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Expert Opinion on Therapeutic Targets

ISSN: 1472-8222 (Print) 1744-7631 (Online) Journal homepage: http://www.tandfonline.com/loi/iett20

Statins and their potential targets in multiplesclerosis therapy

Olaf Stüve, Thomas Prod’homme, Anthony Slavin, Sawsan Youssef, ShannonDunn, Lawrence Steinman & Scott S Zamvil

To cite this article: Olaf Stüve, Thomas Prod’homme, Anthony Slavin, Sawsan Youssef,Shannon Dunn, Lawrence Steinman & Scott S Zamvil (2003) Statins and their potential targetsin multiple sclerosis therapy, Expert Opinion on Therapeutic Targets, 7:5, 613-622

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1. Multiple sclerosis

2. Statins

3. Rationale for the use of statins

in multiple sclerosis

4. Conclusions and expert opinion

Central & Peripheral Nervous Systems

Statins and their potential targets in multiple sclerosis therapyOlaf Stüve, Thomas Prod’homme, Anthony Slavin, Sawsan Youssef,Shannon Dunn, Lawrence Steinman & Scott S Zamvil††Department of Neurology, University of California, San Francisco, 521 Parnassus Avenue, C-440,

San Francisco, CA 94143-0114, USA

Multiple sclerosis (MS) is a CNS-demyelinating disease characterised by relaps-ing and chronic neurological impairment. While traditionally CNS autoanti-gen-specific CD4+ T cells have been considered the culprits in the initial phaseof the disease, recent observations have altered this concept. It is now recog-nised that other T lymphocyte subclasses can initiate CNS demyelination. Inaddition, other cell types and molecules may play an important role inMS pathogenesis. There is overwhelming evidence that MS is a dynamic proc-ess, in which recurrent episodes of blood–brain barrier disruption andCNS inflammation play a crucial role in early disease stages, leading eventu-ally to the largely irreversible changes of demyelination, gliosis and axonaldegeneration. These observations may have important therapeutic implica-tions. Within the last ten years, several medications have been approved forMS treatment. These agents, all of which are given parenterally, are only par-tially effective and are often associated with adverse effects and potentialtoxicities. The number and different types of medications used for MS arelikely to increase in the near future, as several novel therapies are currentlytested in clinical trials. 3-hydroxy-3-methyglutaryl coenzyme A reductaseinhibitors, ‘statins’, are cholesterol-lowering drugs that are given orally, aresafe and have biological effects independent of their cholesterol-reducingproperties. Recent reports have shown that statins have anti-inflammatoryand neuroprotective properties that may be beneficial in the treatment ofMS. This article will outline experimental evidence that suggests potentialclinical benefits of statins for MS patients.

Keywords: experimental autoimmune encephalomyelitis (EAE), 3-hydroxy-3-methyglutarylcoenzyme A (HMG-CoA) reductase inhibitors, inflammation, multiple sclerosis (MS), statins

Expert Opin. Ther. Targets (2003) 7(5):613-622

1. Multiple sclerosis

Multiple sclerosis (MS) is the most common CNS-demyelinating disease, affectingapproximately 350,000 individuals in North America [1] and 1.1 million worldwide.Based on clinical, immunopathological and neuroimaging observations, MS is nowthought to be a multiphasic disease [2]: > 80% of MS patients initially display arelapsing-remitting (RR) disease course with clinical exacerbations of neurologicalsymptoms, followed by recovery that may or may not be complete [3]. Approximately10 years after disease onset, an estimated 50% of patients with RRMS will convert toa progressive clinical course [3]. During this stage, known as secondary-progressiveMS (SPMS), patients’ disability gradually worsens with or without superimposedexacerbations. Another 10% of individuals, usually somewhat older, have an insidi-ous ‘primary’ progressive course from onset with few, if any, exacerbations.

While the cause of MS remains unknown, the disease is considered to have anautoimmune basis [4-6]. Women are affected at least twice as often as men, a

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Figure 1. HMG-CoA reductase catalyses the conversion of HMG-CoA to L-mevalonate. Through competitive inhibition ofHMG-CoA reductase, statins suppress numerous biological activities downstream of L-mevalonate.FPP: Farnesyl pyrophosphate; GGPP: Geranylgeranyl pyrophosphate; HMG-CoA: 3-Hydroxy-3-methyglutaryl coenzyme A.

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gender bias that is typical in other autoimmune diseases,such as rheumatoid arthritis (RA) and systemic lupus ery-thematosus (SLE) [7]. Numerous epidemiological reportsand studies examining identical twins have suggested thatboth genetic and environmental factors, particularly viralinfections, may play a role in MS pathogenesis [6,8,9]. Theprevalence varies between 50 and 100 per 100,000 in high-risk regions, which include the northern European coun-tries, the northern US and Canada, to less than 5 per100,000 in Japan and Africa. Genetic studies have shownthat the risk to develop MS is elevated 10- to 20-fold infirst-degree relatives of individuals with MS and that theconcordance rate among monozygotic twins is 30 – 35% butonly 2 – 5% in dizygotic twins. Chromosome 6p21 is onelocus most consistently identified in MS susceptibility [10,11].This region contains genes that encode the highly polymor-phic human leukocyte antigen (HLA) D molecules. Thesemolecules present peptide antigens to CD4+ T cells. Inter-estingly, individuals with HLA-DR2 (DRB*1501,DQB*0602) alleles carry a 3- to 4-fold risk for MS [12].However, it appears that not one but multiple genes act inconcert to elevate MS susceptibility. Several genes within the

HLA complex, including components of the complementcascade, TNF-α and myelin oligodendrocyte glycoprotein(MOG), may also be involved in MS pathogenesis. In addi-tion, genome-based studies of multiplex MS families (morethan one family member affected) indicate that 10 – 15 cho-mosomal loci, some of them outside of the HLA locus, maycontribute to the risk of developing MS [10,11,13,14].

2. Statins

Statins, 3-hydroxy-3-methyglutaryl coenzyme A(HMG-CoA) reductase inhibitors, are widely used for thetreatment of hypercholesterolaemia [15]. Statins can reduceblood total cholesterol levels by 15 – 40%, low density lipo-protein (LDL) cholesterol levels by 20 – 60% and triglyceridelevels by 10 – 30% [16]. There is now overwhelming evidencethat statins decrease cardiovascular-related morbidity andmortality in individuals with and without coronary artery dis-ease [17-25]. Statins are administered orally and they achievetheir cholesterol-lowering effects through the competitiveinhibition of HMG-CoA reductase (Figure 1). This enzymecatalyses the conversion of HMG-CoA to L-mevalonate

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(Figure 1). Statins bind HMG-CoA reductase at nanomolarconcentrations, which presents an approximately 1000-foldhigher affinity than its physiological substrate, HMG-CoA[26]. Inhibition of cholesterol biosynthesis is accompanied byan increase in hepatic LDL receptor expression, which pro-motes uptake and catabolism of cholesterol. Through theinhibition of L-mevalonate, statins prevent multiple biologicalactivities downstream of L-mevalonate [27].

Statins were originally discovered in 1973 and wereapproved for use in patients with hypercholesterolaemia bythe FDA in 1987. All available statins have been demon-strated to be safe and well-tolerated [17,18,23], including ‘natu-ral statins’ (or ‘fermentation-derived statins’) and ‘syntheticstatins’. The chemical structure of ‘natural statins’, lovasta-tin, mevastatin, pravastatin and simvastatin, is very similar(Figure 2). Alteration of structural characteristics has led tothe development of ‘synthetic statins’, including atorvasta-tin, fluvastatin, cerivastatin and rosuvastatin (Figure 2). Fur-thermore, various second-generation statins are currentlybeing evaluated in clinical trials for their effectiveness inpatients with hypercholesterolaemia. One of them, rosuvas-tatin (Crestor®, Shionogi and Co. Ltd), has been approvedin several countries.

While oral administration leads to rapid absorption of allstatins, the extent of oral absorption varies between 30 and98%, depending on the agent [15,28,29]. Infrequent side effectsof statins currently available include a dose-dependent eleva-tion of hepatic transaminases (∼ 2%) [30,31] and myopathy(∼ 0.1 – 0.5%) [30]. The incidence of these more severe com-plications is much higher in patients with dysfunction of thekidneys or thyroid gland [32,33]. Cerivastatin was recentlyremoved from the market after data confirmed that its useresulted in a significantly higher incidence of myopathies andbreakdown of muscle tissue (rhabdomyolysis) than other stat-ins [34,35]. Some studies have also suggested a minimal risk ofdeveloping a mostly reversible, axonal sensory neuropathy fol-lowing statin use [36,37].

The pleiotropic effects of statins on cell functions can beexplained by their impact on the metabolic pathway ofL-mevalonate; while some metabolites of L-mevalonate aredirectly involved in cholesterol synthesis, others impact dif-ferent biological systems (Figure 1) [38]. Statins interfere withthe synthesis of isoprenoid intermediates of the cholesterolbiosynthesis pathway, including farnesyl pyrophosphate(FPP) and geranylgeranyl pyrophosphate (GGPP) (Figure 1).Prenylation by FPP and GGPP is a requirement for theassembly, subcellular localisation, intracellular trafficking andassembly of plasma and internal membranes [39-41]. In addi-tion, the modification of small ‘Ras-like proteins’, includingRab, Rac, Rap, Ras and Rho, is dependent on FPP andGGPP. Ras-like proteins bind GTP and play an importantrole in mediating cell proliferation, differentiation and migra-tion [42-44]. Statins also have an effect on the activity of certaincell surface molecules including lymphocyte function-associ-ated antigen (LFA)-1 [42,44,45].

3. Rationale for the use of statins in multiple sclerosis

The potential impact of statins as immunomodulators surfacedin 1995, when it was reported that cardiac transplant patientswho were treated with pravastatin had a decreased incidence ofhaemodynamically significant rejection episodes and decreasedmortality compared with placebo-treated organ recipients [46].These effects did not correlate with cholesterol reduction [46]. Anumber of studies have since identified immunoregulatory andanti-inflammatory properties of statins [47,48].

Much of our current knowledge regarding potential mecha-nisms of statins in CNS autoimmune disease has been derivedfrom experiments in experimental autoimmune (allergic)encephalomyelitis (EAE) [43,49-53], the archetypal model forMS [6,54]. In EAE, activated CD4+ T helper 1 (TH1) cells spe-cific for one of the candidate CNS autoantigens cause relaps-ing paralysis and CNS demyelination. Clinically, there may bean acute, chronic or RR phenotype, depending on species andstrain of the recipient animal [54]. Pathologically, there isCNS demyelination and inflammation.

It was recently demonstrated that statin treatment caneither prevent or reverse chronic and relapsing EAE [43,49-53].When treatment was discontinued in one study, only aminority of experimental animals developed clinically verymild EAE [52]. This finding suggests a sustained treatmenteffect of this agent, which may have particular relevance forpatients with MS.

3.1 Effects of statins on the pathogenic cascade of multiple sclerosisThe potential effects of statins on the pathogenic cascade ofMS include T cell activation, leukocyte migration into theCNS and suppression of inflammatory mediators.

3.1.1 Effects of statins on T cell activationThe activation of CD4+ T cells is a prerequisite for theirmigration from the blood into brain and spinal cord tissue[55-57]. The activation of CD4+ T cells requires recognition oflinear peptide antigen (Ag) within the Ag-binding groove ofthe major histocompatibility complex (MHC) Class II.IFN-γ-inducible MHC Class II expression in ‘non-profes-sional’ antigen-presenting cells (APCs), and constitutiveMHC Class II expression in ‘professional’ APCs, are regulatedby a transcriptional co-activator, the MHC Class II transacti-vator (CIITA) [58,59]. CIITA expression is transcriptionallyregulated by differential activation of multiple non-homolo-gous promoters and is controlled in a tissue-specific manner[60]. Originally, it was observed that promoter (p) I (pI) andpIII directed constitutive CIITA expression in dendritic cellsand B cells, respectively [60]. Promoter pIV, which contains anIFN-γ-activating sequence (GAS) site, E-box and interferonregulatory factor (IRF) element [60,61], directs IFN-γ-inducibleCIITA expression in non-professional APCs [60,62-64].Recently, it was demonstrated that statins inhibited

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Figure 2. The chemical structures of the natural statins, lovastatin, mevastatin, pravastatin and simvastatin, are verysimilar. Alteration of structural characteristics has led to the development of the synthetic statins, atorvastatin, fluvastatin,cerivastatin and rosuvastatin.

NH

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IFN-γ-inducible MHC Class II expression on different non-professional APCs [65]. Furthermore, atorvastatin-mediatedinhibition of IFN-γ-inducible MHC Class II expression onmicroglia was reversible by L-mevalonate [52]. Interestingly,atorvastatin prevented IFN-γ-inducible CIITA transcriptiondirected by each CIITA promoter almost completely [52].

In addition to binding of the T cell receptor (TCR) toAg bound in the Ag-binding groove of MHC Class II, a sec-ond costimulatory signal is required for activation ofCD4+ T cells [66,67]. Costimulatory cell–cell interactions arecommonly referred to as ‘Signal 2’. Once activated, T cellsexpress the surface molecule CD40 ligand (CD40L), whichengages to the costimulatory molecule CD40 on APCs(Figure 3). Crosslinking of CD40 with its ligand subse-quently leads to expression of other costimulatory moleculeson APCs, namely, B7-1 (CD80) and B7-2 (CD86), whichbind to CD28 on activated T cells (Figure 3). In addition,binding of CD40L to CD40 leads to expression of IL-12 byAPC, which, in concert with IFN-γ, leads to the develop-ment of pro-inflammatory CD4+ TH1 cells. The inhibitionof IFN-γ-inducible expression of the costimulatory mole-cules including CD40, CD80 (B7-1) and CD86 (B7-2) byatorvastatin was recently reported [52].

3.1.2 The effects of statins on T cell migrationThe migration of T cells from the blood into the CNS is amultistep event, which involves chemoattraction to the site ofinflammation, cell adhesion to the endothelial wall and prote-olytic degradation of the basal lamina and extracellular matrix(ECM) [68,69]. Chemokines (chemoactive cytokines) attractleukocytes to areas of inflammation within the brain and spi-nal cord [70,71]. They are expressed by several cell types rele-vant in EAE and MS pathogenesis, including endothelialcells, microglia, oligodendrocytes and astrocytes [70,72-74].Monocyte chemotactic protein (MCP)-1 is a chemokine thatis expressed late in the acute phase and in the relapsing phaseof EAE. The levels of MCP-1 expression correlate with clini-cal disease severity [75]. In a recent report, lovastatin and simv-astatin reduced the production of MCP-1 in a dose-dependent manner in peripheral blood mononuclear cells andin human endothelial cells [76].

Leukocyte adhesion to the endothelial wall is in part medi-ated by LFA-1, an integrin on leukocytes. LFA-1 binds tointracellular cell adhesion molecule (ICAM)-1 on endothelialcells [77,78]. Both molecules are abundantly present in perivas-cular MS lesions [79-86]. It was recently shown that lovastatinbinds to LFA-1 and directly inhibits LFA-1–ICAM-1-medi-ated cell adhesion [45,87]. This effect is independent of lovas-tatins inhibition of HMG-CoA reductase.

After T cells adhere to the endothelial wall, they still haveto break down other physiological barriers to gain access tobrain and spinal cord tissue, namely, the basement membrane(basal lamina) of blood vessels and ECM in the brain and spi-nal cord. Proteolytic enzymes, known as matrix metalloprotei-nases (MMPs), are regarded as important physiological

mediators of T cell migration through biological membranes[88]. In addition, MMPs degrade myelin basic protein (MBP),a potential CNS autoantigen in MS, into encephalitogenicpeptide fragments [89]. It was recently demonstrated by twoseparate groups that the secretion of MMP-9 by leukocyteswas reduced by statins [90,91].

3.1.3 The effects of statins on the expression of inflammatory mediators by T lymphocytesCD4+ T cells are categorised into TH1 and TH2 subsets, basedon their cytokine profile [92]. EAE is primarily mediated byCD4+ TH1 cells, which secrete the pro-inflammatorycytokines IL-2, IFN-γ and TNF-α . The presence ofTH1 T cells is strongly associated with clinical disease severity[93], and it has been demonstrated in several species thatTH1 cytokines are upregulated during acute disease andrelapses but not during clinical remission [94,95]. In addition,IFN-γ is considered a key player in the pathogenesis of MS[96]. In contrast, the TH2 cytokines IL-4, -5, -10 and -13 arethought to downregulate disease activity in EAE. It wasrecently demonstrated that atorvastatin induces the secretionof several TH2 cytokines, including IL-4, -5, -10 and trans-forming growth factor (TGF)-β [52]. In addition, atorvastatinpromoted differentiation of TH0 cells into TH2 cells [52].

3.1.4 The effects of statins on the expression of inflammatory mediators in the CNSNitric oxide (NO) is a vascular and neuronal messengerabundantly present in the CNS. NO is produced byNO synthase (NOS), an enzyme that is expressed by astro-cytes and has been identified in MS lesions [97]. The induci-ble isoform of NOS (iNOS), which is expressed mainly inastrocytes, can be activated by several pro-inflammatorycytokines, including IFN-γ and TNF-α [98,99]. Decreasedexpression of iNOS in rat primary astrocytes and a reducedproduction of NO after treatment with lovastatin wasrecently reported [49]. It was also shown that systemic admin-istration of lovastatin reduced the expression of iNOS, IFN-γand TNF-α in the CNS of EAE animals, while improvingthe clinical signs of EAE concomitantly [50].

3.2 Clinical considerations in human CNS autoimmune diseaseWhile the reports from animal studies are certainly encourag-ing, there is only preliminary evidence thus far that statins maybe clinically beneficial in the treatment of MS patients. Neu-haus et al. recently confirmed some of the findings describedabove in humans [100]. Following statin treatment, proliferationof polymorphonuclear cells from MS patients was significantlyreduced. In addition, the authors observed a decreased expres-sion of the adhesion molecule ICAM-1 and various chemokinereceptors on T and B cells, a reduced activity of MMP-9 and ashift of the cytokine profile to a TH2 phenotype [100].

There are several reasons why retrospective analysis ofclinical data to evaluate whether statin treatment of

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MS patients with hypercholesterolaemia had any clinicalbenefit with regard to their CNS autoimmune disease hasbeen challenging and is not yet available [1]; the mean age ofMS onset is 32 years, whilst the mean age of individuals tak-ing statins for cholesterol reduction is ∼ 57 years [1,2]. Thestatins currently available for treatment ofhypercholesterolaemia have different potencies [3]. Datafrom in vitro experiments and animal studies suggest that theimmunomodulatory effects of statins may be dose-depend-ent. Thus, there may well be a threshold effect inMS patients, and a relatively low dose used for cholesterolreduction may not provide sufficient immunomodulation.

Ultimately, it will be necessary to conduct prospective clin-ical trials to evaluate the clinical benefits of statins during dif-ferent disease phases of MS. Considering our currentknowledge of the anti-inflammatory properties of statins,these agents may have their most potential benefit in the early,inflammatory phase of MS, rather than during the neurode-generative phase that follows.

While prospective, placebo-controlled, randomised trialshave not yet been conducted to validate how efficacious stat-ins may be in MS therapy, the results of a small open-labeltrial that tested the efficacy of simvastatin in patients withclinically definite RRMS were recently made public [101].During a period of 3 months, which was designated as the‘pre-treatment period’, 30 MS patients received monthlybrain magnetic resonance imaging (MRI) scans, followed by a6-month ‘treatment period’ during which patients received80 mg of simvastatin daily, the highest FDA-approved dose.The patients underwent imaging of their CNS with brainMRI scans at months 4, 5 and 6 during the treatment period.Analysis of pre- and post-treatment MRI data revealed areduction of ∼ 45% in the mean number and volume of gado-linium (Gd)-enhancing MRI lesions during the treatmentperiod. A randomised, placebo-controlled trial that antici-pates to enrol 152 MS patients is planned to start in 2003.The trial will evaluate whether treatment with high-dose ator-vastatin (80 mg) in patients that have experienced their first

Figure 3. Statins decrease the surface expression of MHC Class II molecules on APCs. In addition, statins reduce IFN-γ-inducibleexpression of the costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2). Through the downregulation of CD40, statins reducethe expression of IL-12 by APCs and suppress the secretion of the TH1 cytokines IL-2, IFN-γ and TNF-α. Statins promote the differentiationof TH0 cells into TH2 cells and induce the secretion of several of the TH2 cytokines, including IL-4, -5, -10 and TGF-β. The effects of statinsare indicated by the circular symbols.APC: Antigen-presenting cell; CD40L: CD40 ligand; MHC: Major histocompatibility complex; TCR: T cell receptor; TGF: Transforming growth factor; TH: T helper cell.

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demyelinating attack (a ‘clinically isolated syndrome’) willdecrease the risk of developing MS.

In comparison with immunomodulatory agents that arecurrently approved for the treatment of MS, statins have dif-ferent mechanisms of action. Thus, statins may also be usefulin combination with established therapies. For example, oneof the principal mechanisms of action of glatiramer acetate(Copaxone®, Teva Pharmaceutical Industries Ltd) seems to bethe induction of CD4+ TH2 cells that downregulate T cellresponses to CNS autoantigens through ‘bystander suppres-sion’ [102]. As statins have also been shown to induce aTH2 response and promote differentiation of naive TH0 cellsinto TH2 cells, statin therapy may enhance the effectiveness ofglatiramer acetate. In combination with IFN-β, statins mayhave an additive effect on MMP-9 suppression, one of theprincipal effects of IFN-β in MS therapy [103-105].

Obviously, the occurrence of adverse effects is an importantaspect and concern of combination therapies. Given the sideeffect profile of individual agents, addition, synergism andpotentiation of these adverse effects may occur when theseagents are used in combination. The risk of adverse effectsmay increase in patients taking higher doses of each agent.Some of the overlapping adverse effects, including thepotential hepatotoxicity of IFN-β and statins, will require thecareful monitoring of patients in clinical trials.

4. Conclusions and expert opinion

There have been major advances in the pharmacotherapy ofMS in recent years. All currently available therapies areadministered parenterally and potentially have significant sideeffects. Recent observations from animal studies and in vitroexperiments have demonstrated that statins have numerousimmunomodulatory properties, including the suppression ofcell adhesion, migration and activation. There is also evidencefrom a small clinical trial that statins may have beneficial clin-ical effects in MS [101]. Larger scale, randomised, double-blindtrials are required to evaluate the role of statins as a treatmentfor autoimmune CNS diseases.

Acknowledgements

Support for this study was provided to SSZ by theAlexander M and June L Maisin Foundation (grant no.98-416), the National Institutes of Health (grant no.K02 NS02207), the National Multiple Sclerosis Society(NMSS) (RG 3206-A-3), and the Nancy Davis Foundation.SSZ is a 2002 recipient of a research grant from theWadsworth Foundation. OS is supported by an advanced fel-lowship from the NMSS and is a Boehringer-IngelheimFonds Scholar.

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AffiliationOlaf Stüve MD PhD1, Thomas Prod’homme PhD2, Anthony Slavin PhD3, Sawsan Youssef PhD, Shannon Dunn PhD, Lawrence Steinman MD & Scott S Zamvil MD PhD†4

†Author for correspondence1Department of Neurology, University of California, San Francisco, USA2INSERM U396, Institut Biomédical des Cordeliers, Paris, France3Tularik, Inc., South San Francisco, CA 94080, USA4Department of Neurology, University of California, San Francisco, 521 Parnassus Avenue, C-440, San Francisco, CA 94143-0114, USATel: +1 415 502 7395; Fax: +1 415 502 1331;E-mail: zamvil@ucsf.neuroimmunol.org

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