Zane A. Arp

Optimization of a high shear wet granulation process using focused beam reflectance measurement and particle vision microscope technologies

Application of process analytical technology in the pharmaceutical industry has led to a great number of studies into inline instrumentation. Near-infrared moisture monitoring in fluid bed drying and content uniformity assurance in blending are gaining acceptance for monitoring and quality control of these processes. Although these techniques are a great improvement over traditional methods, each is performed at points in processing wherein processing is well understood and interfacing equipment is relatively easy. More complex unit operations have largely been unexplored due to complexities interfacing inline analytical equipment to unit operations or a lack of methodologies that can be applied to measure attributes of interest. This paper reports results from a study utilizing a focused beam reflectance measurement system equipped with window scraper technology for the inline measurement and control of a high shear wet granulation (HSWG) process. In addition to this, offline results obtained with a particle vision microscope system are compared to verify the results obtained inline. It is shown that using these technologies in monitoring the HSWG process greatly increases process understanding of physical changes occurring during processing through real-time observation of particle size, leading to real-time control of the process.

Longitudinal in vivo tracking of adverse effects following topical steroid treatment.

Topical steroids are known for their anti‐inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two‐photon excitation fluorescence microscopy (TPEF) of reduced nicotinamide adenine dinucleotide (NADH) to visualize the epidermal cell layers and second harmonic generation (SHG) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM) is also utilized to image intracellular NADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP)‐treated, mometasone furoate (MF)‐treated and untreated animals were imaged longitudinally using a custom‐built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime of NADH, suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment.

Noninvasive monitoring of pharmacodynamics in the skin wound healing process using multimodal microscopy (Conference Presentation)

Approximately 29 million Americans have diabetes, and 86 million are living with prediabetes, increasing the risk of developing type 2 diabetes. Complications of wound healing in diabetic patients represent a significant health problem. Impaired diabetic wound healing is characterized by reduced collagen production and diminished angiogenesis. During the proliferative phase of wound healing, the injured tissue undergoes angiogenesis, re-epithelialization, and fibroplasia. Monitoring the development of new blood vessels, metabolic changes, and collagen deposition, is critical to elucidate the process of diabetic wound healing and to improve the development of therapeutic drugs. This study employs a custom-built multimodal microscope where Optical Coherence Tomography Angiography (OCTA) is used for studying neovascularization, Fluorescence Lifetime Imaging Microscopy (FLIM) for NADH/FAD assessment, Second Harmonic Generation (SHG) microscopy for analyzing collagen deposition, and Coherent anti-Stoke’s Raman Scattering (CARS) microscopy for visualizing water/lipid distribution, all together to non-invasively follow closure of a skin wound in healthy diabetic (db/db) mice treated with placebo and angiogenesis-promoting topical formulation (GlaxoSmithKline). The (db/db) mouse model presents hyperglycemia, obesity, and delayed wound healing that is pathologically similar to human type 2 diabetes mellitus. In this ongoing study, the animals are treated once daily for 14 days after wounding. Images of the wound and surrounding areas are taken at different time points for 28 days. In this experiment, the wound healing process is investigated to gain deeper understanding of the drug mechanism. The capability to non-invasively monitor wound healing mechanisms can become a valuable tool in development of new drug compounds for diabetic wound care.

Investigating the healing mechanisms of an angiogenesis-promoting topical treatment for diabetic wounds using multimodal microscopy

Impaired skin wound healing is a significant comorbid condition of diabetes that is caused by poor microcirculation, among other factors. Studies have shown that angiogenesis, a critical step in the wound healing process in diabetic wounds, can be promoted under hypoxia. In this study, an angiogenesis-promoting topical treatment for diabetic wounds, which promotes angiogenesis by mimicking a hypoxic environment via inhibition of prolyl hydroxylase resulting in elevation or maintenance of hypoxia-inducible factor, was investigated utilizing a custom-built multimodal microscopy system equipped with phase-variance optical coherence tomography (PV-OCT) and fluorescence lifetime imaging microscopy (FLIM). PV-OCT was used to track the regeneration of the microvasculature network, and FLIM was used to assess the in vivo metabolic response of mouse epidermal keratinocytes to the treatment during healing. Results show a significant decrease in the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide, suggesting a hypoxic-like environment in the wounded skin, followed by a quantitative increase in blood vessel density assessed by PV-OCT. Insights gained in these studies could lead to new endpoints for evaluation of the efficacy and healing mechanisms of wound-healing drugs in a setting where delayed healing does not permit available methods for evaluation to take place.

Highly integrated label-free multiphoton nonlinear optical microspectroscopy imaging system for biomolecular imaging (Conference Presentation)

A biological sample consists of a variety of complex biomolecules, and fluorescence microscopy enables visualization of specific molecules at the sub-cellular level. However, these fluorescence techniques require certain fluorescence dyes to label the sample, and the fluorophores raise serious problems such as photo toxicity and photobleaching which could affect biological functionality in living systems. Advanced label-free optical imaging techniques based on nonlinear optical phenomena overcome these limitations of fluorescence microscopy. We have developed a novel label-free multimodal multiphoton nonlinear optical imaging system based on a near-IR femtosecond laser with photonic crystal fiber and pulse shaper. This highly integrated system offers numerous label-free techniques including third harmonic generation, three-photon excited fluorescence, second harmonic generation, two-photon excited fluorescence, fluorescence lifetime imaging, and broadband coherent anti-Stokes Raman scattering microspectroscopy in one platform. All of the nonlinear signals are spectrally separated by dichroic filters and simultaneously measured by photomultiplier tubes. Moreover, this system includes phase-variance optical coherence tomography as well to enable vascular imaging. We have applied our system to investigate processes in numerous biological samples. Our imaging technique is highly integrated and time efficient to generate big data, offering an array of biomolecular information at one time without staining, three-dimensional sub-micron resolution with deeper penetration, and less photodamage. The big data output from this system is analyzed by multivariate analysis such as principal component analysis and hierarchical cluster analysis. Therefore, this novel technology and methodology will have a great impact on fast in vivo label-free biomedical imaging as a big data generator.

Detecting testicular toxicity using label-free multimodal nonlinear optical imaging (Conference Presentation)

Toxicology of the male reproductive system has received increased interest in recent years partly fueled by the growing reports of falling sperm counts and rising reproductive disorders in the human population. Testicular toxicity (TT) in pharmaceutical development is a challenging issue due to the lack of simple and robust screening methods. Currently, histopathologic examination and hormonal evaluation are the commonly used methods to assess TT. Improved biomarker or screening platforms that would allow identification of TT at an earlier stage can have a significant impact on the safety evaluation of pharmaceutical candidates. We investigated the potential of label-free optical nonlinear imaging technologies such as fluorescence lifetime imaging microscopy (FLIM), multi-photon microscopy (MPM) and coherent anti-Stokes Raman scattering (CARS) microscopy to identify novel biomarkers for effective detection of TT. In this study, testicular damage was induced in rats by intraperitoneal injection with 3 mg/kg cisplatin, a chemotherapy drug. Multimodal optical images were obtained from the fixed, unstained testicular tissue sections of untreated and treated rats using a custom-built near-infrared multiphoton imaging system. Structural and biochemical parameters extracted from these images were compared between both groups to identify abnormal features associated with TT in the treated group. By analyzing the complimentary information obtained using these label-free optical imaging technologies, it may be possible to develop a novel platform for evaluation of TT in safety assessment of pharmaceuticals on reproduction and fertility, which reveal these changes at the molecular level and allow observation of these changes at an earlier time point than available today.

Imaging bio-distribution of a topically applied dermatological cream on minipig skin using fluorescence lifetime imaging microscopy (Conference Presentation)

Currently there is a lack of in vivo techniques to evaluate the spatial bio-distribution of dermal drugs over time without the need to take multiple serial biopsies. To address this gap, we investigated the use of multi-photon optical imaging methods to non-invasively track drug distribution on miniature pig (Species: Sus scrofa, Strain: Göttingen) skin in vivo. Minipig skin is the standard comparative research model to human skin, and is anatomically and functionally similar. We employed fluorescence lifetime imaging microscopy (FLIM) to visualize the spatial distribution and residency time of a topically applied experimental dermatological cream. This was made possible by the endogenous fluorescent optical properties of the experimental drug (fluorescence lifetime > 3000 ps). Two different drug formulations were applied on 2 minipigs for 7 consecutive days, with the control creams applied on the contralateral side, followed by 7 days of post-application monitoring using a multi-modal optical imaging system (MPTflex-CARS, JenLab, Germany). FLIM images were obtained from the treated regions 24 hr post-application from day 1 to day 14 that allowed visualization of cellular and sub-cellular features associated with different dermal layers non-invasively to a depth of 200 µm. Five punch biopsies per animal were obtained from the corresponding treated regions between days 8 and 14 for bioanalytical analysis and comparison with results obtained using FLIM. In conclusion, utilization of non-invasive optical biopsy methods for dermal drug evaluation can provide true longitudinal monitoring of drug spatial distribution, remove sampling limitations, and be more time-efficient compared to traditional methods.

Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

Investigation of an angiogenesis-promoting topical treatment for diabetic wounds using multimodal microscopy (Conference Presentation)

Impaired skin wound healing is a significant co-morbid condition of diabetes that is caused by poor microcirculation among other factors. Hypoxia-inducible factors (HIFs) are transcription factors that mediate the effects of decreased levels of oxygen in biological environments. Inducing mild hypoxia in the tissue could promote angiogenesis, a critical step in the wound healing process in diabetic wounds. To investigate the relationship between hypoxia and diabetic wound healing, a topical treatment consisting of a HIF-activating prolyl-hydroxylase inhibitor was administered to the wounded skin of diabetic (db/db) mice. Studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed at GSK or by the ethical review process at the institution where the work was performed. The wounded area was tracked in vivo for 28 days utilizing a custom-built multimodal microscopy system. An increase in vascular density around the wounds of treated animals was observed using phase-variance optical coherence tomography (PV-OCT), in comparison to normal controls. In addition, second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) were utilized to examine the collagen regeneration and cellular metabolic activity, respectively, in the wounded skin. The utilization of these light based methods can follow metabolic and morphologic changes in the wound healing process in ways not possible with current evaluation processes. Insights demonstrated in these studies could lead to new endpoints for evaluation of the efficacy of drugs and lead to more direct ways of detecting patient response to treatment.