Several criteria should be considered when selecting immunomodulatory agents for use in combination therapy for MS: (a) medications should have complementary activities, preferentially acting through different mechanisms of action; (b) ideally each medication should have an excellent safety profile; and (c) the combination should not create additional toxicities. In this regard, the 2 FDA-approved medications used in this investigation are well tolerated and have no apparent overlapping toxicities (5
). Data from previous studies indicate that atorvastatin mediates immunomodulatory effects on T cells and APCs primarily through inhibition of synthesis of isoprenoid compounds in the mevalonate pathway (24
). Isoprenylation of small GTP-binding proteins is necessary for their intracellular trafficking and subcellular localization to the cytoplasmic surface of the plasma membrane (33
). Possibly by reducing available ras, which has an important role in ERK activation and Th1 differentiation (34
), statins may facilitate Th2 differentiation (35
). In contrast, GA is considered an antigen-based immunomodulatory agent that has been associated with enhanced secretion of antiinflammatory cytokines by myelin-reactive T cells (12
) and APCs (14
). Thus the mechanisms of action of these 2 drugs are distinct. Having met all of the criteria described above and having demonstrated that optimal doses of atorvastatin and GA did not antagonize one another, we reasoned that these 2 drugs were excellent candidates to be tested in combination therapy.
In this report we established that atorvastatin and GA had synergistic clinical and immunological effects. The combination of suboptimal doses of GA and atorvastatin was as effective in EAE prevention as the optimal dose of either drug alone and promoted Th2 immunomodulation. More importantly, the combination of suboptimal doses of atorvastatin and GA, administered s.c. in aqueous solution, effectively reversed paralysis when daily treatment was initiated after EAE was established. Further, treatment of EAE with this combination was associated with a reduced number of CNS inflammatory lesions and less demyelination. In order to investigate one potential mechanism responsible for the Th2 bias, we examined whether atorvastatin and GA have a combined immunomodulatory effect on APCs. Our results indicate that the combination of GA and atorvastatin facilitated the differentiation of type II (16
) monocytes that secreted an antiinflammatory profile of T cell–polarizing cytokines. Indeed, monocytes matured in the presence of the combination of atorvastatin and GA promoted Th2 differentiation of naive myelin-specific T cells. While other mechanisms may contribute, we have established one potential mechanism that could account for the observed clinical and immunological synergistic effects of this combination. Our results clearly support testing the combination of atorvastatin and GA in clinical MS trials.
Other approved immunomodulatory agents are also being considered for combination therapy in MS (36
). For example, the combination of GA and Avonex, a preparation of interferon β-1a approved for MS treatment, was assessed for safety in a small trial of patients with relapsing-remitting MS (37
) and is now being tested for efficacy in a larger trial (http://www.combirx.org/). Although interferon β acts through a different pathway than statins, these 2 drugs may overlap in their immunomodulatory effects. For example, both interferon β and atorvastatin can induce secretion of antiinflammatory cytokines (20
). Like statins (19
), interferon β also inhibits MHC class II upregulation on APCs (40
). While statins inhibit transcription of the MHC class II transactivator (CIITA) (19
), the “master regulator” for MHC class II expression, interferon β reduces CIITA activity (40
). It is known that interferon β is effective in reducing the number of new radiographically detectable inflammatory MS lesions (41
), presumably by inhibiting secretion of proteases and preventing upregulation of adhesion/costimulatory molecules that participate in leukocyte trafficking into the CNS (43
). Data indicate that through inhibition of rho prenylation in endothelial cells (45
), statin treatment may also inhibit leukocyte trafficking into the CNS (22
). Since it is unclear whether the pleiotropic activities of these 2 drugs will be complementary, studies are necessary to evaluate whether the combination of interferon β and statins will be antagonistic or beneficial (46
As we have shown in this investigation, the EAE model is useful for initial testing of potential combination therapies for MS. One must consider that FDA-approved therapies and several novel therapies, administered individually, potently suppress EAE. As we have described, one strategy to evaluate potential complementary therapeutic benefit is to administer candidate drugs using suboptimal doses. In this regard, atorvastatin and GA were synergistic when administered at doses that had no detectable clinical or immunomodulatory effects alone. The general principles that we have established in this study should be applicable for evaluating other combinations of immunomodulatory agents in the EAE model.