Experimental And Clinical Research On Mycophenolate Mofetil Therapy For Hepatic Failure

Background Hepatitis B virus infection is common in china, witha infection rate of 10%-12%, and approximately 1%-2% of patients withhepatitis B developed hepatic failure, which is named as severe hepatitis.As is know, the pathogenesis of hepatic failure induced by hepatitis virusand other pathogenic factors was described as "two hit injury". Liverinjury induced by various pathogenic factors (such as hepatitis virus,ethanol, drugs and hepatotoxicants, etc.) through their respective specialpathogenesis is referred to as "primary liver injury" (PLI), which isregarded as "the first hit". Liver injury resulted from endotoxin(lipopolysaccharide, LPS) and the activation of Kupffer cells by LPSwhile intestinal endotoxemia (IETM) occurred during the occurrence anddevelopment of hepatitis is named the "secondary liver injury" (SLI),which is regarded as "the second hit". The latter which has lost their ownspecificities of primary pathogenic factors is ascribed to IETM. The"secondary liver injury" is of important action and impact ondevelopment and prognosis of hepatitis. More severe IETM commonlyresults in excessive inflammatory responses, with serious hepaticnecrosis, further severe hepatitis and even induces acute liver failure.Mycophenolate mofetil(MMF), the morpholinoethyl ester of MPA, iscurrently used as an immunosuppressive agent in transplant recipients.After oral administration, MMF is hydrolysed to Mycophenolic acid(MPA), the active compound. MPA is a non-nucleoside inhibitor ofinosine mono-phosphate dehydrogenase(IMP-DH), which limits the rateof de novo synthesis of guanosine nucleotides. Because proliferation of Tand B cells is predominantly dependent on this pathway, its blockadeinhibits their expansion. And MPA has been shown to inhibit theproduction of proinflammation cytokines such as tumour necrosis factoralpha(TNF-α), interferon gamma(IFN-γ), and interleukin-1(IL-1).Recently, MPA has been shown to inhibit the replication of a number ofviruses, including yellow fever virus, Epstein-Barr virus, humanimmunodeficiency virus, and dengue virus.Objective Based on the immunological dysfunction in severehepatitis and MMF's immunomodulatory feature with high effication andlow toxicity, we assumed that MMF might have therapeutic effect onsevere hepatitis. In this study, therefore, we have clarified thetherapeutical effect of MPA on severe hepatitis.Methods(1) In vitro antiviral efficacy of Mycophenolic acid against hepatitisB virus. The different concentrations of the test drugs in DMEM wereadded to the well with the monolayer growth of HepG2 2.2.15 cells. Theculture medium was refreshed with DMEM containing the appropriatedrugs every 3 days. At 3rd, 6th and 9th days, the supernatant wascollected for HBsAg quantitative assay, total RNA was isolated fromcells for RT-PCR and realtime-PCR assays.(2) Prevention of Lethal Acute Hepatic Failure by MMF in MiceTreated With Bacille Calmette-Guerin(BCG) and Lipopolysaccharide(LPS). Acute liver failure was induced in male Kunming strain mice byinjecting the animals with BCG (2.5 mg/mouse), and administering LPS(10 μg/mouse) ten days later. The mice in treatment groups were givenMMF 2 hr before, simultaneous with, or 2 hr after administration of LPS,and the mice in control group was given the same dose of saline. The24-hr survival rate, serum alanine aminotransferase(ALT), aspartateaminotransferase(AST) levels were compared. Serum levels of TNF-α,IFN-γ and IL-6 were measured and the expressions of TNF-α, IFN-γand IL-6 mRNA in the liver were determined by reverse transcriptionpolymerase chain reaction(RT-PCR). Concanavalin A (ConA)-inducedsplenocyte proliferation response were determined by methods of MTT.(3) Preliminary clinical research on treatment of severe hepatitis B byMMF. The treatment group included 25 chronic severe hepatitis Bpatients. Based on routine treatments, they were oral taken with MMF500mg per 8hrs. The control group included 25 chronic severe hepatitis Bpatients. They were only treated with routine treatments for severehepatitis, include drugs for hepatocyte protection, jaundice regression,nutrition support, antibiotics, and artifical liver treatment should be givenwhen it is needed.Results(1) It was found no significant anti-HBV effect of mycophenolicacid on concentration of 2-250 μg/ml, and mini inhibitory efficacy ofHBV replication on concentration of 1,250 μg/ml. There was significantanti-HBV effect of lamivudine on concentration of 5 μg/ml. Whencombined with lamivudine mycophenolic acid can significantlypotentiate anti-HBV activities of lamivudine, and it can get maximalinhibition effect on concentration of 10 μg/ml.(2) Injecting a small dose of LPS into BCG-primed mice caused alethal hepatic injury mimicking fulminant hepatitis, in which 4 of 20mice died within 24 hours (20% survival rate). Massive necrosis ofparenchymal hepatocytes with marked inflammatory cell infiltration wasobserved by histological examination. In parallel, serumaminotransferase and TNF-α, IFN-γ, IL-6 levels were increased.Expression of TNF-α, IFN-γ, and IL-6 mRNA in the liver weresignificantly increased also. Treatment with MMF markedly reduceddeath rate in a dose-dependent manner. It reached its maximal effect atthe dosage of 150mg per kg body weight when pretreated 2 hours beforeLPS injection, with improvement of histological feather and survival rate(84.2%, 16/19). MMF significantly inhibited serum levels of TNF-α,IFN-γ, and IL-6, and significantly reduced TNF-α, IFN-γ, and IL-6expression in the liver, which increased after BCG and LPS injection.Moreover, Splenocyte proliferation response induced by Con A was alsoinhibited by MMF treatment on fulminant hepatic failure.(3) Mean values for ALT, total bilirubin(TB), prothrombin time(PT),days from onset of symptoms to initiation of treatment, and cause of FHFwere similar between treatment group and control group. Theimprovement rate of clinical situation after MMF treatment was80%(20/25), significantly higher than that of control group(52.0%,13/25), and that of other chronic severe hepatitis inpatients in thesame period (58.6%, 98/167 ). The TNF-α and IL-6 concentration inserum of treatment group patients were significantly lower than that ofcontrol group.Conclusion(1) Mycophenolic acid could inhibit HBV replication and enhanceanti-HBV activities of lamivudine.(2) Treatment with the MMF suppresses the endotoxin-induced fatalhepatic failure by regulating production of inflammatory cytokines andT-cell proliferation.(3) MMF was effective and safe for treatment of severe hepatitis B.