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Understanding a drug candidate’s pharmacokinetic (PK) parameters is a challenging but essential aspect of drug development. Investigating the penetration and distribution of a topical drug’s active pharmaceutical ingredient (API) allows for evaluating drug delivery and efficacy, which is necessary to ensure drug viability. A topical gel (BPX-05) was recently developed to treat moderate to severe acne vulgaris by directly delivering the combination of the topical antibiotic minocycline and the retinoid tazarotene to the pilosebaceous unit of the dermis. In order to evaluate the uptake of APIs within human facial skin and confirm accurate drug delivery, a selective visualization method to monitor and quantify local drug distributions within the skin was developed. This approach uses fluorescence lifetime imaging microscopy (FLIM) paired with a multicomponent phasor analysis algorithm to visualize drug localization. As minocycline and tazarotene have distinct fluorescence lifetimes from the lifetime of the skin’s autofluorescence, these two APIs are viable targets for distinct visualization via FLIM. Here, we demonstrate that the analysis of the resulting FLIM output can be used to determine local distributions of minocycline and tazarotene within the skin. This approach is generalizable and can be applied to many multicomponent fluorescence lifetime imaging targets that require cellular resolution and molecular specificity.

Background and objectives Successful outcomes of clinical studies for acne vulgaris depend greatly on achieving statistically significant reduction in acne lesion count and improvement in Investigator's Global Assessment score of the investigational drug product against its vehicle control. To date, there has not been a validated preclinical acne model to evaluate investigational drug products in order to improve the probability of clinical success. An inflammatory acne-like lesion mouse model developed in-house has previously been used for clinical guidance in our drug development program. In this study, we aim to implement and assess the adequacy of swept-source optical coherence tomography (SS-OCT) in quantifying the dynamic changes in inflammatory acne-like lesions. Study design/materials and methods Live Propionibacterium acnes bacteria were injected intradermally resulting in inflammatory acne-like lesions. Topical 1% and 2% minocycline gels were applied to the lesions in separate groups once daily for 2 weeks and compared with vehicle and untreated control groups. The growth of these lesions was monitored and measured with a ruler (height)/microcaliper (width)-an approach previously developed, and with SS-OCT. The reliability of the two methods were assessed. Acquired OCT images across the apex of these inflammatory lesions were statistically analyzed for lesion volume reduction from baseline as well as between the treatment groups and the control groups. Results The OCT technique allowed for reliable lesion volume analysis with varying conic profiles. After 14 days of topical minocycline treatments (1%, 2% minocycline), statistically significant reduction in lesion volume (P ≤ 0.05) based on OCT image analysis was observed compared with untreated and vehicle control groups as well as compared with baseline measurements. Under the right conditions, some morphological aspects of the P. acnes injection site were discernible within the skin in images captured with OCT. Conclusions We demonstrated the first use of SS-OCT in evaluating in vivo inflammatory acne-like lesions in a murine model. Our findings support the use of OCT in assessing lesion size and evolution of P. acnes injection sites non-invasively in preclinical in vivo studies, which could potentially lead to more consistent and predictable outcomes in clinical development. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean phasor analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.

Graphical abstract, Acne vulgaris is a clinically distinct skin condition with evidence suggesting that inflammation plays a critical role in the pathogenesis of this disorder. Treatment of severe inflammatory acne often involves the use of oral antibiotics, sometimes in combination with topical products. Oral antibiotics often result in systemic side effects and the risks of antibiotic resistance, but no commercial topical minocycline is currently available. We have developed a unique, stable, hydrophilic topical gel formulation with fully solubilized minocycline (MNC-H). Minocycline delivered in our hydrophilic gel remained more stable in situ, resulting in less degradation product (4-epiminocycline) than a lipophilic formulation (MNC-L). The hydrophilic nature of our formulation enabled 2–3 fold increase in delivery into the skin ex vivo compared to a lipophilic counterpart, mostly seen in the epidermis and pilosebaceous units. The lipophilic formulation also appeared to be more occlusive, resulting in higher sebum production in minipigs, which may exacerbate acne vulgaris. As our results indicate, a 1, 2% minocycline hydrophilic gel may deliver sufficient drug (>15 μg/g) to potentially demonstrate clinical efficacy. These findings suggest that topical hydrophilic minocycline gel may provide a novel tool for topical acne therapy.

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.

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.

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 common chronic skin disease in young adults caused by infection of the pilosebaceous unit, resulting in pimples and possibly permanent scarring on the skin. Minocycline, a common antibiotic, has been widely utilized as a systemic antimicrobial treatment for acne via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline through the epidermis and into the pilosebaceous unit to achieve localized treatment with lower doses of drug. As the effectiveness of the drug is directly related to its successful delivery, there is a need to evaluate the pharmacokinetics at the cellular level within tissue. Advantageously, minocycline is naturally fluorescent and can be directly visualized using microscopy-based approaches. Due to high endogenous autofluorescence, however, imaging of weakly emitting fluorescent molecules such as minocycline in skin tissue can be challenging. Here, we demonstrate a method for the selective visualization of minocycline within human skin tissue by utilizing two-photon excitation fluorescence (TPEF) microscopy and fluorescence lifetime imaging microscopy (FLIM). To demonstrate the feasibility of this approach, ex vivo human facial skin samples treated with various concentrations of BPX-01 were investigated. From the TPEF analysis, we were able to visualize relatively high levels of drug uptake within facial skin. However, minocycline fluorescence could be overwhelmed by endogenous fluorescence that complicates TPEF quantitative analysis, making FLIM more advantageous for visualizing drug uptake. Importantly, we found a unique signature of minocycline uptake via FLIM analysis that enabled the successful differentiation of the drug and enabled the extraction of drug local distribution from the endogenous fluorescence using a non-Euclidean phasor analysis method. Based on these results, we believe that the drug local distribution visualization method using TPEF and FLIM with phasor analysis can play an important role in studying the pharmacokinetics and pharmacodynamics of a topically applicable drug.

Background and Objectives A novel carbon dioxide (CO2) laser device employing ablative fractional resurfacing was tested on human skin in vivo for the first time. Study Design/Materials and Methods An investigational 30 W, 10.6 µm CO2 laser system was focused to a 1/e2 spot size of 120 µm to generate an array of microscopic treatment zones (MTZ) in human forearm skin. A range of pulse energies between 5 and 40 mJ was tested and lesion dimensions were assessed histologically using hematoxylin & eosin. Wound healing of the MTZ's was assessed immediately-, 2-day, 7-day, 1-month, and 3-month post treatment. The role of heat shock proteins was examined by immunohistochemistry. Results The investigational CO2 laser system created a microscopic pattern of ablative and thermal injury in human skin. The epidermis and part of the dermis demonstrated columns of thermal coagulation that surrounded tapering ablative zones lined by a thin eschar layer. Changing the pulse energy from 5 to 30 mJ resulted in a greater than threefold increase in lesion depth and twofold increase in width. Expression of heat shock protein (hsp)72 was detected as early as 2 days post-treatment and diminished significantly by 3 months. In contrast, increased expression of hsp47 was first detected at 7 days and persisted at 3 months post-treatment. Conclusion The thermal effects of a novel investigational ablative CO2 laser system utilizing fractional resurfacing were characterized in human forearm skin. We confirmed our previous ex vivo findings and show for the first time in-vivo, that a controlled array of microscopic treatment zones of ablation and coagulation could be deposited in human skin by varying treatment pulse energy. Immunohistochemical studies of heat shock proteins revealed a persistent collagen remodeling response lasting at least 3 months. We successfully demonstrated the first in-vivo use of ablative fractional resurfacing (AFR™) treatment on human skin. Lasers Surg. Med. 39:96–107, 2007. © 2007 Wiley-Liss, Inc.

We tested the hypothesis that the mechanism of long pulse erbium:YAG laser lithotripsy is photothermal.Human urinary calculi were placed in deionized water and irradiated with erbium:YAG laser energy delivered through a sapphire optical fiber. Erbium:YAG bubble dynamics were visualized with Schlieren flash photography and correlated to acoustic emissions measured by a polyvinylidene fluoride needle hydrophone. The sapphire fiber was placed either parallel or perpendicular to the calculus surface to assess the contribution of acoustic transients to fragmentation. Stones were irradiated using desiccated stone irradiated in air, hydrated stone irradiated in air and hydrated stone irradiated in water. Ablation crater sizes were compared. Uric acid stones were irradiated in water and the water was assayed for cyanide.During the early phase of vapor bubble expansion, acoustic transients had minimal effects on calculus fragmentation. Fragmentation occurred due to direct absorption of laser energy transmitted to the calculus through the vapor channel between the sapphire fiber tip and calculus. The forward axial expansion of the bubble occurred more rapidly than the radial expansion. A parallel oriented fiber on the calculus surface produced no fragmentation but generated larger amplitude acoustic transients compared to perpendicular orientation. In perpendicular orientation the erbium:YAG laser did not generate any collapse acoustic waves but fragmentation occurred. Crater width was greatest for desiccated stones irradiated in air (p0.03). Cyanide production increased as erbium:YAG irradiation of uric acid calculi increased, (r2 = 0.98).The erbium:YAG laser fragments stones through a photothermal mechanism.

Not AvailableStudy supported by BioPharmX.

Each year, over 1.7 million women are diagnosed with breast cancer worldwide and about 35% of these diagnosed are expected to die from the disease. A significant barrier to improving treatment of breast cancer lies in our incomplete understanding of breast cancer initiation and metastasis. Regular use of molecular iodine has been shown to reduce the sensitivity of breast cells to the proliferative effects of estrogen, resulting in normalization of breast tissue.The aim of the current study was to evaluate the mechanisms of molecular iodine on two common breast cancer sub-types using well-established breast cancer cell lines. In order to study Luminal A and triple-negative breast cancer in vitro, MCF7 (cells from a luminal A subtype) and MDA-MB231 (cells from a triple-negative subtype), were treated with molecular iodine at various concentrations to measure proliferation and cell death. This was subsequently followed by gene expression analysis of key important molecular markers, which are primarily responsible for cell growth and apoptosis. Primary human mammary epithelial cells derived from healthy female donor were used as an internal control. Data from this study indicated that molecular iodine had potent inhibitory effects on cell growth and showed a dramatic increase in cell death in both breast cancer cell lines. Gene expression analysis using quantitative RT-PCR further confirmed that cell cycle genes controlling G1-S phase transition were largely up-regulated. Changes were not seen in Cyclin B expression levels, which further suggest that cells were arrested before entry into cell division. BCL-2, PPAR-α and PPAR-γ were also up-regulated along with down-regulation of Caspase 3, suggesting molecular iodine induced cell death through activation of a caspase-independent apoptosis pathway. Interestingly, mesenchymal-epithelial transition (MET) occurrence was noticed upon molecular iodine treatment as indicated by sharp increase of GATA3 and E-Cadherin and significant down-regulation of Vimentin in invasive MDA-MB231 cells. Our current study enables us to understand a molecular mechanism controlling tumor cell growth and demonstrates potent cellular effects of molecular iodine on breast cancer cell lines. These results also demonstrate promising effects of molecular iodine for regulation of breast cancer EMT differentiation, which is required for tumor initiation and metastasis. Further studies are underway to determine possible effects of molecular iodine in these breast cancer subtypes using in vitro 3D models. Note: This abstract was not presented at the meeting. Citation Format: Zack Xu, Xin Chen, Noymi Yam, kin Chan, Usha Nagavarapu. Elucidating the mechanism of action of molecular iodine on breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2243. doi:10.1158/1538-7445.AM2017-2243

Purpose: We test the hypothesis that erbium:YAG (Er:YAG) lithotripsy is more efficient than holmium:YAG (Ho:YAG) lithotripsy.Materials and Methods: Human calculi composed of greater than 97% calcium oxalate monohydrate and cystine were studied. Calculi were irradiated in water using Er:YAG or Ho:YAG lasers. Er:YAG lithotripsy was done with a 425 μm. sapphire optical fiber at a pulse energy of 50 mJ. at 10 Hz. Ho:YAG lithotripsy was performed with a 365 μm. low hydroxy optical fiber at a pulse energy of 500 mJ. at 10 Hz. or a 425 μm. sapphire optical fiber at a pulse energy of 50 mJ. at 10 Hz. Fragmentation was defined as the initial stone mass minus the final dominant fragment mass and normalized for incident laser fluence (energy per unit area of fiber tip).Results: Mean fragmentation plus or minus standard deviation for calcium oxalate monohydrate was 38 ± 27 mg. for Er:YAG and 22 ± 6 for Ho:YAG (low hydroxy silica fiber) versus 5 ± 1 for Ho:YAG (sapphire fiber, p = 0.001). When fragmentation was normal...

Infrared laser ablation of urinary calculi was investigated as a function of wavelength to determine the relation of ablation threshold fluences, ablation depths, and optical absorption. A simple photothermal ablation model was employed to examine this relationship. Human urinary calculi composed of >95% uric acid, >95% cystine, >95% calcium oxalate monohydrate (COM), and >90% magnesium ammonium phosphate hexahydrate (MAPH) were used. Various wavelengths between 2.1 and 6.5 /spl mu/m were selected to perform threshold fluence and ablation depth measurements. The laser source for this study was the tunable pulsed infrared free electron laser (FEL) at Vanderbilt University. Experimental results indicated a correlation of threshold fluence and ablation depth to the optical absorption properties of the calculi. When calculus optical absorption increased, the threshold fluences decreased. Although the ablation depths increased with calculus optical absorption, results indicated that in certain calculi the ablation depth was affected by optical attenuation through the ablation plume. These observations were in agreement with the photothermal ablation model, but fractures in striated calculi at higher optical absorptions indicated the contribution of a photomechanical mechanism.

Background and Objective: Hyper-osmotic chemical agents were used to study the effects of transient tissue scattering on the remitted fluorescence emission intensity from a target placed under a tissue sample. Study Design/Materials and Methods: A fluorescent film was placed underneath in vitro and in vivo samples of hamster skin, and the remitted fluorescent signal traveling to the tissue surface was monitored over time as the tissue was treated with an osmotically active agent. Results: The detected fluorescent signal increased as the scattering in tissue samples was substantially reduced. The increase was greater for dimethyl sulfoxide than glucose or glycerol. It was not statistically different between in vivo skin and in vitro skin. Conclusion: The study shows how chemical agents can be used to improve the detected signal for a specific optical application. It could be useful in a number of optical therapeutic and diagnostic applications that can benefit from an increase in the penetration depth of light. Lasers Surg. Med. 29:213‐220, 2001. fl 2001 Wiley-Liss, Inc.

A series of experiments were conducted to test the hypothesis that the mechanism of holmium:YAG lithotripsy is photothermal.To show that holmium:YAG lithotripsy requires direct absorption of optical energy, stone loss was compared for 150 J Ho:YAG lithotripsy of calcium oxalate monohydrate (COM) stones for hydrated stones irradiated in water (17+/-3 mg) and hydrated stones irradiated in air (25+/-9 mg) v dehydrated stones irradiated in air (40+/-12 mg) (P0.001). To show that Ho:YAG lithotripsy occurs prior to vapor bubble collapse, the dynamics of lithotripsy in water and vapor bubble formation were documented with video flash photography. Holmium:YAG lithotripsy began at 60 microsec, prior to vapor bubble collapse. To show that Ho:YAG lithotripsy is fundamentally related to stone temperature, cystine, and COM mass loss was compared for stones initially at room temperature (approximately 23 degrees C) v frozen stones ablated within 2 minutes after removal from the freezer. Cystine and COM mass losses were greater for stones starting at room temperature than cold (Por = 0.05). To show that Ho:YAG lithotripsy involves a thermochemical reaction, composition analysis was done before and after lithotripsy. Postlithotripsy, COM yielded calcium carbonate; cystine yielded cysteine and free sulfur; calcium hydrogen phosphate dihydrate yielded calcium pyrophosphate; magnesium ammonium phosphate yielded ammonium carbonate and magnesium carbonate; and uric acid yielded cyanide. To show that Ho:YAG lithotripsy does not create significant shockwaves, pressure transients were measured during lithotripsy using needle hydrophones. Peak pressures were2 bars.The primary mechanism of Ho:YAG lithotripsy is photothermal. There are no significant photoacoustic effects.

Background and Objective Evidence is presented that the fragmentation process of long-pulse Holmium:YAG (Ho:YAG) lithotripsy is governed by photothermal decomposition of the calculi rather than photomechanical or photoacoustical mechanisms as is widely thought. The clinical Ho:YAG laser lithotriptor (2.12 μm, 250 μs) operates in the free-running mode, producing pulse durations much longer than the time required for a sound wave to propagate beyond the optical penetration depth of this wavelength in water. Hence, it is unlikely that shock waves are produced during bubble formation. In addition, the vapor bubble induced by this laser is not spherical. Thus the magnitude of the pressure wave produced at cavitation collapse does not contribute significantly to lithotripsy. Study Design/Materials and Methods A fast-flash photography setup was used to capture the dynamics of urinary calculus fragmentation at various delay times following the onset of the Ho:YAG laser pulse. These images were concurrently correlated with pressure measurements obtained with a piezoelectric polyvinylidene-fluoride needle-hydrophone. Stone mass-loss measurements for ablation of urinary calculi (1) in air (dehydrated and hydrated) and in water, and (2) at pre-cooled and at room temperatures were compared. Chemical and composition analyses were performed on the ablation products of several types of Ho:YAG laser irradiated urinary calculi, including calcium oxalate monohydrate (COM), calcium hydrogen phosphate dihydrate (CHPD), magnesium ammonium phosphate hexahydrate (MAPH), cystine, and uric acid calculi. Results When the optical fiber was placed perpendicularly in contact with the surface of the target, fast-flash photography provided visual evidence that ablation occurred approximately 50 μs after the initiation of the Ho:YAG laser pulse (250–350 μs duration; 375–400 mJ per pulse), long before the collapse of the cavitation bubble. The measured peak acoustical pressure upon cavitation collapse was negligible (< 2 bars), indicating that photomechanical forces were not responsible for the observed fragmentation process. When the fiber was placed in parallel to the calculus surface, the pressure peaks occurring at the collapse of the cavitation were on the order of 20 bars, but no fragmentation occurred. Regardless of fiber orientation, no shock waves were recorded at the beginning of bubble formation. Ablation of COM calculi (a total of 150 J; 0.5 J per pulse at an 8-Hz repetition rate) revealed different Ho:YAG efficiencies for dehydrated calculus, hydrated calculus, and submerged calculus. COM and cystine calculi, pre-cooled at −80°C and then placed in water, yielded lower mass-loss during ablation (20 J, 1.0 J per pulse) compared to the mass-loss of calculi at room temperature. Chemical analyses of the ablated calculi revealed products resulting from thermal decomposition. Calcium carbonate was found in samples composed of COM calculi; calcium pyrophosphate was found in CHPD samples; free sulfur and cysteine were discovered in samples composed of cystine samples; and cyanide was found in samples of uric acid calculi. Conclusion These experimental results provide convincing evidence that long-pulse Ho:YAG laser lithotripsy causes chemical decomposition of urinary calculi as a consequence of a dominant photothermal mechanism. Lasers Surg. Med. 25:22–37, 1999. © 1999 Wiley-Liss, Inc.

467 Background: There is an unmet need for a comprehensive genomic characterization of non muscle invasive bladder cancer (NMIBC). NMIBC comprise over 70% of all bladder cancers at presentation. They have highly variable clinical behavior that is not always adequately predicted on the basis of their histological grade (2004 World Health Organization low and high grade, LG-HG). The discrepancy between phenotype and genotype is compounded further by interobserver variability in pathological grading. We have previously established methods for whole transcriptome RNAseq from formalin fixed paraffin embedded tissues (FFPE). Methods: Whole transcriptomic analysis of 110 NMI FFPE BC both LG and HG was performed incorporating messenger RNA expression, splice variants, gene fusion, and pathway perturbation. We used a discovery (n = 40) and a validation cohort (n = 70). These data were integrated and tested for correlation with both pathological grading and clinical outcomes. Grade Risk Index (GRI) score quantifying how closely a patient's transcriptome is related to a reference set of LG NMIBC samples was established. Conventional pathological grading was reviewed by 3 different expert uro-pathologists and interobserver variability calculated. Results: Unsupervised clustering of data from RNA sequencing uncovered classification of three robust - - nonoverlapping, prognostically significant subtypes of NMIBC with distinct GRIs and signatures. When applied by expert pathologists, interobserver variability in histological grading was observed in 17.5%. In the intermediate group (GRI 0.13 to 0.19), pathologists disagreed in 37.5% whether BC was LG or HG. HG NMIBC clustered with MIBC. LG NMIBC in the intermediate GRI group included either very bulky tumors or extremely rare metastatic LG BC (n = 4). HG disease was associated with a shift in BMP signaling and a germ stem cell-like phenotype. Multiple components of the centromere complex and APOBEC3B were upregulated in HG BC. FGFR3::TACC3 fusion events were observed in LG NMIBC only (11.5%). Conclusions: Whole transcriptomic sequencing data delineated three molecular classes of NMIBC.

Lasers can be used as intracorporeal lithotriptors for urinary calculi. Laser lithotripsy generally involves one of two mechanisms: photoacoustic or photothermal lithotripsy. Photoacoustic lithotripsy produces large fragments but has difficulty in fragmenting ­calcium oxalate monohydrate, cystine, and brushite stones. Photothermal lithotripsy produces small fragments and is effective in fragmenting all stone compositions. Photothermal lithotripsy, such was with the holmium:YAG laser, tends to be slow compared to photoacoustic lithotripsy. The physics of photothermal lithotripsy is reviewed with the objective to enhance fragmentation efficiency and minimization of retropulsion.

Despite the emergence of nonablative fractional resurfacing (NFR) as a new therapeutic modality for skin photoaging, little is known about the molecular events that underlie the heat shock response to different treatment parameters. Human subjects are treated with a scanned 1550-nm fractional laser at pulse energies spanning 6 to 40 mJ and a 140-μm spot size. The heat shock response is assessed immunohistochemically immediately through 7 days posttreatment. At the immediately posttreatment time point, we observe subepidermal clefting in most sections. The basal epidermis and dermal zones of sparing are both found to express HSP47, but not HSP72. By day 1, expression of HSP72 is detected throughout the epidermis, while that of HSP47 remains restricted to the basal layer. Both proteins are detected surrounding the dermal portion of the microscopic treatment zone (MTZ). This pattern of expression persists through day 7 post-NFR, although neither protein is found within the MTZ. Immediately posttreatment, the mean collagen denaturation zone width is 50 μm at 6 mJ, increasing to 202 μm at 40 mJ. The zone of cell death exceeds the denaturation zone by 19 to 55% over this pulse energy range. The two zones converge by day 7 posttreatment.

Light emitted from a femtosecond laser is capable of plasma-induced ablation of various materials. We tested the feasibility of utilizing femtosecond-pulsed laser radiation =800 nm, 140 fs, 0.9 mJ/pulse for ablation of urinary calculi. Ablation craters were observed in human calculi of greater than 90% calcium oxalate monohydrate COM, cystine CYST, or magnesium ammonium phosphate hexahydrate MAPH. Largest crater volumes were achieved on CYST stones, among the most difficult stones to fragment using Holmium:YAG Ho:YAG lithotripsy. Diameter of debris was characterized using optical microscopy and found to be less than 20 m, substantially smaller than that produced by long-pulsed Ho:YAG ablation. Stone retropulsion, monitored by a high-speed camera system with a spatial resolution of 15 m, was negligible for stones with mass as small as 0.06 g. Peak shock wave pressures were less than 2 bars, measured by a polyvinylidene fluoride PVDF needle hydrophone. Ablation dynamics were visualized and charac- terized with pump-probe imaging and fast flash photography and cor- related to shock wave pressures. Because femtosecond-pulsed laser ablates urinary calculi of soft and hard compositions, with micron- sized debris, negligible stone retropulsion, and small shock wave pressures, we conclude that the approach is a promising candidate technique for lithotripsy. © 2010 Society of Photo-Optical Instrumentation Engineers.

Background and Objective: Due to the significant risk profile associated with traditional ablative resurfacing, a safer and less invasive treatment approach known as fractional deep dermal ablation (FDDA™) was recently developed. We report the results of the first clinical investigation of this modality for treatment of photo- damaged skin. Study Design/Materials and Methods: Twenty-four subjects received treatments on the inner forearm with a prototype fractional CO2laser device (Reliant Technologies Inc., Mountain View, CA) at settings of 5-40 mJ/MTZ and 400 MTZ/cm2. Clinical and histological effects were assessed by study investigators 1 week, 1 month, and 3 months following treatment. Thirty subjects were then enrolled in a multi-center study for treatment of photo- damage using the same device. Subjects received 1-2 treatments on the face and neck, with energies ranging from 10 to 40 mJ/MTZ and densities ranging from 400 to 1,200 MTZ/cm2. Study investigators assessed severity of post-treatment responses during follow-up visits 48 hours, 1 week, 1 month, and 3 months following treatment. Using a standard quartile improvement scale (0-4), subjects and investigators assessed improvement in rhytides, pigmentation, texture, laxity and overall appearance 1 and 3 months post-treatment. Results: Clinical and histologic results demonstrated that fractional delivery of a 10,600 nm CO2laser source offers an improved safety profile with respect to traditional ablative resurfacing, while still effectively resurfacing epidermal and dermal tissue. Forearm and facial treatments were well-tolerated with no serious adverse events observed. Eighty-three percent of subjects exhibited moderate or better overall improvement (50-100%), according to study investigator quartile scoring. Conclusions: FDDA™ treatment is a safe and promising new approach for resurfacing of epidermal and deep dermal tissue targets. Lasers Surg © 2009 Wiley-Liss, Inc.

This paper describes in simple terms the physics of laser-calculus interactions and introduces a method with which physicians can understand or evaluate the application of any new laser technique for use in lithotripsy or other medical fields. Tissue optical properties and laser parameters govern the mechanism(s) of fragmentation of urinary or biliary calculi. Laser pulse energies for clinical lithotripsy range from Q0 = 20 mJ to 2 J for short-pulsed lasers to long-pulsed lasers, respectively. Lasers with short pulse durations (i.e., less than a few microseconds) fragment calculi by means of shockwaves following optical breakdown and plasma expansion of ionized water or calculus compositions or by cavitation collapse, thus manifesting a photoacoustical effect. Laser-tissue interactions involving dominant photomechanical or photoacoustical effects are usually stress confined. Long-pulsed lasers (i.e., >100 microsec), on the other hand, generate minimal acoustic waves, and calculi are fragmented by temperatures beyond the thresholds for vaporization of calculus constituents, melting, or chemical decomposition.

The potential application of an Erbium:YAG (Er:YAG) laser (Q 0 = 50 mJ/pulse; τ p = 275 μs; rep. rate = 2, 10 Hz) with a sapphire delivery fiber for intracorporeal laser lithotripsy was explored. Preliminary measurements on calculus mass-loss and fragmentation efficiency were conducted and results were compared with that of Ho:YAG laser lithotripsy. Laser induced bubble and lithotripsy dynamics were investigated to assess the mechanism(s) involved in the fragmentation process. Results showed that the fragmentation efficiency (mass-loss/H o - g.μm 2 /J) in Er:YAG laser lithotripsy was about 2.4 times that of Ho:YAG laser lithotripsy (used: Q 0 = 500 mJ/pulse; Tp = 250 μs; rep. rate = 10 Hz). Acoustic transients were found to have minimal effect during Er:YAG laser lithotripsy. Schlieren flash images suggested a predominantly photothermal mechanism due to direct laser energy absorption, which resulted in recrystallization and plume formation. These events indicated melting and chemical decomposition of the calculus composition. Another observation led to the possibility of a plasma-mediated photothermal mechanism. The 'Moses effect' facilitating pulsed mid-infrared laser delivery appeared more efficient for the Er:YAG laser than for the Ho:YAG laser. With the sapphire fiber, experimental results suggested the potential of an improved treatment modality by the Er:YAG laser for intracorporeal laser lithotripsy.

The objectives of this study were to determine if the optical absorption properties of urinary calculi affect thethreshold fluence for ablation or fragmentation and the ablation efficiency due to laser irradiation. The Vanderbiltfree electron laser (FEL) was tuned to selected wavelengths based on the absorption spectrum of various types ofurinary calculi. The threshold fluences for ablation of the calculi were measured at different wavelengths. Apreliminary study of the ablation efficiency (ablation depth per unit incidence fluence) was performed. The resultswere found to be in agreement with a thermal ablation model for which the threshold fluences were proportional to 1/J.ta. The ablation efficiencies were higher in regions of the infrared spectra in which absorption was higher. For afixed laser irradiation, the lower threshold fluences within regions of high optical absorption allowed more energy toenhance calculus ablation. This study provided insight into determining the optimum wavelengths for ablation andlaser lithotripsy.Keywords: ablation efficiency, absorption coefficient, fragmentation efficiency, free electron laser, infrared, kidneystone, optimum wavelength, threshold radiant exposure, tissue ablation.

To determine the threshold radiant exposures (J/cm2) needed for ablation or fragmentation as a function of infrared wavelengths on various urinary calculi and to determine if there is a relation between these thresholds and lithotripsy efficiencies with respect to optical absorption coefficients.Human calculi composed of uric acid, calcium oxalate monohydrate (COM), cystine, or magnesium ammonium phosphate hexahydrate (MAPH) were used. The calculi were irradiated in air with the free electron laser (FEL) at six wavelengths: 2.12, 2.5, 2.94, 3.13, 5, and 6.45 microm.Threshold radiant exposures increased as optical absorption decreased. At the near-infrared wave-lengths with low optical absorption, the thresholds were1.5 J/cm2. The thresholds decreased below 0.5 J/cm2 for regions of high absorption for all the calculus types. Thresholds within the high-absorption regions were statistically different from those in the low-absorption regions, with P values much less than 0.05.Optical absorption coefficients or threshold radiant exposures can be used to predict lithotripsy efficiencies. For low ablation thresholds, smaller radiant exposures were required to achieve breakdown temperatures or to exceed the dynamic tensile strength of the material. Therefore, more energy is available for fragmentation, resulting in higher lithotripsy efficiencies.

Because of the >= 250 microsecond(s) pulsewidth emitted by the Ho:YAG laser used in clinical lithotripsy, it is unlikely that stress confinement occurs within the irradiated stones. Experimental data supports a thermal mechanism for Ho:YAG laser stone ablation. Previous work has shown that stone fragmentation occurs soon after the onset of the laser pulse, is uncorrelated to cavitation bubble formation or collapse, and is associated with low pressures. Moreover, lithotripsy proceeds fastest with desiccated stones in air (data based on laser ablation of calcium oxalate monohydrate stones), indicating that direct absorption of the laser radiation by the stone material is required for the most efficient ablation. Lowering the initial temperature of calculi reduces the stone mass-loss following 20 J of delivered laser energy: 2.2 +/- 1.1 mg vs 5.2 +/- 1.6 mg for calcium oxalate monohydrate (COM) stones (-80 vs 23 degree(s)C), and 0.8 +/- 0.4 mg vs 2.2 +/- 1.1 mg for cystine stones (-80 vs 23 degree(s)C), p

Because of the greater than or equal to 250 microsecond pulsewidth emitted by the Ho:YAG laser used in clinical lithotripsy, it is unlikely that stress confinement occurs within the irradiated stones. Experimental data supports a thermal mechanism for Ho:YAG laser stone ablation. Stone fragmentation occurs soon after the onset of the laser pulse, is uncorrelated to cavitation bubble formation or collapse, and is associated with low pressures (cf. part I). The mass- loss of desiccated calcium oxalate monohydrate (COM) stones exposed to 150 J from the Ho:YAG laser in air was 40 plus or minus 12 mg (mean plus or minus 1 s.d.); for hydrated stones in air was 25 plus or minus 9 mg; and for hydrated stones in water was 17 plus or minus 3 mg, p less than .001. These differences indicate that direct absorption of the laser radiation by the stone is required for the most efficient ablation. Lowering the initial temperature of COM or cystine stones also reduced the stone mass-loss following 20 J of delivered laser energy: 2.2 plus or minus 1.1 mg vs 5.2 plus or minus 1.6 mg for COM stones (-80 vs 23 degrees Celsius), and 0.8 plus or minus 0.4 mg vs 2.2 plus or minus 1.1 mg for cystine stones (-80 vs 23 degrees Celsius), p less than or equal to .05. Finally, chemical analysis of the laser-induced stone fragments revealed the presence of thermal breakdown products: CaCO 3 from COM; free sulfur and cysteine from cystine; Ca 2 O 7 P 2 from calcium hydorgen phosphate dihydrate, and cyanide from uric acid.

The Ho:YAG laser commonly used for clinical lithotripsy of urinary stones typically emits 250-microsecond pulses at a wavelength of 2.12 micrometer and repetition rates of up to 10 Hz. This pulse duration is longer than the time required for a pressure wave to propagate beyond the optical penetration depth of this wavelength in water. Fast-flash photography was used to study the dynamics of urinary stone fragmentation by the Ho:YAG laser. Stone ablation began approximately 50 microseconds after the onset of the laser pulse, long before the collapse of the cavitation bubble at about 350 microseconds. Pressure measurements, made with a PVDF needle- hydrophone and correlated with the fast-flash images, indicated that the peak acoustical transient was less than 2 bars. Regardless of fiber orientation to the stone, no shockwaves were recorded at the beginning of the bubble, and the maximum pressure waves recorded at bubble collapse were approximately 20 bars. However, no fragmentation occurred during or subsequent to the bubble collapse. The measurements indicated that stone ablation was not due to a photomechanical effect.