Edward R. Lee

Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.

Cationic Lipid Structure and Formulation Considerations for Optimal Gene Transfection of the Lung

Abstract Enhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid : plasmid DN A (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanol-amine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphati-dylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid: pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.