Our group has recently developed a method for characterizing distribution of a topical drug within skin using two-photon fluorescence lifetime imaging (FLIM) and phasor analysis. Here, we expand on this work by describing a multimodal approach for rapidly visualizing multiple components in tissue using FLIM and coherent Raman imaging (CRI). By employing a non-Euclidian FLIM phasor analysis for a three-component system informed with the vibrational signature of one of the components retrieved with CRI, we were able to semi-quantitatively describe the spatial distribution of drugs in tissue with molecular specificity and cellular resolution.
The safety and efficacy of an investigational topical minocycline gel (BPX-01) has recently been studied in a Phase 2b trial for the treatment of acne vulgaris. As part of the drug development process, there was a need to determine if minocycline was delivered to the target tissue compartments, including the epidermis, hair follicle, and the sebaceous gland. While it was easier to demonstrate delivery on an ex vivo human skin model with an infinite dose, it was initially challenging to verify low-dose delivery with conventional fluorescence microscopy due to the high autofluorescence inherent to human skin. An integrating sphere screening approach was implemented along with conventional fluorescence microscopy to quantitatively and qualitatively assess endogenous fluorescence concurrently from numerous human facial skin specimens. Donor tissues were cut into 50-µm frozen sections, mounted onto microscope slides, and positioned on an inverted fluorescence microscope, sandwiched between the microscope’s 40x high NA objective lens and an external integrating sphere. The tissue sections were illuminated with UV excitation centered at 386 nm. For the first time, it was found that random samples from >40 human facial skin donors produced at least 5× differential in measurable autofluorescence. This observation has significant implications for the use of 2PEF microscopy and FLIM to visualize/quantify drug distribution; the endogenous autofluorescence may limit the detectability of the minocycline signature. Our studies indicated that a single daily dose of BPX-01 was detected in low autofluorescence skin specimens with FLIM, thus validating a novel imaging modality for future pharmacokinetic studies.
Oral minocycline has been the standard of care for the treatment of non-nodular moderate to severe inflammatory acne vulgaris due to its inhibitory effects on the acne-causing Propionibacterium acnes bacterium and its anti-inflammatory properties, Despite the availability of an oral dosage form since 1966, a commercial topical minocycline remains elusive because of the challenges in stabilizing the active pharmaceutical ingredient (API) in a liquid/semisolid while ensuring sufficient uptake into targeted lesions. Recently, an investigative topical minocycline gel (BPX-01) has been developed to address the unmet needs for localized and targeted delivery while minimizing the risks of systemic side effects. Earlier preclinical studies pertaining to transepidermal delivery of the API had depended on semi-infinite doses of the 1%, 2% and 4% formulations to elicit enough fluorescence yield. We have subsequently shown evidence of minocycline delivery of 1% and 4% BPX-01 into the pilosebaceous unit of ex vivo human facial skin specimens dosed with about 2.5× daily dose using two-photon excitation fluorescence microscopy. In this study, we demonstrated another novel approach to identifying minocycline fluorescence signature using fluorescence lifetime imaging microscopy (FLIM) with phasor analysis. It was found that for a single daily dose and with FLIM, minocycline was consistently noted in the epidermis and hair follicle, with some incidence in the sebaceous gland for both 1% and 2% BPX-01. These observations corroborated with the recent success of a Phase 2b dose-finding study, with 2% BPX-01 meeting the primary endpoint of lesion reduction at week 12 with statistical significance over the vehicle.
Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the phasor approach to FLIM. The unique signature of minocycline in FLIM phasor analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.
Minocycline is an antibiotic regularly prescribed to treat acne vulgaris. The only commercially available minocycline comes in an oral dosage form, which often results in systemic adverse effects. A topical minocycline composition (BPX-01) was developed to provide localized and targeted delivery to the epidermis and pilosebaceous unit where acne-related bacteria, Propionibacterium acnes (P. acnes), reside. As minocycline is a known fluorophore, fluorescence microscopy was performed to investigate its potential use in visualizing minocycline distribution within tissues. BPX-01 with various concentrations of minocycline, was applied topically to freshly excised human facial skin specimens. Spatial distribution of minocycline and its fluorescence intensity within the stratum corneum, epidermis, dermis, and pilosebaceous unit were assessed. The resulting fluorescence intensity data as a function of minocycline concentration may indicate clinically relevant therapeutic doses of topical BPX-01 needed to kill P. acnes and reduce inflammation for successful clinical outcomes.
Acne vulgaris is a chronic inflammatory skin condition commonly resulting in negative aesthetic and social impacts on those affected. Minocycline, currently available as an oral antibiotic for moderate to severe acne, has a known minimum inhibitory concentration (MIC) for the acne-causing bacterium Propionibacterium acnes (P. acnes) in vitro, with its anti-inflammatory properties also eliciting inhibitory effects on pro-inflammatory molecules. A novel topical gel composition containing solubilized minocycline (BPX-01) has been developed to directly deliver the drug to the skin. Because minocycline is a known fluorophore, fluorescence microscopy and concurrent quantitative measurements were performed on excised human facial skin dosed with different concentrations, in order to determine the spatial distribution of the drug and quantification of its local concentration in the epidermis and the pilosebaceous unit where P. acnes generally reside. Local minocycline delivery confirmed achievement of an adequate therapeutic dose to support clinical studies. Subsequently, a 4-week double-blind, randomized, vehicle controlled clinical study was performed to assess the safety and efficacy of 1% minocycline BPX-01 applied daily. No instances of cutaneous toxicity were reported, and a greater than 1 log reduction of P. acnes count was observed at week 4 with statistical significance from baseline and vehicle control. In addition, no detectable amounts of minocycline in the plasma were reported, suggesting the potential of this new formulation to diminish the known systemic adverse effects associated with oral minocycline. Follow-on clinical plans are underway to further establish the safety of BPX-01 and to evaluate its efficacy against inflammatory acne lesions in a 225 patient multi-center dose-finding study.
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