Using protease inhibitors to improve protein stability in the presence of skin: A case study on the stability of insulin like growth factor 1.

Insulin-like growth factor 1 (IGF-1) is indicated for growth failure in pediatric patients with primary IGF-1 deficiency and for patients with neutralizing antibodies to growth hormone. IGF-1 was cloned, expressed and purified in-house. Preliminary stability studies prior to the transdermal delivery experiments showed that although stable in contact with stratum corneum, the solution concentration of IGF-1 decreased to 23.63 ± 2.48 and 21.58 ± 2.62% of the initial value upon exposure for 8 h to porcine dermis of 250 and 750 µm thickness. This led to an investigation into how it might be possible to improve the stability of IGF-1 in the presence of porcine/human skin. The stability of IGF-1 in the presence of dermis improved upon heating the skin samples at 60 °C for 2 min suggesting that IGF-1 was subject to enzymatic degradation. Although addition of the protease inhibitor, phenylmethanesulfonyl fluoride (PMSF) alone, did not improve stability, the use of a protease inhibitor cocktail completely blocked proteolytic degradation of IGF-1; the solution concentration after an 8 h exposure to porcine skin was equivalent to the initial level (103.87 ± 9.15%). The results obtained with porcine skin were confirmed with human skin (IGF-1 recovery was 99.31 ± 9.98%). These findings suggest that the inclusion of protease inhibitor cocktails may be useful in limiting the degradation of therapeutic proteins during iontophoresis and transdermal delivery in general - this could be of particular interest for local delivery of peptide/protein therapeutics for dermatological applications.

Erratum: Withdrawal notice to 'WITHDRAWN: Specific protein-protein interactions limit the cutaneous iontophoretic transport of interferon beta-1B and a poly-ARG interferon beta-1B analogue' [International Journal of Pharmaceutics 589 (2020) 119913].

[This corrects the article DOI: 10.1016/j.ijpx.2020.100051.].

Specific protein-protein interactions limit the cutaneous iontophoretic transport of interferon beta-1b and a poly-Arg interferon beta-1b analogue.

The first objective was to investigate the transdermal iontophoresis of interferon beta 1b (IFN); the second was to determine whether the addition of 10 Arg residues at the N-terminus, creating a highly charged poly-Arg analogue (Arg10-IFN), increased delivery. Cumulative permeation of IFN and Arg10-IFN after iontophoresis at 0.5 mA/cm2 for 8 h was 6.97 ± 4.82 and 9.55 ± 1.63 ng/cm2, respectively - i.e. >1000-fold less than that of ribonuclease A, cytochrome c and human basic fibroblast growth factor. Co-iontophoresis of acetaminophen showed that, in contrast to lysozyme, neither IFN nor Arg10-IFN interacted with skin to decrease convective solvent flow. Furthermore, there was no statistically significant difference between (i) iontophoretic delivery of IFN across intact or laser porated skin and (ii) passive or iontophoretic delivery of IFN across laser porated skin. Chromatographic characterisation supported the hypothesis that IFN was bound strongly to albumin. The formation of a ~86 kDa complex with albumin was probably responsible for the poor cutaneous delivery of IFN/Arg10-IFN despite the use of iontophoresis and/or laser microporation. Biopharmaceuticals might interact with specific proteins during iontophoretic transport and so decrease their (per)cutaneous delivery without affecting electroosmotic solvent flow, which is usually considered as a reliable marker to report on permeant binding during electrotransport across the skin.

Specific protein-protein interactions limit the cutaneous iontophoretic transport of interferon beta-1B and a poly-ARG interferon beta-1B analogue.

The first objective was to investigate the transdermal iontophoresis of interferon beta 1b (IFN); the second was to determine whether the addition of 10 Arg residues at the N-terminus, creating a highly charged poly-Arg analogue (Arg10-IFN), increased delivery. Cumulative permeation of IFN and Arg10-IFN after iontophoresis at 0.5 mA/cm2 for 8 h was 6.97 ± 4.82 and 9.55 ± 1.63 ng/cm2, respectively - i.e. >1000-fold less than that of ribonuclease A, cytochrome c and human basic fibroblast growth factor. Co-iontophoresis of acetaminophen showed that, in contrast to lysozyme, neither IFN nor Arg10-IFN interacted with skin to decrease convective solvent flow. Furthermore, there was no statistically significant difference between (i) iontophoretic delivery of IFN across intact or laser porated skin and (ii) passive or iontophoretic delivery of IFN across laser porated skin. Chromatographic characterisation supported the hypothesis that IFN was bound strongly to albumin. The formation of a ~ 86 kDa complex with albumin was probably responsible for the poor cutaneous delivery of IFN/Arg10-IFN despite the use of iontophoresis and/or laser microporation. Biopharmaceuticals might interact with specific proteins during iontophoretic transport and so decrease their (per)cutaneous delivery without affecting electroosmotic solvent flow, which is usually considered as a reliable marker to report on permeant binding during electrotransport across the skin.

Transdermal therapeutic systems for memantine delivery. Comparison of passive and iontophoretic transport.

Memantine is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist used in the treatment of moderate to severe dementia including the symptoms of Alzheimer's disease (AD). It is administered orally but compliance, swallowing problems and the routine use of multiple medications in elderly AD patients means that an alternative route of administration would be of interest. The aim of the present study was to develop memantine hydrochloride occlusive transdermal therapeutic systems (TTS) for passive and iontophoretic delivery across the skin. Polyvinyl pyrrolidone (PVP) and a mixture with polyvinyl alcohol (PVA) were employed as polymeric matrices. The study involved the TTS characterization in addition to quantification of the memantine transport across porcine skin in vitro. The evaluation of the TTS physical properties suggested that systems were made more mechanically resistant by including PVA (6%) or high concentrations of PVP (24%). Moreover, a linear correlation was observed between the concentration of PVP and the bioadhesion of the systems. Drug delivery experiments showed that the highest transdermal flux provided by a passive TTS (PVP 24% w/w limonene) was 8.89±0.81µgcm-2h-1 whereas the highest iontophoretic transport was 46.4±3.6µgcm-2h-1. These innovative TTS would enable two dosage regimens that could lead to therapeutic plasma concentrations.

Topical delivery of diclofenac into and across equine skin from a novel liquid diclofenac epolamine formulation.

The aim was to investigate diclofenac delivery into and across equine skin in vitro using Franz diffusion cells from a novel diclofenac epolamine (DIC-EP; 1.3%) formulation and to compare the results to those of Surpass® (1% diclofenac sodium liposomal cream) and a 1% aqueous solution of diclofenac sodium. Skin was harvested from the lower legs of Freiberger geldings immediately after slaughter and sliced to a thickness of ~2 mm. Skin samples were divided into two groups [Group 1: 1 year old (n = 2) and Group 2: 6-8 years old (n = 3)]. Cumulative permeation of diclofenac in Groups 1 and 2 after 24 h using diclofenac sodium solution was 1.91 ± 0.27 and 1.76 ± 0.34 µg/cm2 , respectively. The values for Surpass® and DIC-EP were 3.2 ± 0.8/3.3 ± 0.7 µg/cm2 and 230 ± 59/89.2 ± 32.5 µg/cm2 , respectively. Thus, diclofenac permeation from DIC-EP was significantly greater and appeared to show an age-dependent effect. Mathematical modelling showed that the DIC-EP formulation significantly increased diclofenac partitioning into the skin and a linear correlation was observed between steady-state flux and the partition parameter. Greater skin deposition of diclofenac was also observed with DIC-EP. These preliminary results suggest that the DIC-EP formulation may be effective in treating inflammatory conditions in horses.

Understanding the poor iontophoretic transport of lysozyme across the skin: when high charge and high electrophoretic mobility are not enough.

The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8h at 0.5mA/cm(2) (0.7mM; pH6) was surprisingly low (5.37±3.46µg/cm(2) in 8h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0±496.1 and 170.71±92.13µg/cm(2), respectively) - despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI ~4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12-16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) - suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750µm, tape-stripped skin and heat-separated dermis). Although no difference was seen between permeation across 250 and 750µm dermatomed skin (13.57±12.20 and 5.37±3.46µg/cm(2), respectively), there was a statistically significant increase across tape-stripped skin and heat-separated dermis (36.86±7.48 and 43.42±13.11µg/cm(2), respectively) - although transport was still much less than that seen across intact skin for Cyt c or RNase A. Furthermore, electroosmotic inhibition factors fell to 2.2 and 1.0 for tape-stripped skin and heat-separated dermis - indicating that lysozyme affected convective solvent flow through interactions with the epidermis and predominantly the stratum corneum. Finally, cation exchange and hydrophobic interaction chromatography confirmed that although lysozyme had greater positive charge than Cyt c or RNase A under the conditions used for iontophoresis, it also possessed the highest surface hydrophobicity, which may have facilitated the interactions with the transport pathways and encouraged aggregation in the skin microenvironment. Thus, high charge and electrophoretic mobility seem to be inadequate descriptors to predict the transdermal iontophoretic transport of proteins whose complex three dimensional structures can facilitate interactions with cutaneous transport pathways.

Turning theory into practice: the development of modern transdermal drug delivery systems and future trends.

Despite its remarkable barrier function, the skin remains an attractive site for systemic drug delivery given its easy accessibility, large surface area and the possibility to bypass the gastrointestinal tract and the liver and so modify drug absorption kinetics. The pioneering work of Scheuplein, Higuchi and others in the 1960s helped to explain the processes involved in passive percutaneous absorption and led to the development of mathematical models to describe transdermal drug delivery. The intervening years have seen these theories turned to practice and a significant number of transdermal systems are now available including some that employ active drug delivery. This review briefly discusses the evolution of transdermal therapeutic systems over the years and the potential of newer transdermal technologies to deliver hydrophilic drugs and macromolecules through the skin.

Controlled intra- and transdermal protein delivery using a minimally invasive Erbium:YAG fractional laser ablation technology.

The aim of the study was (i) to investigate the feasibility of using fractional laser ablation to create micropore arrays in order to deliver proteins into and across the skin and (ii) to demonstrate how transport rates could be controlled by variation of poration and formulation conditions. Four proteins with very different structures and properties were investigated - equine heart cytochrome c (Cyt c; 12.4 kDa), recombinant human growth hormone expressed in Escherichia coli (hGH; 22 kDa), urinary follicle stimulating hormone (FSH; 30 kDa) and FITC-labelled bovine serum albumin (FITC-BSA; 70 kDa). The transport experiments were performed using a scanning Er:YAG diode pumped laser (P.L.E.A.S.E.®; Precise Laser Epidermal System). The distribution of FITC-BSA in the micropores following P.L.E.A.S.E.® poration was visualised by using confocal laser scanning microscopy (CLSM). Porcine skin was used for the device parameter and CLSM studies; its validity as a model was confirmed by subsequent comparison with transport of Cyt c and FITC-BSA across P.L.E.A.S.E.® porated human skin. No protein transport (deposition or permeation) was observed across intact skin; however, P.L.E.A.S.E.® poration enabled total delivery after 24h of 48.2±8.9, 8.1±4.2, 0.2±0.1 and 273.3±30.6 µg/cm(2) for Cyt c, hGH, FSH and FITC-BSA, respectively, using 900 pores/135.9 cm(2). Calculation of permeability coefficients showed that there was no linear dependence of transport on molecular weight ((1.6±0.3), (0.1±0.05), (0.08±0.03) and (0.9±0.1)×10(-3) cm/h, for Cyt c, hGH, FSH and FITC-BSA, respectively); indeed, a U-shaped curve was observed. This suggested that molecular weight was not a sufficiently sensitive descriptor and that transport was more likely to be determined by the surface properties of the respective proteins since these would govern interactions with the local microenvironment. Increasing pore density (i.e. the number of micropores per unit area) had a statistically significant effect on the cumulative permeation of both Cyt c (at 100, 150, 300 and 600 pores/cm(2), permeation was 11.2±2.4, 15.3±11.8, 33.8±10.5 and 51.2±15.8 4 µg/cm(2), respectively) and FITC-BSA (at 50, 100, 150 and 300 pores/cm(2), it was 58.5±15.3, 132.6±40.0, 192.7±24.4, 293.3±76.5 µg/cm(2), respectively). Linear relationships were established in both cases. However, only the delivery of FITC-BSA was improved upon increasing fluence (53.3±22.5, 293.3±76.5, 329.6±11.5 and 222.1±29.4 µg/cm(2) at 22.65, 45.3, 90.6 and 135.9 J/cm(2), respectively). The impact of fluence - and hence pore depth - on transport will depend on the relative diffusivities of the protein in the micropore and in the 'bulk' epidermis/dermis. Experiments with Cyt c and FSH confirmed that delivery was dependent upon concentration, and it was shown that therapeutic delivery of the latter was feasible. Cumulative permeation of Cyt c and FITC-BSA was also shown to be statistically equivalent across porcine and human skin. In conclusion, it was demonstrated that laser microporation enabled protein delivery into and across the skin and that this could be modulated via the poration parameters and was also dependent upon the concentration gradient in the pore. However, the role of protein physicochemical properties and their influence on transport rates remains to be elucidated and will be explored in future studies.

Noninvasive transdermal iontophoretic delivery of biologically active human basic fibroblast growth factor.

Human basic fibroblast growth factor (hbFGF; 17.4 kDa) has shown promise in the treatment of several dermatological conditions; symptomatic improvement was also observed in patients with peripheral arterial disease after arterial infusion. The objective of this study was to demonstrate the feasibility of using transdermal iontophoresis to deliver biologically active hbFGF noninvasively into and across the skin. The protein was cloned, expressed and purified in-house. Porcine skin was used to investigate transdermal iontophoretic transport of hbFGF as a function of current density (0.15, 0.3, and 0.5 mA/cm(2)); results were subsequently confirmed using human skin. Cumulative hbFGF permeation and skin deposition were quantified by ELISA. The absence of proteolytic degradation during skin transit was confirmed by SDS-PAGE. Biological activity postdelivery was determined using cell proliferation assays in human foreskin fibroblast (HFF) and NIH 3T3 cell lines. Confocal laser scanning microscopy (CLSM) was used to visualize the distribution of rhodamine-tagged hbFGF in the skin. Cumulative iontophoretic permeation at 0.3 mA/cm(2) was statistically superior to that at 0.15 mA/cm(2); however, there was no further improvement at 0.5 mA/cm(2). Significant skin deposition of hbFGF was observed, and this dominated transport; for example, after iontophoresis for 8 h at 0.5 mA/cm(2), skin deposition (77.74 ± 37.36 µg/cm(2)) was 4.4-fold higher than cumulative permeation (17.64 ± 5.18 µg/cm(2)). The superior skin deposition may be advantageous for dermatological applications. The HFF and NIH 3T3 cell proliferation assays confirmed that biological activity of hbFGF was retained postdelivery. Coiontophoresis of acetaminophen showed that the dominant transport mechanism switched from electroosmosis to electromigration upon increasing current density from 0.15 to 0.3 mA/cm(2). Experiments using human skin confirmed that iontophoretic permeation of hbFGF across porcine and human membranes was statistically equivalent. CLSM images of rhodamine-tagged hbFGF postiontophoresis indicated that the protein was evenly distributed throughout the epidermis and dermis. In conclusion, the results confirmed that transdermal iontophoresis was indeed able to deliver structurally intact, functional hbFGF noninvasively into and across the skin. The amounts of protein delivered were similar to those in reports from preclinical and clinical studies.

Novel micelle formulations to increase cutaneous bioavailability of azole antifungals.

Efficient topical drug administration for the treatment of superficial fungal infections would deliver the therapeutic agent to the target compartment and reduce the risk of systemic side effects. However, the physicochemical properties of the commonly used azole antifungals make their formulation a considerable challenge. The objective of the present investigation was to develop aqueous micelle solutions of clotrimazole (CLZ), econazole nitrate (ECZ) and fluconazole (FLZ) using novel amphiphilic methoxy-poly(ethylene glycol)-hexyl substituted polylactide (MPEG-hexPLA) block copolymers. The CLZ, ECZ and FLZ formulations were characterized with respect to drug loading and micelle size. The optimal drug formulation was selected for skin transport studies that were performed using full thickness porcine and human skin. Penetration pathways and micellar distribution in the skin were visualized using fluorescein loaded micelles and confocal laser scanning microscopy. The hydrodynamic diameters of the azole loaded micelles were between 70 and 165nm and the corresponding number weighted diameters (d(n)) were 30 to 40nm. Somewhat surprisingly, the lowest loading efficiency (<20%) was observed for CLZ (the most hydrophobic of the three azoles tested); in contrast, under the same conditions, ECZ was incorporated with an efficiency of 98.3% in MPEG-dihexPLA micelles. Based on the characterization data and preliminary transport experiments, ECZ loaded MPEG-dihexPLA micelles (concentration 1.3mg/mL; d(n)<40nm) were selected for further study. ECZ delivery was compared to that from Pevaryl® cream (1% w/w ECZ), a marketed liposomal formulation for topical application. ECZ deposition in porcine skin following 6h application using the MPEG-dihexPLA micelles was >13-fold higher than that from Pevaryl® cream (22.8±3.8 and 1.7±0.6µg/cm(2), respectively). A significant enhancement was also observed with human skin; the amounts of ECZ deposited were 11.3±1.6 and 1.5±0.4µg/cm(2), respectively (i.e., a 7.5-fold improvement in delivery). Confocal laser scanning microscopy images supported the hypothesis that the higher delivery observed in porcine skin was due to a larger contribution of the follicular penetration pathway. In conclusion, the significant increase in ECZ skin deposition achieved using the MPEG-dihexPLA micelles demonstrates their ability to improve cutaneous drug bioavailability; this may translate into improved clinical efficacy in vivo. Moreover, these micelle systems may also enable targeting of the hair follicle and this will be investigated in future studies.

Electrically-assisted delivery of an anionic protein across intact skin: cathodal iontophoresis of biologically active ribonuclease T1.

Cathodal iontophoresis of anionic macromolecules has been considered a major challenge owing to (i) the presence of a negative charge on the skin under physiological conditions and (ii) the electroosmotic solvent flow in the (opposite) anode-to-cathode direction. Moreover, electroosmosis, and not electromigration, was considered as the likely electrotransport mechanism for high molecular weight cations. However, it was recently shown that electromigration governed anodal iontophoretic transport of Cytochrome c (12.4 kDa) and Ribonuclease A (RNAse A; 13.6 kDa). Thus, the objective of this study was to investigate the feasibility of iontophoresing a negatively charged protein, the enzyme Ribonuclease T1 (RNAse T1, 11.1 kDa), from the cathode across intact skin. Cumulative permeation and skin deposition of RNAse T1 were investigated as a function of current density (0.15, 0.3 and 0.5 mA/cm(2) applied for 8h) using porcine ear skin and quantified by an enzymatic activity assay. Although RNAse T1 permeation was dependent upon current density (22.41 ± 8.10, 76.41 ± 56.98 and 142.19 ± 62.23µg/cm(2), respectively), no such relationship was observed with respect to skin deposition (9.78 ± 2.39, 7.76 ± 4.34 and 8.70 ± 2.94 µg/cm(2), respectively). MALDI-TOF spectra and the activity assay confirmed that RNAse T1 retained structural integrity and enzymatic function post-iontophoresis. Acetaminophen iontophoresis demonstrated the anode-to-cathode directionality of electroosmotic solvent flow confirming that RNAse T1 electrotransport was due entirely to electromigration. Interestingly, despite its lower net charge and higher molecular weight, electromigration of cationic Ribonuclease A was superior to that of RNAse T1 after iontophoresis at 0.5 mA/cm(2) for 8h. These results provide further evidence that charge to mass ratio and hence electric mobility might not alone be sufficient to predict protein electrotransport across the skin; three dimensional structures and the spatial distribution of physicochemical properties must also be considered. The skin extraction data suggest that negatively charged molecules may have fewer potential binding sites in the skin than their cationic counterparts. This was supported by confocal laser scanning microscopy images which showed that whereas fluorescence from RNAse A was distributed throughout the epidermis and dermis, RNAse T1 appeared to be bound to the epidermis alone. In conclusion, this is the first report demonstrating successful non-invasive cathodal iontophoresis of a negatively charged functional protein (RNAse T1) across intact skin.

Human growth hormone: new delivery systems, alternative routes of administration, and their pharmacological relevance.

The availability of recombinant human growth hormone (GH) has broadened its range of clinical applications. Approved indications for GH therapy include treatment of growth hormone deficiency (in children and in adults), Turner syndrome, Prader-Willi syndrome, chronic renal insufficiency and more recently, idiopathic short stature in children, AIDS-related wasting and fat accumulation associated with lipodystrophy in adults. Therapy with GH usually begins at a low dose and is gradually titrated to obtain optimal efficacy while minimizing side effects. It is usually administered on a daily basis by subcutaneous injection, since this was considered to impact upon patient compliance, extended-release GH preparations were developed and new delivery platforms - e.g., auto-injectors and needle-free devices - were introduced in order to improve not only compliance and convenience but also dosing accuracy. In addition, alternative less invasive modes of administration such as the nasal, pulmonary and transdermal routes have also been investigated. Here, we provide an overview of the different technologies and routes of GH administration and discuss the principles, limitations and pharmacological profiles for each approach.

Effect of controlled laser microporation on drug transport kinetics into and across the skin.

The objectives of this study were to investigate a novel laser microporation technology ( P.L.E.A.S.E. Painless Laser Epidermal System) and to determine the effect of pore number and depth on the rate and extent of drug delivery across the skin. In addition, the micropores were visualized by confocal laser scanning microscopy and histological studies were used to determine the effect of laser fluence (energy applied per unit area) on pore depth. Porcine ear skin was used as the membrane for both the pore characterization and drug transport studies. Confocal images in the XY-plane revealed that the pores were typically 150-200 microm in diameter. Histological sections confirmed that fluence could be used to effectively control pore depth - low energy application (4.53 and 13.59 J/cm(2)) resulted in selective removal of the stratum corneum (20-30 microm), intermediate energies (e.g., 22.65 J/cm(2)) produced pores that penetrated the viable epidermis (60-100 microm) and higher application energies created pores that reached the dermis (>150-200 microm). The effects of pore number and pore depth on molecular transport were quantified by comparing lidocaine delivery kinetics across intact and porated skin samples. After 24h, cumulative skin permeation of lidocaine with 0 (control), 150, 300, 450 and 900 pores was 107+/-46, 774+/-110, 1400+/-344, 1653+/-437 and 1811+/-642 microg/cm(2), respectively; there was no statistically significant difference between 300, 450 and 900 pore data - probably due to the effect of drug depletion since >50% of the applied dose was delivered. Importantly, increasing fluence did not produce a statistically significant increase in lidocaine permeation; after 24h, cumulative lidocaine permeation was 1180+/-448, 1350+/-445, 1240+/-483 and 1653+/-436 microg/cm(2) at fluences of 22.65, 45.3, 90.6 and 135.9 J/cm(2), respectively. Thus, shallow pores were equally effective in delivering lidocaine. Increasing lidocaine concentration in the formulation from 10 to 25mg/ml produced a corresponding increase in permeation (at 24h, 1650+/-437 and 4005+/-1389 microg/cm(2), respectively). The validity of the porcine skin model was confirmed as transport across porcine and human skins was shown to be statistically equivalent (at 24h, 1811+/-642 and 2663+/-208 microg/cm(2), respectively). The clinical potential of the technology and its capacity to provide significantly faster delivery than conventional passive administration was demonstrated in short duration experiments involving application of a marketed lidocaine cream (LMX4) to laser-porated skin; after only 5 min of formulation application, lidocaine deposition was measured at 61.3+/-7.5 microg/cm(2). In conclusion, the results demonstrate the ability of P.L.E.A.S.E.(R) (i) to create well-defined conduits in the skin, (ii) to provide a controlled enhancement of transdermal transport and (iii) to enable improvement in both the rate and extent of drug delivery.

Non-invasive iontophoretic delivery of enzymatically active ribonuclease A (13.6 kDa) across intact porcine and human skins.

The purpose of the study was to demonstrate the feasibility of using transdermal iontophoresis to deliver a functional protein, ribonuclease A (RNAse; 13.6 kDa), non-invasively across the skin. Iontophoretic transport experiments were conducted using porcine skin in vitro and established the effect of current density and protein concentration on delivery kinetics. A methylene blue-based assay was used to quantify RNAse transport and to simultaneously demonstrate that protein functionality was retained post-iontophoresis. The results confirmed that intact functional RNAse was indeed delivered across the skin; cumulative permeation and steady state flux after 8h iontophoresis at 0.3 mA/cm(2) were 224.37+/-72.34 microg/cm(2) and 68.28+/-23.87 microg/cm(2)h, respectively. Significant amounts of protein were also deposited within the membrane (e.g., 1425.13+/-312.09 microg/cm(2) at 0.3 mA/cm(2)). In addition to the evidence provided by the enzymatic assay with regards to RNAse integrity and functionality, SDS-PAGE gels and MALDI-TOF spectra were also used to characterize RNAse present in the receiver phase (MALDI-TOF spectra: RNAse control, 13.690 kDa cf. RNAse from permeation samples, 13.692 kDa). Co-iontophoresis of acetaminophen showed that, despite its molecular weight, electromigration was the predominant electrotransport mechanism, accounting for >80% of RNAse total flux. Increasing RNAse concentration from 0.35 to 0.7 mM in the formulation did not result in a statistically significant increase in delivery. Iontophoretic transport of RNAse across human skin was statistically equivalent to that seen with porcine skin under the same conditions; cumulative permeation across human and porcine skin was 241.48+/-60.01 and 170.71+/-92.13 microg/cm(2), respectively. Laser scanning confocal microscopy was used to visualize the distribution of rhodamine B-labelled RNAse in the epidermis and dermis as a function of depth following 8h iontophoresis (results were compared to control experiments involving passive administration of the same formulation for 8h). Although fluorescence was localized at the skin surface following passive administration, it was visible throughout the membrane after current application. In conclusion, the results demonstrate that non-invasive transdermal iontophoresis can be used to deliver significant amounts of a structurally intact, functional protein across skin.

Transdermal iontophoresis of dexamethasone sodium phosphate in vitro and in vivo: effect of experimental parameters and skin type on drug stability and transport kinetics.

The aim of this study was to investigate the cathodal iontophoresis of dexamethasone sodium phosphate (DEX-P) in vitro and in vivo and to determine the feasibility of delivering therapeutic amounts of the drug for the treatment of chemotherapy-induced emesis. Stability studies, performed to investigate the susceptibility of the phosphate ester linkage to hydrolysis, confirmed that conversion of DEX-P to dexamethasone (DEX) upon exposure to samples of human, porcine and rat dermis for 7 h was limited (82.2+/-0.4%, 72.5+/-4.8% and 78.6+/-6.0% remained intact) and did not point to any major inter-species differences. Iontophoretic transport of DEX-P across dermatomed porcine skin (0.75 mm thickness) was studied in vitro as a function of concentration (10, 20, 40 mM) and current density (0.1, 0.3, 0.5 mA cm(-2)) using flow-through diffusion cells. Increasing concentration of DEX-P from 10 to 40 mM resulted in a approximately 4-fold increase in cumulative permeation (35.65+/-23.20 and 137.90+/-53.90 microg cm(-2), respectively). Good linearity was also observed between DEX-P flux and the applied current density (i(d); 0.1, 0.3, 0.5 mA cm(-2); J(DEX) (microg cm(2) h(-1))=237.98 i(d)-21.32, r(2)=0.96). Moreover, separation of the DEX-P formulation from the cathode compartment by means of a salt bridge - hence removing competition from Cl(-) ions generated at the cathode - produced a 2-fold increase in steady-state iontophoretic flux (40 mM, 0.3 mA cm(-2); 20.98+/-7.96 and 41.82+/-11.98 microg cm(-2) h(-1), respectively). Pharmacokinetic parameters were determined in Wistar rats (40 mM DEX-P; 0.5 mA cm(-2) for 5h with Ag/AgCl electrodes and salt bridges). Results showed that DEX-P was almost completely converted to DEX in the bloodstream, and significant DEX levels were achieved rapidly. The flux across rat skin in vivo (1.66+/-0.20 microg cm(-2) min(-1)), calculated from the input rate, was not statistically different from the flux obtained in vitro across dermatomed porcine skin (1.79+/-0.49 microg cm(-2) min(-1)). The results suggest that DEX-P delivery rates would be sufficient for the management of chemotherapy-induced emesis.

Transdermal delivery of cytochrome C--A 12.4 kDa protein--across intact skin by constant-current iontophoresis.

PURPOSE: To demonstrate the transdermal iontophoretic delivery of a small (12.4 kDa) protein across intact skin. MATERIALS AND METHODS: The iontophoretic transport of Cytochrome c (Cyt c) across porcine ear skin in vitro was investigated and quantified by HPLC. The effect of protein concentration (0.35 and 0.7 mM), current density (0.15, 0.3 or 0.5 mA.cm(-2) applied for 8 h) and competing ions was evaluated. Co-iontophoresis of acetaminophen was employed to quantify the respective contributions of electromigration (EM) and electroosmosis (EO). RESULTS: The data confirmed the transdermal iontophoretic delivery of intact Cyt c. Electromigration was the principal transport mechanism, accounting for approximately 90% of delivery; correlation between EM flux and electrophoretic mobility was consistent with earlier results using small molecules. Modest EO inhibition was observed at 0.5 mA.cm(-2). Cumulative permeation at 0.3 and 0.5 mA.cm(-2) was significantly greater than that at 0.15 mA.cm(-2); fluxes using 0.35 and 0.7 mM Cyt c in the absence of competing ions (J ( tot ) = 182.8 +/- 56.8 and 265.2 +/- 149.1 microg.cm(-2).h(-1), respectively) were statistically equivalent. Formulation in PBS (pH 8.2) confirmed the impact of competing charge carriers; inclusion of approximately 170 mM Na(+) resulted in a 3.9-fold decrease in total flux. CONCLUSIONS: Significant amounts ( approximately 0.9 mg.cm(-2) over 8 h) of Cyt c were delivered non-invasively across intact skin by transdermal electrotransport.

Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations.

It is, sometimes, desirable to maintain a constant plasma drug concentration within the therapeutically effective concentration range. The use of high viscosity hydromiscible vehicles such as hydrophilic gels, is one of various approaches for controlled drug delivery, and represents an important area of pharmaceutical research and development. Of these systems, Pluronic F-127 (PF-127) provides the pharmacist with an excellent drug delivery system for a number of routes of administration and is compatible with many different substances. Gels containing penetration enhancers have proven to be especially popular for administering anti-inflammatory medications since they are relatively easy to prepare and very efficacious.

Skin permeation enhancement by sucrose esters: a pH-dependent phenomenon.

The purpose of the present study was to evaluate the effect of sucrose esters (particularly, sucrose laureate and sucrose oleate in Transcutol) on the percutaneous penetration of a charged molecule as a function of ionization. We have investigated the influence of these sucrose esters on the in vitro diffusion profiles of lidocaine hydrochloride, a weak ionizable base (pKa=7.9), at different pH values, using porcine ear skin as the barrier membrane. As expected, lidocaine flux in the absence of an enhancer, increased from pH 5 to 9 with a corrresponding increase in the level of the unionized base. However, when skin was pretreated with 2% laureate in Transcutol (2% L-TC), drug permeation was higher at pH 5.0 and 7.0 than at 9.0. A different trend was observed in experiments with 2% oleate in Transcutol (2% O-TC), where skin flux was maximal at a more basic pH, when the degree of ionization is low. The results suggest that sucrose laureate enhances the penetration of the ionized form of the drug (12-fold greater flux relative to control), whereas sucrose oleate is more effective in promoting permeation of the unionized species. The structural properties of the sucrose esters as well as the degree of ionization of the drug are important characteristics affecting the transdermal flux of lidocaine.