Periodic Report Summary 1 - HCV NEW INHIBITORS (HCV Progress in Therapy with Novel Sialomimetic-chimeric inhibitors)
Project Context and Objectives:
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. In the EU, in 2013, 32,512 cases of hepatitis C were reported in 26 EU/ EEA Member States, a crude rate of 9.9 per 100 000 population with an estimate number of EU citizens currently affected by hepatitis C of 5.5 millions. Among them, given the naturally history of the disease, approximately 70,000 Europeans may be dying from chronic liver diseases every year. Chronic hepatitis C leads also to higher risks of developing cirrhosis and cancer of the liver. Indeed, hepatitis C is considered to be the leading cause of liver cancer and liver transplants in Europe and the USA.
No vaccine is currently available to prevent hepatitis C, which remains an unmet need; therapy with peg interferon alpha (PEG-IFN alfa) and ribavirin has represented for many years the standard of care in patients infected with hepatitis C virus (HCV) and only very recently direct antiviral agents (DAA) have been licensed allowing the establishment of “interferon-free” regimens. Nevertheless the new anti-HCV drugs are so expensive that many countries (including several in the EU) restrict their use to patients with significant fibrosis progression. Therefore, the search for medicinal products with a novel and different mechanism of action is not over.
The HCV New inhibitors project aimed to perform multidisciplinary studies having both co-operative advantages and also scientific and technical objectives specific for each SMEP.
In the eight months of the project the preliminary set up for the in vitro antiviral testing were developed using two different HCV-cell culture systems (JFH, genotype 1a, and TNcc genotype 2a) using ribavarin.
In the same time the synthesis of the new compounds started and the intermediate molecules 3 and 4 produced.
Project Results:
The results of the preliminary work, done before the decision of early termination of the project, are summarized as follows:
• For the HCV inhibition screening tests we obtained (by means of Material Transfer Agreement with Dr. C Rice, APATH New York, USA and J. Buch Copenhagen University Hospital, respectively) and propagated two HCV clones. JFH1 (genotyoe 2a) and TNcc (genotype 1a). The produced viral particles have been titrated, using the focus forming unit protocol (FFU), and stocked at -80°C.
• A MTA with Dr. A. Patel (University of Glasgow) was signed to obtain a reporter cell line called HuH7-J20 in which viral growths can be determined by a luminometer measuring secreted alkaline phosphatase (SEAP).
• The viral inhibitory test was set up using ribavirin as a reference product while waiting the new inhibitors targeting the glycans. The test was performed using the standard FFU method and the results compared with those obtained with the SEAP production. These experiments gave similar results, with values of 19 and 30 µM for the two models, respectively
• In parallel, the process of chemical synthesis started; an analytical method by Thin Layer Chromatography (TLC) to monitor the process was developed, and the first two intermediates were produced.
• To obtain the intermediate 3 and intermediate 4 proved more difficult than expected. In fact, the intermediates obtained are not stable and decompose when trying to isolate the reaction product. The problem is worse for intermediate 4, possibly due to excess of the oxidizing agent.
Potential Impact:
not applicable
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. In the EU, in 2013, 32,512 cases of hepatitis C were reported in 26 EU/ EEA Member States, a crude rate of 9.9 per 100 000 population with an estimate number of EU citizens currently affected by hepatitis C of 5.5 millions. Among them, given the naturally history of the disease, approximately 70,000 Europeans may be dying from chronic liver diseases every year. Chronic hepatitis C leads also to higher risks of developing cirrhosis and cancer of the liver. Indeed, hepatitis C is considered to be the leading cause of liver cancer and liver transplants in Europe and the USA.
No vaccine is currently available to prevent hepatitis C, which remains an unmet need; therapy with peg interferon alpha (PEG-IFN alfa) and ribavirin has represented for many years the standard of care in patients infected with hepatitis C virus (HCV) and only very recently direct antiviral agents (DAA) have been licensed allowing the establishment of “interferon-free” regimens. Nevertheless the new anti-HCV drugs are so expensive that many countries (including several in the EU) restrict their use to patients with significant fibrosis progression. Therefore, the search for medicinal products with a novel and different mechanism of action is not over.
The HCV New inhibitors project aimed to perform multidisciplinary studies having both co-operative advantages and also scientific and technical objectives specific for each SMEP.
In the eight months of the project the preliminary set up for the in vitro antiviral testing were developed using two different HCV-cell culture systems (JFH, genotype 1a, and TNcc genotype 2a) using ribavarin.
In the same time the synthesis of the new compounds started and the intermediate molecules 3 and 4 produced.
Project Results:
The results of the preliminary work, done before the decision of early termination of the project, are summarized as follows:
• For the HCV inhibition screening tests we obtained (by means of Material Transfer Agreement with Dr. C Rice, APATH New York, USA and J. Buch Copenhagen University Hospital, respectively) and propagated two HCV clones. JFH1 (genotyoe 2a) and TNcc (genotype 1a). The produced viral particles have been titrated, using the focus forming unit protocol (FFU), and stocked at -80°C.
• A MTA with Dr. A. Patel (University of Glasgow) was signed to obtain a reporter cell line called HuH7-J20 in which viral growths can be determined by a luminometer measuring secreted alkaline phosphatase (SEAP).
• The viral inhibitory test was set up using ribavirin as a reference product while waiting the new inhibitors targeting the glycans. The test was performed using the standard FFU method and the results compared with those obtained with the SEAP production. These experiments gave similar results, with values of 19 and 30 µM for the two models, respectively
• In parallel, the process of chemical synthesis started; an analytical method by Thin Layer Chromatography (TLC) to monitor the process was developed, and the first two intermediates were produced.
• To obtain the intermediate 3 and intermediate 4 proved more difficult than expected. In fact, the intermediates obtained are not stable and decompose when trying to isolate the reaction product. The problem is worse for intermediate 4, possibly due to excess of the oxidizing agent.
Potential Impact:
not applicable