Improving the in vivo therapeutic index of siRNA polymer conjugates through increasing pH responsiveness.

Polymer based carriers that aid in endosomal escape have proven to be efficacious siRNA delivery agents in vitro and in vivo; however, most suffer from cytotoxicity due in part to a lack of selectivity for endosomal versus cell membrane lysis. For polymer based carriers to move beyond the laboratory and into the clinic, it is critical to find carriers that are not only efficacious, but also have margins that are clinically relevant. In this paper we report three distinct categories of polymer conjugates that improve the selectivity of endosomal membrane lysis by relying on the change in pH associated with endosomal trafficking, including incorporation of low pKa heterocycles, acid cleavable amino side chains, or carboxylic acid pH sensitive charge switches. Additionally, we determine the therapeutic index of our polymer conjugates in vivo and demonstrate that the incorporation of pH responsive elements dramatically expands the therapeutic index to 10-15, beyond that of the therapeutic index (less than 3), for polymer conjugates previously reported.

Quantitation and characterization of serotype 6A activation for pneumococcal conjugate vaccine by cryo-EM and SEC methods.

Pneumococcal conjugate vaccines (PCV) typically consist of capsular polysaccharides from different S. pneumoniae serotypes which are covalently attached to carrier protein. A well-established process to manufacture PCV is through activating polysaccharide by oxidation of vicinal diols to aldehydes, followed by protein conjugation via reductive amination. Polysaccharide activation is a crucial step that affects vaccine product critical attributes including conjugate size and structure. Therefore, it is highly desired to have robust analytical methods to well characterize this activation process. In this study, using pneumococcal serotype 6A as the model, we present two complimentary analytical methods for characterization of activated polysaccharide. First, a size exclusion chromatography (SEC) method was developed for quantitative measurement of polysaccharide activation levels. This SEC method demonstrated good assay characteristics on accuracy, precision and linearity. Second, a gold nanoparticle labeled cryo-electron microscopy (Cryo-EM) technique was developed to visualize activation site distribution along polysaccharide chain and provide information on activation heterogeneity. These two complimentary methods can be utilized to control polysaccharide activation process and ensure consistent delivery of conjugate vaccine products.

Highly efficient asymmetric synthesis of sitagliptin.

A highly efficient synthesis of sitagliptin, a potent and selective DPP-4 inhibitor for the treatment of type 2 diabetes mellitus (T2DM), has been developed. The key dehydrositagliptin intermediate 9 is prepared in three steps in one pot and directly isolated in 82% yield and >99.6 wt % purity. Highly enantioselective hydrogenation of dehydrositagliptin 9, with as low as 0.15 mol % of Rh(I)/(t)Bu JOSIPHOS, affords sitagliptin, which is finally isolated as its phosphate salt with nearly perfect optical and chemical purity. This environmentally friendly, 'green' synthesis significantly reduces the total waste generated per kilogram of sitagliptin produced in comparison with the first-generation route and completely eliminates aqueous waste streams. The efficiency of this cost-effective process, which has been implemented on manufacturing scale, results in up to 65% overall isolated yield.