Non-Invasive Delivery of Negatively Charged Nanobodies by Anodal Iontophoresis: When Electroosmosis Dominates Electromigration.

Iontophoresis enables the non-invasive transdermal delivery of moderately-sized proteins and the needle-free cutaneous delivery of antibodies. However, simple descriptors of protein characteristics cannot accurately predict the feasibility of iontophoretic transport. This study investigated the cathodal and anodal iontophoretic transport of the negatively charged M7D12H nanobody and a series of negatively charged variants with single amino acid substitutions. Surprisingly, M7D12H and its variants were only delivered transdermally by anodal iontophoresis. In contrast, transdermal permeation after cathodal iontophoresis and passive diffusion was

Cutaneous Delivery and Biodistribution of Cannabidiol in Human Skin after Topical Application of Colloidal Formulations.

The objective of this study was to investigate the cutaneous delivery of cannabidiol (CBD) from aqueous formulations developed for the targeted local treatment of dermatological conditions. CBD was formulated using a proprietary colloidal drug delivery system (VESIsorb®) into an aqueous colloidal solution at 2% (ACS 2%) and two colloidal gels (CG 1% and CG 2%, which contained 1% and 2% CBD, respectively). Two basic formulations containing CBD (5% in propylene glycol (PG 5%) and a 6.6% oil solution (OS 6.6%)) and two marketed CBD products (RP1 and RP2, containing 1% CBD) were used as comparators. Cutaneous delivery and cutaneous biodistribution experiments were performed using human abdominal skin (500-700 µm) under infinite- and finite-dose conditions with 0.5% Tween 80 in the PBS receiver phase. The quantification of CBD in the skin samples was performed using a validated UHPLC-MS/MS method and an internal standard (CBD-d3). The cutaneous deposition of CBD under finite-dose conditions demonstrated the superiority of CG 1%, CG 2%, and ACS 2% over the marketed products; CG 1% had the highest delivery efficiency (5.25%). Cutaneous biodistribution studies showed the superiority of the colloidal systems in delivering CBD to the viable epidermis, and the upper and lower papillary dermis, which are the target sites for the treatment of several dermatological conditions.

Enhancing Oral Delivery of Biologics: A Non-Competitive and Cross-Reactive Anti-Leptin Receptor Nanofitin Demonstrates a Gut-Crossing Capacity in an Ex Vivo Porcine Intestinal Model.

Biotherapeutics exhibit high efficacy in targeted therapy, but their oral delivery is impeded by the harsh conditions of the gastrointestinal (GI) tract and limited intestinal absorption. This article presents a strategy to overcome the challenges of poor intestinal permeability by using a protein shuttle that specifically binds to an intestinal target, the leptin receptor (LepR), and exploiting its capacity to perform a receptor-mediated transport. Our proof-of-concept study focuses on the characterization and transport of robust affinity proteins, known as Nanofitins, across an ex vivo porcine intestinal model. We describe the potential to deliver biologically active molecules across the mucosa by fusing them with the Nanofitin 1-F08 targeting the LepR. This particular Nanofitin was selected for its absence of competition with leptin, its cross-reactivity with LepR from human, mouse, and pig hosts, and its shuttle capability associated with its ability to induce a receptor-mediated transport. This study paves the way for future in vivo demonstration of a safe and efficient oral-to-systemic delivery of targeted therapies.

Finite sample corrections for average equivalence testing.

Average (bio)equivalence tests are used to assess if a parameter, like the mean difference in treatment response between two conditions for example, lies within a given equivalence interval, hence allowing to conclude that the conditions have "equivalent" means. The two one-sided tests (TOST) procedure, consisting in testing whether the target parameter is respectively significantly greater and lower than some pre-defined lower and upper equivalence limits, is typically used in this context, usually by checking whether the confidence interval for the target parameter lies within these limits. This intuitive and visual procedure is however known to be conservative, especially in the case of highly variable drugs, where it shows a rapid power loss, often reaching zero, hence making it impossible to conclude for equivalence when it is actually true. Here, we propose a finite sample correction of the TOST procedure, the α $$ \alpha $$ -TOST, which consists in a correction of the significance level of the TOST allowing to guarantee a test size (or type-I error rate) of α $$ \alpha $$ . This new procedure essentially corresponds to a finite sample and variability correction of the TOST procedure. We show that this procedure is uniformly more powerful than the TOST, easy to compute, and that its operating characteristics outperform the ones of its competitors. A case study about econazole nitrate deposition in porcine skin is used to illustrate the benefits of the proposed method and its advantages compared to other available procedures.

Enhanced topical paromomycin delivery for cutaneous leishmaniasis treatment: Passive and iontophoretic approaches.

Conventional treatments for cutaneous leishmaniasis, a neglected vector-borne infectious disease, can frequently lead to serious adverse effects. Paromomycin (PAR), an aminoglycoside antibiotic, has been suggested for the topical treatment of disease-related lesions, but even when formulated in high drug-loading dosage forms, presents controversial efficacy. The presence of five ionizable amino groups hinder its passive cutaneous penetration but make PAR an excellent candidate for iontophoretic delivery. The objective of this study was to verify the feasibility of using iontophoresis for cutaneous PAR delivery and to propose a topical passive drug delivery system that could be applied between iontophoretic treatments. For this, in vitro iontophoretic experiments evaluated different application durations (10, 30, and 360 min), current densities (0.1, 0.25, and 0.5 mA/cm2), PAR concentrations (0.5 and 1.0 %), and skin models (intact and impaired porcine skin). In addition, 1 % PAR hydrogel had its penetration profile compared to 15 % PAR ointment in passive transport. Results showed iontophoresis could deliver suitable PAR amounts to dermal layers, even in short times and with impaired skin. Biodistribution assays showed both iontophoretic transport and the proposed hydrogel delivered higher PAR amounts to deeper skin layers than conventional ointment, even though applying 15 times less drug. To our knowledge, this is the first report of PAR drug delivery enhancement by iontophoresis. In summary, the association of iontophoresis with a topical application of PAR gel seems appropriate for improving cutaneous leishmaniasis treatment.

Development of an ex vivo porcine skin model for the preclinical evaluation of subcutaneously injected biomacromolecules.

Subcutaneous administration is used to deliver systemically-acting biotherapeutics, e.g. antibodies, and locally-acting biomacromolecules, e.g. hyaluronic acid. However, few preclinical models are available to evaluate post-injection behaviour in the tissue microenvironment. In vivo animal studies are costly, time-consuming, and raise obvious ethical concerns. In vitro models are cost-efficient, high-throughput solutions, but cannot simulate complex skin structure and biological function. An ex vivo model (containing hypodermis) with an extended culture period that enabled longitudinal studies would be of great interest for both the pharmaceutical and cosmeceutical industries. We describe the development of one such ex vivo model, using viable full-thickness porcine skin. Structural integrity was evaluated using a histological scoring system: spongiosis and epidermal detachment were identified as discriminating parameters. Ki67 and Claudin-1 expression reported on epidermal cell proliferation and barrier function, respectively and their expression decreased as a function of incubation time. After optimization, the system was used to investigate the fate/impact of subcutaneously administered hyaluronic acid (HA) formulations. The results showed that HA was localized at the injection site and adjacent adipocytes were well preserved during 5 days' incubation and confirmed that the full-thickness ex vivo porcine skin model could provide a platform for preclinical evaluation of subcutaneously injected biomacromolecules.

Polymeric micelles for cutaneous delivery of the hedgehog pathway inhibitor TAK-441: Formulation development and cutaneous biodistribution in porcine and human skin.

TAK-441 is a potent inhibitor of the hedgehog pathway (IC50 4.4 nM) developed for the treatment of basal cell carcinoma that is active against the vismodegib-resistant Smoothened receptor D473H mutant. The objective of this study was to develop a micelle-based formulation of TAK-441 using D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and to investigate its cutaneous delivery and biodistribution. The micelles were prepared using solvent evaporation and incorporation of TAK-441 in the TPGS micelles increased aqueous solubility ∼40-fold. The optimal formulation, a 3% HPMC hydrogel of TAK-441 loaded TPGS micelles, retained ∼92% of the initial TAK-441 content (2.5 mgTAK-441/g) after storage at 4 °C for 6 months. Finite dose experiments using human skin demonstrated that this formulation resulted in significantly greater cutaneous deposition of TAK-441 after 12 h than a non-micelle control formulation, (0.40 ± 0.11 µg/cm2 and 0.05 ± 0.02 µg/cm2, respectively) - no transdermal permeation was observed. The cutaneous biodistribution profile demonstrated that TAK-441 was predominantly delivered to the viable epidermis and upper dermis. Delivery from the HPMC hydrogel formulation resulted in TAK-441 epidermal concentrations that were several thousand-fold higher than the IC50, with almost negligible transdermal permeation, thereby decreasing the risk of systemic side effects in vivo.

Injectable Hyaluronan-Based Thermoresponsive Hydrogels for Dermatological Applications.

Most marketed HA-based dermal fillers use chemical cross-linking to improve mechanical properties and extend their lifetime in vivo; however, stiffer products with higher elasticity require an increased extrusion force for injection in clinical practice. To balance longevity and injectability, we propose a thermosensitive dermal filler, injectable as a low viscosity fluid that undergoes gelation in situ upon injection. To this end, HA was conjugated via a linker to poly(N-isopropylacrylamide) (pNIPAM), a thermosensitive polymer using "green chemistry", with water as the solvent. HA-L-pNIPAM hydrogels showed a comparatively low viscosity (G' was 105.1 and 233 for Candidate1 and Belotero Volume®, respectively) at room temperature and spontaneously formed a stiffer gel with submicron structure at body temperature. Hydrogel formulations exhibited superior resistance against enzymatic and oxidative degradation and could be administered using a comparatively lower injection force (49 N and >100 N for Candidate 1 and Belotero Volume®, respectively) with a 32G needle. Formulations were biocompatible (viability of L929 mouse fibroblasts was >100% and ~85% for HA-L-pNIPAM hydrogel aqueous extract and their degradation product, respectively), and offered an extended residence time (up to 72 h) at the injection site. This property could potentially be exploited to develop sustained release drug delivery systems for the management of dermatologic and systemic disorders.

Influence of Molecular Structure and Physicochemical Properties of Immunosuppressive Drugs on Micelle Formulation Characteristics and Cutaneous Delivery.

The aim of this study was to investigate whether subtle differences in molecular properties affected polymeric micelle characteristics and their ability to deliver poorly water-soluble drugs into the skin. D-α-tocopherol-polyethylene glycol 1000 was used to prepare micelles containing ascomycin-derived immunosuppressants-sirolimus (SIR), pimecrolimus (PIM) and tacrolimus (TAC)-which have similar structures and physicochemical properties and have dermatological applications. Micelle formulations were prepared by thin-film hydration and extensively characterized. Cutaneous delivery and biodistribution were determined and compared. Sub-10 nm micelles were obtained for the three immunosuppressants with incorporation efficiencies >85%. However, differences were observed for drug loading, stability (at the highest concentration), and their in vitro release kinetics. These were attributed to differences in drug aqueous solubility and lipophilicity. Differences between the cutaneous biodistribution profiles and drug deposition in the different skin compartments pointed to the impact of differences in thermodynamic activity. Therefore, despite their structural similarities, SIR, TAC and PIM did not demonstrate the same behaviour either in the micelles or when applied to the skin. These outcomes indicate that polymeric micelles should be optimized even for closely related drug molecules and support the hypothesis that drugs are released from micelles prior to skin penetration.

Biodistribution of progesterone in the eye after topical ocular administration via drops or inserts.

Progesterone (PG) has been shown to have a slowing effect on photoreceptor cell death in mouse models of retinitis pigmentosa when administered orally. The aim of this study was to investigate whether ophthalmically administered progesterone was able to reach neuroretina and thus, the distribution through ocular tissues of different PG formulations was studied. The effect of different initial PG concentration was also investigated. Different formulations with PG in their composition (drops, a corneal/scleral-insert and scleral-inserts) were prepared and assayed. Using whole porcine eyes, the different formulations were topically administered to the ocular surface. Frozen eyes were dissected, the PG in each tissue was extracted in acetonitrile and the amount of PG quantified by UHPLC-MS/MS. Our results show that after topical administration, PG diffuses from the ocular surface and distributes throughout all tissues of the eye. Lower levels of PG were found in sclera, choroid and neuroretina when PG was applied as drops compared to inserts. Our results also show that an increase in the initial PG concentrations applied, resulted in a statistically significant increase in the amounts of PG in aqueous humour, sclera, choroid and neuroretina.

Erbium:YAG fractional laser ablation improves cutaneous delivery of pentoxifylline from different topical dosage forms.

Topical application of pentoxifylline (PTX) would enable targeted treatment of radiation-induced skin fibrosis. However, PTX is hydrophilic with limited partitioning into the stratum corneum. The objective of this study was to investigate whether use of Erbium:YAG fractional laser ablation and different topical dosage forms (solution, hydrogel and patch) could be used to improve PTX cutaneous delivery as opposed to transdermal permeation. Initial results confirmed that fractional laser ablation significantly increased PTX delivery from each dosage form compared to passive controls. Delivery efficiencies of âˆ¼ 30% were achieved with each dosage form but a large proportion of PTX permeated across the skin; thus, fluences were decreased to create shallower micropores, their depth being linearly dependent on fluence. The hydrogel was selected as the optimal formulation and PTX delivery efficiencies were further increased (44%-67%) by reducing the amount of hydrogel applied (better mimicking conditions of use). As this resulted in PTX depletion in the formulation, a loss of dependence of delivery on laser fluence was observed. These findings suggest that fractional laser ablation at moderate fluences enables an effective and targeted cutaneous delivery of PTX from a hydrogel formulation, which can be easily produced without the need for complex equipment.

Bolus delivery of palonosetron through skin by tip-loaded dissolving microneedles with short-duration iontophoresis: A potential strategy to rapidly relieve emesis associated with chemotherapy.

The objective of this study was to investigate the feasibility of the bolus administration of PLS via skin by using dissolving microneedles of palonosetron hydrochloride (PLS-DMNs). Tip-loaded PLS-DMNs were fabricated by a casting method using sodium hyaluronate (HA) as DMNs-forming polymer. PLS-DMNs were shown to have a content of 118.5 ± 8.7 µg per piece with sufficient mechanical strength for insertion into pig skin ex vivo. In situ dissolution of PLS-DMNs was achieved within 5 min and 83.2 % of PLS was delivered. In vitro studies showed that PLS-DMNs provided much higher PLS permeation than that after passive permeation using a PLS hydrogel. Moreover, the application of 30 min-iontophoresis at the beginning of PLS-DMNs administration further enhanced PLS delivery. In vivo pharmacokinetic studies were carried out in rats. The area under the curve (AUC) and the time to reach the peak (Tmax) after application of PLS-DMNs was not significantly different compared to those after subcutaneous (S.C.) injection. PLS-DMNs plus 30 min-iontophoresis enabled the pharmacokinetic profile to be even closer to that seen after S.C. administration. These results suggest that application of PLS-DMNs with short-duration iontophoresis exhibits promise as an alternative PLS delivery method that can be painlessly self-administered with rapid onset.

Non-Invasive Iontophoretic Delivery of Cytochrome c to the Posterior Segment and Determination of Its Ocular Biodistribution.

The intact porcine eye globe model was used to demonstrate that transscleral iontophoresis could deliver a small protein, cytochrome c (Cyt c), to the posterior segment and to investigate post-iontophoretic biodistribution in the different ocular compartments. The effects of Cyt c concentration (1, 5, and 10 mg/mL), current density (3.5 and 5.5 mA/cm2), and duration of the current application (10 min and 1, 2, and 4 h) were evaluated. The data confirmed that transscleral iontophoresis enhanced the intraocular delivery of Cyt c under all conditions as compared to passive controls (same setup but without the current application). Increasing the Cyt c concentration resulted in a proportional enhancement in the Cyt c delivery. Increasing the current density from 3.5 to 5.5 mA/cm2 increased iontophoretic delivery at a Cyt c concentration of 10 mg/mL but did not appear to do so at 5 mg/mL; this was attributed in part to the effect of melanin binding. Short duration iontophoresis (10 min, 3.5 mA/cm2) of a 10 mg/mL Cyt c solution created a depot in the sclera. When this was followed by a 4 h incubation period, post-iontophoretic Cyt c diffusion from the sclera resulted in a different biodistribution, and Cyt c could be quantified in the posterior segment.

Simultaneous Delivery of Econazole, Terbinafine and Amorolfine with Improved Cutaneous Bioavailability: A Novel Micelle-Based Antifungal "Tri-Therapy".

Lack of accurate diagnosis and the use of formulations designed to address the poor aqueous solubility of antifungal agents, but not optimized for delivery, contribute to unsatisfactory outcomes for topical treatment of cutaneous mycoses. The objective of this study was to develop a micelle-based antifungal formulation containing econazole (ECZ), terbinafine (TBF) and amorolfine (AMF) using D-α-tocopheryl polyethylene glycol succinate (TPGS) for simultaneous cutaneous delivery of three agents with complementary mechanisms of action. The antifungal "tri-therapy" micelle-based formulation containing 0.1% ECZ, 0.1% TBF and 0.025% AMF had a drug loading 10-fold lower than the "reference" marketed formulations (Pevaryl®, 1% ECZ; Lamisil®, 1% TBF; Loceryl®, 0.25% AMF). Finite dose application of the micelle-based formulation for 6 h resulted in a statistically equivalent deposition of ECZ (p > 0.05) and TBF (p > 0.05) from the 2 systems, and a 2-fold higher accumulation of AMF (p = 0.017). Antifungal concentrations above MIC80 against Trichophyton rubrum were achieved in each skin layer with the "tri-therapy", which also exhibited a preferential deposition of each antifungal agent in pilosebaceous unit (PSU)-containing biopsies as compared with PSU-free biopsies (p < 0.05). A planned clinical study will test whether these promising results translate to improved therapeutic outcomes in vivo.

Effect of mRNA Delivery Modality and Formulation on Cutaneous mRNA Distribution and Downstream eGFP Expression.

In vitro transcribed messenger ribonucleic acid (mRNA) constitutes an emerging therapeutic class with several clinical applications. This study presents a systematic comparison of different technologies-intradermal injection, microneedle injection, jet injection, and fractional laser ablation-for the topical cutaneous delivery of mRNA. Delivery of Cy5 labeled mRNA and non-labeled enhanced green fluorescent protein (eGFP) expressing mRNA was investigated in a viable ex vivo porcine skin model and monitored for 48 h. Forty 10 µm-thick horizontal sections were prepared from each skin sample and Cy5 labeled mRNA or eGFP expression visualized as a function of depth by confocal laser scanning microscopy and immunohistochemistry. A pixel-based method was used to create a semi-quantitative biodistribution profile. Different spatial distributions of Cy5 labeled mRNA and eGFP expression were observed, depending on the delivery modality; localization of eGFP expression pointed to the cells responsible. Delivery efficiencies and knowledge of delivery sites can facilitate development of efficient, targeted mRNA-based therapeutics.

Preclinical and clinical studies to evaluate cutaneous biodistribution, safety and efficacy of UV filters encapsulated in mesoporous silica SBA-15.

Innovative technologies have been designed to improve efficacy and safety of chemical UV filters. Encapsulation can enhance efficacy and reduce transdermal permeation and systemic exposure. The aims of this work were (i) to determine the cutaneous biodistribution of avobenzone (AVO), oxybenzone (OXY), and octyl methoxycinnamate (OMC) incorporated in mesoporous silica SBA-15 and (ii) to perform preclinical (in vitro) and (iii) clinical safety studies to demonstrate their innocuity and to evaluate sun protection factor (SPF) in humans. Skin penetration studies showed that deposition of OXY and AVO in porcine and human skin after application of stick formulation with incorporated filters (stick incorporated filters) was significantly lower than from a marketed (non-encapsulated) stick. Cutaneous deposition and transdermal permeation of OXY in and across human skin were 3.8-and 13.4- fold lower, respectively, after application of stick entrapped filters. Biodistribution results showed that encapsulation in SBA-15 decreased AVO and OXY penetration reaching porcine and human dermis. Greater deposition (and permeation) of OXY in porcine skin than in human skin, pointed to the role of follicular transport. Stick incorporated filters had good biocompatibility in vivo and safety profiles, even under sun-exposed conditions. Entrapment of UV filters improved the SPF by 26% and produced the same SPF profile as a marketed stick. Overall, the results showed that SBA-15 enabled safety and efficacy of UV filters to be increased.

DESI-MS imaging to visualize spatial distribution of xenobiotics and endogenous lipids in the skin.

The cutaneous biodistribution method (CBM) yields a high-resolution quantitative profile of drug deposition as a function of skin depth. However, it provides limited details about drug spatial distribution or penetration pathways. Mass spectrometry imaging (MSI) can complement the detailed quantitative data generated by CBM studies. The objectives of this work were to use desorption electrospray ionization (DESI)-MSI to (i) investigate the spatial cutaneous distributions of a topically applied drug and excipient and relate them to skin structures and (ii) image endogenous skin components and combine these results to gain insight into drug penetration routes. Porcine skin was used to compare two bioequivalent creams of econazole nitrate (ECZ) and a micelle formulation based on D-α-tocopheryl succinate polyethylene glycol 1000 (TPGS). DESI-MSI successfully imaged the cutaneous spatial distribution of ECZ and TPGS in 40 µm-thick horizontal sections and vertical cross-sections of the skin. Interestingly, clinically bioequivalent formulations did not appear to exhibit the same molecular distribution of ECZ in XY-horizontal sections. DESI-MSI also enabled visualization of TPGS (m/z 772.4706), mainly in the upper epidermis (≤80 µm). In conclusion, through co-localization of drugs and excipients with endogenous elements of the skin, DESI-MSI could further our understanding of the cutaneous penetration pathways of xenobiotics.

Controlled simultaneous iontophoresis of buflomedil hydrochloride and dexamethasone sodium phosphate to the mucosa for oral submucous fibrosis.

A novel concentric experimental set-up was used to investigate short-duration topical co-iontophoresis of cationic buflomedil hydrochloride (BUF) and anionic dexamethasone phosphate (DEX-P) to the oral mucosa. A constant current of 3.0 mA (0.6 mA/cm2 for BUF and 1.95 mA/cm2 for DEX-P) was applied to porcine esophageal mucosa for 5, 10 and 20 min. Iontophoresis for only 5 min increased total delivery of BUF from 29.8 ± 5.1 nmol/cm2 to 194.3 ± 23.8 nmol/cm2 and DEX-P from 29.4 ± 1.2 nmol/cm2 to 193.3 ± 19.8 nmol/cm2 as compared to passive controls. Quantification of drug between the electrode compartments reported on lateral ion migration. In the absence of current, DEX-P did not migrate laterally; however, iontophoresis for 5 min increased DEX-P delivery >5-fold under the cathodal compartment (its application area) and >8-fold in the adjacent "inter-electrode" area. Similarly, delivery of BUF increased ~6.8-fold under the anodal compartment and ~12.8-fold under the cathode. The results showed that co-iontophoresis enabled the controlled simultaneous delivery of BUF and DEX-P achieving therapeutically relevant concentrations after current application for only 5 min. Short duration topical co-iontophoresis of single or multiple therapeutics to the mucosa increases local bioavailability and presents a patient-friendly treatment for diseases of the oral cavity.

Using Porcine Jejunum Ex Vivo to Study Absorption and Biotransformation of Natural Products in Plant Extracts: Pueraria lobata as a Case Study.

Herbal preparations (HPs) used in folk medicine are complex mixtures of natural products (NPs). Their efficacy in vivo after ingestion depends on the uptake of the active ingredient, and, in some cases, their metabolites, in the gastrointestinal tract. Thus, correlating bioactivities measured in vitro and efficacy in vivo is a challenge. An extract of Pueraria lobata rich in different types of isoflavones was used to evaluate the capacity of viable porcine small intestine ex vivo to elucidate the absorption of HP constituents, and, in some cases, their metabolites. The identification and transport of permeants across the jejunum was monitored by liquid chromatography-mass spectrometry (LC-MS), combining targeted and untargeted metabolite profiling approaches. It was observed that the C-glycoside isoflavones were stable and crossed the intestinal membrane, while various O-glycoside isoflavones were metabolized into their corresponding aglycones, which were then absorbed. These results are consistent with human data, highlighting the potential of using this approach. A thorough investigation of the impact of absorption and biotransformation was obtained without in vivo studies. The combination of qualitative untargeted and quantitative targeted LC-MS methods effectively monitored a large number of NPs and their metabolites, which is essential for research on HPs.

Current profile controlled transdermal delivery of pramipexole from an iontophoretic patch system in vitro and in vivo.

The objective was to evaluate the transdermal iontophoretic delivery of pramipexole using constant and complex multi-phasic current profiles from an iontophoretic patch system in vitro and in vivo. Preliminary in vitro experiments were performed to optimize iontophoretic patch design and configuration. "Single" compartment systems containing only pramipexole dihydrochloride, and designed to maximize delivery efficiency, suffered from an insufficiency of chloride ions with anodal electrochemistry passing from an Ag/AgCl couple to an Ag dissolution electrode. Addition of NaCl to provide more chloride ions decreased pramipexole delivery efficiency due to competition between pramipexole and sodium cations. A "two-compartment" iontophoretic patch where the drug reservoir was separated from the anodal compartment, which now included NaCl, was shown to be a good compromise since it ensured Ag/AgCl electrochemistry at the anode and an acceptable delivery efficiency. In vivo studies using this iontophoretic patch demonstrated that the plasma concentration of pramipexole closely followed the variation of the applied continuous and multi-phasic current profiles and underlined the control provided by iontophoresis and its unique ability to rapidly change drug input rates. The applied current density and duration of current application were also shown to modulate pramipexole delivery to the brain and CSF.