THE JOURNAL OF BIOLOGICAL CHEMISTRV 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 20, Issue of July 15, pp. 13938-13942,1992 Printed in U.S.A.

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

(Received for publication, February 5, 1992)

Chien-Neng Chang, Shin-Lian Doong, James Hua Zhou, J. Warren Beach$, Lak S. JeongS, Chung K. ChuS, Ching-hwa Tsai, and Yung-chi Chenga From the Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 and the $Department of Medicinal Chemistry, The University of Georgia, College of Pharmacy, Athens, Georgia 30602

2’,3’-Dideoxy-3’-thiacytidine ((2)-SddC) was found to have potent activity against human hepatitis B virus as well as human immunodeficiency viruses in culture. The (-)form ((-)-SddC) which is resistant to deoxycy- tidine 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. Sim- ilar 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 converted to (-)-SddCDP and (-)-SddCTP. One additional major metabolite which has been ten- tatively assigned the name “(-)-SddCMP sialaten was also identified. No significant difference in terms of the profiles of the metabolites was found between 4 and 24 h. There is an appreciable amount of (-)- SddCTP detectable 24 h after removal of the drug. (-)- SddCTP was also found to be approximately %fold more potent than (+)-SddCTP in inhibiting human hep- atitis B virus DNA polymerase. This is the first nucle- oside analog with the unnatural sugar configuration demonstrated to have antiviral activity.

Hepatitis B virus (HBV)’ infection is a major health prob- lem. It causes infectious disease and is closely associated with primary hepatocellular carcinoma (1-3). Currently, there are no effective drugs for the treatment of HBV infections without substantial toxicity. Several nucleoside analogs have been

* This work was supported by National Institutes of Health Grants CA44358 and AI25899. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 To whom correspondence should be addressed. Tel.: 203-785- 7119; Fax: 203-785-7129.

The abbreviations used are: HBV, hepatitis B virus; SddC, 2’,3’- dideoxy-3’-thiacytidine; HIV, human immunodeficiency viruses; FSddC, 5-fluoro-2’,3’-dideoxy-3‘-thiacytidine; ddC, 2’,3’-dideoxycy- tidine; SddCMP, 2’,3’-dideoxy-3’-thiacytidine monophosphate; SddCDP, 2’,3’-dideoxy-3’-thiacytidine diphosphate; SddCTP, 2’,3’- dideoxy-3’-thiacytidine triphosphate; dCyd, deoxycytidine; HPLC, high performance liquid chromatography; SddU, 2’,3’-dideoxy-3’- thiauridine; PEG, polyethylene glycol.

found to have activity against HBV (4-10). Among them, 2’,3’-dideoxy-3’-thiacytidine, which was discovered to have potent anti-HIV activity (11-14), and its &fluor0 analog were found to be two of the most potent and selective compounds against HBV replication in culture (14). The drugs examined, however, were the racemic mixture of the cis-isomers (see Fig. 1). There are four possible stereoisomers each for SddC or 5- FSddC: a pair of cis-isomers and a pair of trans-isomers (Fig. 1). With the exception of cis-D-(+)-SddC, the isomers have unnatural sugar configurations. It was not clear whether one, or all of these stereoisomers were responsible for the inhibi- tion of HBV replication. 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 stereoiso- mers. The metabolism of the active isomer as well as the inhibitory effect of its triphosphate metabolite against HBV- associated DNA polymerase is also reported.

MATERIALS AND METHODS

Compounds and Deaminase-The stereoisomers of SddC were syn- thesized by J. W. Beach, L. S. Jeong, and c. K. Chu, Department of Chemistry, University of Georgia. 5-FSddC was synthesized by D. Liotta and sent by R. F. Schinazi (Emory University, Atlanta, GA). 3H-SddC (BCH-189-5, 6-3H, mixture of two cis-isomers, (+)-SddC and (-)-SddC), 20 Ci/mmol, was purchased from Moravek Biochem- icals, Inc., Brea CA. Human deoxycytidine deaminase was partially purified from human liver through a DEAE-cellulose column using a previously published procedure (15) with the specific activity of 2 units/mg. The unit is defined as the amount of enzyme which converts 1 pmol of dCyd in 1 min at 37 “C.

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 desig- nated 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 pre- cipitates and analyzed on Southern blots.

Endogenous HBV Polymerase Assay-For the assay of viral poly- merase activity, the 2215 cells were incubated under conditions de- scribed 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 (16, 17) with some modifications. The assay mixture contained, 42 mM Tris-HC1, pH 7.5, 34 mM MgC12, 340 mM KCl, 22 mM mercaptoeth- anol, 0.4% Nonidet P-40, 70 p~ each of dATP, dTTP, and dGTP,

13938

Inhibition of Hepatitis B Virus by Dideoxycytidine Analogs 13939 C

cls-Dl+)SddC

HO A9 trans-L(+)SddC

C ClS-LI-EddC

C

trans-D(-)SddC

Ifor FSddC analogs)

FIG. 1. Structures of ddC analogs used in this study.

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 phenol- chloroform extraction and ethanol precipitation. The reaction prod- uct 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 de- aminase as described above. The proteins were removed by acetoni- trile 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-buff- ered 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 collec- tor, fractions were collected at 1-min intervals, and used directly for scintillation counting.

Preparation of SddCMP and SddCTP-The procedure was a mod- ification of the procedure described by Ruth and Cheng (18). Approx- imately 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 oxy- chloride (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 equiva- lents 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).

RESULTS

Susceptibilities of cis-(k)-SddC Stereoisomers to Human Deoxycytidine Deaminase-Previous studies (14) using the mixture of cis-isomers of SddC and FSddC showed potent activity against HBV replication. By incubating the (*)-cis- isomers with human dCyd deaminase for 16 h, approximately 50% of the (k)-SddC can be deaminated based on HPLC analysis (Fig. 2 A ) . Longer incubation does not increase the conversion. The deaminated product, SddU, eluted off the HPLC column at approximately 8.5 min while SddC eluted off the column at 7.0 min. The identity of SddU was ensured

I ,

7 0 8.5

B

I , 7 0 8.5

Retentlon tlme (mm)

FIG. 2. HPLC profiles of the deamination of cis-SddC ster- eoisomers by human dCyd deaminase. The reaction mixture contained 25 mM Tris-HC1, pH 8.0, 0.2 mM of (rt)-SddC, (+)-SddC and (-)-SddC and approximately 0.04 units of partially purified human CdR deaminase in a final volume of 50 pl. The reaction time was 16 h, and incubation temperature was 37 "C. The reaction prod- ucts were analyzed by HPLC as described under "Materials and Methods." Under these conditions cis(+)- and (-)-SddC eluted off HPLC column at approximately 7.0 min with a UV Amax = 270 nm; however, the deaminated product SddU has a retention time of 8.5 min with UV Amax of 260 nm.

" The rates o f deamination were determined using H P I X analysis. I ' I'roduct of the reaction was not detected.

C.-N. Chang and Y.-C. Cheng, unpul)lished results.

by spectral analysis. cis-(+)-SddC, synthesized in Dr. C. K. Chu's laboratory, was also subjected to deamination. After 16 h, (+)-SddC was almost completely converted to (+)-SddU as shown in Fig. 2R. T h e chemically synt,hesized cis-(-)-SddC was incubated under the same conditions, with human dCyd deaminase (Fig. 2C), and proved to be completely resistant to the deamination. By taking advantage of the selective deam- ination of (+)-SddC by human dCyd deaminase, (-)-SddC, and ["HI(-)-SddC were subsequently prepared.

The behavior of cis-SddC stereoisomers relative to deoxy- cytidine toward human dCyd deaminase was examined and is summarized in Table I. The reaction rate difference of deam- ination between dCyd and cis-(+)-SddC at 640 p~ was ap- proximately &fold when using human dCyd deaminase as shown in Table I, and the difference of K,, values was ap- proximately 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 deami- nase.

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 de- creased 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. Al- t,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 A 8

RC - u

ss- - ss

FIG. 3. Potency of SddC and FSddC stereoisomers as anti- HBV compounds. T h e 2215 cells were incutmted with varinus con- centrations ( p ~ , as indicated in the figure) of drug and grown for 12 davs. Medium was changed every 3 days. The medium from day 9 t o 12 was harvested, and virions were precipitated with PK;. Surleic acids were extracted from PEG precipitates and analyzed on Southern hlots. I K ' , relaxed circular HHY 1)SA; S S . single-stranded HHV DNA; ST', solvent control. I'nnrls A and H are results from two separate experiments with their own controls.

p .Sf

(+)-SddC 0.05 37 >20 47 (-)-SddC" o.01 (-)-SddC 0.01 50 >'Lo >50 (+)-SddC 0.5 2 1 3 4 (*)-5-F-SddC 0.1 >'OO >zoo

ddC (-)-5-F-SddC" 0.0' > 2 0 0 > ? o r )

2.8 1 0 0,022 " ~

"The compound was purified from HPLC after deamination from their racemic mixtures.

than cis-(+)-SddC with regard to the inhibition of CEM cell growth after 4 days in culture. ci.s-(+)-SddC could also de- crease mitochondrial DNA content in cells at a concentration lower than c is-(+)"X. The t rans- 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 pro- files 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 phosphodi- esterase 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 com- pared 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 phospha- tase and based on the elution position, it was tentativelv

Inhibition of Hepatitis I3 Virus by Dideoxycytidine Analogs 13941

IA)

7000

6000 4 h

5000t A

E SddCTP I

0 10 20 30 40 50 60 70

(E) 7000 1

6ooo 5 0 0 0 t I 24 tu

0 10 20 30 40 50 60 70

(C) 7000

2 4 h after removlnp the arvp

z[ 3000 1 (C)

7000

6000 ~

5000

2 4 h after removlnp the arvp

-

4000

3000 - -

0 10 20 30 4 0 50 60 70

retartom tune h~nJ

FIG. 4. The metabolism studies of (-)-SddC in 2215 cells. Cells were incubated in the presence of 0.5 p~ ,"H(-)-SddC (2.3 mCi/ mmol) for 4 h ( A ) and 24 h (H). Medium was removed after 24 h, replaced with fresh medium without drug, and incubated for an additional 24 h ( C ) . At the time and condition indicated, cells were harvested, extracted, and analyzed by HPLC according to the proce- dures described under "Materials and Methods."

assigned as SddCMP-sialate. This will require further confir- mation. 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 signif- icant 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 concentra- tion was approximately 0.26 FM) the drug was then removed and replaced with fresh medium for another 24 h, and a significant amount of (-)-SddCTP still remained (intracel- lular concentration was approximately 0.1 pM) (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).

(-)SddCTP (OSddC'TP

C C 0 . 0 5 a.1'75 0 . 5 0 0 . 0 5 0 . 1 7 5 ( u M )

FIG. 5. Comparative potency of (+)-SddCTP and (-)- SddCTP as inhibitors of endogenous HRV D N A polymerane. The assay mixture contained the virus preparation (as desrrihed in Materials and Methods). 42 mM Tris-HCI (pH 7 . 5 ) . :14 mM MgCI:. 340 mM KCI, 22 mM mercaptoethanol. O.JrE nonidet p-41). 70 pM earh of dATP, dTTP and dCTl', and 0.175 p~ dCT1' inrluding 1 0 pCi cr."I'-dCTP and appropriate amount of inhihitors in a final volume of 50 p l . After incuhation at 37 "C for 2 hr the reaction was stopped and the "T' labeled viral DNA was isolated then analyzed hy elertrcl- phoresis on a 0.7Ci agarose gel.

Behavior of (-)-SddCTP and (+)-SddCTP Toward HRV- associated 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 approxi- mately 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 differ- ences 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 ten- tatively 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 phos- phorylate (-)-SddC. Whether this is the only enzyme respon- sible 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

13942 Inhibition of Hepatitis B Virus by Dideoxycytidine Analogs

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 enter- tained.

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