Evolution of matrinic ethanol derivatives as anti-HCV agents from matrine skeleton.

Twenty-two novel 12N-substituted matrinic ethanol derivatives were synthesized and evaluated for their antiviral activities against HCV taking compound 1 as the lead. The SAR study indicated that the shortening of the 11-butyl chain to ethyl chain did not affect the activity significantly. Out of the target compounds, matrinic ethanol 6a demonstrated a potential anti-HCV effect with an EC50 value of 3.2µM and a SI value of 96.6. The free hydroxyl arm in 6a made it possible as a parent structure to prepare pro-drug for the potential application in HCV treatment. This study provided powerful information on further strategic optimization and development of this kind of compounds into a novel family of anti-HCV agents.

Structure-activity relationship of N-benzenesulfonyl matrinic acid derivatives as a novel class of coxsackievirus B3 inhibitors.

A novel series of N-benzenesulfonyl matrinic amine/amide and matrinic methyl ether analogues were designed, synthesized and evaluated for their in vitro anti-coxsackievirus B3 (CVB3) activities. The structure-activity relationship (SAR) studies revealed that introduction of a suitable amide substituent on position 4' could greatly enhance the antivirus potency. Compared to the lead compounds, the newly synthesized matrinic amide derivatives 21c-d and 21j exhibited stronger anti-CVB3 activities with lower micromolar IC50 from 2.5 µM to 2.7 µM, and better therapeutic properties with improved selectivity index (SI) from 63 to 67. The SAR results provided powerful information for further strategic optimization, and these top compounds were selected for the next evaluation as novel enterovirus inhibitors.

A zebrafish model for subgenomic hepatitis C virus replication.

Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma. The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model. Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally. In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms. As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter. By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver. The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity. Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver. The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells. To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae. In conclusion, this zebrafish model of HCV may prove to be a novel and simple in vivo model for the study of the mechanisms of HCV replication and may also prove useful in the disovery of new anti-HCV drugs.

Tetrandrine potentiates the hypoglycemic efficacy of berberine by inhibiting P-glycoprotein function.

This study was designed to improve the absorption and hypoglycemic efficacy of berberine (BBR), which is a substrate of P-glycoprotein (P-gp), by combination with a P-gp inhibitor tetrandrine (Tet). Flow cytometry and LC-MS/MS were used to determine the cellular efflux or retention of chemicals. Pharmacokinetic study was performed in ICR mice following oral administration of the study compounds. The hypoglycemic efficacies of the compounds were evaluated in diabetic KK-Ay mice. In the in vitro experiments, Tet significantly inhibited the efflux and increased the uptake of P-gp substrates rhodamine-123 as well as BBR in MCF7/DOX cells and Caco-2 intestinal cells. Meanwhile, Tet greatly reduced the expression of P-gp in Caco-2 cells. The inhibition of BBR efflux by Tet was translated into improved pharmacokinetics in vivo. When co-administered, Tet dose-dependently increased the average maximum concentration (C(max)) and area under concentration-time curve (AUC0₋24) of BBR in mice. Tet itself had no impact on glucose metabolism. However, it greatly potentiated the hypoglycemic efficacy of BBR in diabetic KK-Ay mice. In addition, we found that Tet had moderate inhibitory effect on the catalytic activity of CYP3A4, which played a role in the bio-transformation of BBR, and this may also take part in the improvement of the pharmacokinetics of BBR. In summary, combination with P-gp inhibitors such as Tet can improve the pharmacokinetics and hypoglycemic efficacy of BBR greatly; this implicates a feasible strategy for exploring the therapeutic effects of BBR and other pharmaceuticals which are substrates of P-gp.

Zebrafish as a potential model organism for drug test against hepatitis C virus.

Screening and evaluating anti- hepatitis C virus (HCV) drugs in vivo is difficult worldwide, mainly because of the lack of suitable small animal models. We investigate whether zebrafish could be a model organism for HCV replication. To achieve NS5B-dependent replication an HCV sub-replicon was designed and created with two vectors, one with HCV ns5b and fluorescent rfp genes, and the other containing HCV's 5'UTR, core, 3'UTR and fluorescent gfp genes. The vectors containing sub-replicons were co-injected into zebrafish zygotes. The sub-replicon amplified in liver showing a significant expression of HCV core RNA and protein. The sub-replicon amplification caused no abnormality in development and growth of zebrafish larvae, but induced gene expression change similar to that in human hepatocytes. As the amplified core fluorescence in live zebrafish was detectable microscopically, it rendered us an advantage to select those with replicating sub-replicon for drug experiments. Ribavirin and oxymatrine, two known anti-HCV drugs, inhibited sub-replicon amplification in this model showing reduced levels of HCV core RNA and protein. Technically, this method had a good reproducibility and is easy to operate. Thus, zebrafish might be a model organism to host HCV, and this zebrafish/HCV (sub-replicon) system could be an animal model for anti-HCV drug screening and evaluation.

Berberine lowers blood glucose in type 2 diabetes mellitus patients through increasing insulin receptor expression.

Our previous work demonstrated that berberine (BBR) increases insulin receptor (InsR) expression and improves glucose utility both in vitro and in animal models. Here, we study the InsR-up-regulating and glucose-lowering activities of BBR in humans. Our results showed that BBR increased InsR messenger RNA and protein expression in a variety of human cell lines, including CEM, HCT-116, SW1990, HT1080, 293T, and hepatitis B virus-transfected human liver cells. Accordingly, insulin-stimulated phosphorylations of InsR beta-subunit and Akt were increased after BBR treatment in cultured cells. In the clinical study, BBR significantly lowered fasting blood glucose (FBG), hemoglobin A(1c), triglyceride, and insulin levels in patients with type 2 diabetes mellitus (T2DM). The FBG- and hemoglobin A(1c)-lowering efficacies of BBR were similar to those of metformin and rosiglitazone. In the BBR-treated patients, the percentages of peripheral blood lymphocytes that express InsR were significantly elevated after therapy. Berberine also lowered FBG effectively in chronic hepatitis B and hepatitis C patients with T2DM or impaired fasting glucose. Liver function was improved greatly in these patients by showing reduction of liver enzymes. Our results confirmed the activity of BBR on InsR in humans and its relationship with the glucose-lowering effect. Together with our previous report, we strongly suggest BBR as an ideal medicine for T2DM with a mechanism different from metformin and rosiglitazone.

Synthesis and activity evaluation of benzoylurea derivatives as potential antiproliferative agents.

3-Haloacylamino benzoylureas (3-HBUs) consist of a new family of tubulin ligands that kill cancer cells through mitotic arrest. In exploring the structure-activity relationship (SAR), 17 analogues defined through variations of formylurea at the 1-position of the aromatic ring were synthesized. SAR analysis revealed that (i) the p-pi conjugation between the aromatic ring and formylurea was essential; (ii) suitable aryl substitutions at the N'-end increased anticancer activity with a mechanism different from that of parent compounds; and (iii) introduction of pyridyl at the N'-end provided an opportunity of making soluble salts to improve bioavailability. Among the analogues, 16c bearing 3,4,5-trimethoxyphenyl and 16g bearing 2-pyridyl at the N'-end showed an enhanced activity and were active in hepatoma cells that were resistant to tubulin ligands including the parent compounds. Furthermore, 16c and 16g killed cancer cells with a mechanism independent of mitotic arrest, indicating a change of action mode.