The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (2'-deoxy-3'-thia-5-fluorocytosine [FTC]) were studied by using an HBVtransfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, whfle the (+) isomer was found to be considerably less active. Both isomers showed miimal toxicity to HepG2 cells (50% inhibitory concentration, >200 a.M) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 5'-triphosphate of (-)-FIC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 5'-triphosphate of the (+) isomer was inactive.Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, souggestng that the antiviral activity of the compound is dependent on anabolism to the 5'-triphosphate. Both (-)-and (+)-FTC were anabolized to the corresponding 5'-triphosphates in chronically HBV-infected HepG2 celLs. The rate of accumulation and the steady-state concentration of the 5'-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to d tion and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase.Hepatitis B virus (HBV), the causative agent of acute and chronic hepatitis, directly affects about 5% of the world's population. Chronic carriers of HBV are at an increased risk of liver damage that, in the worst cases, can lead to cirrhosis of the liver and/or to hepatocellular carcinoma. Vaccination against HBV is one way to effectively prevent HBV infection. However, vaccination is not an effective therapy for the estimated 200 million chronic carriers. Although several antiviral agents such as alpha interferon, adenine arabinoside monophosphate, and acyclovir have been tested as therapeutic agents, only alpha interferon has demonstrated some promise (7,8,9,17,23,25).The replication cycle of hepadnaviruses includes the reverse transcription of an RNA template (6). This process is catalyzed by a polymerase that shares significant sequence homology with the reverse transcriptase from retroviruses (17). As a consequence, it has been demonstrated that a number of compounds that inhibit human immunodeficiency virus (HIV) replication in vitro (for example, 2',3'-dideoxycytidine) also inhibit HBV replication in vitro (4,14,15,18,24). These agents await further study to determine their usefulness as therapeutic agents for the treatment of HBV infections.Here we report the anti-HBV activities, cytotoxicities, and anabolism of the resolved enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (FTC) (Fig. 1). The anti-HBV activity of the racemic material has been reported previously (4). Our results show that the * Corresponding authors. antiviral activ...

and its congener 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]-methyl}guanine (BW B759U) exhibit comparable antiviral activity against . However, BW B759U is a more potent inhibitor of both human CMV and Epstein-Barr virus (EBV) in vitro than is ACV (1-3, 5). As is the case with ACV, BW B759U is activated to the triphosphate form in HSV-and VZV-infected cells and the initial step of this phosphorylation is associated with the activity of the virus-specified thymidine kinase (TK) (refs. 4, 6-8; unpublished data Table 1). In the present study, we report that BW B759U, in contrast to ACV, is preferentially activated in human diploid fibroblasts infected with HCMV. MATERIALS AND METHODSCells and Virus. Human foreskin fibroblast (HFF) cells were derived in this laboratory from tissues obtained from Duke University Hospital Pediatrics Department (Durham, NC) and were used up to passage 11. Human diploid embryonic lung fibroblast (MRC-5) cells were obtained from American Type Culture Collection and were used between passage 22 and passage 26. Monolayer cultures were grown in Eagle's minimal essential medium (ME medium) containing 50 units of penicillin per ml and 50 ,g of streptomycin per ml supplemented with 10% fetal bovine serum (Sterile Systems, Logan, Utah) and 1% L-glutamine. HCMV strain AD169 was also obtained from American Type Culture Collection. Kerr strain of HCMV was obtained from E. S. Huang (Cancer Research Center, University of North Carolina, Chapel Hill). The clinical strain Wade was isolated. from a congenitally infected infant by J. Zeller (Duke University Hospital Infectious Diseases Laboratory).Virus stocks of AD169 were stored in liquid nitrogen and were amplified before use by low multiplicity of infection (moi) passage in MRC-5 cells [<0.05 plaque-forming units (pfu) per cell]. Supernatant virus was used at time of peak titer (days 8-11) and was back titrated at the time of use to determine approximate moi.Cells and virus stocks were routinely checked for mycoplasma contamination by electron microscopy and [125i]iododeoxycytidine autoradiography methods.Cell Cytotoxicity Assay. The cytotoxicity of the nucleoside analogs was determined by Naomi Cohn (The Wellcome Research Laboratories) by using a cell growth assay that allows Abbreviations: EBV, Epstein-Barr virus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella zoster virus; ACV, acyclovir or 9-[(2-hydroxyethoxy)methyl]guanine; FIaC, 2'-fluoro-5-iodoarabinosylcytosine; BW B759U, 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine, also abbreviated 2' NDG, BIOLF-62, and DHPG by other investigators; HFF, human foreskin fibroblast; TK, thymidine kinase; pfu, plaque-forming unit(s); moi, multiplicity of infection. 2473The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.