The β-l-2′-deoxynucleoside series is specific for HBV.
The structure-activity relationships (SARs) established among the l-dC, l-dT, and l-dA series are presented in Table . Substitution of a halogen atom at the 5 position (R1) in the pyrimidine ring of l-dC, without modification of the deoxyribose sugar (e.g., β-l-2′-deoxy-5-fluorocytidine [l-5-FdC] and β-l-2′-deoxy-5-chlorocytidine [l-5-CldC]), decreased the potency against HBV but did not affect the specificity for HBV. In contrast, analogs of l-dC which lacked the 3′-OH group (R3) on the deoxyribose sugar (e.g., β-l-2′,3′-dideoxycytidine [l-ddC], 3TC, and β-l-2′,3′-didehydro-2′,3′-dideoxycytidine [l-d4C]) lost antiviral specificity for HBV and showed activity against HIV. Similarly, replacement of the 3′-OH group with a 3′-fluoro- moiety (e.g., β-l-2′,3′-dideoxy-3′-fluorocytidine [l-3′-FddC]) eliminated the antiviral specificity, although antiviral potency against HBV and HIV was retained.
SARs of l-dC, l-dT, and l-dA analogs
In addition, substitutions at the 5 position (R1) of the pyrimidine base of l-ddC lacking the 3′-OH group (e.g., β-l-2′,3′-dideoxy-5-fluorocytidine [l-5-FddC], β-l-2′,3′-dideoxy-5-chlorocytidine [l-5-ClddC], β-l-2′,3′-dideoxy-3′-thia-5-fluorocytidine [FTC], β-l-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine [l-d4FC], β-l-2′,3′-dideoxy-3′-fluoro-5-fluorocytidine [l-3′-F-5-FddC], and β-l-2′,3′-dideoxy-3′-azido-5-fluorocytidine [l-3′-azido-5-FddC]) further affected the antiviral potency of these analogs against HBV as well as HIV. These studies suggest that the 3′-OH of the β-l-2′-deoxyribose of l-dC plays a crucial role in inhibiting virus replication, possibly by specific interaction with the HBV DNA polymerase.
The SARs for the l
-dT and l
-dA series (Table ) were similar to those observed for the l
-dC series. The specific anti-HBV activity of l
-dT and l
-dA was lost upon removal or substitution of the 3′-OH group (R3). β-l
-xylo-dT), which is identical to l
-dT except that the 3′-OH group is in the opposite orientation (R2), also lost anti-HBV activity, further emphasizing the importance of the 3′-OH group in the interaction with the HBV DNA polymerase. An l
-dT analog with a fluorine substitution at the 2′ up position
-2′-deoxy-2′-fluoro-5-methyl-arabinofuranosyl uracil [l
-FMAU]) has been reported to have activity against both HBV and EBV (6
). Thus, it is possible that modification of the 2′ position in addition to the 3′ position of l
-dT may also change antiviral specificity for HBV.
Substitution at the 2 position (R1) on the purine base of l
-dA (e.g., β-l
-2-CldA]) had a negative effect on anti-HBV activity. The analogs of l
-dA lacking the 3′-OH group with or without further modification of the deoxyribose sugar lost specificity and were not as potent against HBV. The marginal antiviral activity of β-l
-ddA), despite its potent inhibitory activity against both HIV reverse transcriptase and WHV DNA polymerase (Placidi et al., unpublished data), can be explained by the low intracellular concentrations of the phosphorylated form due to rapid and extensive catabolism (36
). This conclusion is also supported by recent studies that demonstrated potent antiviral activity of an l
-ddA 5′-monophosphate prodrug (β-l
-ddAMP-bis(terbutyl-SATE)]). The prodrug form decreases the intracellular catabolism of the parent molecule (L. Placidi et al., Proc. 2nd Int. Conf. Ther. Vir. Hepatitis, abstr. A22, 1998 [Antivir. Ther. 3
, Suppl. 3]) and releases the 5′-monophosphate derivative inside the cell. When used in this pronucleotide form, l
-ddA was active against both HIV and HBV, further supporting the importance of the 3′-OH group for antiviral specificity. As in the l
-dC and l
-dT series, unmodified β-l
-2′-deoxyadenosine most potently and specifically inhibited HBV replication.
To further assess their antiviral specificities, l-dC, l-dT, and l-dA were screened against 15 different RNA and DNA viruses. The β-l-2′-deoxynucleosides inhibited hepadnavirus replication as previously defined by the SAR but had no activity against HIV-1, herpes simplex virus types 1 and 2, varicella-zoster virus, EBV, human cytomegalovirus, adenovirus type 1, influenza A and B viruses, measles virus, parainfluenzavirus type 3, rhinovirus type 5, or respiratory syncytial virus type A at concentrations as high as 200 μM. Potent antiviral activity against WHV, determined using an in vivo model of chronic HBV infection, is described below. Thus, the unmodified β-l-2′-deoxynucleosides l-dC, l-dT, and l-dA are uniquely specific for the hepadnaviruses HBV, duck HBV (DHBV), and WHV.
Selectivity of β-l-2′-deoxynucleosides.
Since long-term treatment is expected for chronic HBV infection, drug selectivity is a critical issue. Toxic side effects have been a major problem, limiting the clinical use of some nucleoside analogs (17
). The 5′-triphosphates of l
-dT, and l
-dA did not inhibit human DNA polymerases α, β, and γ at concentrations up to 100 μM. Semizarov and coworkers also reported that the 5′-triphosphates of l
-dC and l
-dT were not substrates for human DNA polymerases (42
-dT, and l
-dA had no cytotoxic effect on the human hepatoma cell line 2.2.15 (50% cytotoxic concentration > 1,000 μM), primary human PBM cells, human foreskin fibroblasts, or other cell types of mammalian and avian origin. In addition, studies by Verri et al. demonstrated that l
-dC was not cytotoxic toward lymphoblastoid T cells (51
). Human bone marrow stem cells in primary culture have been shown to be a good predictor of potential nucleoside analog-induced hematotoxicity in patients (15
). GM-CFU and E-BFU precursors exposed to l
-dT, and l
-dA in clonogenic assays at concentrations up to 10 μM were not affected. These results suggest that l
-dT, and l
-dA are highly selective and that their phosphorylated forms will be nontoxic in vivo.
-dT, and l
-dA were efficiently metabolized (activated) to their respective 5′-triphosphate derivatives in HepG2 cells and human hepatocytes in primary culture (Placidi et al., 3rd Int. Conf. Ther. Vir. Hepatitis, abstr. A122, 1999 [Antivir. Ther. 4
, Suppl. 4]). Earlier studies reported limited intracellular activation of l
). Together with the potent in vitro antiviral activity, these data suggest that like other nucleoside analogs, the intracellular phosphorylated form was responsible for inhibition of the viral polymerase. Furthermore, the 5′-triphosphates of l
-dT, and l
-dA each inhibited WHV DNA polymerase with a 50% inhibitory concentration of 0.24 to 1.82 μM. In addition, exposure of HepG2 cells to l
-dC led to a second 5′-triphosphate derivative, i.e., β-l
-dUTP) which also inhibited WHV DNA polymerase, with a 50% inhibitory concentration of 5.26 μM (Faraj et al. and Placidi et al., 3rd Int. Conf. Ther. Vir. Hepatitis, abstr. A119 and A122, 1999 [Antivir. Ther. 4
, Suppl. 4]). Similar to β-l
-cytidine analogs (4
-dC was not a substrate for cytosolic cytidine deaminase, which suggested that the 5′-monophosphate metabolite of l
-dC may be susceptible to deamination through deoxycytidylate deaminase. The inhibition of HBV replication by these β-l
-2′-deoxynucleosides and inhibition of hepadnaviral polymerase by their corresponding 5′-triphosphates suggested that, like most nucleoside analogs, l
-dT, and l
-dA may act by inhibiting the reverse transcription of HBV pregenomic RNA. Demonstration that l
-deoxynucleoside triphosphate analogs inhibit HBV reverse transcriptase and/or DNA polymerase activity does not preclude other mechanisms of action. Inhibition of other important activities of the polymerase (which include RNase H activity, priming of reverse transcription, and coordination of intracellular virion assembly) and the possibility of internal incorporation of l
-deoxynucleoside monophosphates into viral DNA as a mechanism of inhibition are currently under investigation.
β-l-2′-Deoxynucleosides have no effect on mitochondrial function or morphology.
Nucleoside analogs used in AIDS therapy, such as zidovudine (β-d
-3′-azido-3′-deoxythymidine), stavudine (β-d
-2′,3′-didehydro-2′,3′-dideoxythymidine [d4T]), didanosine (β-d
-2′,3′-dideoxyinosine [ddI]), and zalcitabine (β-d
-2′,3′-dideoxycytidine [ddC]), have shown clinically limiting delayed toxicities such as peripheral neuropathy, myopathy, and pancreatitis (17
). This nucleoside analog-related cellular toxicity has been attributed to decreased mtDNA content and altered mitochondrial function, leading to increased lactic acid production (5
). Concomitant morphological changes in mitochondria (e.g., loss of cristae, matrix dissolution and swelling, and lipid droplet formation) can be observed with ultrastructural analysis using transmission electron microscopy (10
). In HepG2 cells incubated with 10 μM fialuridine (FIAU; 1,2′-deoxy-2′-fluoro-1-β-d
-arabinofuranosly-5-iodo-uracil), a substantial increase in lactic acid production was observed (data not shown). Electron micrographs of these cells showed the presence of enlarged mitochondria with morphological changes consistent with mitochondrial dysfunction. Lamivudine (10 μM) did not affect mitochondrial structure or function. Under similar conditions, exposure of HepG2 cells to 10 μM l
-dT, or l
-dA for 14 days had no effect on lactic acid production, mtDNA content, or morphology (data not shown).
In vivo antiviral activity and toxicity.
The woodchuck model of chronic HBV infection has proven to be a positive predictor of the antiviral activity and safety of antiviral drug candidates for the treatment of human chronic HBV infection (47
-dT, and l
-dA were given orally to woodchucks once daily at 10 mg/kg/day. The levels of WHV DNA in serum during 4 weeks of drug treatment and 8 weeks of posttreatment follow-up were determined by DNA dot blot hybridization (detection limit, approximately 107
genome equivalents/ml of serum) and by quantitative PCR (detection limit, 300 genome equivalents/ml of serum). The WHV DNA replication was significantly inhibited within the first few days of treatment and this inhibition was maintained throughout the treatment period. Notably, serum WHV DNA levels (HBV viremia) decreased in the l
-dT-treated animals by as much as 8 logs (Fig. ). Following drug withdrawal, viral rebound reached near-pretreatment levels between week 4 and week 8. In the l
-dC-treated animals, serum WHV DNA levels decreased by up to 6 logs by the third week of therapy. Viral rebound was detected within the first week posttreatment. Animals receiving l
-dA showed a decrease in serum WHV DNA levels of approximately 1.5 logs within the first week of treatment, and this decrease was also followed by viral rebound. In addition to the determination of viral load, WHV surface antigen (WHsAg), which is assumed to represent the level of intercellular gene expression, was measured using the method of Cote et al. (7
). In general, the serologic profiles paralleled the decrease in viral load and continued to fall for several weeks after drug removal.
FIG. 1 Woodchuck hepatitis virus levels in serum in animals at 4 weeks of treatment with l-dC (A), l-dT (B), or l-dA (C) and 8 weeks posttreatment. Data are presented for individual animals administered 10 mg/kg/day orally (n = 3) and untreated control animals (more ...)
The cytidine analog lamivudine (10 mg/kg/day), used for comparison to the l
-dC treatment group, reduced the number of HBV genome equivalents per milliliter in serum by 0.5 log. This weak effect is consistent with previous studies using similar doses of lamivudine (20
). Much higher doses (40 to 200 mg/kg) are required to produce significant antiviral activity in this model (32
). The low activity of lamivudine in the woodchuck model has been explained in part by the low rate of conversion of lamivudine and other cytidine analogs to their active 5′-triphosphate forms in woodchuck liver compared to that in human liver. In addition, the oral bioavailability of lamivudine in woodchucks was reported to be 18 to 54%, whereas the oral bioavailability observed in humans was 82% (37
The woodchuck model was also valuable for the preclinical toxicological evaluation of nucleoside analogs. This model identified the delayed severe hepatocellular toxicity induced by FIAU in humans not seen in preclinical evaluation in rats, dogs, or monkeys (38
). The FIAU-induced toxicity observed in woodchucks, including significant weight loss, wasting, and hepatocellular damage seen upon liver biopsy, was identified beginning 6 to 8 weeks from onset of treatment and was similar to that observed in the treated HBV-infected patients (33
). Using this model we found in additional studies that the unmodified β-l
-dT, and l
-dA were well tolerated and caused no drug-related toxicity through 12 weeks of treatment and 4 weeks of follow-up (data not shown).
In summary, this is the first report of β-l-2′-deoxynucleosides with potent, selective, and specific activity against HBV replication. This series of drug candidates has in common the presence of a hydroxyl group in the 3′ position that determines specific activity against hepadnavirus. In the woodchuck model of chronic HBV infection, oral administration of these β-l-2′-deoxynucleosides reduced serum viral load by as much as 108 genome equivalents/ml without toxicity. These β-l-2′-deoxynucleosides are highly attractive clinical development candidates for the treatment of chronic HBV infection.