Optimization of an Efficient Cell Culture Hepatitis B Infection System for Assessment of Hepatitis B Virus Neutralizing Monoclonal Antibodies.

BACKGROUND: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. OBJECTIVE: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. METHODS: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. RESULTS: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. CONCLUSION: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.

Bile Acid Conjugation on Solid Nanoparticles Enhances ASBT-Mediated Endocytosis and Chylomicron Pathway but Weakens the Transcytosis by Inducing Transport Flow in a Cellular Negative Feedback Loop.

Bile acid-modified nanoparticles provide a convenient strategy to improve oral bioavailability of poorly permeable drugs by exploiting specific interactions with bile acid transporters. However, the underlying mechanisms are unknown, especially considering the different absorption sites of free bile acids (ileum) and digested fat molecules from bile acid-emulsified fat droplets (duodenum). Here, glycocholic acid (GCA)-conjugated polystyrene nanoparticles (GCPNs) are synthesized and their transport in Caco-2 cell models is studied. GCA conjugation enhances the uptake by interactions with apical sodium-dependent bile acid transporter (ASBT). A new pathway correlated with both ASBT and chylomicron pathways is identified. Meanwhile, the higher uptake of GCPNs does not lead to higher transcytosis to the same degree compared with unmodified nanoparticles (CPNs). The pharmacological and genomics study confirm that GCA conjugation changes the endocytosis mechanisms and downregulates the cellular response to the transport at gene levels, which works as a negative feedback loop and explains the higher cellular retention of GCPNs. These findings offer a solid foundation in the bile acid-based nanomedicine design, with utilizing advantages of the ASBT-mediated uptake, as well as inspiration to take comprehensive consideration of the cellular response with more developed technologies.

Entry inhibition of hepatitis B virus using cyclosporin O derivatives with peptoid side chain incorporation.

Hepatitis B virus (HBV) infection is a serious worldwide health problem causing liver cirrhosis and hepatocellular carcinoma. The development of novel therapeutics targeting distinct steps of the HBV life cycle and combination therapy with approved drugs (i.e., nucleot(s)ides, interferon-α) are considered effective strategies for curing HBV. Among these strategies is the development of entry inhibitors that interfere with the host entry step of HBV to prevent viral infection and transmission. Herein, we generated a novel library of cyclosporin O (CsO) derivatives that incorporate peptoid side chains. Twenty-two CsO derivatives were evaluated for membrane permeability, cytotoxicity, and in vitro HBV entry inhibitory activity. The lead compound (i.e., compound 21) showed the greatest potency in the in vitro HBV entry inhibition assay (IC50 = 0.36 ± 0.01 µM) with minimal cytotoxicity. Our peptide-peptoid hybrid CsO scaffold can readily expand chemical diversity and is applicable for screening various targets requiring macrocyclic chemical entities.

Optimization of a cellular HBV infection model for use in high-throughput drug screening.

Hepatitis B virus (HBV) is a partially double-stranded DNA virus that specifically targets hepatocytes. It is considered a major health issue due to its high prevalence and the life-threatening consequences of chronic infection, including liver cirrhosis and hepatocellular carcinoma. Despite widespread vaccination against HBV, millions of people live with chronic HBV infection. Existing antiviral therapies fail to achieve full HBV elimination, so most patients with the disease require lifelong treatment. The search for new antiviral therapy strategies is hindered by the limited availability of in vitro HBV infection models that are able to support the full HBV life cycle. Therefore, the development and optimization of cellular models are crucial to the search for drugs effective against HBV. In this study, we optimized an in vitro HBV infection model consisting of two cell lines: HepAD38 cells, which are able to produce infectious HBV; and HepG2-NTCP cells, which are susceptible to HBV infection. We showed that prolonged production of HBV in the "donor" cells and HBV inoculation of the "acceptor" cells simultaneously with seeding improves the established procedure. This modified protocol was proven effective in experiments involving compounds with known activity against HBV, suggesting its utility for future high-throughput screening. Keywords: HBV; HBV in vitro models; HepG2-NTCP; HepAD38.

Long-term trans-inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid.

Human hepatic bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) represents the liver-specific entry receptor for the hepatitis B and D viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is downregulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function, and membrane expression of human NTCP were analyzed in HepG2 and human embryonic kidney (HEK293) cells stably overexpressing NTCP. Even after short-pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.NEW & NOTEWORTHY The hepatic bile acid transporter NTCP is a high-affinity receptor for hepatitis B and D viruses. This study shows that TLC rapidly accumulates in NTCP-expressing hepatoma cells and mediates long-lasting trans-inhibition of NTCP's transporter and receptor function via an intracellularly accessible domain, without substantially affecting its membrane expression. This domain is a promising novel NTCP target site for pharmacological long-acting HBV/HDV entry inhibitors.

[The inhibitors of the apical sodium-dependent bile acid transporter (ASBT) as promising drugs]. / Ingibitory natrii-zavisimogo perenoschika zhelchnykh kislot (ASBT) kak perspektivnye lekarstvennye sredstva.

Inhibition of the apical sodium-dependent bile acid transporter (ASBT, also known as IBAT - ileal bile acid transporter, SLC10A2) leads to disruption of the enterohepatic circulation of bile acids and their excretion with fecal masses. This is accompanied by cholesterol utilization for synthesis of new bile acids. ASBT inhibitors are promising drugs for the treatment of such diseases as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes mellitus, necrotic enterocolitis, chronic constipation, atherosclerosis. To date the most known chemically synthesized inhibitors are: A3309, SHP626, A4250, 264W94, GSK2330672, SC-435. All of them are at different stages of clinical trials, which confirm the high efficacy and good tolerance of these inhibitors. Current trends in this field also include directed chemical synthesis of ASBT inhibitors, as well as their search among substances of plant origin.

Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virus.

Hepatitis B virus (HBV) is a blood-borne pathogen responsible for chronic hepatitis, cirrhosis, and liver cancer. The mechanism of HBV entry into hepatocytes remains to be investigated. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was discovered as a major HBV receptor based on an in vitro infection system using NTCP-reconstituted HepG2 cells. However, this infection system relies on the compound polyethylene glycol (4% PEG), which is not physiologically relevant to human infection. High concentration of heparin has been commonly used as an inhibitor control for in vitro infection in the field. Surprisingly, we found that heparin at physiological concentration can enhance HBV infection in a PreS1-peptide sensitive, NTCP-dependent manner in both HepaRG and HepG2-NTCP-AS cells. O-sulfation of heparin is more important for the infection enhancement than N-sulfation. This system based on the HepG2-NTCP-AS cells can support in vitro infection with HBV genotypes B and C, as well as using serum samples from HBeAg positive and negative chronic carriers. In summary, our study provides a PEG-free infection system closely resembling human natural infection. In addition, it points to a future research direction for heparin and heparin-binding host factor(s) in the blood, which are potentially involved in viral entry. To our knowledge, this is the first soluble and circulatory host factor which can enhance HBV in vitro infection.

Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes.

Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and ß-tauromuricholic acid; α/ß TMCA), were profiled in primary rat and human SCH. Using B-CLEAR® technology, BAs were measured in cells+bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells+bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3±5.9 µM in CTL rat and 183±56 µM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16±0.21 µM in CTL rat SCH and 9.61±6.36 µM in CTL human SCH. Treatment of cells for 24h with 10 µM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na⁺-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account.

Randomised clinical trial: The ileal bile acid transporter inhibitor A3309 vs. placebo in patients with chronic idiopathic constipation--a double-blind study.

BACKGROUND: One half of patients with constipation are not satisfied with available therapies, hence there is a need for more effective and well-tolerated drugs. AIM: To evaluate the effects of a specific inhibitor of the Ileal Bile Acid Transporter (IBAT; syn apical sodium-dependent bile acid transporter; ASBT) in patients with chronic idiopathic constipation (CIC) with focus on safety, colonic transit and efficacy signals. METHODS: This was a single-centre, prospective, randomised, double-blind, placebo-controlled study with a dose-escalating design in patients with CIC. In addition to evaluation of conventional safety and tolerability parameters, (i) colonic transit time (CTT) was measured using radio-opaque markers, (ii) metabolic parameters [lipid profile, C4 (7α-hydroxy-4-cholesten-3-one) and FGF19 (Fibroblast Growth Factor 19)] were evaluated, and (iii) constipation parameters, such as changes in stool frequency and consistency, were analysed. RESULTS: Thirty patients were randomised into five dose-levels (range: 0.1-10 mg/day) or to placebo. All patients completed a 14-day treatment period, and the safety/tolerability analysis was favourable. A3309, present in picomolar concentrations in plasma, induced up to a three-fold increase in bile acid synthesis (C4) and a reduction of plasma FGF19, as well as reduction in total and LDL cholesterol. CTT was reduced in the highest dose groups; the main acceleration was identified in the left colon. Efficacy parameters showed trends for increased number of spontaneous bowel movements and improved stool consistency. CONCLUSIONS: Ileal Bile Acid Transporter inhibition is a novel mechanism for treatment of patients with chronic idiopathic constipation and has additional benefits of improving metabolic parameters (EudraCT 2008-003255-72).

The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin alpha-amanitin.

Hepatotoxicity caused by the mushroom poison alpha-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of alpha-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for alpha-amanitin uptake, we tested the hypothesis that Na(+)-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular alpha-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K(m) and V(max). A toxicologically relevant functional assay for alpha-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1beta (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 micro M alpha-amanitin for 6-10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without alpha-amanitin). These findings demonstrate that Ntcp may be an important mediator of alpha-amanitin uptake by the liver.