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Abstract: A synthetically useful protocol has been developed for the preparation of highly functionalized 3,4-fused spiro[isobenzofuran-3-ones], spiro[furo[3,4-b]pyridin-5(7H)-ones], 3-aryl-, and alkylphthalides. Reaction of 2-iodobenzoate esters and 2-iodopyridine carboxylate esters with i-PrMgCl·LiCl in the presence of cyclic ketones under standard Barbier reaction conditions affords 3,4-fused spiro[isobenzofuran-3-ones] and spiro[furo[3,4-b]pyridin-5(7H)-ones] in good to excellent yields. Step-wise addition of i-PrMgCl·LiCl to 2-iodobenzoate esters followed by trapping with various aldehydes yields 3-aryl and 3-alkylphthalides; whereas, under similar conditions access to 3-aryl and 3-alylazaphthalides is also possible. Extension of this methodology toward the preparation of 3-n-butylphthalide and chrycolide, a natural product isolated from the leaves and stems of Chrysanthemum coronarium, is also described. [Copyright &y& Elsevier]

Abstract: There is evidence in the literature that silicone oil, a lubricant, can induce aggregation in protein formulations delivered through prefilled syringes. Surfactants are commonly used to minimize protein–silicone oil and protein–container interactions; however, these interactions are not well characterized and understood. The purpose of this manuscript was to understand the competitive interactions of a fusion protein with the silicone oil in the presence of Tween® 20. An adsorption isotherm for Tween® 20 at the silicone oil/water interface, using silicone oil coated quartz crystals, was generated at 25°C to identify surface saturation concentrations. A concentration of Tween® 20 providing interfacial saturation was selected for protein adsorption studies at the silicone oil/water interface. The surfactant molecules adsorbed at the interface in a monolayer with a reduced viscoelastic character in comparison to the bound protein layer. A significant reduction in protein adsorption was observed when the surfactant was present at the interface. No desorption of the pre-adsorbed protein molecules was observed when Tween® 20 was introduced, suggesting that the protein has strong interactions with the interface. However, both, Tween® 20 and protein, adsorbed to the silicone oil/water interface when adsorption was carried out from a mixture of protein and Tween® 20. [Copyright &y& Elsevier]

Abstract: In this study, we have used quartz crystal microbalance (QCM) to quantitate the adsorption of a protein on silicone oil coated surfaces as a function of protein concentration, pH and ionic strength using a 5MHz quartz crystal. Protein adsorption isotherms were generated at different solution pH and ionic strengths. Surface saturation concentrations were selected from adsorption isotherms and used to generate adsorption profiles from pH 3.0 to 9.0, and at ionic strengths of 10mM and 150mM. At low ionic strength (10mM) and pH 5.0 (close to the isoelectric point of the protein), maximum adsorption of protein to the silicone oil surface was observed. At higher ionic strength (150mM), no significant pH influence on adsorption was observed. QCM could be used as a reliable technique to study the binding of proteins to silicone oil coated surfaces. [Copyright &y& Elsevier]

This study investigated factors associated with vial filling with a positive displacement piston pump leading to formation of protein particles in a formulation of an IgG. We hypothesized that nanoparticles shed from the pump's solution-contact surfaces nucleated protein aggregation and particle formation. Vials of IgG formulation filled at a clinical manufacturing site contained a few visible particles and about 100,000 particles (1.5–3 µm) per mL. In laboratory studies with the same model (National Instruments FUS-10) of pump, pumping of 20 mg/mL IgG formulation resulted in about 300,000 particles (1.5–3 µm) per mL. Pumping of protein-free formulation resulted in 13,000 particles (1.5–15 µm) per mL. More than 99% of the particles were 0.25–0.95 µm in size. Mixing of protein-free pumped solution with an equal volume of 40 mg/mL IgG resulted in 300,000 particles (1.5–15 µm) per mL. Also, mixing IgG formulation with 30,000/mL stainless steel nanoparticles resulted in formation of 30,000 protein microparticles (1.5–15 µm) per mL. Infrared spectroscopy showed that secondary structure of IgG in microparticles formed by pumping or mixing with steel nanoparticles was minimally perturbed. Our results document that nanoparticles of foreign materials shed by pumps can serve as heterogeneous nuclei for formation of protein microparticles. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:94–104, 2009 [ABSTRACT FROM AUTHOR]

Bruton's tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5- a pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5- a pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model. [ABSTRACT FROM AUTHOR]