Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation.

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41-59, BMI 26-38, cold ischemic time < 10 h). Collagen IV, pan-laminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan-laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan-laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.

Predicting skin penetration of actives from complex cosmetic formulations: an evaluation of inter formulation and inter active effects during formulation optimization for transdermal delivery.

Twenty products, containing a radiolabelled form of each active in typical cosmetic formulations, were made and applied to female human epidermal membranes mounted in Franz diffusion cells for 48 h under 'in use' conditions. The products consisted of combinations of five formulations (a hydro-alcoholic gel, an oil in water emulsion, a water in oil emulsion, a microemulsion and an oil) with four model drug actives (testosterone, hydrocortisone, 5-fluorouracil and ketoconazole). Steady-state flux appeared to be reached by 8 h and maintained for all products, other than for the microemulsions, consistent with the actives being present in the residual formulation on the skin at saturation. The recovery for each active at the end of the 48-h study (from a series of stratum corneum tape strips, the remaining skin, cumulative amount penetrating into the receptor solution, product washed from the skin and on the donor chamber cap) ranged from 86.5% to 100.6%. The rank order of the fluxes for the actives from the hydro-alcoholic gel is consistent with the known active molecular size and polarity determinants for maximum epidermal flux. Actives with similar steady-state (maximum) fluxes from a range of formulations had retention in the stratum corneum and similar transport rate constants through the stratum corneum. The microemulsion formulation significantly enhanced both the stratum corneum steady-state flux and transport rate constant for 5-fluorouracil, hydrocortisone and testosterone. The penetration flux of each active could be related to its size and polarity and appeared maximal when the actives in the different cosmetic formulations applied to the skin under 'in use' conditions were likely to remain in the residual product on the skin as a saturated solution after solvent evaporation. Enhanced penetration fluxes can be achieved by formulation selection and an appropriate choice/mix of emollients/adjuvants. The principles described here provide a framework for understanding the delivery of cosmetic ingredients from various formulations.

An integrated pharmacokinetic and imaging evaluation of vehicle effects on solute human epidermal flux and, retention characteristics.

OBJECTIVE: Our understanding of the differential effects of topically applied vehicles on solute partitioning and diffusion within the skin is presently limited. In this work, in vitro epidermal partitioning, penetration and multiphoton laser scanning microscopy (MPLSM) imaging studies were used to assess the distribution of 2-naphthol across human epidermis. MATERIALS AND METHODS: Four commonly used liquid vehicles (100% water, 20% propylene glycol (PG)/water, 50% ethanol (EtOH)/water and 100% isopropyl myristate (IPM)) were used. RESULTS AND DISCUSSION: The maximum flux and membrane retention of 2-naphthol from 50% EtOH/water was almost an order of magnitude or larger than from the other vehicles evaluated whereas IPM resulted in the highest membrane retention and lowest membrane penetration for 2-naphthol than other vehicles. MPLSM studies showed that 2-naphthol solute partitioned favourably into the intercellular lipids and that there was a vehicle-dependent uptake of 2-naphthol into corneocytes. CONCLUSIONS: The integrated evaluation using in vitro penetration, epidermal retention and MPLSM imaging has shown that vehicle effects on skin penetration occurs by an alteration in the distribution of solutes between the corneocytes and intercellular lipids in addition to the well known mechanisms of altered partitioning into the stratum corneum and enhanced epidermal diffusion.

The effects of freezing skin on transdermal drug penetration kinetics.

This study investigated the effects of freezing canine skin on the penetration kinetics of hydrocortisone. Skin samples from three dogs were used for in vitro penetration studies commencing on the day of skin collection (fresh skin) and again after freezing at -20 degrees C for 1, 4, 8 and 12 months. When the data from the dogs was averaged, the pseudo-steady-state flux (Jss) of hydrocortisone through skin frozen for any duration was significantly (P < 0.023) greater than through fresh skin and there was a positive relationship (P < 0.007) between the length of freezing and DeltaJss. For all dogs, the lag times (tlag) calculated for hydrocortisone penetration were significantly (P < 0.029) shorter through skin that had been frozen, compared with fresh skin. However, the shapes of the permeation profiles of hydrocortisone appeared similar through the fresh and frozen dog skins and no differences were detected between the groups on histological examination. The results of this study have shown that freezing dog skin at -20 degrees C can significantly increase the transdermal penetration of hydrocortisone in vitro, and that the extent of this enhancement can increase with duration of freezing.

Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs.

AIMS/HYPOTHESIS: Rapamycin, part of the immunosuppressive regimen of the Edmonton protocol, has been shown to inhibit vascular endothelial growth factor (VEGF) production and VEGF-mediated survival signalling in tumour cell lines. This study investigates the survival-promoting activities of VEGF in human islets and the effects of rapamycin on islet viability. MATERIALS AND METHODS: Levels of VEGF and its receptors in isolated human islets and whole pancreas was determined by western blotting and immunostaining. Islet viability following VEGF or immunosuppressive drug treatment was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Islet VEGF release was measured by ELISA. Mouse islets infected with an adenovirus expressing the gene for VEGF were transplanted syngeneically into streptozotocin-induced diabetic mice, with blood glucose levels measured three times per week. RESULTS: Isolated human islets produced multiple isoforms of VEGF and VEGF receptors 1, 2 and 3 and the coreceptor neuropilin 1. Exogenous VEGF (10 ng/ml) prevented human islet death induced by serum starvation, which suggests that VEGF can act as a survival factor for human islets. Transplantation of mouse islets infected with a VEGF-expressing adenovirus in a syngeneic model, improved glycaemic control at day 1 post-transplantation (p < 0.05). Rapamycin at 10 and 100 ng/ml significantly reduced islet VEGF release (by 37 +/- 4% and 43 +/- 6%, respectively; p < 0.05) and at 100 ng/ml reduced islet viability (by 36 +/- 9%) and insulin release (by 47 +/- 7%, all vs vehicle-treated controls; p < 0.05). Tacrolimus had no effect on islet VEGF release or viability. CONCLUSIONS/INTERPRETATION: Our data suggest that rapamycin may have deleterious effects on islet survival post-transplantation, both through a direct effect on islet viability and indirectly through blockade of VEGF-mediated revascularisation.

Regional differences in transdermal penetration of fentanyl through equine skin.

The rate and regional differences for the penetration of fentanyl through equine skin was investigated in vitro using a commercial transdermal therapeutic system (TTS) or 'patch'. Skin collected from the thorax, groin and leg (dorsal metacarpal) regions of five horses was placed in diffusion cells and a fentanyl TTS applied to each skin sample. Drug penetration through each skin sample over 48 h measured using high performance liquid chromatography (HPLC). Cumulative penetration (microg/cm2) was plotted against time (h) and used to regress the steady state flux (microg/cm2/h) of fentanyl through each skin site. Results showed similar fluxes for both the thorax (2.32+/-0.17 microg/cm2/h and groin (2.21+/-0.11 (microg/cm2/h) regions, but significantly lower flux (P=<0.05) for the leg region (1.56+/-0.120 microg/cm2/h. Interestingly, there was a significantly longer lag time for the penetration of fentanyl through the groin region (7.87+/-0.51 h) compared to the other two sites (5.66+/-0.97 h and 5.75+/-0.43 h for the thorax and leg regions respectively). The results suggest that a fentanyl TTS applied to the leg region may have a small but significantly lower amount of fentanyl available systemically, compared to patches applied to the thorax or groin regions, which may affect the level of analgesia subsequently achieved in the horse.

Regional differences in the in vitro penetration of methylsalicylate through equine skin.

Commercial formulations of non-steroidal anti-inflammatory drugs (NSAIDs) are developed for human use but the extent to which they will pass through equine skin is unknown. Skin was harvested from five Thoroughbred geldings from the thorax, groin and leg (dorsal metacarpal) regions and frozen (-20 degrees C) until required. Two grams of methylsalicylate (MeSa) gel was applied to defrosted full-thickness samples in diffusion cells and the penetration of MeSa and its active metabolite, salicylate (Sa), through skin samples were measured over 24 h. Significantly higher (P < or = 0.02) total salicylate (AUC; MeSa + Sa) penetrated through skin from the leg region (5491.3 h mg/L), compared to thorax (3710.7 h mg/L) and groin (3571.5 h mg/L). In addition, there was a significantly higher (P0.01) rate of penetration of total Sa through leg skin in the first 6h after application. It was concluded that the commercial formulation of MeSa would achieve therapeutic levels of total salicylate beneath sites of topical application, with a faster and more pronounced response through the leg region, compared to the upper body.

Percutaneous absorption of steroids: determination of in vitro permeability and tissue reservoir characteristics in human skin layers.

The skin localization of steroids following topical application is largely unknown. We determined the distribution of five steroids in human skin using excised epidermal, dermal, and full-thickness membranes in vitro. There was no significant difference in steroid maximum flux through epidermal and full-thickness membranes, other than significantly lower fluxes for the most polar steroid, aldosterone. Hydrocortisone had the highest dermal diffusivity and dermal penetration, and the accumulation of hydrocortisone and corticosterone was higher than that of the other steroids. Slower penetration and higher accumulation in the viable epidermis of progesterone in full-thickness skin were consistent with dermal penetration limitation effects associated with high lipophilicity.

Regional differences in the in vitro penetration of hydrocortisone through equine skin.

Little is known about the transdermal penetration of hydrocortisone in the horse and, although commercial formulations containing hydrocortisone are registered for topical use in the horse, there have been no studies investigating the movement of this glucocorticoid through different regions of equine skin. Skin was harvested from the thorax, groin and leg (dorsal metacarpal) regions of five Thoroughbred geldings and frozen (-20 degrees C) until required. Defrosted skin was placed in Franz-type diffusion cells and the amount of radiolabelled ((3)H) hydrocortisone, in a saturated solution of unlabelled hydrocortisone in 50% ethanol (w/w), which penetrated through and remained within skin samples was measured over 24 h. Significantly higher (P < 0.001) maximum flux (J(max); mol/cm(2)/h) was measured when hydrocortisone was applied to skin from the leg, compared to thorax and groin, although significantly less hydrocortisone (P < 0.001) was retained within skin from the leg at 24 h. Topical application of hydrocortisone in a vehicle containing ethanol would penetrate faster through leg skin from the lower leg when compared with the thorax or groin, which depending on cutaneous blood flow, may result in higher systemic drug concentrations or greater efficiency in treating local inflamed tissue.

Transdermal drug delivery: basic principles for the veterinarian.

The use of topical pharmaceutical formulations is increasingly popular in veterinary medicine. A potential concern is that not all formulations are registered for the intended species, yet current knowledge strongly suggests that simple extrapolation of transdermal drug pharmacokinetics and pharmacodynamics between species, including humans, cannot be done. In this review, an overview is provided of the underlying basic principles determining the movement of topically applied molecules into and through the skin. Various factors that may affect transdermal drug penetration between species, between individuals of a particular species and regional differences in an individual are also discussed. A good understanding of the basic principles of transdermal drug delivery is critical to avoid adverse effects or lack of efficacy when applying topical formulations in veterinary medicine.

Sunscreen penetration of human skin and related keratinocyte toxicity after topical application.

Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents (avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration-human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation (DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes.

Factors affecting the formation of a skin reservoir for topically applied solutes.

The reservoir function of the skin is an important determinant of the duration of action of a topical solute. The reservoir can exist in the stratum corneum, in the viable avascular tissue (viable epidermis and supracapillary dermis) and in the dermis. A steroid reservoir in the stratum corneum has been demonstrated by the reactivation of a vasoconstrictor effect by occlusion or application of a placebo cream to the skin some time after the original topical application of steroid. Other solutes have also been reported to show a reservoir effect in the skin after topical application. A simple compartmental model is used to understand why reactivation of vasoconstriction some time after a topical steroid application shows dependency on time, topical solute concentration and the product used to cause reactivation. The model is also used to show which solutes are likely to show a reservoir effect and could be potentially affected by desquamation, especially when the turnover of the skin is abnormally rapid. A similar form of the model can be used to understand the promotion of reservoir function in the viable tissue and in the dermis in terms of effective removal by blood perfusing the tissues.

The effect of region of application on absorption of ethanol and hexanol through canine skin.

The effect of region of application on the percutaneous penetration of solutes with differing lipophilicity was investigated in canine skin. Skin from the thorax, neck, back, groin, and axilla regions was harvested from Greyhound dogs and placed in Franz-type diffusion cells. Radiolabelled (14C) ethanol (Log P 0.19) or hexanol (Log P 1.94) was applied to each skin section for a total of 5h. The permeability coefficient (kP, cm h(-1)) and residue of alcohol remaining in the skin were significantly (P=0.001) higher for hexanol compared to ethanol. In contrast, ethanol had a far greater maximum flux (Jmax, mol (cm2)(-1) h(-1)) than hexanol (P=0.001). A comparison of regional differences shows the kP and Jmax for ethanol in the groin was significantly lower (P=0.035) than the back. The kP and Jmax for hexanol were significantly higher (P=0.001) in the axilla than the other four skin sites. An understanding of factors influencing percutaneous drug movement is important when formulating topical preparations for the dog.

Physical enhancement of transdermal drug application: is delivery technology keeping up with pharmaceutical development?

Advances in molecular biology have given us a wide range of protein and peptide-based drugs that are unsuitable for oral delivery because of their high degree of first-pass metabolism. Though parenteral delivery is the obvious answer, for the successful development of commercial chronic and self-administration usage formulations it is not the ideal choice. Transdermal delivery is emerging as the biggest application target for these agents, however, the skin is extremely efficient at keeping out such large molecular weight compounds and therapeutic levels are never going to be realistically achieved by passive absorption. Physical enhancement mechanisms including: iontophoresis, electroporation, ultrasound, photomechanical waves, microneedles and jet-propelled particles are emerging as solutions to this topical delivery dilemma. Adding proteins and peptides to the list of other large molecular weight drugs with insufficient passive transdermal fluxes to be therapeutically useful, we have a collection of pharmacological agents waiting for efficient delivery methods to be introduced. This article reviews the current state of physical transdermal delivery technology, assesses the pros and cons of each technique and summarises the evidence-base of their drug delivery capabilities.

Effect of solute lipophilicity on penetration through canine skin.

OBJECTIVE: To investigate the effect of lipophilicity on the percutaneous penetration of a homologous series of alcohols through canine skin. DESIGN: Skin harvested from Greyhound thorax was placed in Franz-type diffusion cells and the in vitro passage of radiolabelled (14C) alcohols (ethanol, butanol, hexanol and octanol (Log P 0.19-3.0)) through separate skin sections was measured in replicates of five. Permeability coefficient (kP, cm/h), maximum flux (Jmax, mol/cm2/h) and residue remaining within the skin were determined. RESULTS: The kP increased with increasing lipophilicity (6.2 x 10(-4) +/- 1.6 x 10(-4) cm/h for ethanol to 1.8 x 10(-2) +/- 3.6 x 10(-3) cm/h for octanol). Alcohol residues remaining within each skin sample followed a similar pattern. An exponential decrease in Jmax with increasing lipophilicity was observed. CONCLUSION: Changes in canine skin permeability occur with increasing alcohol lipophilicity. This finding has practical consequences for the design of topical formulations and optimisation of drug delivery through animal skin.

Probing the effect of vehicles on topical delivery: understanding the basic relationship between solvent and solute penetration using silicone membranes.

PURPOSE: In the present study we examined the relationship between solvent uptake into a model membrane (silicone) with the physical properties of the solvents (e.g., solubility parameter, melting point, molecular weight) and its potential predictability. We then assessed the subsequent topical penetration and retention kinetics of hydrocortisone from various solvents to define whether modifications to either solute diffusivity or partitioning were dominant in increasing permeability through solvent-modified membranes. METHODS: Membrane sorption of solvents was determined from weight differences following immersion in individual solvents, corrected for differences in density. Permeability and retention kinetics of 3H-hydrocortisone, applied as saturated solutions in the various solvents, were determined over 48 h in horizontal Franz-type glass diffusion cells. RESULTS: Solvent sorption into the membrane could be related to differences in solubility parameters, MW and hydrogen bonding (r2=0.76). The actual and predicted volume of solvent sorbed into the membrane was also found to be linearly related to Log hydrocortisone flux, with changes in both diffusivity and partitioning of hydrocortisone observed for the different solvent vehicles. CONCLUSIONS: A simple structure-based predictive model can be applied to the sorption of solvents into silicone membranes. Changes in solute diffusivity and partitioning appeared to contribute to the increased hydrocortisone flux observed with the various solvent vehicles. The application of this predictive model to the more complex skin membrane remains to be determined.

Can increasing the viscosity of formulations be used to reduce the human skin penetration of the sunscreen oxybenzone?

The effect of adding thickening agents on the penetration of a sunscreen benzophenone-3 through epidermal and a high-density polyethylene membrane was studied using both very thick (infinite dose) and thin (in use) applications. Contradictory results were obtained. Thickening agents retard skin penetration, in a manner consistent with a diffusional resistance in the formulation, when applied as an infinite dose. In contrast, when applied as in thin (in use) doses, thickening agents promote penetration, most likely through greater stratum corneum diffusivity arising from an enhanced hydration by the thicker formulations. The two key implications from this work are (i) a recognition of the danger in the potential extrapolation of infinite dosing to in use situations, and (ii) to recognize that thicker formulations may sometimes enhance the penetration of other topical agents when applied "in use".

The effect of occlusion on epidermal penetration of parabens from a commercial allergy test ointment, acetone and ethanol vehicles.

The efficacy of topical allergy screening systems relies on the ability of test agents to effectively penetrate the stratum corneum from applied vehicles and reach the viable cells involved in the cutaneous immune response system. There is very little evidence in the dermatologic literature to justify the choice and suitability of vehicles used in many allergy test systems and the effectiveness of occlusion, reported to have variable effects on solute penetration, often employed in combination with these systems. In this study we evaluated the in vitro human epidermal penetration of a mixture of paraben ester preservatives from a commercially available test ointment and two commonly employed solvent vehicles (acetone and ethanol), together with the effect of occlusion on the rate of delivery from these systems. Parabens were applied as finite doses (5 mg per cm2) to epidermal membranes mounted in horizontal Franz-type diffusion cells. At intervals of 2 h for a total of 10 h the receptor phase (20% ethanol in distilled water) was completely removed and replaced. Occlusion was effected by the placement of a piece of high density polyethylene (20 microm) over the application site immediately after dosing. Concentrations of parabens in receptor fluid and remaining in the epidermis at the end of the study were determined by high-performance liquid chromatography. There was a significant change in the epidermal flux of parabens from each of the vehicles following occlusion. Whereas increases were observed for the acetone and ethanol vehicles a decrease was seen following occlusion of the ointment formulation. Changes in flux appeared to result from a significant decrease in the epidermal partitioning of the esters following occlusion of the ointment and primarily by an increase in paraben epidermal diffusivity (estimated from changes in flux/retention) following occlusion of the solvent vehicles. These studies show that the effects of occlusion are strongly vehicle dependent, having wide implications for optimization of this technique with a range of topically applied solutes.

Ion-pair formation as a strategy to enhance topical delivery of salicylic acid.

An in-vitro study was carried out to determine the possibility of improving the efficiency of transdermal delivery of salicylate through human epidermis by ion-pair formers (alkylamines and quaternary ammonium ions). Further, the relationship between the physicochemical properties of the counter-ions and salicylate flux was examined. It was found that flux can be related to the conductivity associated with the penetrant solution, molecular size of the counter-ion and lipophilicity expressed as either octanol/water partition coefficient of the ion pairs or the carbon chain-length of the counter-ions. Equations have been developed to predict salicylate flux from these physicochemical parameters.