Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.

Nanotechnology offers novel delivery vehicles for cancer therapeutics. Potential advantages of nanoscale platforms include improved pharmacokinetics, encapsulation of cytotoxic agents, enhanced accumulation of therapeutics in the tumor microenvironment, and improved therapeutic structures and bioactivity. Here, we report the design of a novel amphiphilic molecule that self-assembles into nanostructures for intracellular delivery of cytotoxic peptides. Specifically, a cationic α-helical (KLAKLAK) 2 peptide that is known to induce cancer cell death by membrane disruption was integrated into a peptide amphiphile (PA) that self-assembles into bioactive, cylindrical nanofibers. PAs are composed of a hydrophobic alkyl tail, a β-sheet forming peptide, and a bioactive peptide that is displayed on the surface of the nanofiber after self-assembly. PA nanostructures that included (KLAKLAK) 2 were readily internalized by breast cancer cells, in contrast to the (KLAKLAK) 2 peptide that on its own was not cell permeable. (KLAKLAK) 2 nanostructures, but not the peptides alone, also induced breast cancer cell death by caspase-independent and Bax/Bak-independent mechanisms associated with membrane disruption. Significantly, (KLAKLAK) 2 nanostructures induced cell death more robustly in transformed breast epithelial cells than in untransformed cells, suggesting a degree of tumor selectivity. Our results provide proof-of-principle that self-assembling PAs can be rationally designed to generate nanostructures that can efficiently deliver cytotoxic peptides to cancer cells. Cancer Res; 70(8); 3020-6. ©2010 AACR.

Self-assembling peptide amphiphile (PA) nanofibers were used to encapsulate camptothecin (CPT), a naturally occurring hydrophobic chemotherapy agent, using a solvent evaporation technique. Encapsulation by PA nanofibers was found to improve the aqueous solubility of the CPT molecule by more than 50-fold. PAs self-assembled into nanofibers in the presence of CPT as demonstrated by transmission electron microscopy. Small-angle X-ray scattering results suggest a slight increase in diameter of the nanofiber to accommodate the hydrophobic cargo. In vitro studies using human breast cancer cells show an enhancement in antitumor activity of the CPT when encapsulated by the PA nanofibers. In addition, using a mouse orthotopic model of human breast cancer, treatment with PA nanofiber encapsulated CPT inhibited tumor growth. These results highlight the potential of this model PA system to be adapted for delivery of hydrophobic therapies to treat a variety of diseases including cancer.

A library of polyurethanes and polyureas with different hydrophobicities containing the same acid-degradable dimethyl ketal moiety embedded in the polymer main chain have been prepared. All polymers were synthesized using an AA-BB type step-growth polymerization by reaction of bis(p-nitrophenyl carbamate/carbonate) or diisocyanate monomers with an acid-degradable, ketal-containing diamine. These polymers were designed to hydrolyze at different rates in mildly acidic conditions as a function of their hydrophobicity to afford small molecules only with no polymeric byproduct. The library of polymers was screened for the formation of microparticles using a double emulsion technique. The microparticles that were obtained degraded significantly faster at acidic pH (5.0) than at physiological pH (7.4) with degradation kinetics related to the hydrophobicity of the starting polymer. In vitro studies demonstrated the ability of the FITC-BSA loaded microparticles to be phagocytosed by macrophages resulting in a 10-fold increase in the protein uptake compared to a free protein control; in addition, the microparticles were found to be nontoxic at the concentrations tested of up to 1 mg/mL. The ease of preparation of the polymers coupled with the ability to tune their hydrophobicity and the high acid sensitivity of the microparticles identify this new class of materials as promising candidates for the delivery of bioactive materials.