In this study, we demonstrate that in vitro exposure of spinal motoneurons to selected statins, especially fluvastatin, resulted in marked cell loss. Shorter term exposures to fluvastatin resulted in neuritic degeneration. Other statins, specifically pravastatin and simvastatin, also exhibited toxic effects towards spinal motor neurons, but at much higher concentrations. The susceptibility of spinal motor neurons was in contrast to primary cultures of cortical neurons and peripheral nerve-derived Schwann cells which did not demonstrate significant deleterious changes. The results of this study provide evidence that specific pathophysiological mechanisms may underlie reports of neuromuscular disease exacerbation with statin exposure, however the clinical implications of this study remain to be determined.
There are multiple factors which might account for the differences between the clear in vitro toxicity reported here and the uncertainty surrounding observations of statin toxicity for spinal motor systems in clinical settings. Among the biological factors proposed to explain the wide clinical tolerability of statins are pharmacokinetics. Statins as a group are subject to high first-pass metabolism by the liver. This means that systemic levels are substantially below what might be predicted based on simple dilutional calculations. Statins do vary in the degree of first pass metabolism with the earlier-developed statins being more extensively metabolized [6
]. Interestingly the low first-pass metabolism of cerivistatin, initially hailed as a breakthrough, may have contributed to the high-rate of serious adverse events [3
]. In addition, most statins are hydrophobic, with several newer statins being especially so. Pravastatin is uniquely hydrophilic and likely requires distinct consideration when considering systemic effects. In our study, there was the suggestion that pravastatin may be somewhat less toxic to cultured spinal motoneurons that fluvastatin, however further studies are needed. Finally, compartmental pharmacokinetics may limit the effects of statins on spinal motor neurons. The extent to which specific statins attain significant concentrations in the spinal fluid is not well understood. Studies in laboratory animals have indicated that the permeability of lipophilic statins, simvastatin and lovastatin into rat brain is orders of magnitude greater than that of pravastatin [20
]. Thus, there are multiple pharmacokinetic reasons why statins may exhibit lower in vivo than in vitro toxicity.
Pharmacokinetic data for fluvastatin from clinical trials of the drug, suggest that a more cautious interpretation may warranted. From the early pharmacokinetic studies in man, it is estimated that the peak daily serum levels of fluvastatin after a 40
mg dose is in the low single micromolar range [21
]. The results of the fluvastatin dose response studies we report here provide evidence that the drug concentration necessary to produce 50% toxicity for spinal motoneurons in culture is less than 1
μM. Taken together, these data suggest that systemic levels of statins may approach levels that are toxic to spinal motoneurons and that particular mechanisms must account for the relatively infrequent occurrence of clinically demonstrable toxicity. Perhaps sequestration of spinal motoneurons in the spinal space is one such mechanism.
The pharmacokinetics of fluvastatinare are such that peak concentrations are present in the body for only a short period of time following single-dose administration [21
]. For this reason, early fluvastatin treatment followed a twice-daily dosing regimen. This approach has subsequently been supplanted by the use of extended release formulations but for reasons that are not known, fluvastatin remains among the least commonly prescribed statins.
Based on these studies, it is not possible to specify a mechanism or mechanisms by which statins produce depletion of spinal motoneurons in culture. Intriguingly, somewhat analogous effects of statins have been reported in myotube cultures, where it was observed that fluvastatin and simvastatin exhibit clearly evident toxicity, the effects of pravastatin were apparently more mild [6
]. Mechanisms by which statins have produced undesirable and desirable effects in other systems continue be explored. The effects of statins are widely acknowledged to be protean. Statins have profound impacts on inflammatory signaling pathways [22
], are known to effect the linkage of G proteins to the various cell membranes [24
], are thought to play a role in cancer signaling [26
], have been shown to have direct actions on carnitine palmitoyl transferase localized to mitochondrial membranes [6
], can disrupt oxidative phosphorylation and mitochondrial membrane potential [6
] as well as interfering with cholesterol synthesis [27
]. As an example of a one potential mechanism, statins have been shown to disrupt the attachment of Rho and Ras-family G proteins to cell membranes. It is interesting that one Ras-family protein, Rab5, is critically important for early neuronal endocytosis [28
] and has been implicated in producing an ALS phenotype resulting from Alsin mutations [29
]. The se animals may represent an especially attractive model system for further exploration of statin effects on motor neurons. Among these studies is a report that statins promote apoptosis of a glioma cell line mediated by ERK1/2 and AKT [25
]. This is in contrast to our observations of robust resistance to statin effects in primary cultures of Schwann cells suggesting that important differences between normal and transformed glia that underlie statin effects on glioma cells. Thus, statins have a variety of effects on cell signaling. We here describe the observation of deleterious effects on the survival of spinal motor neurons in culture and because the mechanisms of statin effects on specific neuronal cell populations remain relatively obscure, additional study is warranted.