Carcinogenicity assessment of the Hedgehog pathway inhibitor, vismodegib in Tg.rasH2 mice and Sprague-Dawley rats.

Vismodegib (also known as GDC-0449) is a novel small molecule inhibitor of the Hedgehog (Hh) signaling pathway currently approved for the treatment of metastatic or locally advanced basal cell carcinoma (BCC) in humans. Its tumorigenic potential was assessed in dedicated carcinogenicity studies in rasH2 transgenic (Tg.rasH2) mice and Sprague Dawley (SD) rats. Tumorigenicity potential of vismodegib was identified in rats only and was limited to benign hair follicle tumors, including pilomatricomas and keratoacanthomas at exposures of ≥0.1-fold and ≥0.6-fold, respectively, of the steady-state exposure (AUC0-24h) of the recommended human dose. No malignant tumors were identified in either species. Overall, the totality of pharmacology and nonclinical safety data (lack of genotoxicity, in vitro secondary pharmacological binding, and immunoregulatory effects, and limited effects on the endocrine system) suggests that the development of the benign hair follicle tumors may be related to pharmacologically-mediated disruption of hair follicle morphogenesis, although the exact mechanism of tumorigenesis is unclear. Hair follicle tumors have not been reported in vismodegib-treated patients. The relevance of this finding in rats to patients is uncertain.

Comparison of the capillary wave method and pressure tensor route for calculation of interfacial tension in molecular dynamics simulations.

We have studied the calculation of surface and interfacial tension for a variety of liquid-vapor and liquid-liquid interfaces using molecular dynamics (MD) simulations. Because of the inherently small scale of MD systems, large pressure fluctuations can cause imprecise calculations of surface tension using the pressure tensor route. The capillary wave method exhibited improved precision and stability throughout all of the simulated systems in this study. In order to implement this method, the interface was defined by fitting an error function to the density profile. However, full mapping of the interface from coordinate files produced enhanced accuracy. Upon increasing the system size, both methods exhibited higher precision, although the capillary wave method was still more reliable.

Spontaneous transport of microparticles across liquid-liquid interfaces.

Transporting micrometer-sized particles through the liquid-liquid interface generally requires high shear force and sometimes surfactant functionalization. Without these aids, particles adhere to the interface due to strong capillary forces (can be on the order of 10(6) kT). Thus, spontaneous transport of microparticles through the liquid-liquid interface has not yet been reported. However, we present a new phenomenon here: some ionic liquids (ILs) possess powerful extraction capabilities and can cause microparticles to migrate across the interface without the aid of any shear forces. Both single particles and clusters of particles were observed to adsorb to, then "jump" across the interface and finally detach. In the absence of external mixing, particles as large as 4 µm (in diameter) could completely penetrate the IL/water interface, despite the significant adhesive forces. We have presented a hypothesis that these forces were overcome by ions dissolved in the non-IL phase, which helped by covering the particle surfaces, allowing for more favorable interactions with the IL.

Nonconvective mixing of miscible ionic liquids.

Ionic liquids (ILs) are ionic compounds that are liquid at room temperature. We studied the spontaneous mixing behavior between two ILs, ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), and observed notable phenomena. Experimental studies showed that the interface between the two ILs was unusually long-lived, despite the ILs being miscible with one another. Molecular dynamics (MD) simulations supported these findings and provided insight into the micromixing behavior of the ILs. We found that not only did the ions experience slow diffusion as they mix but also exhibited significant ordering into distinct regions. We suspect that this ordering disrupted concentration gradients in the direction normal to the interface, thus hindering diffusion in this direction and allowing the macroscopic interface to remain for long periods of time. Intermolecular interactions responsible for this behavior included the O-NH interaction between the EAN ions and the carbon chain-carbon chain interactions between the [BMIM](+) cations, which associate more strongly in the mixed state than in the pure IL state.

Ocular Safety and Toxicokinetics of Bevacizumab-bvzr (Zirabev), a Bevacizumab Biosimilar, Administered to Cynomolgus Monkeys by Intravitreal Injection.

Purpose: Bevacizumab-bvzr (Zirabev®), a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor and a biosimilar to bevacizumab, is approved for intravenous administration for various indications worldwide. The objectives of this study were to evaluate the ocular toxicity, systemic tolerability, and toxicokinetics (TKs) of bevacizumab-bvzr following repeat intravitreal (IVT) injection to cynomolgus monkeys. Methods: Male monkeys were administered saline, vehicle, or bevacizumab-bvzr at 1.25 mg/eye/dose once every 2 weeks (3 doses total) for 1 month by bilateral IVT injection, followed by a 4-week recovery phase to evaluate the reversibility of any findings. Local and systemic safety was assessed. Ocular safety assessments included in-life ophthalmic examinations, tonometry (intraocular pressure, IOP), electroretinograms (ERGs), and histopathology. In addition, concentrations of bevacizumab-bvzr were measured in serum and in ocular tissues (vitreous humor, retina, and choroid/retinal pigment epithelium) and ocular concentration-time profiles and serum TKs were evaluated. Results: Bevacizumab-bvzr was tolerated locally and systemically, with an ocular safety profile comparable to the saline or vehicle control group. Bevacizumab-bvzr was observed in both serum and in the evaluated ocular tissues. There were no bevacizumab-bvzr-related microscopic changes or effects on IOP or ERGs. Bevacizumab-bvzr-related trace pigment or cells in vitreous humor (in 4 of 12 animals; commonly associated with IVT injection) and transient, nonadverse, mild ocular inflammation (in 1 of 12 animals) were noted upon ophthalmic examination and fully reversed during the recovery phase. Conclusions: Bevacizumab-bvzr was well tolerated via biweekly IVT administration in healthy monkeys, with an ocular safety profile comparable to saline or its vehicle control.

The Therapeutic Monoclonal Antibody Bamlanivimab Does Not Enhance SARS-CoV-2 Infection by FcR-Mediated Mechanisms.

As part of the non-clinical safety package characterizing bamlanivimab (SARS-CoV-2 neutralizing monoclonal antibody), the risk profile for antibody-dependent enhancement of infection (ADE) was evaluated in vitro and in an African green monkey (AGM) model of COVID-19. In vitro ADE assays in primary human macrophage, Raji, or THP-1 cells were used to evaluate enhancement of viral infection. Bamlanivimab binding to C1q, FcR, and cell-based effector activity was also assessed. In AGMs, the impact of bamlanivimab pretreatment on viral loads and clinical and histological pathology was assessed to evaluate enhanced SARS-CoV-2 replication or pathology. Bamlanivimab did not increase viral replication in vitro, despite a demonstrated effector function. In vivo, no significant differences were found among the AGM groups for weight, temperature, or food intake. Treatment with bamlanivimab reduced viral loads in nasal and oral swabs and BAL fluid relative to control groups. Viral antigen was not detected in lung tissue from animals treated with the highest dose of bamlanivimab. Bamlanivimab did not induce ADE of SARS-CoV-2 infection in vitro or in an AGM model of infection at any dose evaluated. The findings suggest that high-affinity monoclonal antibodies pose a low risk of mediating ADE in patients and support their safety profile as a treatment of COVID-19 disease.

Molecular dynamics studies on the adaptability of an ionic liquid in the extraction of solid nanoparticles.

Recently, a number of publications have suggested that ionic liquids (ILs) can absorb solid particles. This development may have implications in fields like oil sand processing, oil spill beach cleanup, and water treatment. In this Article, we provide a computational investigation of this phenomenon via molecular dynamics simulations. Two particle surface chemistries were investigated: (1) hydrocarbon-saturated and (2) silanol-saturated, representing hydrophobic and hydrophilic particles, respectively. Employing 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)]) as a model IL, these nanoparticles were allowed to equilibrate at the IL/water and IL/hexane interfaces to observe the interfacial self-assembled structures. At the IL/water interface, the hydrocarbon-based nanoparticles were nearly completely absorbed by the IL, while the silica nanoparticles maintained equal volume in both phases. At the IL/hexane interface, the hydrocarbon nanoparticles maintained minimal interactions with the IL, whereas the silica nanoparticles were nearly completely absorbed by it. Studies of these two types of nanoparticles immersed in the bulk IL indicate that the surface chemistry has a great effect on the corresponding IL liquid structure. These effects include layering of the ions, hydrogen bonding, and irreversible absorption of some ions to the silica nanoparticle surface. We quantify these effects with respect to each nanoparticle. The results suggest that ILs likely exhibit this absorption capability because they can form solvation layers with reduced dynamics around the nanoparticles.

Molecular dynamics simulations of charged nanoparticle self-assembly at ionic liquid-water and ionic liquid-oil interfaces.

Nanoparticle self-assembly at liquid-liquid interfaces can be significantly affected by the individual nanoparticle charges. This is particularly true at ionic liquid (IL) based interfaces, where Coulombic forces play a major role. Employing 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)]) as a model IL, we have studied the self-assembly of hydrophobic nanoparticles with different surface charges at the IL/water and IL/oil (hexane) interfaces using molecular dynamics simulations. In the IL/water system, the nanoparticles were initially dispersed in the water phase but quickly equilibrated at the interface, somewhat in favor of the IL phase. This preference was lessened with increased nanoparticle charge. In the IL/hexane system, all charged nanoparticles interacted with the IL to some extent, whereas the uncharged nanoparticles remained primarily in the hexane phase. Potential of mean force calculations supported the observations from the equilibrium studies and provided new insights into the interactions of the nanoparticles and ionic liquid based interfaces.

Molecular dynamics simulations of nanoparticle self-assembly at ionic liquid-water and ionic liquid-oil interfaces.

We have studied the self-assembly of hydrophobic nanoparticles at ionic liquid (IL)-water and IL-oil (hexane) interfaces using molecular dynamics (MD) simulations. For the 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)])/water system, the nanoparticles rapidly approached the IL-water interface and equilibrated more into the IL phase although they were initially in the water phase. In contrast, when the nanoparticles were dispersed in the hexane phase, they slowly approached the IL-hexane interface but remained primarily in the hexane phase. Consequently, the IL-hexane interface was rather undisturbed by the nanoparticles whereas the IL-water interface changed significantly in width and morphology to accommodate the presence of the nanoparticles. The equilibrium positions of the nanoparticles were also supported and explained by potential of mean force (PMF) calculations. Interesting ordering and charge distributions were observed at the IL-liquid interfaces. At the IL-hexane interface, the [BMIM] cations preferentially oriented themselves so that they were immersed more in the hexane phase and packed efficiently to reduce steric hindrance. The ordering likely contributed to a heightened IL density and a slightly positive charge at the IL-hexane interface. In contrast, the cations at the IL-water interface were oriented isotropically unless in the presence of nanoparticles, where the cations aligned across the nanoparticle surfaces.

Particle self-assembly at ionic liquid-based interfaces.

This review presents an overview of the nature of ionic liquid (IL)-based interfaces and self-assembled particle morphologies of IL-in-water, oil- and water-in-IL, and novel IL-in-IL Pickering emulsions with emphasis on their unique phenomena, by means of experimental and computational studies. In IL-in-water Pickering emulsions, particles formed monolayers at ionic liquid-water interfaces and were close-packed on fully covered emulsion droplets or aggregated on partially covered droplets. Interestingly, other than equilibrating at the ionic liquid-water interfaces, microparticles with certain surface chemistries were extracted into the ionic liquid phase with a high efficiency. These experimental findings were supported by potential of mean force calculations, which showed large energy drops as hydrophobic particles crossed the interface into the IL phase. In the oil- and water-in-IL Pickering emulsions, microparticles with acidic surface chemistries formed monolayer bridges between the internal phase droplets rather than residing at the oil/water-ionic liquid interfaces, a significant deviation from traditional Pickering emulsion morphology. Molecular dynamics simulations revealed aspects of the mechanism behind this bridging phenomenon, including the role of the droplet phase, surface chemistry, and inter-particle film. Novel IL-in-IL Pickering emulsions exhibited an array of self-assembled morphologies including the previously observed particle absorption and bridging phenomena. The appearance of these morphologies depended on the particle surface chemistry as well as the ILs used. The incorporation of particle self-assembly with ionic liquid science allows for new applications at the intersection of these two fields, and have the potential to be numerous due to the tunability of the ionic liquids and particles incorporated, as well as the particle morphology by combining certain groups of particle surface chemistry, IL type (protic or aprotic), and whether oil or water is incorporated.

Understanding droplet bridging in ionic liquid-based Pickering emulsions.

We have studied the unique bridging behavior of solid-stabilized oil-in-ionic liquid (IL) and water-in-ionic liquid emulsions with respect to particle concentration, particle size, and droplet phase using a confocal laser scanning microscope. The emulsions exhibited three morphology regimes: (1) single, sparingly covered droplets, (2) bridged clusters of droplets, and (3) fully covered droplets. The degree of bridging was directly proportional to the total potential bridging area which can be determined from the particle size and concentration. This type of emulsion diverges from much of the conventional wisdom of oil-water Pickering emulsions regarding the particle self-assembly onto droplet interfaces and liquid film stability. While the focus here is the bridging regime, we also report interesting observations, specifically, the deformed oil droplets and the transport of excess solid particles into the water droplets, in the fully covered droplet regime. The work identified new self-assembled particle structure and morphology in solid-stabilized emulsions.

Particle self-assembly in oil-in-ionic liquid Pickering emulsions.

We have studied polydimethylsiloxane (PDMS)-in-1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)]) Pickering emulsions stabilized by polystyrene microparticles with different surface chemistry. Surprisingly, in contrast to the consensus originating from oil/water Pickering emulsions in which the solid particles equilibrate at the oil-water droplet interfaces and provide effective stabilization, here the polystyrene microparticles treated with sulfate, aldehyde sulfate, or carboxylate dissociable groups mostly formed monolayer bridges among the oil droplets rather than residing at the oil-ionic liquid interfaces. The bridge formation inhibited individual droplet-droplet coalescence; however, due to low density and large volume (thus the buoyant effect), the aggregated oil droplets actually promoted oil/ionic liquid phase separation and distressed emulsion stability. Systems with binary heterogeneous polystyrene microparticles exhibited similar, even enhanced (in terms of surface chemistry dependence), bridging phenomenon in the PDMS-in-[BMIM][PF(6)] Pickering emulsions.

26-Week dermal oncogenicity study evaluating pure trans-capsaicin in Tg.AC hemizygous mice (FBV/N).

The objective of this study was to assess the oncogenic potential of trans-capsaicin when administered weekly via topical application to the dorsal skin of Tg.AC mice for 26 weeks. Male and female Tg.AC mice (25 mice/sex/group) received dose formulations containing trans-capsaicin dissolved in diethylene glycol monoethyl ether (DGME). The positive control was tetradecanoylphorbol-13-acetate (TPA) dissolved in DGME. Appropriate controls, including a topical lidocaine local anesthetic pretreatment (4%w/w), were maintained. All groups were dosed once weekly, except for the TPA group, which was dosed twice per week. Analysis of the macroscopic observations after the final sacrifice revealed no noteworthy treatment-related findings, with the exception of dermal masses that were randomly dispersed throughout all treatment groups for both males and females. The frequency of dermal masses in the capsaicin-treated groups (at a dose level of up to 102 mg/kg and an application rate of 25.6 mg/cm2/kg/week) was not elevated in comparison to either concurrent vehicle or untreated controls. In contrast, a notable increase in the frequency of dermal masses was observed in the TPA-treated mice compared to both the concurrent vehicle and untreated controls. Dermal application of capsaicin resulted in no increased incidence of preneoplastic or neoplastic skin lesions. In contrast, over half the male and female mice exposed to TPA had multiple skin papillomas; the majority of the TPA-treated animals either died early or was humanely euthanized due to tumor load. Spontaneously occurring neoplasms were not appreciably increased in capsaicin-treated animals. Capsaicin-related non-neoplastic microscopic findings were seen sporadically in both genders and included acanthosis, hyperkeratosis/parakeratosis (primarily females), epidermal crusts, subepidermal fibrosis, epidermal ulcerations/erosions, and chronic-active inflammation. There was no evidence of a dose response in either the incidence or severity of these findings. The lidocaine- (at a dose level of 162 mg/kg and at an application rate of 40.5 mg/cm2/kg/week) and DGME-treated (at a dose level of 4.0 g/kg and at an application rate of 1 g/cm2/kg/week) control groups also did not display any evidence of increase in dermal masses. Based on these results, trans-capsaicin, lidocaine, and DGME should be considered nononcogenic in the Tg.AC mouse dermal model.