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Abstract. The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased (∼50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

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The beagle dog is a widely used in vivo model to guide clinical formulation development and to explore the potential for food effects. However, the results in dogs are often not directly translatable to humans. Consequently, a physiologically based modeling strategy has been proposed, using the dog as a validation step to verify model assumptions before making predictions in humans. One current weakness in this strategy is the lack of validated tools to incorporate gut wall metabolism into the dog model. In this study, in vitro to in vivo extrapolation factors for CYP2B11 and CYP3A12 mediated metabolism were established based on tissue enzyme abundance data reported earlier. Thereafter, physiologically based modeling of intestinal absorption in beagle dog was conducted in GastroPlus using V(max) and K(m) determined in recombinant enzymes as inputs for metabolic turnover. The predicted fraction of absorbed dose escaping the gut wall metabolism (F(g)) of all five reference compounds studied (domperidone, felodipine, nitrendipine, quinidine, and sildenafil) were within a two-fold range of the value estimated from in vivo data at single dose levels. However, further in vivo studies and analysis of the dose-dependent pharmacokinetics of felodipine and nitrendipine showed that more work is required for robust forecasting of nonlinearities. In conclusion, this study demonstrates an approach for prediction of the gut wall extraction of CYP substrates in the beagle dog, thus enhancing the value of dog studies as a component in a strategy for the prediction of human pharmacokinetics.

Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.