S. Kevin Li

Transscleral iontophoretic and intravitreal delivery of a macromolecule: Study of ocular distribution in vivo and postmortem with MRI

The distribution and clearance of macromolecules in ocular delivery are not well understood. It has been hypothesized that iontophoresis can enhance transscleral delivery of macromolecules. The objective of this study was to investigate the ocular distribution of a macromolecule after transscleral iontophoretic delivery and intravitreal injection in vivo using nuclear magnetic resonance imaging (MRI) and to compare these results. Experiments of constant current transscleral iontophoresis of 4mA or intravitreal injection were performed on New Zealand white rabbits in vivo. Iontophoresis experiments were also performed on rabbits postmortem. Galbumin (Gd-labeled albumin) was the model permeant surrogate to clinical therapeutic agents. MRI was used to monitor the distribution of the molecule in the eye after ocular iontophoresis and intravitreal injection. In addition, the conjunctiva, sclera, choroid, and retina were extracted in the transscleral iontophoresis study to determine the amounts of Galbumin in these tissues using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The results show that iontophoresis enhanced the ocular delivery of Galbumin. The macromolecule was mainly delivered into the conjunctiva and sclera in microgram quantities and then diffused towards the posterior section in the upper hemisphere of the eye in vivo. Both in vivo and postmortem studies show that the iontophoretic delivery of Galbumin into the vitreous was below the detection limit. In the intravitreal injection study, the diffusion coefficient of Galbumin in the vitreous humor was estimated to be close to that of free aqueous diffusion.

Assessment of subconjunctival delivery with model ionic permeants and magnetic resonance imaging.

No HeadingPurpose.The objective was to assess the permeation and clearance of model ionic permeants after subconjunctival injection with nuclear magnetic resonance imaging (MRI).Methods.New Zealand white rabbit was the animal model and manganese ion (Mn2+) and manganese ethylenediaminetetraacetic acid complex (MnEDTA2−) were the model permeants. The current study was divided into three parts: in vitro, postmortem, and in vivo. Transscleral passive permeation experiments were conducted with excised sclera in side-by-side diffusion cells in vitro. Subconjunctival delivery experiments were conducted with rabbits postmortem and in vivo. The distribution and elimination of the probe permeants from the subconjunctival space after subconjunctival injections were determined by MRI.Results.The data of excised sclera in vitro suggest large effective pore size for transscleral transport and negligible pore charge effects upon the permeation of the ionic permeants. The permeability coefficients of Mn2+ and MnEDTA2- across the sclera in vitro were 3.6 × 10-5 cm/s and 2.4 × 10-5 cm/s, respectively. Although relatively high sclera permeability was observed in vitro, subconjunctival injections in vivo did not provide significant penetration of Mn2+ and MnEDTA2- into the globe; permeant concentrations in the eye were below the detection limit, which corresponds to less than 0.05% of the concentration of the injection solution (e.g., less than 0.02 mM when 40 mM injection solution was used). The volume of the subconjunctival pocket and the concentration of the permeants in the pocket were observed to decrease with time after the injection, and this could contribute to the lower than expected subconjunctival absorption in vivo. Different from the results in vivo, experiments with rabbits postmortem show significant penetration of Mn2+ and MnEDTA2- into the globe with the permeants primarily delivered into the anterior segment of the eye. This difference suggests blood vasculature clearance as a main barrier for passive transscleral transport. The data also show that the pars plicata/pars plana is the least resistance pathway for passive transscleral drug delivery of the polar permeants, and there are indications of the presence of another barrier, possibly the retinal epithelium and/or Bruch’s membrane, at the back of the eye.Conclusions.Subconjunctival delivery of the ionic permeants in vivo cannot be quantitatively predicted by the in vitro results. MRI is a noninvasive complementary technique to traditional pharmacokinetic methods. It can provide insights into ocular pharmacokinetics without permeant redistribution that can occur in surgical procedure postmortem in traditional pharmacokinetic studies when the blood vasculature barrier is absent.

Chemical Method to Enhance Transungual Transport and Iontophoresis Efficiency

Transungual transport is hindered by the inherent small effective pore size of the nail even when it is fully hydrated. The objectives of this study were to determine the effects of chemical enhancers thioglycolic acid (TGA), glycolic acid (GA), and urea (UR) on transungual transport and iontophoresis efficiency. In vitro passive and iontophoretic transport experiments of model permeants mannitol (MA), UR, and tetraethylammonium (TEA) ion across the fully hydrated, enhancer-treated and untreated human nail plates were performed in phosphate-buffered saline. The transport experiments consisted of several stages, alternating between passive and anodal iontophoretic transport at 0.1mA. Nail water uptake experiments were conducted to determine the water content of the enhancer-treated nails. The effects of the enhancers on transungual electroosmosis were also evaluated. Nails treated with GA and UR did not show any transport enhancement. Treatment with TGA at 0.5M enhanced passive and iontophoretic transungual transport of MA, UR, and TEA. Increasing the TGA concentration to 1.8M did not further increase TEA iontophoresis efficiency. The effect of TGA on the nail plates was irreversible. The present study shows the possibility of using a chemical enhancer to reduce transport hindrance in the nail plate and thus enhance passive and iontophoretic transungual transport.

Mechanistic Study of Electroosmotic Transport Across Hydrated Nail Plates: Effects of pH and Ionic Strength

The objective of this study was to investigate the effects of pH and ionic strength on electroosmotic transport in transungual iontophoresis. Transungual iontophoretic transport of model neutral permeants mannitol (MA) and urea (UR) across fully hydrated human nail plates in phosphate-buffered saline of different pH and ionic strengths were investigated in vitro. Two protocols were involved in the transport experiments with each protocol divided into stages including passive and iontophoresis transport at 0.1 and/or 0.3 mA. Nail plate electrical resistance and water uptake of nail clippings were measured at various pH and ionic strengths. In the pH study, electroosmosis enhanced the anodal transport of MA at pH 9 and cathodal transport at pH 3. The Peclet numbers of MA were more than two times higher than those of UR under these conditions. No significant electroosmosis enhancement was observed for MA and UR at pH 5. In the ionic strength study, a decrease in solution ionic strength from 0.7 to 0.04 M enhanced electroosmotic transport. Nail electrical resistance increased with decreasing the ionic strength of the equilibrating solution, but reached a plateau when the ionic strength was less than approximately 0.07 M. Solution pH and ionic strength had no significant effect on nail hydration. Under the studied pH and ionic strength conditions, the effects of electroosmosis were small compared to the direct-field effects in transungual iontophoretic transport of small to moderate size permeants.

Iontophoretic transport of charged macromolecules across human sclera

The mechanisms of transscleral iontophoresis have been investigated previously with small molecules in rabbit sclera. The objective of the present study was to examine transscleral iontophoretic transport of charged macromolecules across excised human sclera. Passive and 2mA iontophoretic transport experiments were conducted in side-by-side diffusion cells with human sclera. The effects of iontophoresis upon transscleral transport of model permeants bovine serum albumin (BSA) and polystyrene sulfonic acid (PSS) as well as a model drug bevacizumab (BEV) were determined. Passive and iontophoretic transport experiments of tetraethylammonium (TEA) and salicylic acid (SA) and passive transport experiments of the macromolecules served as the controls. The results of iontophoresis enhanced transport of TEA and SA across human sclera were consistent with those in a previous rabbit sclera study. For the iontophoretic transport of macromolecules BSA and BEV, higher iontophoretic fluxes were observed in anodal iontophoresis as compared to passive and cathodal iontophoresis. This suggests the importance of electroosmosis. For the polyelectrolyte PSS, higher iontophoretic flux was observed in cathodal iontophoresis compared to anodal iontophoresis. Both electroosmosis and electrophoresis affected iontophoretic fluxes of the macromolecules; the relative contributions of electroosmosis and electrophoresis were a function of molecular size and charge of the macromolecules.

Electrically assisted delivery of macromolecules into the corneal epithelium.

Electrically assisted delivery is noninvasive and has been investigated in a number of ocular drug delivery studies. The objectives of this study were to examine the feasibility of electrically assisted delivery of macromolecules such as small interfering RNA (siRNA) into the corneal epithelium, to optimize the iontophoresis and electroporation methods, and to study the mechanisms of corneal iontophoresis for macromolecules. Anodal and cathodal iontophoresis, electroporation and their combinations were the methods examined with mice in vivo. Cyanine 3 (Cy3)-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA and fluorescein isothiocyanate (FITC)-labeled dextran of different molecular weights (4-70 kDa) were the macromolecules studied. Microscopy and histology after cryostat sectioning were used to analyze and compare the delivery of the macromolecules to the cornea. Iontophoresis was effective in delivering siRNA and dextran up to 70 kDa into the cornea. The electroporation method studied was less effective than that of iontophoresis. Although both iontophoresis and electroporation alone can deliver the macromolecules into the cornea, these methods alone were not as effective as the combination of iontophoresis and electroporation (iontophoresis followed by electroporation). The significant enhancement of dextran delivery in anodal iontophoresis suggests that electroosmosis can be a significant flux-enhancing mechanism during corneal iontophoresis. These results illustrate the feasibility of electrically assisted delivery of macromolecules such as siRNA into the cornea.

Efficiency of Fatty Acids as Chemical Penetration Enhancers: Mechanisms and Structure Enhancement Relationship

PurposeThe present study evaluated the effects of fatty acids commonly present in cosmetic and topical formulations on permeation enhancement across human epidermal membrane (HEM) lipoidal pathway when the fatty acids saturated the SC lipid domain without cosolvents (Emax).MethodsHEM was treated with neat fatty acids or fatty acid suspensions to determine Emax. A volatile solvent system was used to deposit fatty acids on HEM surface to compare fatty acid enhancer efficiency in topical volatile formulations with Emax. To elucidate permeation enhancement mechanism(s), estradiol (E2β) uptake into fatty acid-treated SC lipid domain was determined.ResultsEmax of fatty acids was shown to increase with their octanol solubilities and decrease with their lipophilicities, similar to our previous findings with other enhancers. Emax of solid fatty acids was shown to depend on their melting points, an important parameter to the effectiveness of the enhancers. The E2β uptake results suggest that enhancer-induced permeation enhancement across HEM is related to enhanced permeant partitioning into the SC lipid domain.ConclusionsThe results suggest Emax as a model for studying the permeation enhancement effect of the fatty acids and their structure enhancement relationship.

Chemical Enhancer Solubility in Human Stratum Corneum Lipids and Enhancer Mechanism of Action on Stratum Corneum Lipid Domain

Previously, chemical enhancer-induced permeation enhancement on human stratum corneum (SC) lipoidal pathway at enhancer thermodynamic activities approaching unity in the absence of cosolvents (defined as Emax) was determined and hypothesized to be related to the enhancer solubilities in the SC lipid domain. The objectives of the present study were to (a) quantify enhancer uptake into SC lipid domain at saturation, (b) elucidate enhancer mechanism(s) of action, and (c) study the SC lipid phase behavior at Emax. It was concluded that direct quantification of enhancer uptake into SC lipid domain using intact SC was complicated. Therefore a liposomal model of extracted human SC lipids was used. In the liposome study, enhancer uptake into extracted human SC lipid liposomes (EHSCLL) was shown to correlate with Emax. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC) were used to evaluate lipid phase alterations in enhancer-treated intact SC. IR spectra demonstrated an increase in the lipid domain fluidity and DSC thermograms indicated a decrease in the phase transition temperature with increasing Emax. These results suggest that the enhancer mechanism of action is through enhancer intercalation into SC intercellular lipids and subsequent lipid lamellae fluidization related to enhancer lipid concentration.

Iontophoretically Enhanced Ciclopirox Delivery into and Across Human Nail Plate

Transungual delivery of antifungal drugs is hindered by the low permeability of human nail plates, and as such, repeated dosing over a long period of time is necessary for effective treatment. The objectives of this study were to explore the possibilities of (a) enhancing the delivery of ciclopirox (CIC) across human nail plates and (b) sustaining CIC delivery from the larger resultant drug depot in the nail plates with constant voltage iontophoresis. In vitro passive and 9 V cathodal iontophoretic transport experiments of CIC across human nails were performed. Transungual CIC delivery with Penlac was the control. The amounts of CIC released from and deposited in the nails were determined in drug release and extraction experiments, respectively. Iontophoresis increased the flux of CIC permeated across the nail approximately 10 times compared to passive delivery from the same formulation or from Penlac. A significant amount of CIC was loaded into and released from the nails; the CIC concentrations were estimated to be above the minimum inhibitory concentrations of CIC for dermatophytic molds. The apparent transport lag time decreased in iontophoretic transport. The results demonstrate that iontophoresis was able to deliver an effective amount of CIC into and across the nails, and this suggests the feasibility of a constant voltage battery-powered transungual iontophoretic device.

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.