532    

Backgroune:  It has been previously been shown that a MDRN can inhibit the growth of HIV following multiple passages in tissue culture. We have identified a substantially more potent MDRN, SN1212, that demonstrates antiviral activity in a single passage. SN1461 is an oral prodrug of SN1212. SN1212 is not a chain terminator and has an unmodified sugar. Thus, SN1212 is unlikely to be affected by the two major mechanisms of NRTI resistance, lack of affinity of RT for a modified sugar or pyrophosphorolysis. Further, rather than targeting a single protein, SN1212 adversely affects all viral proteins.

 Methods: HIV-1 growth was monitored by p24 ELISA (Beckman-Coulter) and by viral titer. Genotoxicity was tested in CHO and human male lymphoblastoid (TK6) cells by HGPRT assays. HIV (NL4-3) proviral DNA RT and Env genes were PCR amplified, cloned and sequenced after passaging the virus. Mitochondrial toxicity was tested by lactate (Sigma) and mitochondrial (mt) DNA levels, measured by Southern Blot analysis using a mt-specific probe. For acute toxicology studies, SN1461 (a prodrug of SN1212) was administered to dogs and rats under GLP conditions. 

Results: The EC50 (effective concentration inhibiting 50% of viral growth) of SN1212 is 10-100 nM. Passaging HIV in the presence of SN1212 (100 nM) extinguished the virus in 13 passages. Sequencing viral genomes of the penultimate passage prior to extinction showed that SN1212-treated virus had a > 50% increase in mutation rate in RT and Env above control. Passaging experiments failed to yield a SN1212-resistant HIV isolate after several attempts. SN1212 did not exhibit significant genotoxicity in CHO cells or human male B-cells at concentrations up to 1 mM. At 320 mM, SN1212 did not increase lactate levels over control and by southern blot analysis, SN1212 did not inhibit mtDNA over control. Doses of up to 2 g/kg of SN1461 in dogs and 1 g/kg in rats were administered without evidence of toxicity.

Conclusions:  SN1212/1461 is the first MDRN being developed for the treatment of HIV. SN1212/1461 has demonstrated in vitro activity against HIV without evidence of development of de novo resistance by HIV. SN1212/1461 has been shown to have a desirable safety profile in both in vitro and in vivo assays.

Keywords: Antiretroviral; Lethal Mutagenesis