Evaluation of In Vitro Tools to Predict the In Vivo Absorption of Biopharmaceuticals Following Subcutaneous Administration.

PURPOSE: For injectable biopharmaceuticals, the subcutaneous route of administration is increasingly preferred over intravenous administration. However, one of the challenges in the development of subcutaneously administered biopharmaceuticals is a reduced bioavailability, which is difficult to predict. Since animal models do not reliably reflect bioavailability in patients, in vitro models could help to develop drug candidates. The purpose of this study was to evaluate a versatile set of in vitro tools for their suitability to predict bioavailability of biopharmaceuticals after subcutaneous administration. METHODS: We examined seven commercially available biopharmaceuticals using three instruments, i.e., the Subcutaneous Injection Site Simulator (Scissor), the Osmomat 050, and a dialysis system using three artificial extracellular matrices, two dissolution apparatuses, i.e., the USP4 and the USP7, and two evaluation tools, i.e., the affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) and the Developability Index (DI). Results were evaluated for their usefulness to predict the bioavailability and other pharmacokinetic parameters in humans using the Pearson correlation. RESULTS: None of the tested instruments and methods could reliably approximate bioavailability. Only pressure values derived with the Osmomat 050 instrument correlated with Cmax with a Pearson correlation coefficient greater than 0.8. CONCLUSION: No single in vitro method confidently predicted the bioavailability in humans. We only found a correlation to maximum plasma concentration values for one of the tested approaches. However, a more focused evaluation would be necessary to confirm our findings and test combinations of orthogonal methods that may improve the confidence of such a prediction.

Spray-Dried Monoclonal Antibody Suspension for High-Concentration and Low-Viscosity Subcutaneous Injection.

Administration of highly concentrated monoclonal antibodies (mAbs) through injection is often not possible as the viscosity can be readily above 50 mPa·s when the concentration exceeds 150 mg/mL. Besides, highly concentrated mAb solutions always exhibit increased aggregation propensity and lower stability, which raise the difficulty for the successful development of highly concentrated mAb formulations. We hereby explored the possibility of suspension as another formulation form for high-concentration proteins to reduce viscosity and maintain stability. Specifically, we demonstrated that spray drying can serve as a process to prepare particles for suspension. Particles prepared from formulations with different mAb/trehalose mass ratios displayed good physical stability and antibody binding affinity, as indicated by circular dichroism, fluorescence spectroscopy, and surface plasmon resonance (SPR)-based bioassay analyses. During spray drying, a surface tension-dominated enrichment of mAb on the particle surface was observed, but this did not show a significant negative impact on mAb stability. Spray-dried particles were subsequently suspended into benzyl benzoate, and the resulting suspension showed good stability and a lower viscosity when compared to its counterpart solution. Furthermore, mAbs recovered from the suspension maintained their conformational structure. Our study demonstrated that the suspension displayed low viscosity and good physical stability, so it may offer novel opportunities for the preparation of highly concentrated protein formulations.

Quantitative detection of caffeine in human skin by confocal Raman spectroscopy--A systematic in vitro validation study.

For rational development and evaluation of dermal drug delivery, the knowledge of rate and extent of substance penetration into the human skin is essential. However, current analytical procedures are destructive, labor intense and lack a defined spatial resolution. In this context, confocal Raman microscopy bares the potential to overcome current limitations in drug depth profiling. Confocal Raman microscopy already proved its suitability for the acquisition of qualitative penetration profiles, but a comprehensive investigation regarding its suitability for quantitative measurements inside the human skin is still missing. In this work, we present a systematic validation study to deploy confocal Raman microscopy for quantitative drug depth profiling in human skin. After we validated our Raman microscopic setup, we successfully established an experimental procedure that allows correlating the Raman signal of a model drug with its controlled concentration in human skin. To overcome current drawbacks in drug depth profiling, we evaluated different modes of peak correlation for quantitative Raman measurements and offer a suitable operating procedure for quantitative drug depth profiling in human skin. In conclusion, we successfully demonstrate the potential of confocal Raman microscopy for quantitative drug depth profiling in human skin as valuable alternative to destructive state-of-the-art techniques.

Applications of Raman spectroscopy in skin research--From skin physiology and diagnosis up to risk assessment and dermal drug delivery.

In the field of skin research, confocal Raman microscopy is an upcoming analytical technique. Substantial technical progress in design and performance of the individual setup components like detectors and lasers as well as the combination with confocal microscopy enables chemically selective and non-destructive sample analysis with high spatial resolution in three dimensions. Due to these advantages, the technique bears tremendous potential for diverse skin applications ranging from the analysis of physiological component distribution in skin tissue and the diagnosis of pathological states up to biopharmaceutical investigations such as drug penetration kinetics within the different tissue layers. This review provides a comprehensive introduction about the basic principles of Raman microscopy highlighting the advantages and considering the limitations of the technique for skin applications. Subsequently, an overview about skin research studies applying Raman spectroscopy is given comprising various in vitro as well as in vivo implementations. Furthermore, the future perspective and potential of Raman microscopy in the field of skin research are discussed.

Overcoming drug crystallization in electrospun fibers--Elucidating key parameters and developing strategies for drug delivery.

For the development of novel therapeutics, uncontrolled crystallization of drugs within delivery systems represents a major challenge. Especially for thin and flexible polymeric systems such as oral films or dermal wound dressings, the formation and growth of drug crystals can significantly affect drug distribution and release kinetics as well as physical storage stability. In this context, electrospinning was introduced as a fabrication technique with the potential to encapsulate drugs within ultrafine fibers by rapid solvent evaporation overcoming drug crystallization during fabrication and storage. However, these effects could so far only be shown for specific drug-polymer combinations and an in-depth understanding of the underlying processes of drug-loaded fiber formation and influencing key parameters is still missing. In this study, we systematically investigated crystal formation of caffeine as a model drug in electrospun fibers comparing different polymers. The solvent polarity was found to have a major impact on the drug crystal formation, whereas only a minor effect was attributed to the electrospinning process parameters. Based on an in-depth understanding of the underlying processes determining drug crystallization processes in electrospun fibers, key parameters could be identified which allow for the rational development of drug-loaded electrospun fibers overcoming drug crystallization.

Novel in vitro approaches for the simulation and analysis of human skin wounds.

Considering the increasing incidence of chronic wounds and severe wound infections, effective drug delivery to wounded skin is of high importance. The rational development of novel therapeutic systems requires appropriate in vitro testing methodologies. In this context, suitable and reliable in vitro models simulating human wounds and advanced analytical techniques for precise wound characterization are urgently needed. In this study, we introduce a novel in vitro model based on excised human skin. In contrast to the established wound models, our novel approach has a coffin-shaped, linear, rectangular geometry with defined wound edges exhibiting consistent appearance along the entire wound bed. In addition, we introduce optical profilometry as a novel technique for nondestructive wound analysis. We successfully demonstrate the applicability of this optical imaging method based on white light reflection for three-dimensional visualization of different wound models. Furthermore, we create virtual noninvasive cross sections of these wounds to assess wound geometry in direct comparison to conventional histological analysis. Imaging analysis of our novel coffin-shaped model resulted in reproducible virtual sections along the entire wound bed. Our findings indicate the potential of our novel in vitro model for improved simulation of human wounds. Further, we successfully overcome the limitations of conventional histological analysis by the employment of optical profilometry for nondestructive three-dimensional wound characterization.

Combining confocal Raman microscopy and freeze-drying for quantification of substance penetration into human skin.

In the area of dermatological research, the knowledge of rate and extent of substance penetration into the human skin is essential not only for evaluation of therapeutics, but also for risk assessment of chemicals and cosmetic ingredients. Recently, confocal Raman microscopy emerged as a novel analytical technique for analysis of substance skin penetration. In contrast to destructive drug extraction and quantification, the technique is non-destructive and provides high spatial resolution in three dimensions. However, the generation of time-resolved concentration depth profiles is restrained by ongoing diffusion of the penetrating substance during analysis. To prevent that, substance diffusion in excised human skin can instantly be stopped at defined time points by freeze-drying the sample. Thus, combining sample preparation by freeze-drying with drug quantification by confocal Raman microscopy yields a novel analytical platform for non-invasive and quantitative in vitro analysis of substance skin penetration. This work presents the first proof-of-concept study for non-invasive quantitative substance depth profiling in freeze-dried excised human stratum corneum by confocal Raman microscopy.

Freeze-drying as a preserving preparation technique for in vitro testing of human skin.

In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze-drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze-drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze-drying. Furthermore, we show that freeze-drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze-drying as a preserving preparation technique for in vitro testing of human skin.

Towards drug quantification in human skin with confocal Raman microscopy.

Understanding the penetration behaviour of drugs into human skin is a prerequisite for the rational development and evaluation of effective dermal drug delivery. The general procedure for the acquisition of quantitative drug penetration profiles in human skin is performed by sequential segmentation and extraction. Unfortunately, this technique is destructive, laborious and lacks spatial resolution. Confocal Raman microscopy bares the potential of a chemically selective, label free and nondestructive analysis. However, the acquisition of quantitative drug depth profiles within skin by Raman microscopy is impeded by imponderable signal attenuation inside the tissue. In this study, we present a chemical semi-solid matrix system simulating the optical properties of human skin. This system serves as a skin surrogate for investigation of Raman signal attenuation under controlled conditions. Caffeine was homogeneously incorporated within the skin surrogate, and Raman intensity depth profiles were acquired. A mathematical algorithm describing the Raman signal attenuation within the surrogate was derived from these profiles. Human skin samples were incubated with caffeine, and Raman intensity depth profiles were similarly acquired. The surrogate algorithm was successfully applied to correct the drug profiles in human skin for signal attenuation. For the first time, a mathematical algorithm was established, which allows correction of Raman signal attenuation in human skin, thus facilitating reliable drug quantification in human skin by confocal Raman spectroscopy.

Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy.

Hair follicles have recently gained a lot of interest for dermal drug delivery. They provide facilitated penetration into the skin and a high potential to serve as a drug depot. In this area of research, excised pig ear is a widely accepted in vitro model to evaluate penetration of drug delivery into hair follicles. However, a comparison of human and porcine follicles in terms of chemical composition has not been performed so far. In this study, we applied confocal Raman microscopy as a chemically selective imaging technique to compare human and porcine follicle composition and to visualize component distribution within follicle cross-sections. Based on the evaluation of human and porcine Raman spectra optical similarity for both species was successfully confirmed. Furthermore, cyanoacrylate skin surface biopsies, which are generally used to determine the extent of follicular penetration, were imaged by a novel complementary analytical approach combining confocal Raman microscopy and optical profilometry. This all-encompassing analysis allows investigation of intactness and component distribution of the excised hair bulb in three dimensions. Confocal Raman microscopy shows a high potential as a noninvasive and chemically selective technique for the analysis of trans-follicular drug delivery.