Exploring an animal model of amodiaquine-induced liver injury in rats and mice.

Amodiaquine (AQ) is associated with a relatively high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. A previous study reported that a combination of high dose AQ and glutathione (GSH) depletion led to liver injury. However, the characteristics of this toxicity were very different from AQ-induced liver injury in humans. We developed a model of AQ-induced liver injury with characteristics similar to the injury in humans by treating mice with lower doses of AQ for several weeks. In this study we found that not only did GSH depletion not increase AQ covalent binding to hepatic proteins at this lower dose, but also it paradoxically prevented the liver injury. We extended the model to rats and found AQ treatment led to a mild delayed onset liver injury that resolved despite continued treatment with AQ. Immunohistochemistry indicated the presence of Kupffer cell activation, apoptosis and hepatocyte proliferation in the liver. There was also an increase in serum IL-2, IL-5, IL-9, IL-12, MCP-1 and TGFß, but a decrease in leptin. Coincident with the elevated serum ALT, the number of liver CD4(+) T-cells, IL-17 secreting cells and TH17/Treg cells increased at Week 3 and decreased during continued treatment. Increases in NK1.1+ cells and activated M2 macrophages were also observed during liver injury. These results suggest that the outcome of the liver injury was determined by the balance between effector and regulatory cells. Co-treatment with cyclosporin prevented AQ-induced liver injury, which supports an immune mechanism. Retinoic acid (RA), which has been reported to enhance natural killer (NK) cell activity, exacerbated AQ-induced liver injury. These results suggest that AQ-induced IDILI is immune mediated and the subsequent adaptation appears to represent immune tolerance.

Amodiaquine, an antimalarial drug, inhibits dengue virus type 2 replication and infectivity.

Dengue virus serotypes 1-4 (DENV1-4) are transmitted by mosquitoes which cause most frequent arboviral infections in the world resulting in ∼390 million cases with ∼25,000 deaths annually. There is no vaccine or antiviral drug currently available for human use. Compounds containing quinoline scaffold were shown to inhibit flavivirus NS2B-NS3 protease (NS2B-NS3pro) with good potencies. In this study, we screened quinoline derivatives, which are known antimalarial drugs for inhibition of DENV2 and West Nile virus (WNV) replication using the corresponding replicon expressing cell-based assays. Amodiaquine (AQ), one of the 4-aminoquinoline drugs, inhibited DENV2 infectivity measured by plaque assays, with EC50 and EC90 values of 1.08±0.09µM and 2.69±0.47 µM, respectively, and DENV2 RNA replication measured by Renilla luciferase reporter assay, with EC50 value of 7.41±1.09µM in the replicon expressing cells. Cytotoxic concentration (CC50) in BHK-21 cells was 52.09±4.25µM. The replication inhibition was confirmed by plaque assay of the extracellular virions as well as by qRT-PCR of the intracellular and extracellular viral RNA levels. AQ was stable for at least 96h and had minor inhibitory effect on entry, translation, and post-replication stages in the viral life cycle. DENV protease, 5'-methyltransferase, and RNA-dependent RNA polymerase do not seem to be targets of AQ. Both p-hydroxyanilino and diethylaminomethyl moieties are important for AQ to inhibit DENV2 replication and infectivity. Our results support AQ as a promising candidate for anti-flaviviral therapy.

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.