Deoxycytidine Deaminase-resistant Stereoisomer Is the Active Form of (f)-2’,3’-Dideoxy-3’-thiacytidine in the Inhibition of Hepatitis B Virus Replication*

found to have potent activity human hepatitis B as human immunodeficiency The (-)form ((-)-SddC) which is resistant to deoxycytidine deaminase was found to be the more active an- tiviral stereoisomer than the (+)-form ((+)-SddC). The (+)-SddC is susceptible to deamination by deoxycyti- dine deaminase and is 25- and 12-fold more toxic than (-)-SddC in CEM cells in terms of anti-cell growth and anti-mitochondrial DNA synthesis, respectively. Similar results were obtained using a mixture of their 6-fluoro analogs ((+)-FSddC). Unlike 2‘,3’-dideoxycyti- dine, which is a potent inhibitor of mitochondrial DNA synthesis and results in such delayed toxicity as pe- ripheral neuropathy with long term usage, (-)-SddC does not affect mitochondrial DNA synthesis. The (-)form is phosphorylated to (-)-SddCMP and is sub- sequently

In this report, a novel methodology to prepare (-)-SddC and (-)-FSddC from (+)-SddC and (+)-FSddC, respectively, is described. This allowed us to address the issue of whether one or both of the two forms of the stereoisomers were responsible for anti-HBV activity and cytotoxicity. This was investigated further using chemically synthesized stereoisomers. The metabolism of the active isomer as well as the inhibitory effect of its triphosphate metabolite against HBVassociated DNA polymerase is also reported.

MATERIALS AND METHODS
Compounds and Deaminase-The stereoisomers of SddC were synthesized by J. W. Beach, L. S. Jeong, and c. K. Chu, Department of Chemistry, University of Georgia. 5-FSddC was synthesized by D.
Assay for Antiviral Activity-The procedure was essentially the same as that published previously (14). The 2215 (HBV-transfected cell line) cells were incubated with various concentrations of drug and grown for 12 days with medium changes every 3 days. At designated times, the cells were removed by low speed centrifugation, and polyethylene glycol (PEG) was added to the supernatant media to precipitate the virions. Nucleic acids were extracted from PEG precipitates and analyzed on Southern blots.
Endogenous HBV Polymerase Assay-For the assay of viral polymerase activity, the 2215 cells were incubated under conditions described previously (14). Cells were grown to confluence with changes of medium every 3 days. The medium was centrifuged (2,000 X g, 10 min), and an equal volume of 20% polyethylene glycol solution containing 1 M NaCl was added to the supernatant. After 1 h of incubation at 4 'C, virions were pelleted by centrifugation (10,000 X g, 10 min). The pellet was resuspended in 50 mM Tris-HC1, pH 7.5. Endogenous DNA polymerase activity was measured as described 0.175 p~ dCTP including 10 pCi of [a3'P]dCTP (3,000 Ci/mmol, Amersham Corp.), inhibitor and virus suspension in a final volume of 50 pl. After incubation at 37 "C for 2 h, the reaction was stopped by the addition of sodium dodecyl sulfate to a final concentration of 1%, then together with 10 pg of yeast tRNA, and 20 pg of proteinase K with a final total volume of 100 pl. It was incubated at 50 "C for 30 min. The "'P-labeled viral DNA was then isolated by phenolchloroform extraction and ethanol precipitation. The reaction product was analyzed by electrophoresis on a 0.7% agarose gel, dried onto 3MM paper, and subjected to autoradiography. The radioactive areas were then cut from the gel and quantitated in a liquid scintillation counter.
published (14). CEM or MT2 (T-lymphoblastoid cells) cells were Cytotoxicity-The procedure was essentially the same as previously grown in 5 ml of RPMI 1640 medium supplemented with 5% fetal bovine serum at an initial cell number of 2 X 10' cells/ml. The doubling time was approximately 20 h. The cells were incubated with various concentrations of the compounds for 4 days. On day 4, the cell number was determined by using either a Coulter counter or a hemacytometer. Susceptibility to Deoxycytidine Deaminase-The reaction mixture contained 25 mM Tris-HC1, pH 8.0, 0.2 mM of nucleoside, and approximately 0.04 units of human dCyd deaminase in a final volume of 50 p1 and was incubated at 37 "C for the period indicated. At the end of incubation, 100 pl of acetonitrile was added to stop the reaction, and the proteins were removed by centrifugation. The supernatant was lyophilized to dryness and reconstituted with HPLC mobile phase buffer. Nucleosides were separated by HPLC using an Alltech RP-18 column with the detector monitor at wave length 260 and 270 nm. The mobile phase was 8% acetonitrile in 100 mM ammonium acetate, pH 6.8, and the flow rate was 1 ml/min.
Preparation of fHI(-)-SddC-[3H]SddC (mixture of (rt)-SddC, 2.3 mCi/mmol) was incubated in the presence of human dCyd deaminase as described above. The proteins were removed by acetonitrile precipitation and the material which is resistant to deamination, that is (-)-SddC, was separated from (+)-SddU (the deamination product of (+)-SddC) by HPLC. The purification of [3H](-)-SddC was achieved by a second run of HPLC using 10% methanol in H,O as the mobile phase.
Extraction of Metabolites from Cells-CEM or 2215 cell lines (5 X 106 cells) were incubated in 0.5 or 2.0 p~ of 3H-labeled SddC (2.3 mCi/mmol) for the period of time indicated. At the indicated time, medium was removed, cells were washed twice with phosphate-buffered saline, and extracted with 1.5 M perchloric acid at 4 "C. The acid-soluble material was prepared by centrifuging the extract at 10,000 X g for 10 min at 4 'C. The supernatant was neutralized with 4 N KOH, and the KClO, was removed by another centrifugation.
HPLC Analysis of the Acid-soluble Metabolites-The acid-soluble cell extracts were analyzed using HPLC with an anion-exchange column (Partisil 10-SAX, 4.6 mm X 25 cm, Whatman). The solvent used was potassium phosphate buffer, pH 6.5, with a flow rate of 1 ml/min. A step gradient system was employed using 0.03 M buffer from 0-12 min followed by 0.15 M buffer from 12-52 min. At 52 min, the buffer concentration and flow rate were increased to 0.3 M and 2 ml/min, respectively. The HPLC was connected to a fraction collector, fractions were collected at 1-min intervals, and used directly for scintillation counting.
Preparation of SddCMP and SddCTP-The procedure was a modification of the procedure described by Ruth and Cheng (18). Approximately 5 mg of SddC (22 pmol) was dissolved in 50 pl of trimethyl phosphate (Aldrich 13, 219-5) 10 pl/mg, and stirred on methanol-ice for approximately 10 min. Then one equivalent of phosphorus oxychloride (Fisher P106) was added. The progress of the monophosphate formation was monitored using HPLC analysis with a Whatman SAX column using 0.03 M potassium phosphate buffer, pH 6.5, as solvent at a flow rate of 1 ml/min. When the reaction was maximized, three to four volumes of dimethyl formamide containing 5-8 equivalents of the tris-tributyl ammonium pyrophosphate was added. The formation of triphosphate nucleotide was monitored by HPLC with a Whatman SAX column and 0.15 M potassium phosphate buffer, pH 6.5, as the solvent with a flow rate of 2 ml/min. When the amount of triphosphate nucleotide appeared to be at a maximum the reaction was stopped by the addition of ice-cold water and neutralized to approximately pH 7.0 by the addition of triethylamine.
Incorporation into Nucleic Acid-DNA and RNA were purified from 2 X lo7 cells treated for 24 h with 0.5 pM 3H-SddC (1000 mCi/ mmol). They were centrifuged in CS~SO, gradients as previously described (19), except that the rate of centrifugation was at 40,000 rpm for 44 h. Gradients were fractionated from top to bottom, and the positions of the DNA and RNA peaks were determined by ethidium bromide fluorescence as described (20).
The behavior of cis-SddC stereoisomers relative to deoxycytidine toward human dCyd deaminase was examined and is summarized in Table I. The reaction rate difference of deamination between dCyd and cis-(+)-SddC at 640 p~ was approximately &fold when using human dCyd deaminase as shown in Table I, and the difference of K,, values was approximately 13-fold. Our unpublished studies? also indicate that the deaminase from Escherichia coli has a preference for dCyd more than 80-fold above the cis-(+)-SddC at 640 p M , however, cis-(-)-SddC as well as the other two trans-isomers (data not shown) are not substrates for human dCyd deaminase.
Identification of the Active Stereoisomers of SddC and FSddC as Anti-HRV Compounds-The 2215 cell line was used to evaluate the antiviral activities of stereoisomers of SddC and FSddC. The antiviral effects were measured by analysis of extracellular HRV DNA (Fig. 3). The experiments revealed that the amount of extracellular HRV DNA decreased in a dose-dependent manner for each compound. Each dose was done in duplicat,e including the solvent control. The cis-(-)-SddC and cis-(-)-FSddC were prepared from cis-(+)-SddC and cis-(+)-FSddC, respectively, after dCyd deaminase treatment and purification by HPLC. At 0.1 p~ cis-(-)-SddC almost completely inhibited the replication of HRV, however, less than 10% of the inhibition was observed by chemically synthesized cis-(+)-SddC under the same conditions. Alt,hough cis-(+)-FSddC was not availahle, the identification of cis-(-)-FSddC as the active stereoisomer in the mixture of cis-(+)-FSddC can be deduced from the result shown in Fig.  3R. At 0.05 p~ cis-(-)-FSddC was much more potent than cis-(+)-FSddC. If cis-(+)-FSddC was the same potency as cis-(-)-FSddC, one would expect to see a similar intensity for the two lanes treated with 0.1 p~ of (+)-FSddC and 0.05 p~ of (-)-FSddC. The concentrations of the stereoisomers of SddC that inhibit 50% (ID:,ll, p~) of the secreted HRV DNA, t h e growth of CEM cells or M T 2 cells, and the content of mitochondrial DNA from CEM cells are presented in Table  11. cis-(-)-SddC had an HRV ID,,, of 0.01 p~, 50-fold more potent than cis-(+)-SddC (HRV ID,,, = 0.5 p~) (see Table 11). Whereas, cis-(-)-SddC was approximately 25-fold less toxic 1s by Didcoxycytidinc Analo2.s than cis-(+)-SddC with regard to the inhibition of CEM cell growth after 4 days in culture. ci.s-(+)-SddC could also decrease mitochondrial DNA content in cells at a concentration lower than cis-(+)"X. The trans-isomers (see Fig. 1) are relatively inactive as anti-HRV or anti-cell growth compounds (data not shown).
Metabolism of cis-(-)-SddC in 221. 5 Cells-The HPLC profiles of the acid-soluble fraction of 2215 cells treated with 0.5 p~ 3H-cis-(-)-SddC for 4 or 24 h are shown in Fig. 4, A and R, respectively. Peak A was confirmed to be SddC. The identity of other "H-labeled peaks was assessed by subjecting them to alkaline phosphatase and snake venom phosphodiesterase digestion. Peaks E, D, and C can all be digested to the SddC nucleoside (results are not shown). Peak E was also partially digested using alkaline phosphatase and found to regenerate peaks A, C, and D. A similar experiment was done using peak D, and it was found to also regenerate peaks A and C. The elution positions of these peaks were also compared with chemically synthesized SddCMP, SddCDP, and SddCTP nucleotides, and it was concluded that peak C was (-)-SddCMP, peak D was (-)-SddCDP, and peak E was (-)-SddCTP. Peak R was a poor substrate for alkaline phosphatase and based on the elution position, it was tentativelv assigned as SddCMP-sialate. This will require further confirmation. Under these conditions the cellular ATP eluted off the HPLC column at approximately 40 min. In the 2215 cell line, formation of (-)-SddCTP showed a dose response from 0.2 to 2 p~ (-)-SddC (data not shown). There was no significant difference between 4 and 24 h in terms of the profiles of the metabolites. When 2215 cells were incubated with 0.5 p~ (-)-SddC for 24 h (intracellular (-)-SddCTP concentration was approximately 0.26 F M ) the drug was then removed and replaced with fresh medium for another 24 h, and a significant amount of (-)-SddCTP still remained (intracellular concentration was approximately 0.1 p M ) (Fig. 4c). There was no detectable incorporation of (-)-SddCMP into either DNA or RNA after 24 h of 0.5 p~ (-)-SddC treatment in 2215 cells using Cs,SO., gradient analysis (results not shown). Behavior of (-)-SddCTP and (+)-SddCTP Toward HRVassociated DNA Polymerase-Chemically synthesized ( -) -SddCTP and (+)-SddCTP were examined as inhibitors of the HBV endogenous DNA polymerase activity. At 0.175 p M dCTP (-)-SddCTP could inhibit HRV DNA synthesis in a dose-dependent manner (Fig. 5). Less inhibitory action of (+)-SddCTP was indicated. (-)-SddCTP exhibited approximately a 3-fold higher potency than (+)-SddCTP in inhibiting HBV DNA replication with 50% inhibitory dosage less than 0.05 pM.

DISCUSSION
Nucleoside analogs are a major chemical entity in the field of viral chemotherapy because they utilize the subtle differences between the viral DNA synthesis apparatus and the host cellular apparatus. It has always been assumed that the active stereoisomer of these analogs would be the one which most closely mimicked the natural nucleoside. Since most enzymes are stereospecific with respect to their substrates, many stereoisomers were found to have excellent selectivities against certain types of enzymes. In this report, it has been demonstrated that although cis-(+)-SddC, the natural form of the stereoisomer, could be deaminated by dCyd deaminase and was more cytotoxic, it was the cis-(-)-SddC which was the active stereoisomer against HRV. The metabolism studies of (-)-SddC indicate the formation of (-)-SddCMP, (-)-SddCDP, (-)-SddCTP, and an unidentified metabolite tentatively assigned as SddCMP-sialate in (-)-SddC-treated cells. The intracellular half-life of (-)-SddCTP is complicated due to the interconversion of SddC nucleotides and may require further studies. Basically, the kinetics of the drug metabolism seemed to be different between before and after removing the drug. The enzymes responsible for the formation of these metabolites have yet to be identified. Preliminary studies indicate the cytoplasmic dCyd kinase could phosphorylate (-)-SddC. Whether this is the only enzyme responsible for the phosphorylation of (-)-SddC to (-)-SddCMP is not clear. The active metabolite of (-)-SddC is likely to be (-)-SddCTP since this metabolite was shown to have potent inhibitory activity against HRV-associated DNA polymerase. The mode of the inhibition of (-)-SddCTP is competitive with dCTP. A detailed study will be published later. (+)-SddCTP is less active than (-)-SddCTP, but this may not be sufficient to account for the 50-fold difference of inhibition of HBV replication between (-)-and (+)-SddC. Differences in the metabolism between these two stereoisomers could also account for their inhibition. It was noted that the difference in antiviral activity between (-)-and (k)-SddC or (-)-and (+)-FSddC is more than %fold. This suggests the possibility of an interaction of the (-)-and (+)-forms of SddC or FSddC in terms of metabolism. This will be further investigated. Currently, one stereoisomer of SddC is under phase I clinical trial for HIV chemotherapy, however, it is not evident which isomer is being used. The unpublished results' indicate the (-)-isomer is 5-fold more active than the (+)-isomer in terms of anti-HIV effect, therefore, the potential use of (-)-isomer for both anti-HIV and anti-HBV therapy should be entertained.