No abstract

The inhibitory effects of the 9-(2-phosphonylmethoxyethyl)adenine-related compounds (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-adenine, (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)adenine, (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine, and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine on human hepatitis B virus replication in the human hepatoma cell line HepG2 2.2.15 and duck hepatitis B virus infection in primary duck hepatocytes were investigated. (R)-9-(2-phosphonylmethoxypropyl-2,6-diaminopurine had the lowest 50%o inhibitory concentrations against hepatitis B virus and duck hepatitis B virus, 0.22 and 0.06 ,iM, respectively, i.e., two-to fivefold lower concentrations than required for (R)-9-(2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxyethyl)adenine. All compounds were not toxic in vitro at a concentration of 100 ,uM.In chronic hepatitis B virus (HBV) infection the basic therapy is the administration of interferon, although complete disappearance of virus markers is seldom observed. Hepatitis B e antigen seroconversion, reflecting a drastic decline of viral replication, is seen in only 20 to 40% of patients with HBV infections.More potent antiviral drugs are eagerly awaited, and in light of this, the acyclic nucleoside phosphonates described earlier (3, 4) were considered adequate candidates to be further pursued for the treatment of HBV infections.In an earlier study (6) we investigated the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in two hepatoma cell lines and primary duck hepatocytes. The 50% inhibitory concentrations (IC50) for human HBV and duck HBV (DHBV) were found to be 1.2 and 0.2 ,uM as measured in HepG2 2.2.15 cells and primary duck hepatocytes, respectively.Among the acyclic nucleoside phosphonates several other derivatives were found to have high anti-DNA virus activity (herpes group) or antiretrovirus (human immunodeficiency virus [HIV] and Moloney murine sarcoma virus) activity (1). The most effective HIV inhibitors among the acyclic nucleoside phosphonates were evaluated for their anti-HBV activities in the human hepatoma cell line HepG2 2.2.15. Two of the compounds, candidates for application with humans, were also assayed in primary duck hepatocytes infected with DHBV, in anticipation of studies with chronically DHBV-infected ducklings and chronic hepatitis B patients.PMEA, (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine [(S)-FPMPA], (R)-9-(2-phosphonylmethoxypropyl)-2,6-diaminopurine [(R)-PMPDAP], (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], (R)-9-(2-phosphonylmethoxypropyl)adenine [(R)-PMPA], and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine were synthesized by

2′,3′‐Dideoxyadenosine (ddA), 2′,3′‐didehydro‐2′,3′‐dideoxyadenosine (d4A) and their lipophilic 5′‐monophosphate triester (aryloxyphosphoramidate) prodrugs were evaluated for their anti‐retrovirus and anti‐hepatitis B virus activity in various cell culture models. The aryloxyphosphoramidate derivatives of ddA (Cf 1093) and d4A (Cf 1001) showed markedly superior (100–1000‐fold) efficacies than the parent drugs against human immunodeficiency virus type 1 (HIV‐1), HIV‐2, simian immunodeficiency virus (SIV), Moloney murine sarcoma virus (MSV) and human hepatitis B virus (HBV) replication regardless of the cell type in which the virus replication was studied (i.e., human T‐lymphocyte CEM, MT‐4, Molt/4 and C8166 cells, peripheral blood lymphocytes (PBL), monocyte/macrophages (M/M), murine embryo fibroblasts and human hepatocyte cells). Also the selectivity index (ratio of cytotoxic concentration/antivirally effective concentration) of both aryloxyphosphoramidate prodrugs was markedly increased. In particular the d4A prodrug Cf 1001 showed a selectivity index of 300–3000 as compared with 2–3 for the parental d4A in established laboratory cell lines. Also Cf 1001 had a selectivity index of 400–650 in HIV‐1‐infected PBL and M/M, respectively. Both Cf 1001 and Cf 1093 were equally efficient as 3TC (lamivudine) in inhibiting HBV replication in hepatocytes, and rank among the most potent HIV and HBV inhibitors reported so far in cell culture.

A novel membrane-soluble prodrug of the 5-monophosphate derivative of 3TC containing a phenyl group and the methyl ester of L-alanine linked to the phosphorus through a phosphoramidate bond with the primary amino moiety (designated Cf 1109) was prepared. The 3TC prodrug proved less potent an inhibitor of HIV-1 and HIV-2 replication in CEM cell cultures than 3TC, but lost only 20-fold antiviral potency in 2-deoxycytidine kinase-deficient CEM/dCK 0 cells compared with a more than 2,000-fold decrease of activity of 3TC. In contrast, 3TC and Cf 1109 proved equally highly effective in inhibiting HBV release in supernatants of HBV-transfected Hep G2 2.2.15 cell cultures (50% effective concentration Ç 0.02 mM). Both compounds easily selected for highly resistant HIV-1 strains at a comparable speed of breakthrough. The mutant viruses contained an 184-Ile and/or 184-Val amino acid change in their reverse transcriptase. Our data are suggestive for a relatively poor delivery of 3TC-MP in the intact CEM cells but a remarkably high delivery of 3TC and/or 3TC-MP in the intact Hep G2 2.2.15 cells. ᭧ 1996 Academic Press, Inc.The most recently approved 2,3-dideoxynucleoside (ddN) derivative for treatment of HIV infections is 2,3-dideoxy-3-thiacytidine (3TC, lamivudine) (Fig. 1). This drug is also subject of clinical trials in human hepatitis B virus (HBV)-infected patients. In contrast with the other approved (b-D) ddN derivatives has 3TC the b-L(0) isomeric conformation. 3TC needs to be converted to its 5-triphosphate derivative by cellular enzymes to be inhibitory to the reverse transcriptase (RT) of HIV, and is endowed with low if any cytotoxicity (1-5). However, the first activation (phosphorylation) step is catalysed by cytosolic 2-deoxycytidine kinase (dCK) which has a relatively poor affinity for 3TC (K i /K m Ç 50) resulting in a moderate efficiency of conversion of 3TC to its 5-monophosphate derivative (6). In addition, a potential drawback of 3TC is the rapid emergence of drug-resistant HIV-1 strains (7-11). Indeed, mutant virus strains that emerged in the presence of 3TC were shown to contain an amino acid change from methionine (Met) to valine (Val) or isoleucine (Ile) at position 184 of the RT. Such virus is highly resistant to the inhibitory effects of 3TC in cell culture (7-11), and is reported to show low-level cross-resistance to 2,3-dideoxycytidine (ddC) (Fig. 1) and 2,3-dideoxyinosine (ddI).In an attempt to circumvent the first intracellular phosphorylation step of 3TC, we synthesized the arylphosphoramidate derivative of 3TC containing a methylester of L-alanine linked to the 5-phosphate of 3TC-MP through its primary amino group (Fig. 1). The corresponding 2,3-didehydro-2,3-dideoxythymidine (d4T, stavudine) analogue (So324) was previously shown to be able to deliver directly the 5-monophosphate derivative of d4T into intact cells and to efficiently bypass the first (thymidine kinase-directed) phosphorylation of d4T (12,13). The antiviral potency and resistance profile of Cf 1109 is described compared to 3TC.

The antiviral and cytotoxic effects of ara-arahinoside monophosphate, 2',3', dideoxy-cytidine, ganciclovir, 9-2(-phosphonyhnethoxyethyl) adenine, 2',3'dideoxy3'-thiacytidine and recombinant interferon-alpha were studied using two human hepatitis B virus transfected hepatoma cell lines, HepGZ 2.2.15 and HB 611. After 9 days of exposure, starting on day 3 after seeding, inhibition of extracellular HBV-DNA expressed as IDss was in the 0.1-1.0 /cM range for 2',3'-dideoxy3'-thiacytidine and 9-Z(-phosphonyhuethoxyethyl) adenine and >lO PM for dideoxy-cytidine, araarabinoside monophosphate and ganciclovir in both cell lines At 2.500 U/ml recombinant interferon-alpha showed less than 20% inhibition in both cell lines. The HBV-DNA inhibitory effects of 2',3'dideoxy-3'-thiacytidine and 9-2(-phosphonyhnethoxyethyl) adenine were also investigated after 1 and 3 days of exposure. In that setting IDsa's were 10 and 3.3 PM for 2',3'-dideoxy-3'-thiacytidine and >lOO and 30 PM for 9-2(-phosphonyhuethoxyethyl) adenine, respectively. No major inhibitory effect on hepatitis B surface antigen and hepatitis B e antigen secretion was observed for any agent in this study, except for 9-Z(-phosphonylmethoxyethyl) adenine in HB 611 cells. Cytotoxicity ESEARCH R on hepatitis B virus has been hampered by the absence of an in vitro cell culture system for hepatitis B infection. Infection of hepatitis B virus in primary cultures of human hepatocytes has been reported, but great variability has been observed in levels of HBV replication (1).As an alternative, transfection of various hepatoma cell lines with hepatitis B virus DNA has been introduced. Stable transfection was established for the measured by inhibition of [3H-methyl] deoxythymidine incorporation and expressed as CDs,, on day 4 was in the 10-100 PM range for ara-arabinoside monophosphate; in the 100-1000 /IM range for 9-Z(-phosphonylmethoxyethyl) adenine, ganciclovir and dideoxy-cytidine; and >lOOO PM for 2',3'-dideoxy-3'-thiacytidine. This CDs0 decreased considerably (7-100 fold) when measured on day 12 for dideoxy-cytidine, ganciclovir, 9-2(-phosphonyhuethoxyethyl) adenine and 2',3'-dideoxy-3'-thiacytidine, but was similar for ara-arabinoside monophosphate. Since the order of antiviral HBV activity and cytotoxicity of nucleoside analogues was similar in the two transfected hepatoma cell lines, we conclude that 2',3'dideoxy-3'-thiacytidine and 9-2(-phosphonylmethoxyethyl) adenine are very potent inhibitors of HBV-DNA, with a longlasting effect. In view of the progressive toxicity with continuous administration, intermittent administration might be an alternative mode of therapy.Key words: Antiviral agents; Cell culture; Cytotoxicity; Hepatitis B virus. 0 Journal of Hepatology.HepG2 2.2.15 and the HB 611 cell lines. Both cell lines excrete hepatitis B surface antigen, hepatitis B e antigen and complete Dane particles (2,3). These two cell lines are now widely used as model systems for screening of antiviral agents with activity against HBV In previous studies various experimental de...