Alisporivir Inhibition of Hepatocyte Cyclophilins Reduces HBV Replication and Hepatitis B Surface Antigen Production
Sandra Phillips∗
,
Shilpa Chokshi∗
,
Udayan Chatterji
,
Antonio Riva
,
Michael Bobardt
,
Roger Williams
,
Philippe Gallay
,
Nikolai V. Naoumov correspondence email
∗Authors share co-first authorship.
Received: December 15, 2013; Accepted: October 5, 2014; Published Online: October 08, 2014
DOI: http://dx.doi.org/10.1053/j.gastro.2014.10.004
Background & Aims
Cyclophilins are host factors required for hepatitis C virus replication. Cyclophilin inhibitors such as alisporivir have shown strong anti–hepatitis C virus activity in vitro and in clinical studies. However, little is known about whether hepatocyte cyclophilins are involved in the hepatitis B virus (HBV) life cycle. We investigated the effects of 2 cyclophilin inhibitors (alisporivir and NIM811) on HBV replication and hepatitis B surface antigen (HBsAg) production in cell lines.
Methods
Liver-derived cell lines producing full-length HBV and HBsAg particles, owing to stable (HepG2215) or transient (HuH-7) transfection, or infected with HBV (HepaRG cells; Invitrogen [Carlsbad, CA]), were incubated with alisporivir or NIM811 alone, or alisporivir in combination with a direct antiviral (telbivudine). The roles of individual cyclophilins in drug response was evaluated by small interfering RNA knockdown of cyclophilin (CYP)A, CYPC, or CYPD in HepG2215 cells, or CYPA knockdown in HuH-7 cells. The kinetics of antiviral activity were assessed based on levels of HBV DNA and HBsAg and Southern blot analysis.
Results
In HepG2215, HuH-7, and HepaRG cells, alisporivir reduced intracellular and secreted HBV DNA, in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD (reduced by 80%) significantly reduced levels of HBV DNA and secreted HBsAg. Knockdown of CYPA significantly reduced secretion of HBsAg, leading to accumulation of intracellular HBsAg; the addition of alisporivir greatly reduced levels of HBsAg in these cells. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone.
Conclusions
Alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of HBV DNA and HBsAg production and secretion. These effects are potentiated in combination with direct antiviral agents that target HBV-DNA polymerase.
Keywords:
Cyclophilin Inhibition, Direct-Acting Antiviral Agent, Peptidyl-Prolyl Isomerase, Viral Replication 作者: StephenW 时间: 2015-1-27 21:18
NVP018, A CYCLOPHILIN INHIBITOR FOR TREATMENT OF CHRONIC HBV INFECTION
J. Nilsson1, S. Moss2, N. Coates2, M. Bobardt3, P. Gallay3, M. Gregory2
1Neurovive Pharmaceutical AB, Lund, Sweden, 2Isomerase Therapeutics Ltd., Cambridge, United Kingdom, 3The Scripps Research Institute, La Jolla, CA, United States
E-mail: matt.***@****
Background and aims: Whilst cyclophilin (Cyp) is a clinically validated target for chronic HCV infection, recent work has shown that inhibition of Cyps by either siRNA knockdown or therapeutically can also inhibit hepatitis B virus in vitro. NVP018 (previously known as BC556) is an orally available sangamide-based cyclophilin inhibitor (CI), with a well-differentiated preclinical profile when compared to cyclosporine-based CIs such as alisporivir and SCY-635. It has a very different structure and does not have the liabilities associated with these cyclosporine-based compounds.
Methods: Analysis of NVP018 viral inhibition, including mechanism of action studies and immune modulation in models of hepatitis B virus infection.
Results: NVP018 is a potent inhibitor of HBV replication distinct from previously studied inhibitors. It has a dual mechanism of action including both direct inhibition of viral pathways which require cyclophilins and immune modulation via IRFs, including potent inhibition of an interaction between cyclophilin A and IRF9, a key component of the Jak/Stat pathway.
Conclusions: NVP018 inhibits HBV infection in various models via new mechanisms and represents a novel combination partner for nucleotide inhibitors or interferons for treatment of chronic HBV infection, with the potential to inhibit viral replication and increase seroconversion rates.作者: newchinabok 时间: 2015-1-28 15:41
NVP018，A环素抑制剂治疗慢性乙肝病毒感染
J. Nilsson1，S. Moss2，N. Coates2，M. Bobardt3，P. Gallay3，M. Gregory2
1Neurovive制药AB，瑞典隆德，2Isomerase治疗有限公司，剑桥，英国，3The斯克里普斯研究所，拉霍亚，CA，美国
电子邮件：matt.***@****
A New Approach to Hepatitis B Therapy?
165 Liver/Biliary 2 days ago Kristine Novak 1
Cyclophilins are involved in multiple steps of the hepatitis B virus (HBV) life cycle in hepatocytes—cyclophilin inhibitors reduce viral replication and HBV envelope protein production and secretion, researchers report in the February issue of Gastroenterology. The cyclophilin inhibitor alisporivir, combined with the HBV polymerase inhibitor, reduces markers of HBV infection and HBV replication in cells, revealing a possible new therapeutic approach for chronic hepatitis B.
Hepatitis B virions. Source: CDC
Cyclophilins have peptidyl-prolyl isomerase activity, and catalyze the cis to trans conversion of proline-containing peptides and facilitate protein folding. There are several human genes that encode cyclophilins, including CYPA, CYPB, CYPC, and CYPD. Their products localize to different areas of the cell. For example, CYPA and CYP40 are present in the cytosol, CYPB and CYPC in the lumen of the endoplasmic reticulum, and CYPD is found in the mitochondria.
Cyclophilins regulate the life cycle of a range of viruses including HCV, HIV, vaccinia virus, coronaviruses, and polyomavirus BK, and are required for virus replication. CYPA is the main cyclophilin that regulates the life cycle of HCV; cyclophilins reduce HCV replication and secretion from infected cells.
Sandra Phillips et al investigated whether hepatocyte cyclophilins are involved in HBV replication, hepatitis B surface antigen (HBsAg) production, and secretion in liver-derived cell lines that produce full-length HBV and HBsAg particles.
They found that the cyclophilin inhibitor alisporivir reduced intracellular and secreted HBV DNA in liver cells lines in a dose-dependent manner. Knockdown of CYPA, CYPC, or CYPD significantly reduced levels of HBV DNA and secreted HBsAg—a marker of HBV infection.
Small molecule inhibitors of cyclophilins also reduced levels of HBsAg produced by cells, and caused a dose-dependent reduction in HBV DNA. The combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. The authors propose that the cyclophilin inhibitors alisporivir and NIM811 block multiple cyclophilins, affecting not only HBV replication and HBsAg production, but multiple stages of the HBV life cycle.
CYPA is one of the most abundant cytosolic proteins (approximately 0.1% of cell proteins). Phillips et al showed that CYPA participates in HBV replication and envelope protein secretion from hepatocytes. They say that CYPA is important for the formation of intracellular, nucleocapsid-associated HBV DNA and viral secretion and for HBsAg secretion from cells.
The authors propose a new approach for treating chronic hepatitis B that involves a combination of a direct antiviral agent (such as the HBV-DNA polymerase inhibitors) and agents that act directly on liver cells, such as those that block cyclophilins.
Alisporivir is in development for the treatment of chronic hepatitis C. Phillips et al say that it has been evaluated in more than 2000 patients and seems to be well tolerated, with a better safety profile when given as interferon-free treatment than with interferon.
The authors conclude that combination treatment with an HBV nucleos(t)ide polymerase inhibitor plus a cyclophilin inhibitor would interfere with multiple sites of the HBV life cycle, reducing HBsAg production and secretion as well as blocking HBV entry into non-infected hepatocytes. 作者: StephenW 时间: 2015-1-28 21:58
"The FDA has notified Novartis to put the program on clinical hold," Naoumov said. "The reason is that in the last few months we had a cluster of three patients who developed acute pancreatitis and one last week who died, unfortunately. So, as a result, this is to assess the risk. All these patients had been treated with alisporivir plus PR [pegylated interferon and ribavirin]. We know pancreatitis is on the label of interferon, and we are working to see if adding alisporivir potentiated a known side effect."
“美国食品药物管理局已通知诺华把节目临床搁置，”Naoumov说。 “究其原因是，在过去的几个月里，我们不得不谁开发急性胰腺炎，谁死了，遗憾的是最后一个星期三个病人的集群。所以，作为一个结果，这是评估风险，这些病人经治疗后已与alisporivir加PR [聚乙二醇干扰素和利巴韦林。我们知道胰腺炎是干扰素的标签上，我们正在努力，看看是否添加alisporivir增效已知的副作用。作者: tonychant 时间: 2015-1-29 15:18