normality and log-normality. Kpe and ER data, obtained from 5-FU experiments employing 22 different terpene enhancer formulations and one vehicle control, were pooled using standardised values, to obtain 188 replicate measurements for statistical analysis. Similarly, Kpe and ER data, collected from ES experiments using 10 different terpene treatments and two vehicle controls, were pooled to obtain 69 replicate measurements. Statistical analysis revealed that Kp, Kpe and ER data for 5-FU tended to follow a log-normal distribution. In contrast, the Kp, Kpe, and ER data for ES followed, more closely, a Gaussian-normal distribution. Furthermore, the variability of the ES data was smaller in comparison with 5-FU data. It is possible that differences in data distribution may be linked to the physico-chemical properties of 5-FU and ES. The results of this study suggest that, when control permeabilities follow a log-normal distribution, as was the situation for 5-FU, it is likely that enhancer treatment will produce permeabilities and enhancement factors which are also log-normally distributed. In these instances, all data are most accurately represented by geometric means and geometric standard errors.

Raman spectra have been obtained from normal, healthy human stratum corneum, callus tissue and hyperkeratotic psoriatic plaques. Assignments of the spectra have been made and changes in the 1000–1500 and 2800–3100 cm−1 wavenumber regions have been related to lipid loss in the abnormal tissues but the keratin component is structurally unaltered. The potential of the FT-Raman spectroscopic technique for diagnosis of hyperkeratotic conditions in human tissue is assessed.

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.

The activation energies for ion movement and 5-fluorouracil diffusion across human epidermis in vitro have been measured to be 4.08 ± 0.16 and 20.6 ± 1.56 (mean ± SE) kcal mol−1, respectively. These data imply that ions travel across human epiderm largely through aqueous shunt routes and that 5-fluorouracil, a polar non-electrolyte, travels mainly through the intercellular lipid bilayers in the stratum corneum. Treatment of human epidermis with terpene penetration enhancers has been shown to increase electrical conductivity. The increase in ion transport suggests that terpenes open new polar pathways across the stratum corneum. A correlation between increases in ion transport and previously reported increases in 5-fluorouracil penetration suggests that terpene enhancers may create micro-pores in the intercellular lipids through which both ions and polar drugs may pass.

Synopsis Skin penetration enhancers reversibly decrease the barrier resistance of the stratum corneum and allow drugs to penetrate more readily to the viable tissues and the systemic circulation. This paper presents a general theory for accelerant activity based on possible alterations at the molecular level of the components of the horny layer. Within the intercellular route, enhancers may interact at the polar head groups of the lipids, within aqueous regions between lipid head groups, and between the lipophilic tails of the bilayer. Inside the corneocyte it is the keratin fibrils and their associated water that are the targets. High concentrations of solvents may also alter partitioning processes. Because of the involvement of lipid and protein modifications, together with partitioning phenomena, the scheme of possible enhancer interactions has been termed the Lipid Protein Partitioning (LPP) theory. This theory is applied specifically to water, Azone, dimethylsulphoxide, dimethylformamide, 2–pyrrolidone, N-methyl-2–pyrrolidone, oleic acid, decylmethylsulphoxide, sodium lauryl sulphate and propylene glycol. Resume Action des produits augmentant la penetration de la peau Les produits qui augmentent la penetration de la peau par les medicaments diminuent la resistance de la barriere representee par la couche cornee. Cet effet est reversible et permet aux medicaments de penetrer plus facilement et plus rapidement pour acceder aux couches sub-cutanees et a la circulation systemique. Cet article presente une theorie d'ordre general pour l'activite des produits accelerants, basee sur des modifications moleculaires des composants de la couche cornee. Au cours du trajet intercellulaire, les produits accelerant peuvent reagir dans les groupements polaires a la těte des lipides et entre les queues lipophiles de l'interface cellulaire. A l'interieur de la corneocyte, les cibles sont les fibriles de keratine et leur eau associee. De fortes concentrations de solvents peuvent egalement modifier les processus de Partition. En raison des modifications des Lipides et Proteines, accompagnees par un phenomene de RePartition, le schema des interactions possibles a ete denomme theorie LPP. Cette theorie est appliquee specifiquement a l'eau, Azone, dimethylsulphoxide, dimethylformamide, 2–pyrrolidone, N-methyl-2–pyrrolidone, acide oleique, decylmethylsulphoxide, lauryl sulfate de sodium et propylene glycol.

The Lipid-Protein-Partitioning theory of skin penetration enhancement suggests that accelerants usually act by one or more of three main mechanisms; they can alter the intercellular lipid or intracellular protein domains of the homy layer and they may also increase partitioning into the skin of a drug, a co-enhancer, water or any combination of these. This paper discusses these concepts, quoting examples of the three mechanisms as assessed by differential scanning calorimetry and permeation studies and relates such investigations to our ideas of the molecular architecture of the stratum coraeum.

The early eighties saw the introduction of liposomes as skin drug delivery systems, initially promoted primarily for localised effects with minimal systemic delivery. Subsequently, a novel ultradeformable vesicular system (termed "Transfersomes" by the inventors) was reported for transdermal delivery with an efficiency similar to subcutaneous injection. Further research illustrated that the mechanisms of liposome action depended on the application regime and the vesicle composition and morphology. Ethical, health and supply problems with human skin have encouraged researchers to use skin models. Traditional models involved polymer membranes and animal tissue, but whilst of value for release studies, such models are not always good mimics for the complex human skin barrier, particularly with respect to the stratum corneal intercellular lipid domains. These lipids have a multiply bilayered organization, a composition and organization somewhat similar to liposomes. Consequently researchers have used vesicles as skin model membranes. Early work first employed phospholipid liposomes and tested their interactions with skin penetration enhancers, typically using thermal analysis and spectroscopic analyses. Another approach probed how incorporation of compounds into liposomes led to the loss of entrapped markers, analogous to "fluidization" of stratum corneum lipids on treatment with a penetration enhancer. Subsequently scientists employed liposomes formulated with skin lipids in these types of studies. Following a brief description of the nature of the skin barrier to transdermal drug delivery and the use of liposomes in drug delivery through skin, this article critically reviews the relevance of using different types of vesicles as a model for human skin in permeation enhancement studies, concentrating primarily on liposomes after briefly surveying older models. The validity of different types of liposome is considered and traditional skin models are compared to vesicular model membranes for their precision and accuracy as skin membrane mimics.

The penetration of triamcinolone acetonide (TACA) through human epidermis following the evaporation of a finite dose of a hydroalcoholic tincture correlates positively with the applied volume and the penetrated amount of ethanol. The incorporation of salicylic acid into such a tincture extends the lag time and linear region of the TACA penetration curve. Covering with Actiderm following evaporation of a hydroalcoholic vehicle increases 2–3-fold the TACA flux with respect to uncovered skin or to occlusion with Saran Wrap. Solubility and partition coefficient determinations indicate similar thermodynamic activities for the drug in the three tinctures tested; however, diffusion experiments under in vivo mimic conditions show a higher TACA flux for the vehicle composed of isopropyl palmitate and ethanol. This phenomenon probably arises from different kinetics of the diffusion process in circumstances mimicking the in vivo conditions where vehicle components evaporate.

An important step in liposome characterization is to determine the location of a drug within the liposome. This work thus investigated the interaction of dipalmitoylphosphatidylcholine liposomes with drugs of varied water solubility, polar surface area (PSA) and partition coefficient using high sensitivity differential scanning calorimetry. Lipophilic estradiol (ES) interacted strongest with the acyl chains of the lipid membrane, followed by the somewhat polar 5-fluorouracil (5-FU). Strongly hydrophilic mannitol (MAN) showed no evidence of interaction but water soluble polymers inulin (IN) and an antisense oligonucleotide (OLG), which have very high PSAs, interacted with the lipid head groups. Accordingly, the drugs could be classified as: hydrophilic ones situated in the aqueous core and which may interact with the head groups; those located at the water-bilayer interface with some degree of penetration into the lipid bilayer; those lipophilic drugs constrained within the bilayer.

Optimisation of drug delivery through human skin is important in modern therapy. This review considers drug-vehicle interactions (drug or prodrug selection, chemical potential control, ion pairs, coacervates and eutectic systems) and the role of vesicles and particles (liposomes, transfersomes, ethosomes, niosomes). We can modify the stratum corneum by hydration and chemical enhancers, or bypass or remove this tissue via microneedles, ablation and follicular delivery. Electrically assisted methods (ultrasound, iontophoresis, electroporation, magnetophoresis, photomechanical waves) show considerable promise. Of particular interest is the synergy between chemical enhancers, ultrasound, iontophoresis and electroporation.

FT-Raman spectra have been recorded from human stratum corneum in vivo and in vitro using a fibre optic coupling. Spectra from both tissues were near identical illustrating the validity of in vitro studies for examining the mechanisms of action of penetration enhancers in vivo. Treatment of excised human skin with the penetration enhancer 1,8-cineole showed that the permeation promoter induced increased order in the barrier bilayered lipids domains of the stratum corneum in most of the samples, but caused a decrease in order of these domains in some of the samples investigated. This may correlate with a mechanism of action where the enhancer exists as a discrete domain within the stratum corneum lipids which would provide defects in the barrier nature of the permeation limiting lipid domains.

The discovery in September 1991 of a Late Neolithic man in a glacial field in Oetzal between Austria and Italy at a height of 3200m has provided uniquely preserved samples for archaeological studies. The Ice-man (or Similaun man) body is that of a male between 25 and 40 years old who probably died of natural causes some 5200 years ago. Of importance to the present study is the state of preservation which has arisen without the use of exogenous chemicals, as is usually the case in many Egyptian mummified tissues. Ice-man is remarkably preserved in that adipocere had not formed and the body was intact on discovery [1].

Biomedical studies of hard and soft human tissue using Raman spectroscopy have now become well established and there is quite an extensive literature on normal and diseased skin and organs, single cells, implants and the presence of foreign inclusions following implantation, and the interaction of chemical agents with soft tissues. The advancement in technology provided by far red and near infrared Raman excitation sources and the application of new detection and data acquisition techniques has provided an impetus for the study of complex biomaterials with minimisation of fluorescence interference and resulting improved spectral quality. The use of fibre optical coupling to CCD- or FT-Raman spectrometers facilitates in vivo studies of human tissue and has created the potential for diagnostic Raman systems.

The permeation characteristics through human skin of 8-methoxypsoralen (8-MOP) and its physical attributes were investigated. The log octanol/water partition coefficient and saturated aqueous solubility of 8-MOP at 32°C were 1.98 and 55.8 μg mL−1 respectively. 8-MOP showed Fickian diffusion, with its flux being linearly related to the concentration of drug in the donor solution. The permeability coefficient of 8-MOP through human skin from different concentrations of aqueous solutions and a 2.6 μg mL−1 bath lotion (as used in clinics) were statistically identical with mean values of 1.76 ± 0.12 × 10−2 and 1.70 ± 0.32 × 10−1 cm h−1 respectively (P ≥ 005). An ethanol/water (1:1 w/v) receptor solution did not improve the clearance of 8-MOP from the dermis when compared with an aqueous vehicle. Complete removal of the stratum corneum by tape stripping from full-thickness membranes produced a threefold increase in the flux of 8-MOP thus suggesting that the main barrier to 8-MOP permeation resides in the stratum corneum although the aqueous epidermal and dermal tissue provide a significant resistance to transdermal drug permeation. The equilibrium uptake of 8-MOP into psoriatic plaques and the 8-MOP aqueous/plaque partition coefficient were found to be more than twofold greater than for normal stratum corneum. The absorption of 8-MOP from the total applied topical dose (396 mg) was assessed as ∼0.25% and only 2.5% of an oral dose, a significant reduction in the possible toxic hazard. The peak concentration of 8-MOP permeating through the skin was observed at about 35 min after limited exposure for 15 min. Our results suggest that following a 15 min bath in the drug solution, there may be a need for an interval of about 20 min before patients are irradiated to ensure the optimization of photosensitizer with UVA irradiation (PUVA) therapy. Alternatively, UV irradiation could be applied at a lower flux over a longer time.

The effects of propylene glycol/water co-solvent systems and terpene penetration enhancers (1,8-cineole, menthone, (+)-limonene and nerolidol) on the absorption rate of the model hydrophilic permeant, 5-fluorouracil, were investigated using excised human skin. Similar fluxes for 5-fluorouracil were obtained from saturated enhancer-free co-solvent systems. Coapplication of each terpene with the drug, both at saturation, in propylene glycol co-solvent systems increased drug flux significantly. Terpene activity depended on the propylene glycol content in the vehicles. Maximum fluxes were obtained from formulations containing the terpenes in 80% propylene glycol systems (highest concentration used), which when normalized to the flux from the pure vehicles yielded enhancement ratios of about 24, 21, 4 and 18, with 1,8-cineole, menthone, (+)-limonene and nerolidol, respectively. Combining the permeation studies with differential scanning calorimetry (DSC) and partitioning experiments revealed that increased lipid disruption is probably an important mechanism involved in the enhancing ability of formulations containing 1,8-cineole, menthone and nerolidol. This was clearly demonstrated by applying thermodynamic principles to interpret DSC results. This approach has indicated that these terpenes are probably able to disrupt stratum corneum lipids at physiological temperature as manifested by reductions in the entropy changes associated with the lipid-related transitions, particularly T2, the first major lipid transition. Additionally, increased drug partitioning contributed to the effect of the high propylene glycol content formulations. (+)-Limonene, as interpreted from DSC results, produced a freezing point-depression effect on stratum corneum lipids, suggesting little interaction with lipids at skin temperature; its small enhancement effect may involve phase separation of the oil in stratum corneum lipids. Terpenes in co-solvent systems such as propylene glycol/water at appropriate propylene glycol content might thus be useful vehicles for the delivery of drugs from topical formulations.

Wide-angle X-ray-diffraction experiments were used to investigate the molecular organization of barrier components of human stratum corneum. Diffraction lines related to the side-by-side lipid packing arrangements in the intercellular bilayers were identified as were patterns arising from secondary protein structures in intracellular keratin. Reflections were also identified which may be produced by proteins in the corneocyte envelopes. The effects of hydration on stratum corneum structure were monitored using 0, 20–40, 40–60, 60–80 and approximately 300% hydrated samples. The packing arrangements in the intercellular lipid bilayers remained the same over the entire hydration range, as did keratin structures. A new diffraction ring, attributable to liquid water, was produced by 300% hydrated samples with a repeat spacing of 0.35 to 0.30-0.29 nm. The effects of three terpene enhancers, (+)-limonene, nerolidol and 1,8-cineole, on stratum corneum structure were monitored. Treatment with each of the terpenes produced additional reflections which were attributed to the presence of the respective liquid enhancers within the stratum corneum. (+)-Limonene produced an additional reflection at 0.503-0.489 nm, nerolidol, an additional reflection at 0.486-0.471 nm and 1,8-cineole, an intense reflection at 0.583–0.578 nm. Reflections characteristic of gel-phase lipids and crystalline lipids also remained after all terpene treatments. These results provide no clear evidence of lipid bilayer disruption by the terpenes and suggest that areas of liquid terpene exist within the stratum corneum. The mechanisms underlying propylene glycol synergy with terpene enhancers were investigated. Treatment of stratum corneum with each terpene mixed with propylene glycol gave rise to two additional reflections. One reflection, always positioned at 0.452–0.448 nm, had been observed in control studies following propylene glycol treatment and may have been associated with bilayer structures disrupted by propylene glycol or altered keratin structures. The second reflection was developed by the respective terpene enhancer. For example, treatment with a 1,8-cineole/propylene glycol mixture produced reflections at 0.457—0.451 nm (propylene glycol-disrupted lipids or altered keratin) and 0.591—0.578 nm (liquid 1,8-cineole). Since the reflection at 0.452—0.448 nm was unaffected by co-application of propylene glycol with terpene enhancers, this study offers no evidence to support the theory that propylene glycol synergy with the terpenes occurs through enhanced lipid disruption.

The bio-availabilities and activities of commercial creams (Metosyn, Synalar, Betnovate, Locoid, Alphaderm, Dioderm and Efcortelan 1%) were assessed using a multiple dosage regimen vasoconstrictor assay in ten volunteers. The blanching response first increased but then diminished with continued application over a 5-day period: following a 2-day 'resting period', when no steroid was applied, the application sites recovered considerably but tachyphylaxis again resulted from subsequent repeated applications. Acute tolerance occurred with the hydrocortisone formulations (Alphaderm, Dioderm and Efcortelan 1%) in a manner similar to that observed with the potent preparations. Metosyn was significantly more active (P less than 0.05), and Efcortelan 1% was significantly less active (P less than 0.05), than the other formulations. Apart from the reversal of Dioderm and Alphaderm the rank order of preparations was similar to that previously noted in the 6 h occluded single-application vasoconstrictor test. The results may have significance in dosage from design and application for topical corticosteroid formulations.

The bioavailabilities and activities of three amcinonide preparations and Betnovate cream were assessed using three multiple-dosage regimen vasoconstrictor assays in 10 volunteers. Applications were made once daily, twice daily and every alternate day with an initial three times daily loading dose applied on the first day only. Blanching responses first increased and then decreased due to tachyphylaxis. It is proposed that clinically the most advantageous dosage regimen is a once daily application with no loading dose.

Mannitol, hydrocortisone and progesterone were selected as model penetrants to assess the mode of action of eight potential penetration enhancers in human skin. Their partition coefficients, octanol: water and stratum corneum: water were measured and correlated with their postulated routes of penetration through human skin. The results suggest that mannitol penetrated via a polar route, hydrocortisone by a mainly lipid route and progesterone via a lipid pathway but its penetration rate was probably affected by aqueous layers. From permeation studies through cadaver skin in which an in-vivo mimic method was used, it was concluded that the penetration enhancers fell into three main categories: (1) solvents which enhanced permeation of polar and non-polar compounds e.g. 2-pyrrolidone, N-methylpyrrolidone, N-methylformamide and propylene glycol plus Azone; (2) enhancers which preferentially affected the polar route e.g. propylene glycol plus decylmethylsulphoxide, and (3) accelerants which mainly modified the non-polar route e.g. propylene glycol plus oleic acid, propylene glycol alone and, to a limited extent, water.

A. Introducrion _ . . . _ . _ _ _ _ _ _ _ _ _ _ . . . . . . . B. The nature of viscoelastic behavior . . . . . . (i) Model representation of linear viscoelastic behavidr : : : : : : : : : (ii) The Maxwell model (iii) The Voigt (Kelvin) model’ : : : : : : : : : : : 1 : : : : 1 C. Methods of viscoelastic analysis . . _ _ _ _ . . _ . . . . . . . (i)The creep test . . . . . . . . . . . . . . . . . . . . . (ii) Dynamic analysis _ . . . . . . . . . . . _ _ _ _ _ _ _ _ (iii) Transformation of creep to dynamic parameters . . . _ _ . . . . . D. Self-bodied oil-in-water emulsions . . _ . . . . . . . . . . . _ (i) Nature of self-bodied emulsions _ _ _ __ . _ , , . . . . . (ii) Ternary systems and emulsions based on surfaetant/lbng edain alcohol/water. (iii) Kinetics of gel network formation _ . _ . _ . . . . _ . . . . (iv) Influence of temperature (v) Effect of mixed emulsifier cdnckt_ration_ : : : : : 1 : : : : 1 1 (vi) Influence of surfactant chain length . . . . _ _ _ . . . . . . . (vii) Influence of alcohol chain length . . . . . . . . . . . . . . . (viii) Correlation of v-kcoelastic functions from creep and oscillation . . . . . (ix) Summary. . . . . . . . . . . . . . . . . . . . . . . E. Water-in-oil emulsions . . . _ . . _ _ . . _ . _ . _ . . . . . F. Conclusion _ . . . . . . . . . . . . . . . . . . . . . . References _ . . . . . . _ _ . . . . . _ . . . . . . . _ . . 39 40 40 41 -12 44 44 48 54 57 57 58 59 61 62 66 69 70 71 71 73 74

Summary Theoretically two corticosteroid preparations should show the same clinical activity even though they possess different medicament concentrations, provided that both preparations are at or above saturation e.g. they are suspensions. To test this theory, the non-fluorinated corticosteroid desonide prepared as two suspensions in the same cream formulation (Apolar Cream 0.1% and 0.05%) were evaluated for vasoconstriction using an occluded blanching test. Included in the vasoconstriction test were also another desonide 0.05% preparation (Tridesilon Cream®) and Betnovate Cream® (betamethasone 17-valerate 0.1%). The study was extended into patients by comparing the effects of Apolar Cream 0.1% and 0.05, in a double-blind, randomized, right-left comparative study including 40 patients with bilateral, non-infected hand eczemas, No significant difference could be demonstrated between the blanching responses of the four preparations tested in the vasoconstriction study. Betnovate Cream provided the lowest blanching response. Thirty-eight patients completed the clinical trial. All patients improved after 11 days of treatment with Apolar Cream 0.1%. With the 0.05% cream, 37 improved and, in one, the condition remained unchanged. No statistically significant difference could be demonstrated between treatments. It is concluded that, for practical therapeutic purposes, Apolar Cream 0.05% will equate with the corresponding 0.1% preparation.

Air/water interfacial adsorption parameters and CMC values were determined for C 16 polyoxyethylene nonionic surfactants from surface tension data. Temperature (10–40°C) and polyoxyethylene chain length (EO = 17–63) effects were studied. Surface activity increased with temperature but decreased with hydrophilic chain length. The areas per surfactant molecules at the air/water interface increased with temperature and hydrophilic chain length. Thermodynamic parameters for adsorption and micellization were calculated at different temperatures. Adsorption occurred with positive entropic and enthalpic changes. Free energy of the process decreased with temperature but increased with polyoxyethylene chain length. Free energy of micellization was resolved into hydrophobic and hydrophilic components to study the effect of the polyoxyethylene chain length.

Skin penetration enhancers are molecules which reuersibly remove the barrier resistance of the stratum corneum. They allow drugs to penetrate more readily to the viable tissues and thus enter the systemic circulation. This paper presents a general theory for enhancer activity based on possible alterations at the molecular level of the stratum corneum. Within the intercellular route, accelerants may interact at the polar head groups of the lipids, within aqueous regions between lipid head groups, and between the hydrophobic tails of the bilayer. Within the corneocyte the keratin fibrils and their associated water provide the target. High concentrations of solvents may also alter partitioning phenomena. The theory has been applied specifically to water, Azone, dimethylsulfoxide, dimethylformamide, 2-pyrrolidone, N-methyl-2-pyrrolidone, oleic acid, decylmethylsulfoxide, sodium lauryl sulfate and propylene glycol. The main techniques which supplied experimental support for the theory were permeation studies through human skin, the vasoconstrictor assay and differential scanning calorimetry; the results from DSC are mainly considered here.

Experiments were conducted to: (a) compare the bioavailability of betamethasone benzoate in a quick-break aerosol foam and semisolid dosage forms, (b) compare the activity of betamethasone benzoate, betamethasone valerate, clobetasol propionate, triamcinolone acetonide, desonide, flumethasoid reservoir formation in skin, and (d) assess the effect of a natural moisturizer. Efficacy was determined by a graded response 6-hr occluded vasoconstriction test with subsequent reocclusion for reservoir demonstration. Moisturizer effect was assessed by a nonoccluded vasoconstriction test using "plain" and sodium 2-pyrrolidone-5-carboxylate-containing concentrates on arms pretreated with water or moisturizer. The activities of betamethasone benzoate concentrate, collapsed foam, ointment, and gel were similar and significantly better than the activity of the cream. Clobetasol propionate was significantly better than the other medicated concentrates, which were equivalent. Steroid-induced blanching decreased in the presence of a moisturizer.

The effect of physical and chemical variables on consistency of Carbopol 940 and 941 gels was investigated by continuous shear rheometry. Gels provided plastic flow curves with negligible hysteresis. Continuous shear properties were not greatly affected by centrifuging, milling, temperature cycling, and ageing. Initial addition of neutralizing agent (0–6% w/w for 3% gels) markedly increased consistency; further addition caused a more gradual increase. Daylight reduced consistency. Apparent viscosities varied exponentially with concentration. Increased temperature gradually decreased apparent viscosity. Solvents had a variable effect on apparent viscosities which could not be correlated with solvent viscosity nor molecular weight. Apparent viscosities for gels neutralized with different bases were independent of type of base used. Small strain testing investigated fundamental properties of Carbopol gels and determined the effect of polymer concentration, temperature, neutralization, different solvents and neutralizers. Gels were linear viscoelastic and to a first approximation were elastic solids. Creep compliance varied exponentially with concentration and showed no correlation with solvent or neutralizer type.

Viscoelastic properties of aqueous Carbopol gels were examined at frequencies of 7.91 × 10 −3 to 25 Hz using a Weissenberg rheogoniometer. Neutralized gels changed little from 10 to 50dgC and were similar to unneutralized gels. Data confirmed the dominant elastic nature of Carbopol gels (phase lags of 2–14°) and the relative small change of the storage modulus with frequency indicated that the polymer was strongly entangled and the gels were examined in their plateau region of viscoelastic behaviour. Some correlation of oscillatory data with continuous shear properties was observed. Oscillatory data obtained at different temperatures were combined with creep data using the method of reduced variables; satisfactory composite curves over a slightly increased frequency range were obtained. When creep data were transformed into dynamic data and combined with experimental oscillatory results, using exact viscoelastic relationships, curves were obtained which described the behaviour of Carbopol gels over seven frequency decades. From this behaviour it was suggested that Carbopols were high molecular weight amorphous polymers with long side chains. The practical aspects of the transformation procedure were discussed and it was shown that neither small strain technique alone covered the entire frequency or time range necessary for an exhaustive study of Carbopol gels.

An apparatus was developed for investigating water absorption and desorption patterns in stratum corneum in vitro. To validate this technique, absorption and desorption of water vapour by human abdominal stratum corneum was studied using relative humidity changes of 0–91% and 91–0%. These changes were attained by suspending the test samples in a chamber above a drying agent (Drierite) or a saturated salt solution (stationary system), or air was passed in different proportions through either water or Drierite and then recombined before contacting the stratum corneum (flow system). These two methods produced different absorption and desorption patterns and the stratum corneum samples attained 2–3-fold higher water content levels if the stationary system was used. Changes in the rate of air flow during absorption affected the results obtained an effect not observed during desorption. Initial diffusion coefficients and rate constants for absorption and desorption were calculated. The apparatus should prove useful in fundamental studies of the hydration of the stratum corneum and in applied investigations such as treatment of the corneum with non-volatile agents, for example, moisturizers and penetration enhancers.

SUMMARY Eight grades of white soft paraffin BP were investigated for their ability to produce pallor of normal skin in a test designed originally for topical corticosteroid activity. All samples produced definite but transient blanching, the extent of which was determined by the grade of white soft paraffin and the test method employed. Pallor was often more pronounced under non-occlusive test conditions and when the sample contained 5% w/w propylene glycol. Blanching may have been due to the presence of a pallor-inducing substance transported into the skin by propylene glycol, rather than to a simple hydration effect.

Gel networks formed in the continuous phases of oil-in-water emulsions stabilized by cetostearyl alcohol combined with alkyltrimethylammonium bromides (of C 12 C 18 chain lengths) have similar mechanical properties to those of ternary systems formed by dispersing the appropriate quaternary bromide and alcohol in water. The rheological changes with temperature of such emulsions and ternary systems were examined by continuous shear and creep experiments. Variations in the viscosities and compliances were correlated with thermal phase transitions determined microscopically. The C 12 quaternary networks were tenuous. Continuous shear results were uninterpretable, and in creep the networks became nonlinear viscoelastic as the temperature rose. The consistency of each ternary system and emulsion prepared from higher quaternary surfactants increased to a maximum as the temperature rose. These temperatures of maximum consistency were close to the transition temperature at which each network melted from frozen smectic to liquid crystalline phase. They correlated well with penetration temperatures of approximately 1% w/w aqueous solutions of the appropriate alkyltrimethylammonium bromide into cetostearyl alcohol. The transition temperatures of the emulsions were lower than those of the corresponding ternary systems, owing to the presence of solubilized liquid paraffin in the networks. Above the transition temperatures the consistencies of the ternary systems and emulsions decreased as the networks weakened and finally dissolved to form isotropic solutions.

Gel networks in the continuous phases of emulsions of liquid paraffin in water stabilized by cetomacrogol 1000-cetostearyl alcohol mixed emulsifier have similar properties to those of ternary systems which form on dispersing the mixed emulsifier in water. The rheological changes with temperature of a typical emulsion and ternary system were examined by continuous shear (cone/plate viscometer) and creep (concentric cylinder viscometer) experiments. Variations in the viscosities and compliances were correlated with thermal-phase transitions determined microscopically. The nonionic network was tenuous. At high rates of shear, it fractured in the cone- and-plate gap, and further investigations were performed at lower shear rates. In creep, the extent of viscoelastic linearity was restricted, especially at high temperatures. The consistency of the ternary system increased to a maximum, as the temperature rose. The temperature of maximum consistency was close to the temperature at which the gel melted to form the liquid crystalline phase. At higher temperatures, the consistency decreased as the network weakened and finally dissolved to form isotropic solution. The emulsion plots did not reveal a temperature of maximum consistency. Abrupt changes in the compliance and viscosity plots vs temperature indicated that the emulsion network melted at a lower temperature than the ternary system. This was due to the liquid paraffin which lowered the temperature of phase transitions.

Liquid paraffin emulsion stabilized by the mixed emulsifier sodium dodecyl sulfate/cetyl alcohol have been examined by microscopy, particle size analysis, continuous shear rheometry, and creep. The disperse phase was aggregated and included some nonspherical globules containing crystals of cetyl alcohol. Emulsions were thixotropic and the extent increased with emulsifier concentration. In creep, systems suggested a mechanical model of three Voigt units in series with a Maxwell unit; the compliances decreased and the viscosities increased with emulsifier concentration. Rheological properties are explained by the presence of a viscoelastic network in the continuous phase.

Liquid paraffin emulsions stabilized by sodium dodecyl sulfate with long-chain alcohols have been examined by microscopy, particle-size analysis, continuous shear rheometry, and creep. The disperse phases were aggregated and emulsions were viscoelastic and thixotropic and developed crystals on storage. Emulsions were firm and rheologically stable when cetostearyl alcohol was used. They were similar but slightly less stable when formulated with (a) a mixture of C 16 and C 18 alcohols in the ratio present in cetostearyl alcohol and (b) a mixture of C 10 , C 12 , C 14 , C 16 , and C 18 alcohols in similar ratios. Emulsions were less stable when formulated either with C 16 or with mixtures of C 16 and C 18 alcohols in ratios 3 1 , 1 1 , and 1 3 . A more mobile, less stable emulsion was obtained with C 18 alone, but a stiff emulsion was formed provided that the ratio of C 16 to total alcohol (C 16 plus C 18 ) was greater than approximately 0.07. Differences between emulsions were not related to particle-size distribution but to the nature and content of the C 18 alcohol, which influenced the strength of the viscoelastic network formed. Trace amounts of unidentified compounds exerted a minor effect.

Emulsions of liquid paraffin in water stabilized by the mixed emulsifier cetomacrogol 1000-cetostearyl alcohol have been examined using rheological, microscopical, and particle size analytical techniques. The rheological properties of the emulsions were related to gel networks formed in the continuous phases. These networks had similar properties to ternary systems formed by dispersing the mixed emulsifier in water. The networks were not fully formed immediately after preparation; they built up over a period of time at 25°C, and thus the creep compliances fell and the viscosities rose on storage. The mechanisms responsible for the formation of the nonionic networks are discussed and compared with the mechanisms which function in gels formed by ionic surfactant/long-chain alcohol mixed emulsifiers.

Liquid paraffin in water emulsions stabilized by alkyltrimethylammonium bromides (of C 12 C 18 chain lengths) with cetostearyl alcohol have been examined by microscopy, particle size analysis, continuous shear rheometry, and creep. The rheological properties of emulsions prepared from each quaternary mixed emulsifier were related to gel networks formed in the continuous phases. These networks were shown to have properties similar to those of ternary systems formed by dispersing the corresponding quaternary surfactants and cetostearyl alcohol in water. All four quaternary mixed emulsifiers exhibited the self-bodying phenomenon. Ternary systems and emulsions of equivalent mixed emulsifier concentration increased in consistency as the surfactant chain length increased. Emulsions prepared with quaternaries of medium chain length (C 14 and C 16 ) formed strong viscoelastic networks. C 12 emulsions possessed tenuous networks and when C 18 quaternary was used, the structures were not as rigid as predicted by the trend of the lower homologs. When C 12 and C 18 emulsions were stored, oil globules which were hexagonal in cross section were formed. These reverted to a spherical shape when diluted. These results are discussed at a molecular level by considering the effect of variation in surfactant chain length on the formation of smectic structures.

A technique widely used in dermatological research deposits drugs onto the skin from volatile solvents and often uses hairless mouse skin as a model for human tissue. We therefore compared the damaging effect of acetone on hairless mouse and human abdominal skins using an in vitro steady-state technique. The permeabilities of the two skin types to 5-fluorouracil were measured, both untreated and after acetone application. For untreated skins, the permeability coefficients of 5-fluorouracil were similar in the two skin types, suggesting a limited usefulness of the hairless mouse as a model for human skin. After acetone treatment, however, hairless mouse skin increased 16-fold in permeability (P = 0.975), whereas human abdominal skin was not significantly affected (P

SUMMARY Thirty proprietary corticosteroid creams and gels were evaluated for vasoconstriction using an occluded blanching test. The method differed from previous investigators' in employing a more sensitive estimation of pallor and more reading times in order to determine the complete blanching profile. The results were expressed as three parameters: (1) area under the blanching curve, (2) summed % total possible score, and (3) square root transformation of sum of scores divided by number of volunteers; the last permitted statistical differentiation between the preparations. Experiments were performed to (a) compare the bio-availability of the formulations, (b) determine the effect of antimicrobial additives, and (c) assess the retention of steroids in the skin. Scores for the additive-containing and ‘plain’ preparations were within 90% except for Tri-Adcortyl/Adcortyl (78%) and Locorten-Vioform/ Locorten (53%). A steroid reservoir in the skin was demonstrated for up to 14 days. Selected preparations were re-tested by a non-occluded method and the results compared with those obtained by the occluded procedure.

This work investigates vapour and liquid permeation through human skin of model penetrants benzyl alcohol, benzaldehyde, aniline, anisole and 2−phenylethanol applied in model vehicles butanol, butyl acetate, isophorone, isopropyl myristate, propylene carbonate, toluene, n-heptane and water. Vapour permeation was a linear function of thermodynamic activity as measured by headspace gas chromatography, except when the vehicle was n-heptane. Liquid permeation did not always follow simple thermodynamic predictions, e.g. for the penetrant, benzyl alcohol, when the vehicle damaged the skin (toluene, n-heptane) or when propylene carbonate produced low fluxes and isopropyl myristate, high values. At comparable thermodynamic activities, liquid fluxes were often ten-fold higher than vapour fluxes, and these differences were reflected by the partition coefficients and the amount of penetrant entering the stratum corneum membrane. The conclusion was that liquid fluxes were membrane controlled, whereas an interfacial effect probably contributed to low vapour permeation.