Mechanism for effective lymphoid cell and tissue loading following oral administration of nucleotide prodrug GS-7340.

GS-7340 is a prodrug of tenofovir (TFV) that more efficiently delivers TFV into lymphoid cells and tissues than the clinically used prodrug TFV disoproxil fumarate, resulting in higher antiviral potency at greatly reduced doses and lower systemic TFV exposure. First-pass extraction by the intestine and liver represents substantial barriers to the oral delivery of prodrugs designed for rapid intracellular hydrolysis. In order to understand how GS-7340 reduces first-pass clearance to be an effective oral prodrug, its permeability and stability were characterized in vitro and detailed pharmacokinetic studies were completed in dogs. GS-7340 showed concentration-dependent permeability through monolayers of caco-2 cells and dose-dependent oral bioavailability in dogs, increasing from 1.7% at 2 mg/kg to 24.7% at 20 mg/kg, suggesting saturable intestinal efflux transport. Taking into account a 65% hepatic extraction measured in portal vein cannulated dogs, high dose GS-7340 is nearly completely absorbed. Consistent with the proposed role of intestinal efflux transport, coadministration of low dose GS-7340 with a transport inhibitor substantially increased GS-7340 exposure. The result of effective oral absorption and efficient lymphoid cell loading was reflected in the high and persistent levels of the pharmacologically active metabolite, TFV diphosphate, in peripheral blood mononuclear cells following oral administration to dogs. In conclusion, GS-7340 reaches the systemic circulation to effectively load target cells by saturating intestinal efflux transporters, facilitated by its high solubility, and by maintaining sufficient stability in intestinal and hepatic tissue.

Targeting macrophages with baculovirus-produced lysosomal enzymes: implications for enzyme replacement therapy of the glycoprotein storage disorder galactosialidosis.

Lysosomal storage diseases (LSDs) are monogenic disorders of metabolism caused by deficiency of hydrolytic enzymes. In several LSDs, cells of the reticuloendothelial (RE) system are the primary targets of the disease. Exogenous administration of recombinant enzymes overproduced in mammalian cells has proved effective for treating the systemic phenotype in nonneuropathic patients with LSDs. However, for the treatment of diseases with primary involvement of the RE system, the production of the therapeutic enzyme in insect cells could be an alternative and advantageous method because glycoproteins expressed in insect cells carry carbohydrates of the pauci-mannose or core-type. These recombinant enzymes are in principle already poised to be internalized by cells that express mannose receptors, including macrophages. Here, we demonstrate that three baculovirus-expressed enzymes, protective protein/cathepsin A (PPCA), neuraminidase (Neu1), and beta-glucosidase, were readily taken up and restored lysosomal function in enzyme-deficient mouse macrophages. The capacity of recombinant PPCA and Neu1 to clear the lysosomal storage in target cells was assessed in PPCA-/- mice, a model of galactosialidosis. Intravenously injected PPCA-/- mice efficiently internalized the corrective enzymes in resident macrophages of many organs. In addition, treated mice showed overall clearance of lysosomal storage in the most affected systemic organs, kidney, liver, and spleen. Our results suggest that ERT with baculovirus-expressed enzymes might be an effective treatment for nonneuropathic patients with galactosialidosis and possibly for others with LSDs that primarily involve the RE system.