Visualizing and quantifying drug uptake in skin

The study of drug uptake, distribution, and activity within skin is a necessary but problematic requirement in the development and translation of compounds from the bench to the bedside. Drug delivery into the skin is highly complex, due in part to the natural barrier function of the stratum corneum in addition to the many different routes of transdermal entry of drugs. Moreover, skin is not uniform throughout the body or across age groups. For example, epidermal thickness changes 30-fold from the thick skin of the fingertips (485μm) to the thin skin of the face and eyelids (17μm).¹ Transdermal delivery can occur over a wide range of timescales (from seconds to hours), and the number of potential cellular targets necessitates quantification on the micrometer scale.²

Optical imaging tools are well-suited to meet these challenges, in particular for the uptake of drugs within the first millimeter of skin. Fluorescence, Raman, and nonlinear optical imaging techniques offer subcellular resolution, rapid real-time 3D image acquisition, and the ability to quantitatively analyze imaging data for both pharmacokinetic and pharmacodynamic information. Optical tools are unique in that they also offer the ability to quantify drugs via phenomena that emerge from their structure, including light absorption, fluorescence, and molecular vibrations. This is particularly useful as most pharmaceuticals are small molecules, where modification to include a reporter can completely change the behavior and thus uptake of the compound.

Fluorescence imaging methods can be particularly powerful in measuring the uptake and distribution of drugs. We have been developing a topical acne gel, BPX-01, that is currently in a clinical Phase 2b dose-finding study. BPX-01 is an anhydrous hydrophilic topical gel with solubilized minocycline for enhanced cutaneous delivery and bioavailability to target lesions, with acne vulgaris as the intended indication for use. Because minocycline hydrochloride, the active ingredient, is a fluorophore, fluorescence microscopy can be used to visualize BPX-01 delivery and distribution post-topical application.³ We have recently demonstrated visualization of this drug on excised facial skin specimens as well as in in vivo minipig skin studies.

Conventional fluorescence microscopy showed minocycline to accumulate in the stratum corneum, the epidermis, and the hair follicle, as expected, as well as in the sebaceous glands—where acne lesions typically developed—after 24 hours (see Figure 1). The main route of delivery appeared to be both transfollicular and through the stratum corneum. Most recently, two-photon fluorescence microscopy demonstrated a similar trend of drug delivery at doses an order of magnitude lower after only 4 hours of application (see Figure 2).^4,5 The multiphoton imaging approach provided much higher detection sensitivity.

Figure 1. Conventional fluorescence microscopy images of ex vivo human facial skin specimens. (a) Control, and those treated with (b) 1% BPX-01 (a topical acne gel) and (c) 4% BPX-01 at 24 hours. Minocycline fluorescence is shown in red.

Figure 2. Two-photon fluorescence microscopy images of ex vivo human facial skin specimens. Left: Control with endogenous fluorescence in the absence of minocycline in epidermis (EP), hair follicle (HF), and sebaceous gland (SG). Center: 1% BPX-01, and Right: 4% BPX-01 minocycline fluorescence in EP, HF, and SG, after only 4 hours of application.

In other cases, topical compounds—whether the active ingredient itself or the vehicle carrying it—can be visualized in situ without the need for any exogenous labels. One such method makes use of a molecule's intrinsic molecular vibrations via Raman scattering. For rapid imaging and quantification of drugs and actives, coherent Raman imaging can be particularly powerful for rapid, high-resolution, and deep imaging in skin. For example, we have followed the uptake and distribution of compounds such as mineral oil, used in many cosmetic compounds, in real time by visualizing the stretching vibrations of methylene (CH₂) within the molecule via coherent anti-Stokes Raman scattering (CARS) microscopy.⁶ Similarly, the uptake of drugs such as retinoic acid into skin was quantified by X. Sunney Xie and colleagues using stimulated Raman scattering (SRS) microscopy.⁷

Coherent Raman scattering can also be useful in monitoring the state of lipid membranes by probing CH₂ stretching vibrations, which fundamentally control topical drug delivery at the stratum corneum level. Many drug-delivery-enhancement strategies are focused on altering this lipid/protein barrier, primarily by chemical or mechanical means. The effects of these enhancing technologies on the lipid barrier of the stratum corneum can be studied with both CARS and SRS within living human skin. In this way, image analysis can be used to quantify the relative amount of traceable compound being released from the surface of advanced microdevices, such as microneedles or elongated microparticles, where signal intensity changes with depth in the skin and can be quantified over time. This approach gives a far more granular view of molecular delivery and skin structure interactions than the conventional diffusion cell methodologies that dominate the drug delivery literature. One example is the capacity to monitor drug-loaded nanoemulsion release from dry-coated elongated microparticles into living human skin. Combining multiphoton microscopy with CARS and SRS makes it possible to observe these lipid-based nanoparticles in concert with the lipid component of corneocytes and keratinocytes, alongside the microparticles, the fluorescence of NADH and FAD co-factors, and the second-harmonic signal of collagen.

Such multifaceted, parallel imaging protocols yield far more quantifiable data than current drug delivery analytical assays. Successes in applying advanced optical imaging technologies to visualize and quantify targeted drug delivery could provide a competitive and economic advantage necessary for accelerating and optimizing drug development over current available tools such as radiolabeling, which are cumbersome to use, time-consuming, and expensive. Our focus is now on translating these toolkits from the lab into the dermatology clinic to power a new generation of drug development studies.

Figures courtesy of BioPharmX, Inc. Caution: BPX-01 is a new drug limited by US law to investigational use.