Susceptibility to simvastatin-induced toxicity is partly determined by mitochondrial respiration and phosphorylation state of Akt.

Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10µM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.

Cyclic nucleotide phosphodiesterase profiling reveals increased expression of phosphodiesterase 7B in chronic lymphocytic leukemia.

Cyclic nucleotide phosphodiesterase (PDE) isoforms can influence disease pathogenesis and be novel therapeutic targets. Because lower cAMP levels may contribute to the decreased apoptosis that occurs in chronic lymphocytic leukemia (CLL), we assessed the expression levels of PDE isoforms in peripheral blood mononuclear cells (PBMC) of healthy adults and patients with CLL. We found a unique PDE mRNA signature in CLL: higher levels than in normal PBMC of PDE7B (increased approximately 23-fold) and lower levels of PDE3B, 4D, 5A, and 9A mRNA (each decreased approximately 30-fold). Increased PDE7B mRNA in CLL correlates with a 10-fold-higher expression of PDE7B protein and results in an increased contribution of PDE7 to total PDE activity. Consistent with the higher level of PDE7B expression, inhibitors of PDE7 (BRL-50481, IR-202) and a dual PDE4/PDE7 inhibitor (IR-284) selectively increase apoptosis in CLL cells compared with normal PBMC or B cells. Apoptosis of CLL cells promoted by inhibitors of PDE7 and PDE4/7 is attenuated by PKA inhibition, occurs via a mitochondrial-dependent process, and is associated with increased cAMP accumulation and down-regulation of the antiapoptotic protein survivin and of PDE7B. The increase in PDE7B expression and PDE7 inhibitor-promoted apoptosis implicates PDE7B as a drug target in CLL. Our findings identify a unique PDE signature in CLL and illustrate the utility of broad analyses of PDE isoform expression in human disease.

Hepatocellular toxicity of clopidogrel: mechanisms and risk factors.

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100 µM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100 µM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100 µM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100 µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100 µM) in HepG2/CYP3A4 supersome. Co-incubation with 1 µM ketoconazole or 10mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100 µM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100 µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.

The role of CYP3A4 in amiodarone-associated toxicity on HepG2 cells.

Amiodarone is a class III antiarrhythmic drug with potentially life-threatening hepatotoxicity. Recent in vitro investigations suggested that the mono-N-desethyl (MDEA) and di-N-desethyl (DDEA) metabolites may cause amiodarone's hepatotoxicity. Since cytochrome P450 (CYP) 3A4 is responsible for amiodarone N-deethylation, CYP3A4 induction may represent a risk factor. Our aim was therefore to investigate the role of CYP3A4 in amiodarone-associated hepatotoxicity. First, we showed that 50µM amiodarone is more toxic to primary human hepatocytes after CYP induction with rifampicin. Second, we overexpressed human CYP3A4 in HepG2 cells (HepG2 cells/CYP3A4) for studying the interaction between CYP3A4 and amiodarone in more detail. We also used HepG2 wild type cells (HepG2 cells/wt) co-incubated with human CYP3A4 supersomes for amiodarone activation (HepG2 cells/CYP3A4 supersomes). Amiodarone (10-50µM) was cytotoxic for HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes, but not for HepG2 cells/wt or less toxic for HepG2 cells/wt incubated with control supersomes without CYP3A4. Co-incubation with ketoconazole, attenuated cytotoxicity of amiodarone incubated with HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes. MDEA and DDEA were formed only in incubations containing HepG2 cells/CYP3A4 or HepG2 cells/CYP3A4 supersomes but not by HepG2 cells/wt or HepG2 cells/wt with control supersomes. Metabolized amiodarone triggered the production of reactive oxygen species, induced mitochondrial damage and cytochrome c release, and promoted apoptosis/necrosis in HepG2 cells/CYP3A4, but not HepG2 cells/wt. This study supports the hypothesis that a high CYP3A4 activity is a risk factor for amiodarone's hepatotoxicity. Since CYP3A4 inducers are used frequently and amiodarone-associated hepatotoxicity can be fatal, our observations may be clinically relevant.

Effects of drug interactions on biotransformation and antiplatelet effect of clopidogrel in vitro.

BACKGROUND AND PURPOSE: The conversion of clopidogrel to its active metabolite, R-130964, is a two-step cytochrome P450 (CYP)-dependent process. The current investigations were performed to characterize in vitro the effects of different CYP inhibitors on the biotransformation and on the antiplatelet effect of clopidogrel. EXPERIMENTAL APPROACH: Clopidogrel biotransformation was studied using human liver microsomes (HLM) or specific CYPs and platelet aggregation using human platelets activated with ADP. KEY RESULTS: Experiments using HLM or specific CYPs (3A4, 2C19) revealed that at clopidogrel concentrations >10 microM, CYP3A4 was primarily responsible for clopidogrel biotransformation. At a clopidogrel concentration of 40 microM, ketoconazole showed the strongest inhibitory effect on clopidogrel biotransformation and clopidogrel-associated inhibition of platelet aggregation with IC(50) values of 0.03 +/- 0.07 microM and 0.55 +/- 0.06 microM respectively. Clarithromycin, another CYP3A4 inhibitor, impaired clopidogrel biotransformation and antiplatelet activity almost as effectively as ketoconazole. The CYP3A4 substrates atorvastatin and simvastatin both inhibited clopidogrel biotransformation and antiplatelet activity, less potently than ketoconazole. In contrast, pravastatin showed no inhibitory effect. As clopidogrel itself inhibited CYP2C19 at concentrations >10 microM, the CYP2C19 inhibitor lansozprazole affected clopidogrel biotransformation only at clopidogrel concentrations < or =10 microM. The carboxylate metabolite of clopidogrel was not a CYP substrate and did not affect platelet aggregation. CONCLUSIONS AND IMPLICATIONS: At clopidogrel concentrations >10 microM, CYP3A4 is mainly responsible for clopidogrel biotransformation, whereas CYP2C19 contributes only at clopidogrel concentrations < or =10 microM. CYP2C19 inhibition by clopidogrel at concentrations >10 microM may explain the conflicting results between in vitro and in vivo investigations regarding drug interactions with clopidogrel.