Biochemical Pharmacology of (+)- and (-)-2’,3’-Dideoxy-3’-thiacytidine as Anti-hepatitis B Virus Agents*

2’,3’-Dideoxy-3‘-thiacytidine (cis-(2)-SddC) was found to have potent activity against hepatitis B virus and human immunodeficiency viruses in culture. Re- cent studies by us identified (-)-SddC as the stereoisomer responsible for the antiviral effect and showed that the cytotoxicity was mainly caused by (+)-SddC. Metabolism studies showed that these drugs were con- verted to their monophosphates, diphosphates, and triphosphates. The enzyme responsible for the formation of monophosphates was identified to be cytoplasmic deoxycytidine kinase in CEM cells. Uptake studies showed that the intracellular concentration of (-)- SddC and its metabolites was approximately &fold higher than that of (+)-SddC metabolites. (-)-SddCTP was more potent than (+)-SddCTP in inhibiting hepatitis B virus replication; (+)- and (-)-SddCTP exhibited minimal inhibition on polymerases (Y and 6, more inhibition on B, and strong inhibition on y. In all cases, (+)- SddCTP was found to be more inhibitory than (-)- SddCTP to all four polymerases. (+)-SddCMP com- peted with dCTP for incorporation into DNA by DNA polymerase y and @ and served as a chain terminator; however, similar incorporation was not detected using in a total volume of 0.1 ml. The reaction was carried out at 37 "C for 40 min and terminated by the addition of 1.5 N perchloric acid. The acid-insoluble material was washed three times, resuspended and neutralized as previously described (11). The radioactivity was measured using a liquid scintillation counter.

By incubating the racemic mixture of cis-SddC stereoisomers ( Fig. 1) with human deoxycytidine deaminase at 37 "C for 16 h, approximately 50% of the mixture could be deaminated (9). No further deamination were observed upon prolonged incubation. A detailed kinetic study showed that only the (+)-SddC could be deaminated; the other stereoisomers were not substrates for the human deoxycytidine deaminase (9). (-)-SddC, which was resistant to deoxycytidine deaminase, was found to be a more potent antiviral stereoisomer than (+)-SddC (HBV ID5,., = 0.5 pM, 50-fold higher than that of (-)-SddC). The (+)-form, which was susceptible to the deamination, was 25-fold and 12-fold more toxic than the (-)-SddC in CEM cells in terms of anti-cell growth and antimitochondrial DNA synthesis, respectively (9). Compared with ddC, which had an anti-mitochondrial DNA IDSO of approximately 0.022 p~, the (+)-and (-)-SddC were relatively ineffective in inhibiting the mitochondrial DNA (mtDNA) synthesis (9). Peripheral neuropathy has been suggested to be the result of inhibition of mitochondrial DNA synthesis (10). Therefore, delayed toxicity, such as peripheral neuropathy, observed in patients treated with ddC, may not be an issue using (-)-SddC for antiviral drug therapy. Once (-)-SddC enters the cell, it can be phosphorylated to SddCMP and subsequently converted to SddCDP and SddCTP. The identification of each of the (-)-SddC metabolites has been published previously (9). In this report, identification of cytoplasmic deoxycytidine kinase as the enzyme responsible for the monophosphorylation of (+)-and (-)-SddC and their kinetic studies are described. Detailed comparison of nucleoside uptakes and the efflux of metabolites using (+)-and (-)-SddC as well as the inhibition of human DNA polymerases and mitochondrial DNA synthesis using (+)-and (-)-SddCTP are presented. The antiviral effects and the cytotoxicities of these two SddC stereoisomers are explained based on these studies. The possible stereospecificity of the whole cell and the mitochondria membranes for these stereoisomers at either nucleoside or nucleotide level is also discussed. inal Chemistry, University of Georgia (12). The (+)-SddCTP and (-)-SddCTP were synthesized from their nucleoside counterparts and purified from HPLC using a previously published method (9).
Enzymes-Human DNA polymerases a, 6, and y were purified from either K562 (chronic myelogenous leukemia cell line) or CEM (T-lymphoblastoid cell line) cells; polymerase p was purified from AML (acute myelogenous leukemia cell line) cells. In general, 10 ml of cell pellet was resuspended in 5 volumes of a buffer containing 10 mM potassium phosphate, pH 7.5, 2 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM (PMSF), 1 M KC1, and 10% glycerol. The cells were frozen and thawed three times, then sonicated three times, for 30 s/ sonication, a t a setting of 4 on a Bronson model 200 cell disruptor, followed by centrifugation at 40,000 X g for 30 min. The supernatant fraction was passed through a 50-ml DEAE-cellulose column equilibrated with buffer A (400 mM potassium phosphate, pH 7.5, 2 mM DTT, 1 mM EDTA, 1 mM PMSF, and 10% glycerol). Unabsorbed fractions were pooled and dialyzed overnight against buffer B (substituted 400 mM potassium phosphate with 50 mM Tris-HC1, p H 7.5, from buffer A). The dialysate was applied to a 25-ml single-stranded DNA (ssDNA) cellulose column equilibrated with buffer B. The , respectively. The first several fractions that contain polymerase a from ssDNA column were pooled, and a second ssDNA column was repeated to assure that possible contamination of polymerase 6 was removed. The specific activities of these partially purified DNA polymerases CY, @, y, and 6 were 290, 25, 40, and 32 units/mg, respectively. One unit of enzyme activity is defined as the amount which catalyzes the incorporation of 1 nmol of ["HIdTMP into acidinsoluble DNA within 60 min a t 37 "C. The unabsorbed fractions from ssDNA column were pooled and directly applied to a 15-ml hydroxylapatite column preequilibrated with buffer C (10 mM potassium phosphate, p H 7.5,5 mM MgC12, 2 mM DTT, 1 mM PMSF, and 20% glycerol), and eluted with a 100 ml linear gradient of 10-200 mM potassium phosphate. Deoxycytidine kinase activity eluted off the column at approximately 75 mM potassium phosphate. The peak activities were pooled and applied to a thymidine kinase affinity column using published procedures (14). The specific activity of the partially purified dCyd kinase was 240 units/mg. The unit definition was the amount of enzyme which produced 1 nmol of product/h a t 37 "C under our assay condition.
Assay for the Inhibition of DNA Polymerases and Endogenous HBV DNA Polymerase and the Determination of K, and K,-The assays for the human DNA polymerase activity were performed in a reaction mixture (50 pl) of 50 mM Tris-HC1, pH 8.0,8 mM MgC12, 2 mM DTT, 0.2 mg/ml bovine serum albumin, 150 pg/ml activated calf thymus DNA, 50 p~ each dATP, dTTP, and dGTP, 0.175 pM ['HIdCTP, and 0.01 unit of DNA polymerases (J' , y, and 6, and 0.05 units of 01 a t 37 "C for 30 min. Assay mixtures for polymerases y and were supplemented with 150 and 100 mM KCl, respectively. Reactions were stopped by spotting the assay mixtures onto Whatman GF/A filter discs, which were then processed to determine the trichloroacetic acid-insoluble radioactivity. The inhibition of endogenous HBV DNA polymerase activity was performed as previously described (9). In general, the virus was precipitated by polyethylene glycol from culture medium of 2215 cells. The assay mixtures contained the virus preparation, 42 mM Tris-HC1 (pH 7.5), 34 mM MgCl,, 340 mM KC1, 22 mM mercaptoethanol, 0.4% Nonidet P-40, 70 p~ each dATP, dTTP, and dGTP, and the appropriate concentration of dCTP including [ a -"'PIdCTP and appropriate amounts of inhibitors in a final volume of 50 pl. After incubation a t 37 "C for 2 h, the reaction was stopped and the "'P-labeled viral DNA was isolated and analyzed by electrophoresis on a 0.7% agarose gel. The K, and K, values were determined using a previously published procedure (15). Polyacrylamide Gel Assay for the Inhibition of DNA Polymerase Activity Using M13 mp19 DNA-The assay was done as was previously described by our laboratory (16). The M13 primer was labeled by incubating 0.005 (A2,J of the primer with 50 pCi of ['"PJATP (5,000 Ci/mmol) and 9 units of T4 polynucleotide kinase (Boehringer Mannheim) in 50 mM Tris-HC1 (pH 7.5), 50 mM NaCl, 10 mM MgCl,, and 5 mM DTT in a total volume of 30 11 for 15 min a t 37 'C, followed by boiling for 5 min. The primer was annealed to 75 pg of M13 mp19(+) ssDNA by heating a t 65 "C for 10 min followed by gradual cooling to room temperature, usually overnight. The sequence of the primer-template substrate for DNA elongation assays is shown below.
The DNA elongation reactions were done with 0.005 units of DNA polymerases a and 6,0.0005 units of y, and 0.0003 units of a t 37 "C for 40 min. The reaction mixture contained, in a total volume of 10 pl, 25 mM Tris-HC1, pH 8.0, 8 mM MgCl,, 2 mM DTT, 5 or 50 p M each G T P , dGTP, and dTTP, 100 pg of BSA, 0.167 pg of "'P-labeled M13 template/primer, and the indicated concentration of dCTP or analog. The reactions were terminated by the addition of 4.0 p1 of 98% formamide, 10 mM EDTA, and 0.25% bromphenol blue; samples were denatured by boiling for 3 min and loaded onto 15% polyacrylamide/urea DNA sequencing gels. Electrophoresis was carried out at 65 watts constant power for 2 h. The radioactivity was analyzed by autoradiography using Kodak XRP-5 x-ray film.
Standard Assay for the Analysis of the Acid-soluble Metabolites-The assay condition was virtually the same as the procedure described by Chang et al. (9). In general, the cells were incubated in a specified concentration of "H-labeled SddC for the period of time indicated. Medium was removed, and cells were washed and extracted with 1.5 M perchloric acid a t 4 "C. The acid-soluble material was neutralized and analyzed by HPLC with an anion exchange column. The HPLC was connected to a fraction collector, fractions were collected at 1min intervals, and the radioactivities were quantified by scintillation counting.
DNA Synthesis in Isolated Mitochondria-Mitochondria from CEM cells were prepared by the "two-step" procedure as described by Bogenhagen and Clayton (17). Incorporation of ["HIdATP into mitochondrial DNA was performed as described by Chen and Cheng (11). The assay mixture contained mitochondria (0.05 mg of protein), of (&)-SddC as Anti-HB VAgents in a total volume of 0.1 ml. The reaction was carried out at 37 "C for 40 min and terminated by the addition of 1.5 N perchloric acid. The acid-insoluble material was washed three times, resuspended and neutralized as previously described (11). The radioactivity was measured using a liquid scintillation counter.

RESULTS
Nucleoside Uptake of (+)and (-)-SddC in 2215 Cells-The uptakes of (+)-and (-)-SddC nucleosides in 2215 (HBV transfected cell line) cells at different time points were compared, and the results are shown in Fig. 2A. The nucleoside concentrations used was 0.2 p~. The differences of the intracellular concentrations between (+)-and (-)-SddC metabolites increased with time, and at 8 h, a difference of approxi-mately &fold was observed. The HPLC profiles of the acidsoluble fraction of 2215 cells treated with 0.5 PM (-)-and (+)-SddC for 24 h are shown in Fig. 2 ( B and C, respectively).
There is no qualitative difference between the two profiles except that the intracellular concentration of (-)-SddC nucleotides is much higher than that of (+)-SddC nucleotides. It is interesting to note that the retention time of mono-(13 min), di-(23 min), and triphosphates (50 min (Fig. 3B), the production of acid-soluble metabolites of (-)-SddC was significantly inhibited as compared with the metabolites observed in the absence of deoxycytidine (Fig. 3A). Similar results were also obtained when the incubations were extended to 3 h (results not shown); however, the extent of inhibition was slightly lower. The HPLC profile of the acid-soluble metabolites of CEM cells treated with 0.5 FM (-)-[3H]SddC for 8 h is shown in Fig. 3C. The metabolites obtained from wild type CEM cells were identical to those observed in 2215 (Fig. 2B) or HepG2 (data not shown) cells based on the retention time of each metabolite. However, when Ara-Cyd-resistant CEM cells (dCyd kinase-deficient) were used, no acid-soluble metabolites other than the (-)-SddC nucleoside were observed intracellularly (results not shown). These results indicate that dCyd kinase is responsible for the monophosphorylation of (-)-SddC nucleoside in both 2215 and CEM cell lines. "-Labeled dCyd, dideoxycytidine, (+)-SddC, and (-)-SddC were used to determine the kinetic constants of monophosphorylation by partially purified cytoplasmic dCyd kinase. The K , and V, , , values for these substrates are summarized in Table  I. Whereas the K, values were virtually the same, differences of 15-and 30-fold in the V, , , values were obtained for (-)-SddC and (+)-SddC, respectively, relative to the V, , , value of dCyd.
Accumulation and Degradation of (-)-Sddc Metabolites in 2215 Cells after Drug Removal-Upon measuring the half-life of (-)-SddCTP after drug removal, an interesting result was observed (Fig. 4, A-D). The experiment was done by incubating 0.5 PM 3H-labeled (-)-SddC at 37 "C for 24 h (Fig. 4A). Fresh medium without drug was replaced at the end of 24 h, and the incubation was continued. The acid-soluble metabolites were analyzed at different time points using HPLC (Fig.  4, B-D). In the first 2 h, most of the intracellular (-)-SddC nucleoside (retention time, 5 min) was converted to (-)-SddCDP (retention time, 24 min) (Fig. 4B). At the 4-h time point (Fig. 4C), most of the diphosphate was converted to (-)-SddCTP (retention time, 50 min). A t later time points, the triphosphate and diphosphate were gradually degraded (Fig. 4 0 )  A primer extention assay utilizing 3"-labeled M13 primer annealed to M13 mp19 ssDNA was employed to assess the effect of the dCTP analogs ddCTP, (+)-SddCTP, and (-)-SddCTP on partially purified human DNA polymerases. In the case of polymerase 01, reducing the concentration of dCTP from 50 pM to 0.175 FM results in an increased accumulation of DNA products terminated with dTTP one base before the incorporation of dCTP (Fig. 6, lanes 1-3). In the absence of dCTP, there is a small amount of misincorporation indicated by DNA products 26 bases long (lane 4 ) . All analogs inhibit DNA elongation past the first "C" site in reactions without dCTP. Under these conditions, ddCTP, (+)-SddCTP, and (-)-SddCTP are not incorporated into the DNA by polym-Biochemical Pharmacology of (k)-SddC as Anti-HBVAgents erase CY (lanes 5 , 7, and 9). The DNA elongation is restored t o control levels when equimolar dCTP is included in the assay (compare lune 3 with lunes 6, 8, and IO). Similar observations were made with DNA polymerase 6.
In the case of DNA polymerase y, the activity was processive on the M13 primer/template under optimal conditions (lane I ). There was a substantial amount of misincorporation by this enzyme when dCTP was left out of the reaction (lane 4 ) . The results in lune 5 show that (+)-SddCTP is incorporated into the DNA and results in the accumulation of prod- ucts that are terminated at the first "C" site. This effect is partially reversed in the presence of dCTP, indicating that (+)-SddCTP competes with dCTP as a substrate for polymerase y (lune 6). Similar observations were made with (-)-SddCTP; however, this stereoisomer is less inhibitory as indicated by a lower extent of DNA termination. Furthermore, in the presence of dCTP, there is minimal incorporation of the analog at the first "C" site (lane 8). The incorporation of the ddCTP into the DNA proceeds to the same extent as that of (+)-SddCTP and is also partially inhibited by dCTP (lanes 9 and IO).
In the case of DNA polymerase p, the assay conditions had to be changed in order to determine whether this enzyme can incorporate any of the three analogs. Assays utilizing 0.175 p~ dCTP resulted in DNA products which were elongated to 22 bases just prior to the first "C" site (results not shown).
Since the K, for dCTP is 2.6 p~, the concentration of dCTP was increased to 5 pM to attain sufficient reaction velocity.  (+Iassays and the determinations of kinetic constants were performed dependent manner (Fig. 7). APhidicolin (25 Pg/ml), a specific using standard methods as described under "Materials and Methods." inhibitor of DNA polymerase CY, was also used to demonstrate that the nuclear DNA synthesis was absent in the assay conditions. The inhibition of mtDNA synthesis by (+)-SddCTP at 0.1 PM was more potent than the inhibition by (-)-SddCTP at 3 ELM. The relative potencies among these three nucleoside triphosphates were in the following order:

DISCUSSION
Our unpublished studies" showed that dipyridamole (a potent inhibitor of nucleoside transport system) effectively suppressed the transport of SddC, indicating that the nucleoside transport system is operative. However, a majority of the SddC nucleoside enters the cell through nonfacilitated passive diffusion or other mechanisms after prolonged incubation. The intracellular concentration of (-)-SddC acid-soluble metabolites were approximately 5-fold higher than that of (+)-SddC metabolites at steady state levels. Nevertheless, the HPLC profiles of the metabolites from (-)-and (+)-SddC show no intrinsic differences.
Deoxycytidine inhibits the formation of SddC acid-soluble metabolites in 2215 cells, suggesting that SddC and deoxycytidine share a common kinase for their monophosphate phosphorylation. Further investigations using wild type and Ara-Cyd-resistant (deoxycytidine kinase-deficient) CEM cell lines showed that only the wild type CEM cells produced SddC metabolites with a profile similar to the one obtained from 2215 cells. Ara-Cyd-resistant CEM cells have no capability of converting SddC nucleoside to its corresponding nucleotides, indicating that the deoxycytidine kinase is responsible for the SddC monophosphorylation. Although the V, , , values of (-)and (+)-SddC examined from monophosphorylation of cytoplasmic dCyd kinase were approximately 30-and 15-fold lower than that of dCyd, their K,,, values were virtually the same.
The stability study on the acid-soluble metabolites, when monitored by HPLC profiles after drug removal (Fig. 4), showed that (-)-SddCTP had an extended half-life. However, after removing the drug, instead of effluxing out of the cell, SddC nucleoside was further converted to SddCDP and diphosphate accumulation occurred. This observation suggests that the rate-limiting step is the conversion of SddCDP to SddCTP. Phosphorylations of SddC to SddCMP and SddCMP to SddCDP were relatively rapid inside the cell. Further conversion from SddCDP to SddCTP continued, and a net accumulation of SddCTP was observed, indicating that the rate of degradation of SddCTP to SddCDP was slower than the rate of conversion of SddCDP to SddCTP. According to Fig. 4, the apparent half-life of SddCTP was approximately 24 h; however, the real half-life was estimated to be approximately 12 h.
The relative potencies of (+)-and (-)-SddCTP in terms of inhibition of human DNA polymerases were also assessed. (+)-SddCMP and (-)-SddCMP can be incorporated into M13 DNA in competition with dCTP by DNA polymerase y and /? and served as chain terminators; however, similar incorporation was not detected for polymerases 01 and 6. These results are consistent with the cytotoxicity data published previously (9).
There is an approximate 12-fold difference in the IDso values between (+)-(4 FM) and (-)-SddCTP (50 PM) in terms of anti-mitochondrial DNA synthesis (9). Since the only DNA polymerase responsible for the mitochondrial DNA synthesis is DNA polymerase 7, and since the intracellular concentration of (+)-SddCTP was 5-fold less than that of (-)-SddCTP, one would expect that (+)-SddCTP should be approximately 60-fold more inhibitory than (-)-SddCTP to DNA polymerase y activity. However, inhibition of DNA polymerase y by (+)-SddCTP was not that dramatic compared with that by (-)-SddCTP, according to the K, values in Fig. 5. The results of experiments to examine DNA synthesis in isolated mitochondria (Fig. 7) provided a logical explanation. The inhibition of mitochondrial DNA synthesis by (+)-SddCTP at 0.1 PM was more potent than the inhibition by (-)-SddCTP at 3 PM. This result accounts for the above apparent discrepancy, and, more importantly, it also suggests a potential stereospecificity of the transport on the membrane of mitochondria. It should be noted that we have shown previously that ddCTP is able to enter the mitochondria without going through the process of dephosphorylation (11).
Several groups reported the investigation of the antiviral effect using pure (+)-and (-)-SddC nucleosides as antihuman immunodeficiency virus (HIV) agents in various cell systems (13, 18,19). A 2-fold difference in the anti-HIV activity between (+)-and (-)-SddC has been reported by Coates et al. (18), whereas (-)-SddC was found to be 4-100fold more potent than (+)SddC by Schinazi et al. (13). We previously reported that (+)-SddC could be deaminated by human deoxycytidine deaminase with a K,,, value approximately 15-fold higher than that of deoxycytidine, while (-)-SddC is not a substrate for the deaminase (9). Therefore, variable intracellular deoxycytidine deaminase levels may explain the variations obtained by these groups. However, in our studies, we did not observe the SddU type of metabolite with incubation up to 24 h in any of the cells examined. Thus, the variations above may reflect differences in the potential selectivities of various cell membranes and/or differences in cellular metabolisms.
In summary, the information presented here shows that (-)-SddC is the active stereoisomer against HBV DNA replication, whereas (+)-SddC is mainly responsible for the cytotoxicity observed with the racemic mixture. Metabolism studies as well as direct examinations of the inhibition of endogenous HBV DNA replication and that of human DNA polymerases using chemically synthesized (+)-and (-)-SddCTP elucidated the detailed mechanisms of action by these drugs. Both the whole cell and the mitochondrial membranes exhibited stereospecificities for these stereoisomers at nucleoside or nucleotide levels. The potential competitive interactions between these stereoisomers with their membrane transports and metabolisms should be investigated in greater detail.