Research
Gene therapy for liver cirrhosis
T Ueki MD 1, J Fujimoto MD 1, T Hirano MD 1, K Tei MD 1, M Takeuchi MD1,
Y Kaneda MD 2, K Matsumoto PhD 3, T Nakamura PhD 3 and E Okamoto MD. 1
1 First dept. of Surgery, Hyogo College of Medicine, Nishinomiya, Japan. 2 Institute for Mol. and Cellular Biology, and 3 Biomedical Research Center, Osaka University Medical School, Suita, Japan.
AIM
Liver cirrhosis is a major cause of morbidity and mortality worldwide with no effective therapy. Liver cirrhosis often complicates hepatocellular carcinoma (HCC). We show here a therapeutic approach for established rat liver cirrhosis by muscle directed transgene of hepatocyte growth factor (HGF). METHODS/ RESULTS: To achieve persistent in vivo HGF gene expression in rats, we used hemagglutinating virus of Japan (HVJ)-liposome. Liver cirrhosis induced by dimethylnitrosamine(DMN) is characterized by parenchymal collapse followed by hyper-accumulation of extracellular matrix components which mimic human liver cirrhosis. All cirrhotic rats died of liver dysfunction by 7 weeks after the initial injection. In contrast, repetitive transfer of the HGF gene into skeletal muscle achieved a sustained high level in the plasma HGF level, and markedly activated c-Met/ HGF-receptor in the cirrhotic liver. HGF gene therapy markedly suppressed the expression of transforming growth factor-ß 1 (TGF-ß 1), a major growth factor which induce liver fibrosis and apoptosis of hepatocytes. Northern blot revealed the suppression of expression of mRNA of TGF-ß 1. HGF inhibited hepatocyte apoptosis, and stimulated hepatocyte mitosis. Azan-Mallory/ hematoxillin staining revealed that the fibrous component in the liver disappeared after the repetitive HGF gene therapy, and mortality was completely abrogated. CONCLUSIONS: Our present results indicate that HGF gene therapy improved experimental rat liver cirrhosis and may hold promise for the treatment of patients with liver cirrhosis.
Hepatocyte Cell Culture System
Hepatocytes are epithelial cells found in the liver. They perform important functions, such as helping to detoxify blood, and to synthesize transport proteins, such as lipoprotein, albumin and transferrin. Primary and secondary cultures of hepatocytes are useful for studying the mechanisms of liver regeneration and differentiation. Historically, primary hepatocytes have exhibited a limited replicating lifespan in culture. In addition, when stimulated to divide in culture they have generally lost differentiated functions such as the ability to synthesize and secrete albumin and transferrin. While substantial progress has made in understanding the factors which affect these characteristics of hepatocytes in culture, primary and secondary hepatocytes are still not suitable for use in some emerging medical technologies and applications. Among the emerging potential applications for cultured hepatocytes are gene therapy, bio-artificial organs, cell transplants, drug production, and drug and chemical testing. These technologies may benefit from the use of cloned and immortalized hepatocytes. Hence, there is a significant effort to develop hepatocyte cell lines for this purpose.
Hepatocyte Cell Culture Medium
Many classical media (see web-pages in our "Classical Media" section) have been used to culture epithelial hepatocytes. Some examples of these are; Liebovitz L-15, DMEM/F-12, RPMI 1640, Waymouth's MB 752/1 and Williams Medium E. Growth of hepatocytes in these media requires the use of of serum.
Our most advanced formulation for culturing primary, secondary, and cloned immortalized epithelial hepatocytes is H 1777, which is an enriched variation of Leibovitz L-15 formulation. It may be successfully used with reduced concentrations of FBS.
FDA Issues "Talk Paper" on Approval of Hepsera
The FDA announced on Friday (9-20/02) its approval of Hepsera, the new anti-HBV drug from Gilead Sciences for the treatment of chronic hepatitis B. The approval follows a unanimous recommendation for accelerated approval of the drug in August from the FDA's Antiviral Advisory Committee. Hepsera becomes only the third drug that is FDA-approved for the treatment of hepatitis B, a potentially life-threatening disease that infects approximately 1.2 million Americans.
The Food and Drug Administration (FDA) opn 9/20/02 announced the approval of Hepsera (adefovir dipivoxil) tablets for the treatment of chronic hepatitis B in adults with evidence of active viral replication and either elevations in serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST), or histologically active disease.
Chronic hepatitis B is a serious disease caused by a virus that attacks the liver. The virus, which is called hepatitis B virus (HBV), can cause lifelong infection, cirrhosis (scarring) of the liver, cancer, liver failure, and death. According to the Centers for Disease Control and Prevention, approximately 1.25 million Americans are chronically HBV infected.
Hepsera slows the progression of chronic hepatitis B by interfering with viral replication and causing DNA chain termination after its incorporation into viral DNA.
FDA based its approval of Hepsera on the results of two randomized, double-blind, placebo-controlled studies. At week 48 of the studies, 53% of patients receiving Hepsera in one study and 64% of patients in the other study showed significant improvement in the liver inflammation caused by HBV compared to 25% and 35% of patients receiving placebo. A statistically significant improvement in the degree of liver fibrosis (scarring) was observed in the patients who received Hepsera. Moreover, Hepsera has been shown to be effective in treating patients with clinical evidence of HBV that is resistant to another approved antiviral therapy called lamivudine.
The major adverse events associated with the use of Hepsera include severe, acute exacerbation of hepatitis B after discontinuation of Hepsera and kidney toxicity. Patients who have discontinued other approved products for the treatment of chronic hepatitis B have also experienced severe, acute exacerbation of hepatitis. This adverse event occurred in up to 25% of clinical trial participants after discontinuation of Hepsera. The labeling for Hepsera states that patients who discontinue Hepsera should be monitored at repeated intervals over a period of time for hepatic function.
Kidney toxicity was reported in patients at risk of or having underlying kidney dysfunction. In addition, there is a theoretical concern associated with Hepsera that HIV resistance could emerge in chronic hepatitis B patients with unrecognized or untreated HIV infection.
Entecavir Trial for Chronic Hepatitis B
Bristol-Myers Squibb is conducting Phase 3 studies of entecavir, a once-daily oral antiviral agent, in adults with chronic hepatitis B infection. The studies are being conducted in approximately 130 sites in more than 30 countries worldwide.
Goal of Phase 3 Studies
Phase 3 studies are being conducted in a broad range of patients with chronic hepatitis B to evaluate the safety and efficacy of entecavir and determine its ability to achieve and sustain histologic improvement in the liver and reductions in viral levels relative to the current standard of therapy Lamivudine).
Three different studies are being conducted based on the results of patients’ serological status (hepatitis B e-antigen positive or negative), and whether the patient is currently on lamivudine therapy and has evidence of resistance to lamivudine.
Who is Eligible?
Individuals 16 years of age and older who:
- Have a history of chronic hepatitis B for at least 6 months
- Have liver disease caused by the hepatitis B virus
- Do not have HIV, hepatitis C, or hepatitis D
- Do not have liver disease caused by alcohol, biliary disease, or cancer
Additional study eligibility criteria will be reviewed with the patient by the investigator.
What Happens in the Study?
Eligible patients will receive a study-related medical evaluation to determine if they qualify for study participation. If the patient chooses to participate in the study, he/she will receive investigational medication, and be under a doctor’s supervision throughout the duration of the study. Patients will be randomized to receive either entecavir or standard therapy with lamivudine. Neither the patient nor the doctor will be aware of which treatment the patient is receiving. Participants will be asked to participate for a minimum of 64 weeks and to make at least 19 study related visits.
Participants will be required to have a pre-treatment liver biopsy within the past year of randomization into the study or during the screening phase for the study and a second biopsy after receiving 48 weeks of study therapy. After completing the study, all participants will be monitored periodically for up to 5 years for survival and incidence of hepatitis B-related complications.
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