Investigating the Hydrodynamics of Intravenous Drug Infusions.

One of the most common reported adverse events for intravenous (IV) infusions are infusion site reactions, ranging from redness and pain at the site of infusion to thrombophlebitis.  The connection between drug infusion and what drives these adverse events is not well understood. To aid in understanding these phenomena, it is crucial to accurately characterize the evolving hemodynamic environment of the infusion site when developing new intravenous formulations, as too rapid dilution may cause precipitation in the vein, while too little dilution might contribute to phlebitis. In this study, a Large-Eddy Simulation (LES) turbulence modeling inside a Computational Fluid Dynamics (CFD) framework has been used to simulate the flow and mixing characteristics of an infusion entering the bloodstream. This work represents the first such study reporting transient flow fields for intravenous infusions using LES CFD simulations with a realistic non-Newtonian blood model. The output of the CFD model closely resembled the flow and mixing patterns generated in benchtop tests for infusions into a blood analogue and water as the venous fluid across a wide range of flow rates.  These models were then investigated further to compare how changes to the fluid rheology model, needle orientation and needle position within the vein resulted in altered mixing regimes at different flow rates.

Biopharmaceutical Modeling of Food Effect─Exploring the Role of Dietary Fat.

In preclinical drug discovery, simple fraction absorbed calculators are exceptionally valuable tools to better understand the potential limitations to drug absorption and how different formulation approaches may address them. These tools often struggle to accurately capture the impact of food on drug absorption. One possible reason for this is that these models overlook the potential role of dietary fat in altering drug absorption. Herein, we present a novel approach to incorporate the fat content from diet within an absorption model as an additional set of particles that can accumulate in the mucus and act to reduce the effective thickness of the unstirred water layer. Using this approach, we demonstrate improved model prediction on the extent of food effect for a range of marketed compounds comparing two historical absorption models and the new model developed in this work using published food effect data for 21 marketed compounds. We extended this work to investigate each model's ability to predict the reported food effect across a range of dose levels for Venetoclax. Finally, we investigate the new model's capability to predict food effect in both low-fat and high-fat fed states and compare these predictions to the two historical models using three model compounds: Albendazole, Pazopanib, and Venetoclax.

Preclinical modeling of intravitreal suspensions.

There are a number of obstacles that complicate the development of intravitreal delivered small molecules therapies. One serious complication is the potential need for complex polymer depot formulations early in the drug discovery process. The development of such formulations often requires substantial investment of time and material which may not be readily available in preclinical development. Herein I present a diffusion limited pseudo-steady state model to provide prediction of drug release from an intravitreally administered suspension formulation. By using such a model, a preclinical formulator may be able to more confidently determine if development of a complex formulation is required or if a simple suspension may work to support a study design. In this report, the model is used to predict the intravitreal preformance of two different molecules (triamcinolone acetonide and GNE-947) at multiple dose levels in rabbit eyes as well as provide a prediction for the performance of a marketed formulation of Trimacinolone Acetonide in humans.

High-throughput kinetic turbidity analysis for determination of amorphous solubility and excipient screening for amorphous solid dispersions.

Many poorly water-soluble active pharmaceutical ingredients (APIs) rely on supersaturating formulations, such as amorphous solid dispersions (ASDs), to enhance oral bioavailability. ASDs kinetically trap amorphous solid drugs within polymer excipient matrices to maintain the amorphous drug states. The maximum solution concentration of the API in these formulations is known as the amorphous solubility. In early drug development with scarce material and time, high-throughput approaches to measuring amorphous solubility and screening excipient effects on crystallization risk offer significant benefits to preclinical formulation scientists. Here, we developed a high-throughput screening (HTS) workflow to quantify amorphous solubility and screen ASD excipients by automated kinetic turbidity analysis. Testing 20 model APIs with a wide range of biorelevant solubility, we demonstrated their apparent amorphous solubility determined by the HTS approach strongly correlated with quantification results using conventional liquid chromatography; while the real-time analysis significantly saved analytical time and experimental efforts. Furthermore, kinetic turbidity profiles elucidated distinct excipient effects on the precipitation process of APIs. These results were successfully translated to dissolution and precipitation behaviors of ASD formulations composed of the tested polymers. The high-throughput kinetic turbidity workflow presents a facile and information-rich approach for amorphous solubility screenings against excipients, and helps guide enabling formulation development.

Tissue Distribution of Mercury in the Bodies of Wild American Alligators (Alligator mississippiensis) from a Coastal Marsh in Louisiana (USA).

Total mercury (THg) concentrations were measured in wild alligators inhabiting a coastal marsh in southern Louisiana, to determine the tissue distribution of THg among various body organs and tissue compartments. Concentrations of THg in claws and dermal tail scutes were compared to those in blood, brain, gonad, heart, kidney, liver, and skeletal muscle to determine if the former tissues, commonly available by non-lethal sampling, could be used as measures of body burdens in various internal organs. Mercury was found in all body organs and tissue compartments. However, overall, THg concentrations measured in alligators were below the FDA action level for fish consumption and were comparable to previous data reported from southwestern Louisiana. Our results suggest consumption of meat from alligators found in this region may be of little public health concern. However, the extended period of time between sampling (in this study) and the present-day highlight the need for continuous, additional, and more recent sampling to ensure consumer safety. Total mercury concentrations were highest in the kidney (3.18 ± 0.69 mg/kg dw) and liver (3.12 ± 0.76 mg/kg dw). THg levels in non-lethal samples (blood, claws, and dermal tail scutes) were positively correlated with all tissue THg concentrations (blood: R2 = 0.513-0.988; claw: R2 = 0.347-0.637, scutes: R2 = 0.333-0.649). Because THg concentrations from blood, claws, and scutes were correlated with those of the internal organs, non-lethal sampling methods may be a viable method of estimating levels of THg in other body tissues.

Characterization of the Hydrodynamics in the USP Basket Apparatus Using Computational Fluid Dynamics.

Computational fluid dynamics (CFD) has been extensively used for the USP paddle apparatus II, but limited CFD studies have been conducted on the USP basket apparatus I. We expanded on past CFD basket studies to consider the presence of a tablet inside the basket, compared predictions to in vivo conditions, and confirmed observations around the complexity of nonuniform hydrodynamics. Tablets near the basket perimeter experienced near 5-fold increase in maximum velocity and surface shear stress compared to tablets placed at the center of the basket. At higher basket speeds, the predicted velocities at the center of the basket were closer to in vivo predictions but the surface shear stress was about 2 orders of magnitude lesser. Simulations with a high viscosity fluid (1 Pa-s) showed a 10-fold increase in shear stress on a tablet but a decrease in strain rate compared to low-viscosity medium (0.001 Pa-s) which could impact dissolution rates. Also presented are the insights into turbulent energy dissipation rates that could help in a priori prediction of dissolution rates. Overall, the CFD analysis presented in this work reveals significant differences between the basket and in vivo conditions and will help inform relevant in vitro testing.

Invertebrate community response to coarse woody debris removal for bioenergy production from intensively managed forests.

Increased market viability of harvest residues as forest bioenergy feedstock may escalate removal of coarse woody debris in managed forests. Meanwhile, many forest invertebrates use coarse woody debris for cover, food, and reproduction. Few studies have explicitly addressed effects of operational-scale woody biomass harvesting on invertebrates following clearcutting. Therefore, we measured invertebrate community response to large-scale harvest residue removal and micro-site manipulations of harvest residue availability in recently clearcut, intensively managed loblolly pine (Pinus taeda) forests in North Carolina (NC; n = 4) and Georgia (GA; n = 4), USA. We captured 39,794 surface-active invertebrates representing 171 taxonomic groups using pitfall traps situated among micro-site locations (i.e., purposefully retained piles of hardwood stems and piles of conifer stems and areas without coarse woody debris in NC; windrows and no windrows in GA). Micro-site locations were located within six, large-scale treatments (7.16-14.3 ha) in clearcuts. Large-scale treatments represented intensive harvest residue removal, 15% and 30% harvest residue retention, and no harvest residue removal. In NC, ground beetles (Coleoptera: Carabidae) and crickets (Orthoptera: Gryllidae) were three times more abundant in treatments with no harvest residue removal than those with the most intensive harvest residue removal and were reduced in treatments that retained 15% or 30% of harvest residues, although not significantly. Invertebrate taxa richness was greater at micro-site locations with retained hardwood and pine (Pinus spp.) harvest residues than those with minimal amounts of coarse woody debris. In both states, relative abundances of several invertebrate taxa, including cave crickets (Orthoptera: Rhaphidophoridae), fungus gnats (Diptera: Mycetophilidae and Sciaridae), millipedes (Diplopoda), and wood roaches (Blattodea: Ectobiidae), were greater at micro-site locations with retained harvest residues than those with minimal coarse woody debris. Intensified woody biomass harvesting without retention of ≥15% of harvest residue volume may reduce invertebrate taxa richness and abundances of some key invertebrate taxa in regenerating stands. Further, harvest residue management during and after woody biomass harvesting may be an important consideration for maintaining invertebrate diversity and conserving invertebrates that are influential in the maintenance of ecosystem function and integrity in young forests.

Mechanistic Physiologically Based Pharmacokinetic Modeling of the Dissolution and Food Effect of a Biopharmaceutics Classification System IV Compound-The Venetoclax Story.

Venetoclax, a selective B-cell lymphoma-2 inhibitor, is a biopharmaceutics classification system class IV compound. The aim of this study was to develop a physiologically based pharmacokinetic (PBPK) model to mechanistically describe absorption and disposition of an amorphous solid dispersion formulation of venetoclax in humans. A mechanistic PBPK model was developed incorporating measured amorphous solubility, dissolution, metabolism, and plasma protein binding. A middle-out approach was used to define permeability. Model predictions of oral venetoclax pharmacokinetics were verified against clinical studies of fed and fasted healthy volunteers, and clinical drug interaction studies with strong CYP3A inhibitor (ketoconazole) and inducer (rifampicin). Model verification demonstrated accurate prediction of the observed food effect following a low-fat diet. Ratios of predicted versus observed Cmax and area under the curve of venetoclax were within 0.8- to 1.25-fold of observed ratios for strong CYP3A inhibitor and inducer interactions, indicating that the venetoclax elimination pathway was correctly specified. The verified venetoclax PBPK model is one of the first examples mechanistically capturing absorption, food effect, and exposure of an amorphous solid dispersion formulated compound. This model allows evaluation of untested drug-drug interactions, especially those primarily occurring in the intestine, and paves the way for future modeling of biopharmaceutics classification system IV compounds.

Polymer conjugated retinoids for controlled transdermal delivery.

All-trans retinoic acid (ATRA), a derivative of vitamin A, is a common component in cosmetics and commercial acne creams as well as being a first-line chemotherapeutic agent. Today, formulations for the topical application of ATRA rely on creams and emulsions to incorporate the highly hydrophobic ATRA drug. These strategies, when applied to the skin, deliver ATRA as a single bolus, which is immediately taken up into the skin and contributes to many of the known adverse side effects of ATRA treatment, including skin irritation and hair loss. Herein we present a new concept in topical delivery of retinoids by covalently bonding the drug through a hydrolytically degradable ester linkage to a common hydrophilic polymer, polyvinyl alcohol (PVA), creating an amphiphilic nanomaterial that is water-soluble. This PVA bound ATRA can then act as a pro-drug and accumulate within the skin to allow for the sustained controlled delivery of active ATRA. This approach was demonstrated to release active ATRA out to 10days in vitro while significantly enhancing dermal accumulation of the ATRA in explant pig skin. In vivo we demonstrate that the pro-drug formulation reduces application site inflammation compared to free ATRA and retains the drug at the application site at measurable quantities for up to six days.

Breeding, Early-Successional Bird Response to Forest Harvests for Bioenergy.

Forest regeneration following timber harvest is a principal source of habitat for early-successional birds and characterized by influxes of early-successional vegetation and residual downed woody material. Early-successional birds may use harvest residues for communication, cover, foraging, and nesting. Yet, increased market viability of woody biomass as bioenergy feedstock may intensify harvest residue removal. Our objectives were to: 1) evaluate effects of varying intensities of woody biomass harvest on the early-successional bird community; and (2) document early-successional bird use of harvest residues in regenerating stands. We spot-mapped birds from 15 April- 15 July, 2012-2014, in six woody biomass removal treatments within regenerating stands in North Carolina (n = 4) and Georgia (n = 4), USA. Treatments included clearcut harvest followed by: (1) traditional woody biomass harvest with no specific retention target; (2) 15% retention with harvest residues dispersed; (3) 15% retention with harvest residues clustered; (4) 30% retention with harvest residues dispersed; (5) 30% retention with harvest residues clustered; and (6) no woody biomass harvest (i.e., reference site). We tested for treatment-level effects on breeding bird species diversity and richness, early-successional focal species territory density (combined and individual species), counts of breeding birds detected near, in, or on branches of harvest piles/windrows, counts of breeding bird behaviors, and vegetation composition and structure. Pooled across three breeding seasons, we delineated 536 and 654 territories and detected 2,489 and 4,204 birds in the North Carolina and Georgia treatments, respectively. Woody biomass harvest had limited or short-lived effects on the early-successional, breeding bird community. The successional trajectory of vegetation structure, rather than availability of harvest residues, primarily drove avian use of regenerating stands. However, many breeding bird species used downed wood in addition to vegetation, indicating that harvest residues initially may provide food and cover resources for early-successional birds in regenerating stands prior to vegetation regrowth.

A benchtop capillary flow layer-by-layer (CF-LbL) platform for rapid assembly and screening of biodegradable nanolayered films.

Capillary flow layer-by-layer (CF-LbL) is a microfluidic platform for high throughput preparation and screening of nanolayered polymer films. Using a simple benchtop version of CF-LbL, we systematically studied the effects of various flow conditions and channel geometries on the thickness and surface roughness of the resulting films. We also investigated the biocompatibility and degradation behaviors of a series of enzymatically-degradable films made from naturally derived polymers, i.e. either alginate or hyaluronic acid as the anionic species and poly-l-arginine as the positive species. Furthermore, using one optimized film formulation for coating on the inside walls of a microfluidic chip, we successfully demonstrated the ability of this film to capture and rapidly release cancer cells from whole blood. This simple platform is expected to be a powerful tool to increase the accessibility of the LbL film assembly to a broader scientific community.

Do biomass harvesting guidelines influence herpetofauna following harvests of logging residues for renewable energy?.

Forests are a major supplier of renewable energy; however, gleaning logging residues for use as woody biomass feedstock could negatively alter habitat for species dependent on downed wood. Biomass Harvesting Guidelines (BHGs) recommend retaining a portion of woody biomass on the forest floor following harvest. Despite BHGs being developed to help ensure ecological sustainability, their contribution to biodiversity has not been evaluated experimentally at operational scales. We compared herpetofauanal evenness, diversity, and richness and abundance of Anaxyrus terrestris and Gastrophryne carolinensis among six treatments that varied in volume and spatial arrangement of woody biomass retained after clearcutting loblolly pine (Pinus taeda) plantations in North Carolina, USA (n = 4), 2011-2014 and Georgia (n = 4), USA 2011-2013. Treatments were: (1) biomass harvest with no BHGs, (2) 15% retention with biomass clustered, (3) 15% retention with biomass dispersed, (4) 30% retention with biomass clustered, (5) 30% retention with biomass dispersed, and (6) no biomass harvest. We captured individuals with drift fence arrays and compared evenness, diversity, and richness metrics among treatments with repeated-measure, linear mixed-effects models. We determined predictors of A. terrestris and G. carolinensis abundances using a priori candidate N-mixture models with woody biomass volume, vegetation structure, and groundcover composition as covariates. We had 206 captures of 25 reptile species and 8710 captures of 17 amphibian species during 53690 trap nights. Herpetofauna diversity, evenness, and richness were similar among treatments. A. terrestris abundance was negatively related to volume of retained woody biomass in treatment units in North Carolina in 2013. G. carolinensis abundance was positively related with volume of retained woody debris in treatment units in Georgia in 2012. Other relationships between A. terrestris and G. carolinensis abundances and habitat metrics were weak or absent. The lack of consistent community or population responses suggests the addition of a woody biomass harvest to a clearcut in pine plantations does not impact herpetofauna use of Coastal Plain loblolly plantations in the southeastern United States. We recommend additional research to examine relationships between woody biomass harvesting and rarer species or amphibians with high desiccation risk, particularly in other regions and harvesting systems.

Color perception influences microhabitat selection of refugia and affects monitoring success for a cryptic anuran species.

Perceptual-biases are important for understanding an animal's natural history, identifying potential ecological traps, and for developing effective means to monitor individuals and populations. Despite research demonstrating anurans having a positive phototactic response towards blue colors, we do not yet understand if color cues are used functionally beyond sexual selection. The aim of our study was to determine if color cues are used in selecting microhabitat, and if anuran's blue-positive phototactic response could increase selection of artificial PVC refugia used to monitor cryptic camouflaging anuran species. We captured 32 Cope's Gray Treefrogs and placed them in mesh enclosures with three PVC tubes painted blue, brown, and white. Concurrently, we placed blue, brown, or unpainted white PVC tubes in stratified arrays around a treefrog breeding pond, and counted the number of occasions treefrogs occupied different colored PVC tubes. In the confined choice experiment, treefrogs selected blue tubes (48.3%) significantly more often than brown (28.5%) or white (23.2%) tubes. Our field experiment mirrored these findings (52.0% of capture events in blue, 29.0% in brown, and 19.0% in unpainted white tubes). Our results suggest color influences Cope's Gray Treefrog microhabitat selection, and they utilize color vision when choosing refugia. We demonstrate simple, small changes based on perceptual-biases can induce behaviors that may in turn have large impacts on sampling techniques used in monitoring and inventorying. Incorporating non-traditional physiological measures into animal inventorying and monitoring programs can be used in the future to improve conservation efforts.

Nanolayered siRNA delivery platforms for local silencing of CTGF reduce cutaneous scar contraction in third-degree burns.

Wound healing is an incredibly complex biological process that often results in thickened collagen-enriched healed tissue called scar. Cutaneous scars lack many functional structures of the skin such as hair follicles, sweat glands, and papillae. The absence of these structures contributes to a number of the long-term morbidities of wound healing, including loss of function for tissues, increased risk of re-injury, and aesthetic complications. Scar formation is a pervasive factor in our daily lives; however, in the case of serious traumatic injury, scars can create long-lasting complications due to contraction and poor tissue remodeling. Within this report we target the expression of connective tissue growth factor (CTGF), a key mediator of TGFß pro-fibrotic response in cutaneous wound healing, with controlled local delivery of RNA interference. Through this work we describe both a thorough in vitro analysis of nanolayer coated sutures for the controlled delivery of siRNA and its application to improve scar outcomes in a third-degree burn induced scar model in rats. We demonstrate that the knockdown of CTGF significantly altered the local expression of αSMA, TIMP1, and Col1a1, which are known to play roles in scar formation. The knockdown of CTGF within the healing burn wounds resulted in improved tissue remodeling, reduced scar contraction, and the regeneration of papillary structures within the healing tissue. This work adds support to a number of previous reports that indicate CTGF as a potential therapeutic target for fibrosis. Additionally, we believe that the controlled local delivery of siRNA from ultrathin polymer coatings described within this work is a promising approach in RNA interference that could be applied in developing improved cancer therapies, regenerative medicine, and fundamental scientific research.

Self-Assembled Wound Dressings Silence MMP-9 and Improve Diabetic Wound Healing In Vivo.

The direct local delivery of short interfering RNA (siRNA) into target tissues presents a real solution to several complex medical conditions that today lack efficacious therapies. The development of an ultrathin polymer coating is described to sustain the delivery of siRNA for up to 2 weeks in vitro and in vivo. This technology successfully reduces the expression of MMP-9 within the wounds of diabetic mice, significantly accelerating the wound healing process and improving the quality of tissue formed.

Three-dimensional multilayered fibrous constructs for wound healing applications.

Electrospun materials are promising scaffolds due to their light-weight, high surface-area and low-cost fabrication, however, such scaffolds are commonly obtained as ultrathin two-dimensional non-woven meshes, lacking on topographical specificity and surface side-dependent properties. Herein, it is reported the production of three-dimensional fibrous materials with an asymmetrical inner structure and engineered surfaces. The manufactured constructs evidence fibrous-based microsized conical protrusions [length: (10 ± 3) × 10(2) µm; width: (3.8 ± 0.8) × 10(2) µm] at their top side, with a median peak density of 73 peaks per cm(2), while their bottom side resembles to a non-woven mesh commonly observed in the fabrication of two-dimensional electrospun materials. Regarding their thickness (3.7 ± 0.1 mm) and asymmetric fibrous inner architecture, such materials avoid external liquid absorption while promoting internal liquid uptake. Nevertheless, such constructs also observed the high porosity (89.9%) and surface area (1.44 m(2) g(-1)) characteristic of traditional electrospun mats. Spray layer-by-layer assembly is used to effectively coat the structurally complex materials, allowing to complementary tailor features such as water vapor transmission, swelling ratio and bioactive agent release. Tested as wound dressings, the novel constructs are capable of withstanding (11.0 ± 0.3) × 10(4) kg m(-2) even after 14 days of hydration, while actively promote wound healing (90 ± 0.5% of wound closure within 48 hours) although avoiding cell adhesion on the dressings for a painless removal.

Combination Growth Factor Therapy via Electrostatically Assembled Wound Dressings Improves Diabetic Ulcer Healing In Vivo.

Chronic skin ulcerations are a common complication of diabetes mellitus, affecting up to one in four diabetic individuals. Despite the prevalence of these wounds, current pharmacologic options for treating them remain limited. Growth factor-based therapies have displayed a mixed ability to drive successful healing, which may be due to nonoptimal delivery strategies. Here, a method for coating commercially available nylon dressings using the layer-by-layer process is described to enable both sustained release and independent control over the release kinetics of vascular endothelial growth factor 165 and platelet-derived growth factor BB. It is shown that the use of strategically spaced diffusion barriers formed spontaneously by disulfide bonds enables independent control over the release rates of incorporated growth factors, and that in vivo these dressings improve several aspects of wound healing in db/db mice.

Biodegradable nano-films for capture and non-invasive release of circulating tumor cells.

Selective isolation and purification of circulating tumor cells (CTCs) from whole blood is an important capability for both clinical medicine and biological research. Current techniques to perform this task place the isolated cells under excessive stresses that reduce cell viability, and potentially induce phenotype change, therefore losing valuable information about the isolated cells. We present a biodegradable nano-film coating on the surface of a microfluidic chip, which can be used to effectively capture as well as non-invasively release cancer cell lines such as PC-3, LNCaP, DU 145, H1650 and H1975. We have applied layer-by-layer (LbL) assembly to create a library of ultrathin coatings using a broad range of materials through complementary interactions. By developing an LbL nano-film coating with an affinity-based cell-capture surface that is capable of selectively isolating cancer cells from whole blood, and that can be rapidly degraded on command, we are able to gently isolate cancer cells and recover them without compromising cell viability or proliferative potential. Our approach has the capability to overcome practical hurdles and provide viable cancer cells for downstream analyses, such as live cell imaging, single cell genomics, and in vitro cell culture of recovered cells. Furthermore, CTCs from cancer patients were also captured, identified, and successfully released using the LbL-modified microchips.

Tunable nanostructured coating for the capture and selective release of viable circulating tumor cells.

A layer-by-layer gelatin nanocoating is presented for use as a tunable, dual response biomaterial for the capture and release of circulating tumor cells (CTCs) from cancer patient blood. The entire nanocoating can be dissolved from the surface of microfluidic devices through biologically compatible temperature shifts. Alternatively, individual CTCs can be released through locally applied mechanical stress.

Multifunctional Self-Assembled Films for Rapid Hemostat and Sustained Anti-infective Delivery.

Uncontrolled bleeding and infection are the major causes of death and morbidity from traumatic wounds during military conflicts, disasters, and accidents. Because immediate treatment is critical to survival, it is desirable to have a lightweight and rapidly applicable bandage-one capable of delivering a hemostat that can quickly resolve bleeding while addressing infection over short and longer time frames. It is challenging to design thin film coatings capable of multidrug release, particularly when the drugs are quite different in nature (biologic versus small molecule, charged versus neutral) and the desired release profiles are different for each drug. Herein we have adopted a layer-by-layer film assembly technique to create a linear combination of two independently functional films capable of rapidly releasing thrombin within minutes while sustaining vancomycin elution for more than 24 h. By conjugating vancomycin to a hydrolytically degradable polyacid, poly(ß-L-malic acid), we were able to create a robust thin film with loading and release kinetics that remain unaffected by the additional deposition of a thrombin-based film, demonstrating the possibility for future multitherapeutic films with independently tunable release kinetics.