Evaluation of chemical disposition in skin by stimulated Raman scattering microscopy.

Tracking drug disposition in the skin in a non-destructive and at least semi-quantitative fashion is a relevant objective for the assessment of local (cutaneous) bioavailability. Confocal Raman spectroscopy has been shown potentially useful in this regard and, importantly, recent advances have enabled the presence of applied chemicals in the viable epidermis below the stratum corneum (SC) to be determined without ambiguity and having addressed the challenges of (a) background signals from endogenous species and noise and (b) signal attenuation due to absorption and scattering. This study aimed to confirm these observations using a different vibrational spectroscopy approach - specifically, stimulated Raman scattering (SRS) microscopy - and the more conventional in vitro skin penetration test (IVPT). SRS is a nonlinear optical imaging technique which enables more precise location of the skin surface and enhanced skin depth resolution relative to confocal Raman microscopy. The method can also probe larger areas of the sample under investigation and identify the localization of the permeating chemical in specific structural components of the skin. Here, SRS was shown capable of tracking the uptake and distribution of 4-cyanophenol (CP), the same model compound used in the recent confocal Raman investigation, at depths beyond the SC following skin treatment with different vehicles and for different times. The SRS results correlated well with those from the confocal Raman experiments, and both were consistent with independent IVPT measurements. Acquired images clearly delineated CP preference for the intercellular lipid layers of the SC relative to the corneocytes. The stage is now set to apply these and other correlative techniques to examine commercial drug products.

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.

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.

Affinity of Lipophilic Drugs to Mixed Lipid Aggregates in Simulated Gastrointestinal Fluids.

Mixed lipid aggregates, comprising of bile salts and phospholipids, present in the small intestine assist in drug solubilization and subsequent drug dissolution and absorption through the intestinal epithelium. The increased variability in their levels, observed physiologically, may create challenges not only for in vivo bioavailability and bioequivalence studies, but also for in vitro bio-predictive studies as correlations between in vitro and in vivo data are not always successful. The current study investigated the impact of biorelevant dissolution media, with physiologically relevant sodium taurocholate and lecithin levels, on the apparent solubility and affinity of lipophilic compounds with a wide range of physicochemical properties (drug ionization, drug lipophilicity, molecular weight) to mixed lipid aggregates. Apparent solubility data in biorelevant dissolution media for the studied neutral drugs, weak bases and weak acids were compared against a phosphate buffer pH 6.5 in the absence of these lipidic components. Presence of mixed lipid aggregates enhanced the apparent solubility of the majority of compounds and the use of multivariate data analysis identified the significant parameters affecting drug affinity to mixed lipid aggregates based on the chemical class of the drug. For neutral drugs, increasing bile salt concentrations and/or drug lipophilicity resulted in greater enhancement in apparent solubility at 24-hr. For weak bases and weak acids, the effect of increasing bile salt levels on apparent solubility depended mostly on an interplay between drug lipophilicity and drug ionization.

Investigation of drug partition kinetics to fat in simulated fed state gastric conditions based on drug properties.

The presence of fat in the gastric environment can affect the pharmacokinetic behaviour of drugs with mechanisms which have not been yet fully understood. The objective of the current study was to assess the drug partition to the lipid part of the fed gastric content under different emulsification conditions, using in vitro discriminating setups. The model drugs used in the study were selected on the basis of different physicochemical properties (lipophilicity, ionization, molecular weight and aqueous solubility) and different food effect observed in in vivo human studies. Fed State Simulated Gastric Fluid prepared with skimmed milk (FeSSGFsk) and anhydrous milk fat were used as surrogates for the aqueous and fat portions of the fed gastric environment respectively. An optimized biphasic model was developed so as to predict the differences in partition rate constants to fat, for model drugs of a wide range of the properties mentioned above. The experimental data and the use of statistical analysis revealed that molecular weight, molecular weight and log D pH 5 interaction and negative food effect act as negative factors to the rate constants of fat partition, while absence of food effect and logD pH 5 interaction with aqueous solubility affect the rate constants of partition to fat favourably.

Biopharmaceutical Understanding of Excipient Variability on Drug Apparent Solubility Based on Drug Physicochemical Properties. Case Study: Superdisintegrants.

The presence of different excipient types/brands in solid oral dosage forms may affect product performance and drug bioavailability. Understanding the biopharmaceutical implications of superdisintegrant variability (changes in material properties), variation (changes in excipient amount) and interchangeability (use of different excipient types with the same intended functionality) in oral drug performance would be beneficial for the development of robust final dosage forms. The current study investigated the impact of superdisintegrants (sodium starch glycolate, croscarmellose sodium, crospovidone) on the apparent solubility of drugs with different physicochemical properties (drug ionisation, drug lipophilicity, drug aqueous solubility). Compendial and biorelevant media were used to assess the impact of gastrointestinal conditions on the effects of excipient on drug apparent solubility. For the majority of compounds, changes in drug apparent solubility were not observed in superdisintegrant presence, apart from the cases of highly ionised compounds (significant decrease in drug solubility) and/or compounds that aggregate/precipitate in solution (significant increase in drug solubility). Excipient variability did not greatly affect the impact of excipients on drug apparent solubility. The use of multivariate data analysis identified the biopharmaceutical factors affecting excipient performance. The construction of roadmaps revealed that superdisintegrants may be of low risk for the impact of excipients on oral drug performance based on drug solubility alone; superdisintegrants activity could still be a risk for oral bioavailability due to their effects on tablet disintegration.