Published online Aug 15, 2003. doi: 10.3748/wjg.v9.i8.1840
Revised: January 10, 2003
Accepted: February 17, 2003
Published online: August 15, 2003
AIM: To explore the inhibition of β-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome.
METHODS: 2.2.15 cells were plated at a density of 5 × 104 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end, 5 μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindIII. Both DNAs were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a 32P-labeled mtDNA-specific probe, and effect of β-L-D4A on mitochondrial DNA was studied. 2. 2.15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. ID50 was calculated. Structure-activity relationships between D2A and D4A were also studied as above.
RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. ED50 was 0.2 μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.
CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxicity and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.