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.

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.

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.

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.

Defining a model to predict the distribution of topically applied growth factors and other solutes in excisional full-thickness wounds.

Controversy over the efficacy of many topical wound treatments, particularly growth factors, is common, with many clinical practitioners still confused as to the real value of these agents. A serious lack of knowledge appears to exist concerning the diffusion and distribution of topically applied solutes in wounds. Without this basic understanding there seems little chance of accurately predicting the therapeutic window of drugs targeted at cellular activities, such as division and chemotaxis, and processes, such as collagen lattice deposition and contraction, occurring below the surface of the granulating layer. This study was designed to determine the absorption and distribution of a number of radiolabeled solutes (water, sodium chloride, lidocaine) and growth factors (basic fibroblast growth factor, epidermal growth factor) applied topically to full-thickness excisional wounds in rats during the early (2 d), mid (7 d), and late (12 d) stages of repair. Results showed that water and sodium penetrated deepest into wound sites and that changes in water distribution and retention in the wound paralleled the healing process. Multiple stepwise regression showed that molecular weight and tissue depth, but not day of healing, were significant factors in predicting the concentration of each solute in wound and underlying tissue sites. This finding was consistent with a tissue diffusion model developed in this study. Basic fibroblast growth factor and epidermal growth factor only penetrated slightly into the upper granulating layers of the wound site, and calculation of therapeutic doses, based on the percentage of applied solute reaching the deeper granulating layers, is presented.

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".

Models of the self: self-construals and gender.

The authors first describe individual differences in the structure of the self. In the independent self-construal, representations of others are separate from the self. In the interdependent self-construal, others are considered part of the self (H. Markus & S. Kitayama, 1991). In general, men in the United States are thought to construct and maintain an independent self-construal, whereas women are thought to construct and maintain an interdependent self-construal. The authors review the psychological literature to demonstrate that many gender differences in cognition, motivation, emotion, and social behavior may be explained in terms of men's and women's different self-construals. Recognition of the interdependent self-construal as a possible alternative conception of the self may stimulate new investigations into the ways the self influences a person's thinking, feeling, and behaving.

The relational-interdependent self-construal and relationships.

Three studies describe the development and validation of a measure of the relational-interdependent self-construal, which is defined as the tendency to think of oneself in terms of relationships with close others. Study 1 reports the development, psychometric properties, and tests of validity of this new measure. Individuals who scored high on the Relational-Interdependent Self-Construal (RISC) Scale characterized their important relationships as closer and more committed than did individuals who scored low on this measure (Study 1) and were more likely to take into account the needs and wishes of others when making decisions (Study 2). In Study 3, using a dyadic interaction paradigm with previously unacquainted participants, the partners of persons who scored high on the RISC scale viewed them as open and responsive to their needs and concerns; these perceptions were related to positive evaluations of the relationship.

Importance of dermal blood supply and epidermis on the transdermal iontophoretic delivery of monovalent cations.

The effect of dermal blood flow on the transdermal iontophoresis of five monovalent cationic solutes has been investigated using an in vivo rat model. The iontophoretic flux of solutes from topical application sites was shown, using anesthetized and sacrificed rats, to be independent of the dermal blood supply. The presence of a viable blood supply significantly affected the extent of penetration of solutes into deeper underlying tissues during iontophoresis. Higher tissue concentrations of solutes were found in the upper tissue layers of sacrificed rats, with no blood supply, compared to those in anesthetized rats. In all animals the highest concentration of solute after 2 h iontophoresis was found in the epidermis. Iontophoretic application of solutes to skin with the epidermis removed resulted in significantly lower fluxes than from sites where the skin was intact. These findings suggest that local blood flow determines systemic and underlying tissue solute absorption but not epidermal penetration fluxes during iontophoretic delivery. Finally, the dependence of iontophoretic transport of a solute on the solute's size in vivo was similar to relationships previously reported for excised human skin studies.

Influence of physicochemical parameters and perfusate flow rate on the distribution of solutes in the isolated perfused rat hindlimb determined by the impulse-response technique.

The relationship between solute distribution, physicochemical properties, and tissue physiology was determined by the impulse-response (IR) technique and statistical moment analysis in the isolated perfused rat hindlimb. The concentration of bovine serum albumin (BSA; 2, 4.7, and 7%, w/v), perfusate flow rate (4 and 8 mL/min), and solute physicochemical properties (lipophilicity, P, fraction unbound to protein, fu; fraction ionized, fi; and molecular weight MW) were varied to better understand the underlying determinants of solute distribution. An apparent low availability was found for a number of the solutes as a consequence of tissue sequestration. This low availability precludes the estimation of an apparent volume of distribution (V) for these solutes. The V of solute and tissue (skin, fat, and muscle) blood flow increased with perfusion flow rate (p < 0.01). The unbound distribution volume (Vu) of basic solutes was significantly linear with respect to P. Multiple linear regression analysis showed that the distribution volume of solute in tissue was significantly related to fu (p < 0.01), but not improved by including relationships to P, MW, and fi. Data obtained with this IR technique yield results consistent with in vivo studies in terms of the importance of fu as a determinant of V. This work has shown that the estimations of solute V by the IR technique in a single-pass preparation are unreliable for solutes with a low availability due to apparent solute sequestration into tissue. The parameter V may also be affected by changes in the perfused limb physiology associated with the perfusion conditions used. The Vs for lidocaine and diazepam vary with fu in accordance with deductions based on the results of steady-state studies.

Subcutaneous absorption kinetics and local tissue distribution of interferon and other solutes.

The subcutaneous absorption and consequent tissue distribution of interferon g was studied in an anaesthetized rat model. Interferon g showed a biphasic disappearance profile from a solution in a subcutaneous absorption cell. Both the initial rapid distribution phase and slower removal phase followed first order kinetics. The steady-state clearance of interferon g from the cell was 1.41 +/- 0.38 x 10(-3) mL min-1, and the absorption half-life (t1/2) was 3.8 +/- 1.1 h (n = 4). Noradrenaline did not significantly alter either the clearance or absorption of interferon g (1.18 +/- 0.44 x 10(-3) mL min-1, P > 0.05, absorption t1/2 4.96 +/- 1.9 h, P > 0.05). Given that the clearance of smaller solutes, such as tritiated water, is significantly reduced when noradrenaline is coadministered, it is suggested that interferon g is removed via the lymphatic system rather than by the local blood supply. The amount of interferon g recovered in the plasma, urine and muscle is minimal relative to other solutes where the recovery is almost complete.

Effect of perfusion flow rate on the tissue uptake of solutes after dermal application using the rat isolated perfused hindlimb preparation.

The rat perfused hindlimb preparation was investigated as a model to study the dermal clearance and tissue distribution of three solutes with differing physicochemical properties ([3H]water, [3H]diazepam, and [14C]lignocaine). The dermal clearance of each solute increased and the depth of tissue preparation decreased with increasing perfusion flow rate. The half-life for solute efflux from tissues into perfusate, and the peak times for amount of solute in tissue were in the order water > lignocaine > diazepam. Topical iontophoresis, which has previously been shown to achieve similar clearances from topical sites to those seen following passive dermal application, resulted in a clearance of lignocaine in the perfused hindlimb comparable with that obtained in the preparation following passive absorption through exposed dermis. The distribution of capillary blood flow in the perfused hindlimb was quantified using 51Cr-labelled 15-microns microspheres, with greater increases in flow found in deeper muscle tissues with increasing perfusion flow rate compared with superficial layers. Microsphere studies in anaesthetized rats also demonstrate slight differences in the distribution of flow within the hindlimb compared with the perfused preparation. We conclude that the rat perfused hindlimb preparation successfully combines many of the advantages of in-vitro and in-vivo methods and has potential for routine use in the study of solute dermal absorption kinetics.

Availability and mean transit times of phenol and its metabolites in the isolated perfused rat liver: normal and retrograde studies using tracer concentrations of phenol.

Phenolic compounds are frequently detoxified by the formation of sulphate and glucuronic acid conjugates in the liver. These conjugates are formed in the hepatocytes and then either transported into the bile or back into the blood. In this study, we examined the transport kinetics of phenol and its metabolites in the isolated perfused rat liver by monitoring the outflow profiles of these compounds after a bolus input in a single pass preparation. Phenol was almost exclusively metabolized to phenyl sulphate (97%) at the trace concentrations used, with the amount of phenol and metabolites excreted into the bile being minimal (3.5%). The metabolite formed was rapidly transported back into the perfusate, with mean transit times of 17.4 and 12.3 s anterograde and 24.9 and 24.2 s retrograde at flow rates of 15 and 30 mL min-1 respectively, which were intermediate between those of Evans blue and water. The outflow concentration-time profile for phenyl sulphate formation was unaffected by the addition of another organic anion (bromosulphophthalein). The effect of enzyme zonation on outflow concentration-time profiles was also investigated using retrograde perfusions. The transit time ratios for generated metabolite to water for anterograde perfusions (0.6) was found to be more than twice that for retrograde perfusions (0.23) at 15 mL min-1 and approximately 1.6 times greater at 30 mL min-1, being 0.58 and 0.37 respectively. The relative ratios obtained are consistent with previous findings that normalized variance of solutes in the retrograde perfusions is greater than that for anterograde perfusions.

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.

Effect of ion pairing with alkylamines on the in-vitro dermal penetration and local tissue disposition of salicylates.

Hydrophilic ionic drugs can be rendered lipophilic by ion-pair formation with hydrophobic counter-ions. This study examines the value of forming ion pairs between anionic salicylate and a series of amines as model cationic counter-ions to facilitate topical delivery and skin penetration. The in-vitro translocation of salicylate ions from a nonaqueous vehicle through human epidermis was estimated in the presence or absence of amines. The distribution into, and accumulation of the salicylate ion in various tissues following topical application to anaesthetised rats were also investigated. Although the epidermal permeation constants of the salicylate-amine ion pairs were lower than that of salicylate itself (enhancement ratios: 0.74-0.87), salicylate retention and localisation in the underlying rat tissues increased in the presence of some of the counter-ions studied. Salicylate concentrations (microg (g tissue)(-1)) in the dermis were 877.2+/-78.6 for salicylate alone and 1098+/-121.9-2586+/-332.5 for salicylate-amine ion pairs. The levels of salicylate in tissues up to the top muscle layer were 1.2-3.7-fold higher in the presence of the counter-ions. It is concluded that, although amine counter-ions have the ability to influence the penetration of salicylate, in-vitro permeability studies do not reflect the in-vivo increases in tissue concentrations resulting from probable changes in systemic clearance.

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.

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.

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.