Alkoxyalkyl esters of 9-(s)-(3-hydroxy-2-phosphonomethoxypropyl) adenine are potent and selective inhibitors of hepatitis B virus (HBV) replication in vitro and in HBV transgenic mice in vivo

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Antimicrob Agents Chemother



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Alkoxyalkyl esters of acyclic nucleoside phosphonates have previously been shown to have increased antiviral activity when they are administered orally in animal models of viral diseases, including lethal infections with vaccinia virus, cowpox virus, ectromelia virus, murine cytomegalovirus, and adenovirus. 9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] was previously shown to have activity against hepatitis B virus (HBV) in vitro. To assess the effect of alkoxyalkyl esterification of (S)-HPMPA, we prepared the hexadecyloxypropyl (HDP), 15-methyl-hexadecyloxypropyl (15M-HDP), and octadecyloxyethyl (ODE) esters and compared their activities with the activity of adefovir dipivoxil in vitro and in vivo. Alkoxyalkyl esters of (S)-HPMPA were 6 to 20 times more active than unmodified (S)-HPMPA on the basis of their 50% effective concentrations in 2.2.15 cells. The increased antiviral activity appeared to be due in part to the increased uptake and conversion of HDP-(S)-HPMPA to HPMPA diphosphate observed in HepG2 cells in vitro. HDP-(S)-HPMPA retained full activity against HBV mutants resistant to lamivudine (L180M, M204V), but cross-resistance to a mutant resistant to adefovir (N236T) was detected. HDP-(S)-HPMPA is orally bioavailable and provides excellent liver exposure to the drug. Oral treatment of HBV transgenic mice with HDP-(S)-HPMPA, 15M-HDP-(S)-HPMPA, and ODE-(S)-HPMPA for 14 days reduced liver HBV DNA levels by roughly 1.5 log units, a response equivalent to that of adefovir dipivoxil.

9-(S)-(3-Hydroxy-2-phosphonomethoxypropyl)adenine [(S)-HPMPA] is an acyclic nucleoside phosphonate which Holý and coworkers first reported in 1986 (7, 8, 26). (S)-HPMPA was the first acyclic nucleoside phosphonate, a growing and important class of antiviral compounds which now includes cidofovir, adefovir [9-(2-phosphonomethoxyethyl)adenine], and tenofovir [9-(2-phosphonomethoxypropyl)adenine], which are used for the treatment of cytomegalovirus (CMV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV) infections, respectively (6). In this paper, we report on the synthesis and evaluation of several alkoxyalkyl ester prodrugs of (S)-HPMPA in an attempt to find oral treatments for HBV infection more effective than the currently approved antivirals.

(S)-HPMPA is a broad-spectrum antiviral which was shown to inhibit the replication of a wide variety of double-stranded DNA viruses, including orthopoxviruses, herpesviruses, adenoviruses, iridoviruses, and papovaviruses (6). (S)-HPMPA was also reported to be active in vitro against HBV replication in HB611 cells (29) and 2.2.15 cells (11) and to have 50% effective concentrations (EC50s) of 1.15 and 1.5 μM, respectively. Numerous reports have indicated that (S)-HPMPA lacks activity against HIV type 1 (HIV-1) (2, 5, 12). However, alkoxyalkyl esters of (S)-HPMPA, such as hexadecyloxypropyl-(S)-HPMPA [HDP-(S)-HPMPA] and octadecyloxyethyl-(S)-HPMPA [ODE-(S)-HPMPA], exhibit EC50s against HIV-1 in the low nanomolar range, while unmodified (S)-HPMPA is virtually inactive in vitro (13). HDP-(S)-HPMPA exhibits multiple-log increases in antiviral activity in vitro compared with the activity of unmodified (S)-HPMPA against vaccinia virus, cowpox virus, human CMV and murine CMV (3), and adenovirus (10). HDP-(S)-HPMPA is orally bioavailable and is active in vitro against lethal vaccinia virus and cowpox virus infections (24) and against lethal murine CMV infections (25). To assess the effect of alkoxyalkyl esterification of (S)-HPMPA on its in vitro and in vivo anti-HBV activity, we synthesized HDP-(S)-HPMPA, 15-methyl-HDP-(S)-HPMPA [15M-HDP-(S)-HPMPA], and ODE-(S)-HPMPA and evaluated their in vitro activities against HBV replication as well as the cellular uptake and conversion of HDP-(S)-[8-14C]HPMPA to (S)-HPMPA diphosphate (HPMPApp) in HepG2 cells. Using HDP-(S)-[8-14C]HPMPA, we also evaluated the oral pharmacokinetics and the level of drug exposure in the plasma, livers, and spleens of mice. Finally, the oral activities of the HDP, ODE, and 15M-HDP esters of (S)-HPMPA were assessed in HBV transgenic mice (15, 17, 21); and their activities were compared with the in vivo activity of adefovir dipivoxil, a compound licensed for use for the treatment of HBV infection.