9-(2-(phosphonomethoxy)ethyl)adenine-diphosphate and adefovir-dipivoxil

Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

Topics: Adenine; Adenylate Cyclase Toxin; Animals; Anti-Bacterial Agents; Bordetella pertussis; Caco-2 Cells; Cell Line, Tumor; Cell Survival; Humans; Inhibitory Concentration 50; Macrophages; Mice; Microbial Sensitivity Tests; Organophosphonates; Prodrugs

Adefovir dipivoxil (Hepsera), a first-line therapy for chronic hepatitis B, is an esterase-activated prodrug of PMEA. Dose-limiting nephrotoxicity necessitates suboptimal dosing at 10 mg/day. Remofovir mesylate (MB06866Q) (Hepavir B) is a CYP3A4-activated prodrug of PMEA based on the HepDirect technology that targets PMEA to the liver. In a whole body autoradiography study in rats after oral dosing (30 mg/kg) of [14C]adefovir dipivoxil or [14C]remofovir mesylate, remofovir yielded 15 times higher concentrations of radioactivity in the liver than adefovir dipivoxil, but only one-third of the concentrations in the kidney. After oral dosing (4 mg/kg) of the same radiolabelled agents in cynomolgus monkeys, remofovir mesylate yielded 60 times higher levels of total radioactivity in the liver, but only two-thirds of total radioactivity levels in the kidney. Thus, remofovir mesylate may provide better efficacy and reduced nephrotoxicity. In portal vein-cannulated rats (30 mg/kg) after a single oral dose of [14C]adefovir dipivoxil or [14C]remofovir mesylate, no PMEA was detectable in rat portal plasma early after dosing, indicating that intestinal CYP3A4 does not play a role in conversion of remofovir mesylate to PMEA. The portal/systemic extraction ratio was quite high in both models, suggesting good liver-targeting properties. Portal and systemic remofovir/PMEA ratio indicates that the liver is the site of conversion of remofovir to PMEA. 28-Day toxicity studies demonstrated renal toxicity in rats at doses of 100 mg/kg or higher with no safety concerns at 30 mg/kg and acceptable safety in monkeys at doses up to 60 mg/kg. Thus, in rats and non-human primates, remofovir mesylate has liver-targeting properties and is safer than adefovir dipivoxil.

Topics: Adenine; Animals; Antiviral Agents; Autoradiography; Carbon Radioisotopes; Dose-Response Relationship, Drug; Drug Delivery Systems; Haplorhini; Hepatitis B, Chronic; Kidney; Liver; Mesylates; Organophosphonates; Organophosphorus Compounds; Prodrugs; Rats; Species Specificity; Tissue Distribution

Edema factor (EF), a key virulence factor in anthrax pathogenesis, has calmodulin (CaM)-activated adenylyl cyclase activity. We have found that adefovir dipivoxil, a drug approved to treat chronic infection of hepatitis B virus, effectively inhibits EF-induced cAMP accumulation and changes in cytokine production in mouse primary macrophages. Adefovir diphosphate (PMEApp), the active cellular metabolite of adefovir dipivoxil, inhibits the adenylyl cyclase activity of EF in vitro with high affinity (K(i) = 27 nM). A crystal structure of EF-CaM-PMEApp reveals that the catalytic site of EF forms better van der Waals contacts and more hydrogen bonds with PMEApp than with its endogenous substrate, ATP, providing an explanation for the approximately 10,000-fold higher affinity EF-CaM has for PMEApp versus ATP. Adefovir dipivoxil is a clinically approved drug that can block the action of an anthrax toxin. It can be used to address the role of EF in anthrax pathogenesis.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Antigens, Bacterial; Antiviral Agents; Bacterial Toxins; Binding Sites; Cell Line; CHO Cells; Cricetinae; Cyclic AMP; Exotoxins; Hepatitis B, Chronic; Kinetics; Models, Molecular; Organophosphonates; Protein Conformation; Recombinant Proteins; Spodoptera; Transfection