Reverse Iontophoresis: Noninvasive Assessment of Topical Drug Bioavailability.

Assessing drug disposition in the skin after the application of a topical formulation is difficult. It is hypothesized that reverse iontophoresis (RI), which can extract charged/polar molecules for monitoring purposes, may provide a noninvasive approach for the assessment of local drug bioavailability. The passive and RI extraction of salicylic acid (SA) and nicotine (NIC) from porcine skin in vitro was assessed after a simple solution of the former and a transdermal patch of the latter had been applied for 24 and 8 h, respectively. Immediately after this "passive skin loading", the amount of drug in the stratum corneum (SC) and "viable" tissue (VT) was measured either (a) after tape-stripping and subsequent solvent extraction of both skin layers or (b) following RI extraction over 4 h. Parallel experiments were then performed in vivo in healthy volunteers; in this case, the VT was not sampled and the skin loading period for NIC was only 4 h. RI extraction of both drugs was significantly higher (in vitro and in vivo) than that achieved passively, and the cumulative RI extraction profiles as a function of time were mathematically analyzed using a straightforward compartmental model. Best-fit estimates of drug amounts in the SC and VT (ASC,0 and AVT,0, respectively) at the end of "loading" and two first-order rate constants describing transfer between the model compartments were then determined. The in vitro predictions of ASC,0 and AVT,0 were in excellent agreement with the experimental results, as was the value of the former in vivo. The rate constants derived from the in vitro and in vivo results were also similar. In summary, the results provide proof-of-concept that the RI method has the potential to noninvasively assess relevant metrics of drug bioavailability in the skin.

Quantification of Chemical Uptake into the Skin by Vibrational Spectroscopies and Stratum Corneum Sampling.

Evaluation of the bioavailability of drugs intended to act within the skin following the application of complex topical products requires the application of multiple experimental tools, which must be quantitative, validated, and, ideally and ultimately, sufficiently minimally invasive to permit use in vivo. The objective here is to show that both infrared (IR) and Raman spectroscopies can assess the uptake of a chemical into the stratum corneum (SC) that correlates directly with its quantification by the adhesive tape-stripping method. Experiments were performed ex vivo using excised porcine skin and measured chemical disposition in the SC as functions of application time and formulation composition. The quantity of chemicals in the SC removed on each tape-strip was determined from the individually measured IR and Raman signal intensities of a specific molecular vibration at a frequency where the skin is spectroscopically silent and by a subsequent conventional extraction and chromatographic analysis. Correlations between the spectroscopic results and the chemical quantification on the tape-strips were good, and the effects of longer application times and the use of different vehicles were clearly delineated by the different measurement techniques. Based on this initial investigation, it is now possible to explore the extent to which the spectroscopic approach (and Raman in particular) may be used to interrogate chemical disposition deeper in the skin and beyond the SC.

Confocal Raman Spectroscopic Characterization of Dermatopharmacokinetics Ex Vivo.

Confocal Raman spectroscopy is being assessed as a tool with which to quantify the rate and extent of drug uptake to and its clearance from target sites of action within the viable epidermis below the skin's stratum corneum (SC) barrier. The objective of this research was to confirm that Raman can interrogate drug disposition within the living layers of the skin (where many topical drugs elicit their pharmacological effects) and to identify procedures by which Raman signal attenuation with increasing skin depth may be corrected and normalized so that metrics descriptive of topical bioavailability may be identified. It was first shown in experiments on skin cross-sections parallel to the skin surface that the amide I signal, originating primarily from keratin, was quite constant with depth into the skin and could be used to correct for signal attenuation when confocal Raman data were acquired in a "top-down" fashion. Then, using 4-cyanophenol (CP) as a model skin penetrant with a strong Raman-active C≡N functionality, a series of uptake and clearance experiments, performed as a function of time, demonstrated clearly that normalized spectroscopic data were able to detect the penetrant to at least 40-80 µm into the skin and to distinguish the disposition of CP from different vehicles. Metrics related to local bioavailability (and potentially bioequivalence) included areas under the normalized C≡N signal versus depth profiles and elimination rate constants deduced post-removal of the formulations. Finally, Raman measurements were made with an approved dermatological drug, crisaborole, for which delivery from a fully saturated formulation into the skin layers just below the SC was detectable.

Non-destructive, reverse iontophoretic extraction of phytochemicals from Mangifera indica, Centella asiatica, Punica granatum, and Citrus sinensis.

For the commercial-scale isolation of phytochemicals, a suitable plant biomass source (including species, origin, growing season, etc.) must be identified, and frequent analytical verification is required to ensure that the phytochemicals are present at predefined minimum threshold concentrations. While the latter are typically assessed in the laboratory, a more efficient and less resource-intensive approach would involve non-destructive and environmentally friendly measurements in situ. Reverse iontophoretic (RI) sampling offers a potential solution to this challenge. OBJECTIVE: We aimed to demonstrate the non-destructive, RI sampling of phytochemicals of interest from biomass from four different sources. MATERIALS AND METHODS: RI experiments were performed in side-by-side diffusion cells using a current density of 0.5 mA/cm2 , for a predetermined time in a defined pH environment, using (1) fresh leaves from Mangifera indica and Centella asiatica and (2) isolated peel from Punica granatum and Citrus sinensis. RESULTS: Mangiferin, madecassoside, punicalagin, ellagic acid, and hesperidin were extracted from the different biomasses by RI. The amounts extracted ranged from 0.03 mg/100 mg of biomass for the cathodal extraction of madecassoside to 0.63 mg/100 mg of biomass for the anodal extraction of punicalagin. A linear relationship (r2  = 0.73) between the RI-extracted quantities of punicalagin and those determined using conventional methods was demonstrated. CONCLUSION: The non-destructive, in situ measurement of phytochemical levels by RI represents a feasible approach for timing the harvesting process.

Raman Spectroscopic Tools to Probe the Skin-(Trans)dermal Formulation Interface.

Medicines designed to deliver the active pharmaceutical ingredient either into or through the skin─often referred to as topicals and transdermals, respectively─are generally considered to be complex drug products. A particular challenge faced by these formulations is identifying a suitable method (ideally, in terms of specificity, accuracy, precision, and robustness) or combination of methods with which to assess the amount and rate of drug delivery to the target site. Significant research currently aims to identify and validate relevant and minimally invasive techniques that can be used to quantify both the levels of the drug attained within different parts of the skin and the kinetics with which the drug is taken up into the skin and cleared therefrom into the systemic circulation. Here, the application of confocal Raman microspectroscopy and imaging to interrogate events integral to the performance of topical and transdermal drug products at the formulation-skin interface is illustrated. Visualization, depth slicing, and profiling are used (a) to elucidate key chemical properties of both the delivery system and the skin that have impact on their interaction and the manner in which drug transfer from one to the other may occur, (b) for the transformation of a drug product from that manufactured into a residual phase post-application and inunction into the skin (including the potential for important changes in solubility of the active compound), and (c) for drug absorption into the skin and its subsequent '"clearance" into deeper layers and beyond. Overall, the Raman tools described offer both qualitative and potentially semi-quantitative insights into topical and transdermal drug product performance and provide information useful for formulation improvement and optimization.

Skin Pharmacokinetics of Transdermal Scopolamine: Measurements and Modeling.

Prediction of skin absorption and local bioavailability from topical formulations remains a difficult task. An important challenge in forecasting topical bioavailability is the limited information available about local and systemic drug concentrations post application of topical drug products. Commercially available transdermal patches, such as Scopoderm (Novartis Consumer Health UK), offer an opportunity to test these experimental approaches as systemic pharmacokinetic data are available with which to validate a predictive model. The long-term research aim, therefore, is to develop a physiologically based pharmacokinetic model (PBPK) to predict the dermal absorption and disposition of actives included in complex dermatological products. This work explored whether in vitro release and skin permeation tests (IVRT and IVPT, respectively), and in vitro and in vivo stratum corneum (SC) and viable tissue (VT) sampling data, can provide a satisfactory description of drug "input rate" into the skin and subsequently into the systemic circulation. In vitro release and skin permeation results for scopolamine were consistent with the previously reported performance of the commercial patch investigated. New skin sampling data on the dermatopharmacokinetics (DPK) of scopolamine also accurately reflected the rapid delivery of a "priming" dose from the patch adhesive, superimposed on a slower, rate-controlled input from the drug reservoir. The scopolamine concentration versus time profiles in SC and VT skin compartments, in vitro and in vivo, taken together with IVRT release and IVPT penetration kinetics, reflect the input rate and drug delivery specifications of the Scopoderm transdermal patch and reveal the importance of skin binding with respect to local drug disposition. Further data analysis and skin PK modeling are indicated to further refine and develop the approach outlined.

Regulatory Science and Innovation Programme for Europe (ReScIPE): A proposed model.

Regulatory science underpins the objective evaluation of medicinal products. It is therefore imperative that regulatory science and expertise remain at the cutting edge so that innovations of ever-increasing complexity are translated safely and swiftly into effective, high-quality therapies. We undertook a comprehensive examination of the evolution of science and technology impacting on medicinal product evaluation over the next 5-10 years and this horizon-scanning activity was complemented by extensive stakeholder interviews, resulting in a number of significant recommendations. Highlighted in particular was the need for expertise and regulatory science research to fill knowledge gaps in both more fundamental, longer-term research, with respect to technological and product-specific challenges. A model is proposed to realise these objectives in Europe, comprising a synergistic relationship between the European Medicines Agency, the European Medicines Regulatory Network and academic research centres to establish a novel regulatory science and innovation platform.

Simultaneous Transdermal Delivery of Buprenorphine Hydrochloride and Naltrexone Hydrochloride by Iontophoresis.

The opioids buprenorphine hydrochloride (BUP) and naltrexone hydrochloride (NTX) show promise as a combination treatment for addiction, but no means of delivering the two compounds in one medicine currently exist. In this paper, we report sufficient input rates of both these drugs from one iontophoretic transdermal drug delivery system. Experiments were performed using dermatomed pig skin mounted in glass side-bi-side cells. BUP and NTX were iontophoretically delivered together from the anode using direct constant current from Ag/AgCl electrodes. The transdermal drug fluxes and the masses of drugs in both the stratum corneum and the underlying epidermis/dermis were measured. The apparent electroosmotic flow was quantified using a neutral marker (acetaminophen). The effects of donor composition (drug concentration/molar fraction and pH), current density and profile, and the choice of receptor solution were assessed. Iontophoresis dramatically increased the flux of both drugs compared to passive control values. Target fluxes (calculated from literature clearance values and required therapeutic plasma concentrations) were greatly exceeded for NTX and were met for BUP. The latter accumulated in the skin and suppressed electroosmotic flow, inhibiting both its own flux and that of NTX. NTX, in turn, negatively influenced the flux of BUP via co-ion competition. Lowering current density by increasing the delivery area resulted in increased electroosmotic flow but did not significantly affect current-normalized drug fluxes. Delivering the drugs from both electrodes and reversing the polarity for every 2 h did not increase the flux of either compound. In summary, during iontophoresis, BUP and NTX inhibited each other's flux by two distinct mechanisms. While the more complex behavior of BUP complicates the optimization of this drug combination, iontophoresis nevertheless appears to be a feasible approach for the controlled codelivery of NTX and BUP through the skin.

Dermal Absorption of Pesticide Residues.

Current guidance for dermal exposure assessment of plant protection products typically uses in vitro skin penetration data for the active ingredient when applied as both the concentrated product and relevant spray dilutions thereof. However, typical re-entry scenarios involve potential skin exposure to a "dried residue" of the spray dilution, from which the absorption of a pesticide may be quite different. The research reported in this paper has shown: (1) The method to assess the transfer of dried pesticide residues from a surface to the skin is reproducible for four active ingredients of diverse physicochemical properties, after their application in commercially relevant formulations. (2) Skin absorption of all four pesticides examined was significantly less from a dried residue than from a spray dilution; the difference, in general, was of the order of a factor of 2. (3) Decontamination experiments with one of the active ingredients tested (trinexapac-ethyl) showed that, post-exposure to a spray dilution, skin surface cleaning must be performed within 1 h to significantly reduce potential systemic exposure (relative to continual contact for 24 h); in contrast, after contact with a dried residue, the sooner decontamination was performed, the greater the decrease in exposure achieved, even when the time of contact was as long as 8 h.

Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy.

The effective treatment of diseases of the nail remains an important unmet medical need, primarily because of poor drug delivery. To address this challenge, the diffusion, in real time, of topically applied chemicals into the human nail has been visualized and characterized using stimulated Raman scattering (SRS) microscopy. Deuterated water (D2O), propylene glycol (PG-d8), and dimethyl sulphoxide (DMSO-d6) were separately applied to the dorsal surface of human nail samples. SRS microscopy was used to image D2O, PG-d8/DMSO-d6, and the nail through the O-D, -CD2, and -CH2 bond stretching Raman signals, respectively. Signal intensities obtained were measured as functions of time and of depth into the nail. It was observed that the diffusion of D2O was more than an order of magnitude faster than that of PG-d8 and DMSO-d6. Normalization of the Raman signals, to correct in part for scattering and absorption, permitted semiquantitative analysis of the permeation profiles and strongly suggested that solvent diffusion diverged from classical behavior and that derived diffusivities may be concentration dependent. It appeared that the uptake of solvent progressively undermined the integrity of the nail. This previously unreported application of SRS has permitted, therefore, direct visualization and semiquantitation of solvent penetration into the human nail. The kinetics of uptake of the three chemicals studied demonstrated that each altered its own diffusion in the nail in an apparently concentration-dependent fashion. The scale of the unexpected behavior observed may prove beneficial in the design and optimization of drug formulations to treat recalcitrant nail disease.

Bioequivalence Methodologies for Topical Drug Products: In Vitro and Ex Vivo Studies with a Corticosteroid and an Anti-Fungal Drug.

OBJECTIVE: To examine whether in vitro and ex vivo measurements of topical drug product performance correlate with in vivo outcomes, such that more efficient experimental approaches can be reliably and reproducibly used to establish (in)equivalence between formulations for skin application. MATERIALS AND METHODS: In vitro drug release through artificial membranes, and drug penetration into porcine skin ex vivo, were compared with published human in vivo studies. Two betamethasone valerate (BMV) formulations, and three marketed econazole nitrate (EN) creams were assessed. RESULTS: For BMV, the stratum corneum (SC) uptake of drug in 6 h closely matched data observed in vivo in humans, and distinguished between inequivalent formulations. SC uptake of EN from the 3 creams mirrored the in vivo equivalence in man (both clinically and via similar tape-stripping experiments). However, EN clearance from SC ex vivo did not parallel that in vivo, presumably due to the absence of a functioning microcirculation. In vitro release of BMV from the different formulations did not overlap with either ex vivo or in vivo tape-stripping data whereas, for EN, a good correlation was observed. No measurable permeation of either BMV or EN was detected in a 6-h in vitro skin penetration experiment. CONCLUSIONS: In vitro and ex vivo methods for topical bioequivalence determination can show correlation with in vivo outcomes. However, these surrogates have understandable limitations. A "one-size-fits-all" approach for topical bioequivalence evaluation may not always be successful, therefore, and the judicious use of complementary methods may prove a more effective and reliable strategy.

Reverse Iontophoretic Extraction of Metabolites from Living Plants and their Identification by Ion-chromatography Coupled to High Resolution Mass Spectrometry.

INTRODUCTION: The identification and characterisation of cellular metabolites has now become an important strategy to obtain insight into functional plant biology. However, the extraction of metabolites for identification and analysis is challenging and, at the present time, usually requires destruction of the plant. OBJECTIVE: To detect different plant metabolites in living plants with no pre-treatment using the combination of iontophoresis and ion-chromatography with mass spectrometry detection. METHODOLOGY: In this work, the simple and non-destructive method of reverse iontophoresis has been used to extract in situ multiple plant metabolites from intact Ocimum basilicum leaves. Subsequently, the analysis of these metabolites has been performed with ion chromatography coupled directly to high resolution mass spectrometric detection (IC-MS). RESULTS: The application of reverse iontophoresis to living plant samples has avoided the need for complex pre-treatments. With this approach, no less than 24 compounds, including organic acids and sugars as well as adenosine triphosphate (ATP) were successfully detected. CONCLUSION: The research demonstrates that it is feasible to monitor, therefore, a number of important plant metabolites using a simple, relatively fast and non-destructive approach. Copyright © 2016 John Wiley & Sons, Ltd.

Assessing the safety of cosmetic chemicals: Consideration of a flux decision tree to predict dermally delivered systemic dose for comparison with oral TTC (Threshold of Toxicological Concern).

Threshold of Toxicological Concern (TTC) aids assessment of human health risks from exposure to low levels of chemicals when toxicity data are limited. The objective here was to explore the potential refinement of exposure for applying the oral TTC to chemicals found in cosmetic products, for which there are limited dermal absorption data. A decision tree was constructed to estimate the dermally absorbed amount of chemical, based on typical skin exposure scenarios. Dermal absorption was calculated using an established predictive algorithm to derive the maximum skin flux adjusted to the actual 'dose' applied. The predicted systemic availability (assuming no local metabolism), can then be ranked against the oral TTC for the relevant structural class. The predictive approach has been evaluated by deriving the experimental/prediction ratio for systemic availability for 22 cosmetic chemical exposure scenarios. These emphasise that estimation of skin penetration may be challenging for penetration enhancing formulations, short application times with incomplete rinse-off, or significant metabolism. While there were a few exceptions, the experiment-to-prediction ratios mostly fell within a factor of 10 of the ideal value of 1. It can be concluded therefore, that the approach is fit-for-purpose when used as a screening and prioritisation tool.

In Vitro Method to Quantify Dermal Absorption of Pesticide Residues.

All pesticides must go through a rigorous risk assessment process in order to show that they are safe for use.With respect to dermal risk assessment for re-entry workers, the absorption value applied to predict systemic dose from this external exposure is obtained by testing liquid forms of the pesticide in vivo and/or in vitro. However, in a real exposure scenario, the worker would be exposed to a dried residue, for which little or no absorption data are available. This study has developed a novel methodology for assessing the dermal absorption of pesticides from dried residues and aims ultimately to use this methodology to obtain more realistic absorption values for the risk assessment.

Characterization of topical film-forming systems using atomic force microscopy and Raman microspectroscopy.

Polymeric film-forming systems for dermal drug delivery represent an advantageous alternative to more conventional topically applied formulations. Their mechanical properties and homogeneity can be characterized with atomic force microscopy (AFM), using both imaging and nanoindentation modes, and Raman microspectroscopy mapping. Film-forming polymers, with and without a plasticizer and/or betamethasone 17-valerate (a representative topical drug), were dissolved in absolute ethanol. Polymeric films were then cast on glass slides and examined in ambient air using AFM imaging and Raman microspectroscopy. Using nanoindentation, the elastic moduli of various films were determined and found to decrease with increasing plasticizer content. Films with 20% w/w plasticizer had elastic moduli close to that of skin. AFM images showed little difference in the topography of the films on incorporation of plasticizer. Raman microspectroscopy maps of the surface of the polymeric films, with a spatial resolution of approximately 1 µm, revealed homogeneous distributions of plasticizer and drug within the films.

Characterisation of skin barrier function using bioengineering and biophysical techniques.

PURPOSE: To characterise skin barrier function in vivo at two distinct anatomic sites using minimally invasive bioengineering and biophysical tools. METHODS: In healthy human volunteers, the quantities of stratum corneum (SC) per unit area of skin on the forearm and forehead were quantified by gravimetric and imaging techniques. Organisation of the SC intercellular lipids was evaluated as a function of position using attenuated total reflectance infrared spectroscopy (ATR-IR). The constituents of natural moisturising factor (NMF) were extracted from tape-stripped samples of the SC and by reverse iontophoresis; 21 components were identified and quantified by liquid chromatography with mass spectrometric detection. RESULTS: SC was quantified more accurately by imaging and was significantly thinner on the forehead than on the forearm. Intercellular lipids were more disordered near the skin surface at both sites; however, throughout forearm SC, the lipids were substantially better organised than those in the forehead. Compositionally, the NMF from forearm and forehead SC was similar, but the total amount extractable from the forehead was smaller. CONCLUSION: Taken together, the bioengineering and biophysical techniques employed demonstrate, in a complementary, objective and quantitative fashion, that SC barrier function on the forehead is less competent than that on the forearm.

Iontophoretic transdermal sampling of iohexol as a non-invasive tool to assess glomerular filtration rate.

PURPOSE: To explore the potential of non-invasive reverse iontophoresis transdermal extraction of iohexol as a marker of glomerular filtration rate. METHODS: A series of in vitro experiments were undertaken to establish the feasibility of iohexol reverse iontophoresis and to determine the optimal conditions for the approach. Subsequently, a pilot study in paediatric patients was performed to provide proof-of-concept. RESULTS: The iontophoretic extraction fluxes of iohexol in vitro were proportional to the marker subdermal concentration and the reverse iontophoretic technique was able to track changes dynamically in simulated pharmacokinetic profiles. Reverse iontophoresis sampling was well tolerated by the four paediatric participants. The deduced values of the iohexol terminal elimination rate constant from transdermal reverse iontophoresis sampling agreed with those estimated by conventional blood sampling. CONCLUSIONS: Reverse iontophoretic transdermal extraction fluxes mirrored the subdermal concentration profiles of iohexol, a relatively large neutral marker of glomerular filtration both in vitro and in vivo. The efficiency of extraction in vivo was well predicted by the in vitro model used.

A non-rewarding, non-aversive buprenorphine/naltrexone combination attenuates drug-primed reinstatement to cocaine and morphine in rats in a conditioned place preference paradigm.

Concurrent use of cocaine and heroin is a major public health issue with no effective relapse prevention treatment currently available. To this purpose, a combination of buprenorphine and naltrexone, a mixed very-low efficacy mu-opioid receptor agonist/kappa-opioid receptor antagonist/nociceptin receptor agonist, was investigated. The tail-withdrawal and the conditioned place preference (CPP) assays in adult Sprague Dawley rats were used to show that naltrexone dose-dependently blocked the mu-opioid receptor agonism of buprenorphine. Furthermore, in the CPP assay, a combination of 0.3 mg/kg buprenorphine and 3.0 mg/kg naltrexone was aversive. A combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone was neither rewarding nor aversive, but still possessed mu-opioid receptor antagonist properties. In the CPP extinction and reinstatement method, a combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone completely blocked drug-primed reinstatement in cocaine-conditioned rats (conditioned with 3 mg/kg cocaine, drug prime was 3 mg/kg cocaine) and attenuated drug-primed reinstatement in morphine-conditioned rats (conditioned with 5 mg/kg morphine, drug prime was 1.25 mg/kg morphine). These data add to the growing evidence that a buprenorphine/naltrexone combination may be protective against relapse in a polydrug abuse situation.