David F. Mercer

Hepatitis C virus replication in mice with chimeric human livers

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti‐hepatitis C virus activity In Vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus

Anti‐hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC50) values of 0.01 to 0.14 μM for genotype 1, with half maximal effective concentration (EC50s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ± 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV‐796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA‐dependent RNA polymerase inhibitor HCV796 demonstrated potent anti‐HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose‐related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti‐HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment. (HEPATOLOGY 2009.)

Anti-HCV therapies in chimeric scid-Alb/uPA mice parallel outcomes in human clinical application†

Compounds with in vitro anti‐hepatitis C virus (HCV) activity are often advanced directly into clinical trials with limited or no in vivo efficacy data. This limits prediction of clinical efficacy of compounds in the HCV drug pipeline, and may expose human subjects to unnecessary treatment effects. The scid‐Alb‐uPA mouse supports proliferation of transplanted human hepatocytes and subsequent HCV infection. Cohorts of genotype 1a HCV‐infected mice were treated with interferon α‐2b(IFN‐α), BILN‐2061 (anti‐NS3 protease), or HCV371 (anti‐NS5B polymerase). Mice treated with 1350IU/g/day IFN‐α intramuscularly for 10 to 28 days demonstrated reduced viral titers compared with controls in all five experiments (P < .05, t test); viral titers rebounded after treatment withdrawal. A more pronounced antiviral effect with IFN‐α was seen in genotype 3a–infected mice. Pilot studies with BILN2061 confirmed exposure to 10X replicon EC50 at trough and reduced viral titer over 2 log at 4 days. In a second 7‐day study, mean HCV RNA titers dropped 1.1 log in BILN2061‐treated animals, 0.6 log in IFN‐treated mice, and rose 0.2 log in controls (P = .013, ANOVA). Pre‐existing mutants with partial resistance to BILN2061 were identified by sequencing both the human inoculum and sera from treated mice. The polymerase inhibitor HCV371 yielded a decline in HCV titers of 0.3 log relative to vehicle‐treated controls (P = NS). Performance of all three antiviral regimens in the chimeric mouse model paralleled responses in humans. In conclusion, this system may help selection of lead compounds for advancement into human trials with an increased likelihood of clinical success while broadening the tools available for study of the biology of HCV infection. (HEPATOLOGY 2006;43:1346–1353.)

Mice with chimeric human livers: Who says supermodels have to be tall?

In 1990 a group of scientists led by Ralph Brinster, while developing a model of neonatal bleeding disorders, generated a novel murine line carrying a urokinase-type plasminogen activator transgene controlled by an albumin promoter (Alb-uPA).1 Urokinase overproduction (targeted to the liver by the Alb promoter) yielded pups with a bleeding phenotype. While early perinatal mortality rates were high due to bleeding complications, some animals survived this early period only to succumb to subacute liver failure at 2-3 weeks of age; expression of the Alb-uPA transgene had proven to be toxic to the mouse hepatocytes. Not all animals died at this point, however, and when the investigators examined these late survivors, they found that the bleeding phenotype had been lost.2 An elegant series of experiments showed that hepatocytes within the transgenic liver had spontaneously deleted a portion of the transgene. Freed from the constraints of its expression, these nontransgenic hepatocytes began to proliferate and replace the diseased hepatic parenchyma with “normal” functioning hepatocytes, an advantage they recognized could be shared by transplanted hepatocytes also lacking the transgene. Demonstration of the ability to “rescue” the mice from liver failure with congenic hepatocyte transplants3 and subsequently xenografted rat hepatocytes followed.4 Petersen et al.5 and Dandri et al.6 moved forward with woodchuck and human hepatocyte transplantation respectively into mice hemizygous for the Alb/uPA transgene (on an immunodeficient RAG background). They were able to generate modest repopulation of livers, and support for infection with woodchuck hepatitis B virus and human hepatitis B virus (HBV). Our own group began working to develop an animal model of hepatitis C (HCV) infection in 1994, and successfully established HCV infections in scid/Alb-uPA mice carrying the transgene in homozygous fashion where liver cell repopulation frequently exceeded 50% of liver area on immunohistological analysis.7 The chimeric scid/Alb-uPA mouse in homozygous form, is a challenging model. Immunodeficiency and the bleeding tendency inherent in the transgene result in significant risk from infection and death due to perinatal trauma respectively. Optimal repopulation requires transplantation with high quality human hepatocytes within a short period after birth and so creates significant challenge for many laboratories due to difficulty accessing suitable tissue. Not all mice transplanted achieve high level repopulation, and not all those with high level repopulation develop high titre HCV infection after delivery of standardized inocula. Variations in transgene expression or activity of residual immune cells could be some of the answers as to why such variation occurs. Clearly more development and refinement of this model is needed to facilitate broader application in the research community. Nevertheless, we believe the model is the best small animal model of HCV infection currently available, and holds promise of useful application in many other fields. In the current issue of this journal, Meuleman et al. present a detailed series of experiments that more fully characterizes the repopulation of the mouse liver with human hepatocytes in homozygous scid-Alb/uPA mice.8 Histological analysis of the chimeric liver reveals direct communication between biliary radicals of mouse and human origin. Clearly these are not isolated nodules of human hepatocytes in a mouse liver, but rather the human hepatocytes have become an integrated component of the mouse liver and maintain life-sustaining function. Aside from accumulation of glycogen, the human hepatocytes appear normal. Function appears to follow form, with the human proteome in the sera of mice inclusive of human albumin and 21 other human specific proteins. The paper also reports infection of chimeric mice with HBV and HCV, providing important independent confirmation of previous reports.6,7 The authors have provided insight into critical details of the model, information of the type that should help focus study to generate a more reliable and reproducible system, a model simpler to use and yet equally as valid in application. A particularly interesting finding is the presence of cells with markers of human hepatic progenitor cells within the parenchyma of the mouse livers; progressive differentiation into mature hepatocytes was suggested by immuAddress reprint requests to: Norman M. Kneteman, University of Alberta Hospitals—Surgery, 2D4.44 WCM Centre HSC University of Alberta Hospital, Mackenzie Hlth Sc. Ctr., Edmonton T6G 2B7 Canada. E-mail: nkneteman@cha.ab.ca; fax: 780-407-7374. Copyright

The role of protein kinase A in anaerobic energy production during liver storage

BACKGROUND/AIM During cold liver storage in University of Wisconsin solution, glycolysis is inhibited by declining intracellular pH and a reduction in glycogen phosphorylase activity. The current study investigated the effects of a histidine-buffered, modified University of Wisconsin solution with cyclic-AMP analogue plus phosphodiesterase inhibitors to optimize both pH and PK A-mediated limits on glycolytic energy production. METHODS In an isolated rodent-liver system, dioctanoyl-cAMP was supplemented with each phosphodiesterase inhibitor (isobutylmethylxanthine, papaverine, Ro 20-1724, dipyridamole). Once the most efficacious combination was determined, a separate group of livers was cold-stored for 24 h and then reperfused at 37 degrees C to examine regeneration of high energy adenylates. RESULTS Lactate accumulation in the histidine-lactobionate-raffinose group was 8.7 micromol/g; net increases were greater with all four phosphodiesterase inhibitors with dioctanoyl-cAMP; dipyridamole resulted in a maximum increase of 16.7 micromol/g. ATP was consistently higher in all treatment groups with phosphodiesterase inhibitors throughout 24 h; even after 10-24 h, levels with dipyridamole-treatment were 250-280% higher than with University of Wisconsin (p<0.05). Assessment of glycogen phosphorylase activity in the dipyridamole-treatment group indicated that increased glycolytic activity over the first 4 h was a direct consequence of elevated enzyme levels. However, between 4-10 h, phosphofructokinase underwent a phosphorylation, leading to an inhibition at this point in glycolysis. Upon reperfusion, the higher ATP/ADP and ADP/ AMP ratios found with phosphodiesterase inhibitor treatment suggested that adenylate regeneration was superior with dipyridamole+dioctanoyl-cAMP. CONCLUSION Dipyridamole plus dioctanoyl-cAMP treatment achieved increased glycogenolysis throughout 24 h storage by maintaining glycogen phosphorylase in a phosphorylated (active) state; however, a PK A-mediated phosphorylation (inhibition) of phosphofructokinase resulted in decreased glycolytic ATP production between 4-10 h.