6-Aminonicotinamide (6-AN) is a well-established inhibitor of the NADP+-dependent enzyme.

The EC50 of 6-AN on the inhibition of surface antigen (HBsAg) is approximately 23.89 μM in HepAD38 cells with a sensitive index (SI) > 20[1]

6-AN decreases HBsAg production but not blocks its secretion[1].

The cytotoxic effects of 6-AN on various cells were assessed by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay .?Cells were seeded into 96-well?plates and incubated with different concentrations (multiple proportion dilution from 500?μM to 0.98?μM) of 6-AN for 72 h. Next, 40?ug/ml MTT were added into cell culture media and incubated for 4 h under the condition of protection from light.?After that, dimethyl sulfoxide (DMSO) (Solarbio) was added and incubated overnight in the dark to lyses the cells and solubilize formazan dissolving in media.?Fifty-percent cytotoxicity concentrations (CC50) and Fifty-percent effective concentrations (EC50) were determined with a microplate reader and calculated via non-linear regression using GraphPad Prism 5.0[1].

HBV-transgenic mice (HBV-Tg C57 BL/6),Mice were selected as age (6 to 8 weeks), weight (21±1 g), and possess of basically the same serum HBsAg and HBV DNA levels.?Next, the mice were assigned to four groups of 10 individuals per group at random: negative control (0.9% saline), positive control (0.02 mg/kg Entecavir (ETV)), low concentration test group (6-AN 2.5 mg/kg), high concentration test group (6-AN 5 mg/kg).?Animals received 0.9% saline or 6-AN via intraperitoneal injection and animals received ETV via oral gavage every two days.?The serum samples were collected via orbital blood every four days after injection.?All blood was collected for analysis of serum HBsAg and HBV markers.?6 mice from each group were sacrificed by cervical dislocation at day 20 and the liver samples were used for intrahepatic HBV DNA and RNA determination.?The remaining animals were monitored without treatment up to day 32[1].