Trileucine and Pullulan Improve Anti-Campylobacter Bacteriophage Stability in Engineered Spray-Dried Microparticles.

Spray drying biologics into a powder can increase thermal stability and shelf-life relative to liquid formulations, potentially eliminating the need for cold chain infrastructure for distribution in developing countries. In this study, process modelling, microparticle engineering, and a supplemented phase diagram were used to design physically stable fully amorphous spray-dried powder capable of stabilizing biological material. A greater proportion of anti-Campylobacter bacteriophage CP30A remained biologically active after spray drying using excipient formulations containing trehalose and a high glass transition temperature amorphous shell former, either trileucine or pullulan, as compared to the commonly used crystalline shell former, leucine, or a low glass transition temperature amorphous shell former, pluronic F-68. Particle formation models suggest that the stabilization was achieved by protecting the bacteriophages against the main inactivating stress, desiccation, at the surface. The most promising formulation contained a combination of trileucine and trehalose for which the combined effects of feedstock preparation, spray drying, and 1-month dry room temperature storage resulted in a titer reduction of only 0.6 ± 0.1 log10(PFU mL-1). The proposed high glass transition temperature amorphous formulation platform may be advantageous for stabilizing biologics in other spray drying applications in the biomedical engineering industry.

Spray-dried anti-Campylobacter bacteriophage CP30A powder suitable for global distribution without cold chain infrastructure.

Campylobacter jejuni is a leading cause of foodborne illness globally. In this study, a spray drying and packaging process was developed to produce a thermally-stable dry powder containing bacteriophages that retains biological activity against C. jejuni after long distance shipping at ambient temperature. Spray drying using a twin-fluid atomizer resulted in significantly less (p < 0.05) titer reduction than spray drying using a vibrating mesh nebulizer. The use of centrifugation and dilution of filtered bacteriophage lysate in the formulation step resulted in a significantly greater (p < 0.05) proportion of bacteriophages remaining active relative to use of no centrifugation and dilution. The spray-dried bacteriophage powder generated using leucine and trehalose as excipients was flowable, non-cohesive, and exhibited a high manufacturing yield. The powder retained its titer with no significant differences (p > 0.05) in biological activity after storage in suitable packaging for at least 3 weeks at room temperature and after ambient temperature shipping a total distance of approximately 19,800 km, including with a 38 °C temperature excursion. The bacteriophage powder therefore appears suitable for global distribution without the need for cold chain infrastructure.

Delivering Phage Products to Combat Antibiotic Resistance in Developing Countries: Lessons Learned from the HIV/AIDS Epidemic in Africa.

The antimicrobial resistance (AMR) crisis and HIV/AIDS epidemic exhibit many parallels. In both, infectious diseases have caused millions of deaths worldwide, with AMR expected to kill even more people each year than HIV/AIDS did at its peak. In addition, both have required or will require new classes of drugs for control. For HIV/AIDS, development of vital antiretroviral drugs (ARVs) was accomplished in several stages: expanding public awareness about the disease, gathering commitment from the international community to tackle the problem, and eventually establishing policies and global funds to deliver new therapeutics. For AMR, the pursuit of new antimicrobials appears to be following a similar trajectory. This paper examines how lessons and processes leading to ARVs might be applied to developing AMR drugs, in particular bacteriophages (phages). These possess many essential characteristics: inexpensive manufacture, rapid drug development, and a ready means to prevent phage-resistant microbes from emerging. However, the broad application of phage-based products has yet to be fully demonstrated, and will require both international coordination and modified regulatory policies.

RAF inhibitors activate the MAPK pathway by relieving inhibitory autophosphorylation.

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis.

Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice.

Tumor growth, tumor angiogenesis, and vascular endothelial growth factor (VEGF)-specific angiogenesis are all enhanced in beta(3)-integrin-null mice. Furthermore, endothelial cells isolated from beta(3)-null mice show elevated levels of Flk1 (VEGF receptor 2) expression, suggesting that beta(3)-integrin can control the amplitude of VEGF responses by controlling Flk1 levels or activity. We now show that Flk1 signaling is required for the enhanced tumor growth and angiogenesis seen in beta(3)-null mice. Moreover, beta(3)-null endothelial cells exhibit enhanced migration and proliferation in response to VEGF in vitro, and this phenotype requires Flk1 signaling. Upon VEGF stimulation, beta(3)-null endothelial cells exhibit higher levels of phosphorylated Flk1 and extracellular-related kinases 1 and 2 than wild-type endothelial cells. Furthermore, signaling via ERK1/2 is required to mediate the elevated responses to VEGF observed in beta(3)-null endothelial cells and aortic rings in vitro. These data confirm that VEGF signaling via Flk1 is enhanced in beta(3)-integrin-deficient mice and suggests that this increase may mediate the enhanced angiogenesis and tumor growth observed in these mice in vivo.