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`doi:10.1093/brain/awl083
`
`Brain (2006), 129, 1940–1952
`
`R E V I E W A R T I C L E
`The value of animal models for drug development
`in multiple sclerosis
`
`Manuel A. Friese,1 Xavier Montalban,4 Nick Willcox,2 John I. Bell,1,3 Roland Martin4 and Lars Fugger1,5
`
`1MRC Human Immunology Unit and Department of Clinical Neurology, 2Neurosciences Group, Weatherall Institute of
`Molecular Medicine and 3Office of the Regius Professor, John Radcliffe Hospital, University of Oxford, Oxford, UK,
`4Edifici Escola D’infermeria, 2a Planta, Unitat de Neuroimmunologia Clı´nica, Hospital Universitari Vall d’Hebron, Barcelona,
`Spain and 5Department of Clinical Immunology, Aarhus University Hospital, Skejby Sygehus, Denmark
`
`Correspondence to: Lars Fugger, MRC Human Immunology Unit and Department of Clinical Neurology, Weatherall
`Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom or
`Roland Martin, Edifici Escola D’infermeria, 2a Planta, Unitat de Neuroimmunologia Clı´nica,
`Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain
`E-mail: lars.fugger@molecular-medicine.oxford.ac.uk
`
`The rodent model for multiple sclerosis, experimental allergic (autoimmune) encephalomyelitis (EAE), has been
`used to dissect molecular mechanisms of the autoimmune inflammatory response, and hence to devise and test
`new therapies for multiple sclerosis. Clearly, artificial immunization against myelin may not necessarily reproduce
`all the pathogenetic mechanisms operating in the human disease, but most therapies tested in multiple sclerosis
`patients are nevertheless based on concepts derived from studies in EAE. Unfortunately, several treatments, though
`successful in pre-clinical EAE trials, were either less effective in patients, worsened disease or caused unexpected,
`severe adverse events, as we review here. These discrepancies must, at least in part, be due to genetic and
`environmental differences, but the precise underlying reasons are not yet clear. Our understanding of EAE patho-
`genesis is still incomplete and so, therefore, are any implications for drug development in these models. Here, we
`suggest some potential explanations based on new thinking about key pathogenic concepts and differences that may
`limit extrapolation from EAE to multiple sclerosis. To try to circumvent these rodent–human dissimilarities more
`systematically, we propose that pre-clinical trials should be started in humanized mouse models.
`
`Keywords: animal models; experimental allergic encephalomyelitis; immunomodulation; multiple sclerosis; treatments
`
`Abbreviations: CFA = complete Freund’s adjuvant; EAE = experimental allergic (autoimmune) encephalomyelitis;
`HSCT = haematopoietic stem cell transplantation; MBP = myelin basic protein; PDEs = phosphodiesterases; PML = progressive
`multifocal leukoencephalopathy; PPARs = peroxisome proliferator-activated receptors; TCR = T-cell receptor;
`TH1 = T helper 1; TNF = tumour necrosis factor
`
`Received November 30, 2005. Revised March 5, 2006. Accepted March 14, 2006. Advance Access publication April 24, 2006
`
`Introduction
`Multiple sclerosis is the commonest neurological disease of
`young adults, afflicting at least 350 000 individuals in North
`America and 500 000 in Europe (Hafler et al., 2005; Sospedra
`and Martin, 2005). Although multiple sclerosis does not
`usually shorten life expectancy, its socio-economic burden
`in young adults is second only to trauma (Sospedra and
`Martin, 2005). Its clinical signs and symptoms are very
`variable and depend on the parts of the CNS it affects,
`
`that is, the brain and spinal cord, and include motor, sensory,
`autonomic and cognitive disabilities (Noseworthy et al.,
`2000a).
`It can run at
`least
`three clinical courses: (i)
`relapsing–remitting (RR) multiple sclerosis, which is most
`frequent (85%) and characterized by discrete attacks
`(exacerbations) and subsequent periods of clinical stability.
`In most relapsing multiple sclerosis patients, (ii) a secondary
`progressive (SP) phase ensues, with continuously increasing
`deficits. About 10–15% of multiple sclerosis patients develop
`
`# The Author (2006). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
`
`Coalition Exhibit 1048
`Coalition v. Biogen
`IPR2015-01993
`
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`The value of animal models for drug development in multiple sclerosis
`
`Brain (2006), 129, 1940–1952
`
`1941
`
`steadily increasing neurological deficits from onset, (iii) the
`primary-progressive subtype (Noseworthy et al., 2000a).
`Neuropathologically, CNS tissue from multiple sclerosis
`patients shows discrete lesions (predominantly in the white
`matter) with inflammatory infiltrates, demyelination, astro-
`gliosis and early axonal damage. Again, there is considerable
`heterogeneity in composition of cellular infiltrates and in
`involvement of antibodies and complement (Lassmann
`et al., 2001). Multiple sclerosis is widely considered an
`autoimmune demyelinating disease, and the inflammatory
`infiltrates as pathogenically primary events. Its aetiology
`remains a mystery, but infectious agents have long been
`suspected as triggers (Marrie, 2004). The evidence for an
`autoimmune reaction targeting myelin is strong but not
`definitive. There are, for example, descriptions of primary
`oligodendrocyte apoptosis with microglial activation in
`early multiple sclerosis lesions in the absence of lymphocytes
`or myelin phagocytosis (Barnett and Prineas, 2004). Further,
`the decreasing inflammatory activity that is seen by MRI during
`the SP phase has led to the assumption that the pathology is
`inflammatory at first and degenerative later. Despite these uncer-
`tainties, it is generally accepted that multiple sclerosis involves an
`autoimmune reaction by myelin-specific CD4+ T helper 1 (TH1)
`cells, which initiate the neuropathology (Hafler et al., 2005;
`Sospedra and Martin, 2005). This notion is based on the
`cellular composition of CNS- and CSF-infiltrating cells
`(Hauser et al., 1986), on genetic studies in multiple sclerosis
`(Dyment et al., 2004) and on one animal model of multiple
`sclerosis, experimental allergic (autoimmune) encephalomye-
`litis (EAE) (Zamvil and Steinman, 1990).
`Dissecting the pathogenesis of a complex disease in man is
`fraught with many problems, particularly those associated
`with clinical and genetic heterogeneity. Not surprisingly,
`most of our current
`thinking about multiple sclerosis
`stems from EAE. This model originated from vaccination
`with rabies-infected rabbit spinal cord by Louis Pasteur
`(from 1885). About 1 in 1000 vaccinees had ‘neuroparalytic
`incidents’; this acute demyelinating disorder later proved to
`be due to ‘contamination’ by spinal cord components in the
`inoculum. The EAE model has since evolved a long way;
`different variants, mice, rats or non-human primates are
`immunized with whole spinal cord, myelin proteins or
`even defined peptides, usually in complete Freund’s adjuvant
`(CFA). This immunization leads to a disease that shares clin-
`ical and neuropathological changes with multiple sclerosis
`(Steinman, 1999). The course it takes ranges from acute
`monophasic (or even lethal) to chronic progressive or
`relapsing–remitting (Steinman, 1999). Typical CD4+ TH1
`myelin-specific T cells have been implicated as
`the
`disease-initiating subset. In almost all models, they are suffi-
`cient to induce EAE; they can be isolated, cloned and used to
`transfer disease to naı¨ve healthy animals (Zamvil and
`Steinman, 1990). These various EAE models have been
`used to dissect molecular mechanisms of the autoimmune
`inflammatory response, and hence to devise and test new
`therapies for multiple sclerosis. It is clear, however, that
`
`the artificial induction of a myelin-specific immune response
`may by-pass key pathogenetic mechanisms operating in
`human disease, as we do not even know the key target auto-
`antigens in multiple sclerosis.
`
`Limitations of current EAE models
`Without doubt, EAE models are vital for studying general
`concepts as well as specific processes of autoimmunity, how-
`ever rarely they predict success in clinical trials (see below).
`Nevertheless, their value is further challenged by our rudi-
`mentary understanding of the key pathogenetic mechanisms
`in EAE models, and their failure to forewarn us of adverse
`effects (reviewed below). As with other murine disease
`models,
`including the NOD model of
`type 1 diabetes
`(Roep et al., 2004), it appears much easier to prevent, reverse
`or ameliorate EAE in mice than multiple sclerosis in man.
`Furthermore, since EAE almost always has to be induced, it
`cannot mimic a spontaneous disease. The most important
`component in the inducing adjuvant CFA is heat-inactivated
`Mycobacterium tuberculosis, which always induces a promi-
`nent CD4+ TH1 response by activating certain toll-like recep-
`tors (Su et al., 2005). This leaves little room for variability in
`disease pathways and certainly does not reflect heterogeneous
`inducing mechanisms in multiple sclerosis. Also, demyelina-
`tion is not obvious in all models. Moreover, the time courses
`are very different. Since EAE develops over days in most
`models, they seem more similar to post-infectious acute
`demyelinating events (Steinman, 1999). Indeed, the mice
`are rarely monitored for late relapses and fatal adverse effects,
`such as those noted in marmosets (Genain et al., 1996).
`Nevertheless, the same treatment can have a different degree
`of efficacy or even opposite effects at different stages in EAE,
`as has also been reported for other autoimmune models such
`as in NOD mice (Shoda et al., 2005). In contrast, multiple
`sclerosis usually manifests insidiously over years, for example,
`in its
`relapsing–remitting
`and later
`chronic
`forms
`(Noseworthy et al., 2000a), by when antibodies and comple-
`ment may also be more important than in most mouse mod-
`els.
`Indeed, many patients present after much more
`protracted epitope spreading than is usually seen in EAE
`mice (Vanderlugt and Miller, 2002). These and other obvious
`mouse : human differences are summarized in Table 1.
`Many aspects of pathology and immunology differ between
`multiple sclerosis and EAE. These differences are fundamen-
`tal, as ongoing imbalances in immune regulation must be
`crucial for the progression of multiple sclerosis; such orders
`of complexity have not yet been recapitulated in EAE models.
`
`What can we learn from failures or
`successes in adapting therapies from
`EAE to multiple sclerosis?
`Only very few therapeutics that were successful in pre-clinical
`EAE trials have shown similar efficacy in multiple sclerosis
`
`Page 2 of 13
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`Brain (2006), 129, 1940–1952
`
`M. A. Friese et al.
`
`Table 1 Immunological differences between mouse and human relevant for testing multiple sclerosis therapeutics
`
`Mouse
`
`Human
`
`References
`
`General
`
`Inbred; homozygous
`Short lifespan: high fecundity
`Fixed diet; pathogen-free
`
`Clean environment
`
`Outbred; heterozygous
`Long lifespan: low fecundity
`Varied diet; carriers of potential pathogens,
`e.g. EBV, JCV etc
`Open access to new infections
`
`EAE and multiple sclerosis
`
`Induction
`Testing new therapeutics
`
`May be monophasic
`Mice tested while epitopes are
`spreading
`Usually with CFA
`Induction of EAE studied much
`more than ongoing disease
`Only a few dozen mice tested
`
`Less detailed
`Scrutiny
`Often short-term only
`Follow-up
`Molecular differences in immune response
`T-cell responses
`Often stereotypical
`
`75–90%
`
`Different subtypes, usually relapsing
`Epitopes must often have spread long
`before diagnosis
`Spontaneous
`Ongoing disease
`
`Hundreds of multiple sclerosis patients;
`some side-effects are too rare to be seen
`in mice
`Detailed, would be missed in mice
`Several years or life-long
`
`Usually idiosyncratic, even to
`recurring epitope(s)
`30–50%
`
`Lymphocytes in
`peripheral blood
`CD4+ expression
`CD8+ expression
`IL-10 expression
`IFN-a response
`IL-4 and IFN-g
`expression by TH
`CD28 expression
`MHC class II expression
`
`CD52 expression
`Glucocorticoid-sensitivity
`
`Lymphocytes
`Lymphocytes, dendritic cells
`TH2
`No preferential TH differentiation
`Exclusively one or the other
`100% of CD4+ and CD8+ T cells
`Absent on T cells and
`endothelial cells
`Not found in mice
`High
`
`Lymphocytes, macrophages
`Lymphocytes
`TH1 and TH2
`Promotes TH1 response
`Sometimes both
`80% of CD4+ T cells, 50% of CD8+ T cells
`Present on T cells and
`endothelial cells
`Lymphocytes
`Low and variable
`
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`
`Vanderlugt and Miller
`(2002)
`
`Doeing et al. (2003)
`
`Crocker et al. (1987)
`Banchereau et al. (2000)
`Del Prete et al. (1993)
`Farrar et al. (2000)
`Gor et al. (2003)
`
`Lenschow et al. (1996)
`Choo et al. (1997),
`Taams et al. (1999)
`Tone et al. (1999)
`Claman (1972)
`
`patients; the majority of new treatments were either less
`effective in these patients, worsened disease or caused severe
`adverse events. In Table 2 we list a subset of these therapies
`reflecting this discrepancy.
`
`Antigen-specific therapies
`Only one licensed multiple sclerosis therapy (Glatiramer
`acetate, GA), a synthetic amino acid copolymer (Glu, Ala,
`Lys and Tyr), emerged from findings in EAE (Teitelbaum
`et al., 1971). It was designed to mimic encephalitogenic mye-
`lin basic protein (MBP) epitopes, but instead it suppresses
`EAE by other mechanisms in several species, and it reportedly
`reduces multiple sclerosis relapses by 30% (Johnson et al.,
`1995). GA has many biological activities including bystander
`suppression via induction of TH2 cells that partly cross-react
`with MBP, and/or upregulation of CNS growth factors
`(Arnon and Aharoni, 2004). However, its in vivo mechanisms
`are not clear and even its beneficial effects on the main out-
`come measures in multiple sclerosis (disease progression)
`have now been questioned in a systematic Cochrane review
`(Munari et al., 2004).
`A more specific therapeutic approach in EAE and multiple
`sclerosis has been based on an altered peptide ligand of MBP
`
`85–99 that was modified at its main T-cell receptor (TCR)
`contact sites (Brocke et al., 1996). Despite promising effects
`in EAE, subcutaneous administration of altered peptide
`ligand at high doses led to multiple sclerosis exacerbations
`in some patients, which could be linked to this treatment
`(Bielekova et al., 2000). A trend towards improved MRI
`parameters was observed in another phase II trial (Kappos
`et al., 2000), and an additional phase II study is under way. Its
`success in EAE may depend on the stereotyped TH responses
`of inbred mice.
`Oral administration of myelin antigens leads to specific
`immune hyporesponsiveness in mice. Different doses and
`feeding regimes have been demonstrated to induce different
`types of ‘oral tolerance’/degrees of immune suppression in
`different EAE models (Faria and Weiner, 2005). ‘Bystander
`suppression’ directed against one tolerogen may suppress
`reactions against other myelin antigens in situ, a major advan-
`tage where the key autoimmunizing antigen(s) are not
`known. However, a large double-blind phase III trial of a
`single oral dose of bovine myelin in RR multiple sclerosis
`did not show differences in the number of relapses between
`placebo and treated groups (Faria and Weiner, 2005). Treat-
`ment failure could have been due to the unexpectedly strong
`
`Page 3 of 13
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`

`

`The value of animal models for drug development in multiple sclerosis
`
`Brain (2006), 129, 1940–1952
`
`1943
`
`Table 2 Some immunomodulatory approaches of multiple sclerosis and their development from EAE or in vitro studies to
`clinical application*
`
`Treatment
`approach
`
`Based on
`clear
`hypothesis
`
`Rationale
`confirmed
`
`Efficacy
`in EAE
`
`Efficacy
`in multiple
`sclerosis
`
`Adverse
`event
`profile
`
`Status of
`development
`
`Reference
`
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`
`Approved
`No
`
`Johnson et al. (1995)
`Bielekova et al. (2000),Kappos
`et al. (2000)
`Faria and Weiner (2005)
`
`Not continued after phase III
`owing to lack of efficacy
`Miller et al. (2003)
`Taken off the market
`Dumont (2002)
`No
`van Oosten et al. (1997)
`Halted in phase II
`Approved for other indication Coles et al. (1999b)
`Approved for other indication Bielekova et al. (2004)
`In phase III
`Kremer (2004)
`Approved
`Paty and Li (1993)
`Stopped in phase I
`Panitch et al., 1987)
`Approved for other indication van Oosten et al. (1996)
`
`Approved for other indication The Lenercept Multiple Sclerosis
`Study Group and The University
`of British Columbia multiple
`sclerosis/MRI Analysis Group
`(1999)
`Calabresi et al. (1998)
`Wiendl et al. (2000)
`Frank et al. (2002)
`R. Martin et al. (unpublished data)
`Diab et al. (2002), Feinstein et al.
`(2002)
`Vollmer et al. (2004), Youssef
`et al. (2002)
`Hartung et al. (2002)
`Noseworthy et al. (2000c)
`
`Stopped in phase I
`Stopped in phase II
`Phase IIa, not continued
`Halted in phase II
`Not yet tested in multiple
`sclerosis
`Approved for other
`indication
`Approved
`Phase III stopped due to
`cardiotoxicity
`In phase II
`In phase II
`
`Polman et al. (2005)
`Gonsette (2004), Rausch et al.,
`2004)
`Wiendl and Hohlfeld (2002)
`
`Noseworthy et al. (1998)
`Hommes et al. (2004), Sorensen
`et al. (2002)
`Mancardi et al. (2005), Tyndall
`and Saccardi (2005)
`
`++
`++
`
`++; i.d.
`+
`++(+); i.d. +
`
`
`+
`
`6
`6
`
`+
`++
`
`After phase II stopped
`owing to lack of efficacy
`In phase III
`In phase II
`
`++;i.d.
`
`6; i.d.
`
`In phase III
`
`+++
`6
`
`+++
`
`6†
`6
`6
`+
`++
`n.k.
`++
`
`‡
` ‡
`
`
`i.d.
`++
`+
`n.a.
`
`+
`
`6
`6
`
`No
`No; i.d.
`
`Yes
`
`Yes
`Yes
`No
`Yes
`No
`No; i.d.
`No
`No
`No
`
`No
`
`++
`++
`
`++
`
`+
`; i.d.
`
`
`+++
`+++
`n.k.
`+++
`6
`+++
`n.a.
`++; i.d.
`6
`+++; i.d.
`n.k.
`+++
`+
`+
`
`++
`++(?)
`++(?)
`
`++
`6
`+(+)
`++
`++
`
`i.d.
`i.d.
`; i.d.
`; i.d.
`n.t.
`
`+++
`
`++; i.d.
`
`Yes
`No
`Yes
`Yes
`Yes
`
`Yes
`
`No§
`No
`
`No
`No
`
`No
`
`No
`No
`
`Yes
`
`++
`++
`
`++
`+++
`
`+
`
`6
`6
`
`+
`
`Glatiramer acetate No
`Altered peptide
`Yes
`ligand
`Oral myelin
`
`Yes
`
`Anti-a4 integrin
`Anti-CD40L
`Anti-CD4
`Anti-CD52
`Anti-CD25
`CTLA-4-Ig
`IFN-b
`IFN-g
`Anti-TNF
`antibodies
`TNFR-Ig fusion
`protein
`
`TGF-b2
`IL-10
`IGF-1
`PDE4 inhibitors
`PPARg agonists
`
`Statins
`
`Mitoxantrone
`Linomide
`
`Laquinimod
`FTY720/SP-1
`agonist
`Deoxyspergualin
`
`Sulphasalazine
`IVIG
`
`Haematopoietic
`stem cell transplant
`
`Yes
`Yes
`Yes
`Yes
`Yes
`Yes
`No
`No
`Yes
`
`Yes
`
`Yes
`Yes
`Yes
`Yes
`Yes
`
`Yes
`
`No§
`No
`
`No
`No
`
`No
`
`Yes
`No
`
`Yes
`
`i.d., insufficient data; n.a., not applicable; n.k., not known; n.t., not tested; *The table depicts whether a therapeutic approach was developed
`for multiple sclerosis on the basis of a clear and pre-formed hypothesis, whether the rationale for its clinical/EAE testing had later been
`shown and whether the therapy was effective in EAE and/or multiple sclerosis. Both the clinical efficacy and the tolerability and safety are
`depicted by + or signs. In the context of the adverse events, + indicates a favourable profile. The relative weighting reflects the subjective
`perception of the authors either from own experience or the published literature; †Reasonable safety profile, but one specific severe
`adverse event (PML). ‡Development of demyelinating episodes and diseases in RA- and Crohn’s patients; §Broad immunosuppressant; no
`specific target.
`
`effects in the placebo group, wrong dose or type of antigen, or
`route of administration.
`
`Adhesion molecules
`Another promising strategy, using a blocking anti-a4 integrin
`humanized antibody (natalizumab), emerged from EAE
`evidence that a4b1 integrin is critical for T cell and monocyte
`
`homing to the CNS (Yednock et al., 1992). This mAb was
`highly effective in pre-clinical EAE studies and successfully
`completed phase II and III testing in large numbers of multi-
`ple sclerosis patients. Because of its remarkable efficacy in
`multiple sclerosis (Miller et al., 2003), natalizumab was
`approved by the Food and Drug Administration even before
`phase III trial data had been published, but was taken off the
`market four months later because of rare but very severe
`
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`
`adverse events. Three patients had developed progressive
`multifocal
`leukoencephalopathy (PML), an often lethal
`opportunistic infection of the CNS; two died, and one is
`recovering, though with considerable neurological deficits
`(Kleinschmidt-DeMasters and Tyler, 2005; Langer-Gould
`et al., 2005; Van Assche et al., 2005). A large post facto analysis
`estimated the risk of PML for a 2-year treatment period to
`1 in 1000 patients (Yousry et al., 2006). PML is caused by
`reactivation and mutation of the highly prevalent polyoma-
`virus JC (JCV), which destroys oligodendrocytes. PML is
`almost exclusively observed in immunosuppressed indivi-
`duals, and it is not clear what initiated its unexpected deve-
`lopment under natalizumab treatment.
`JCV persists
`in
`kidneys and lymphoid organs,
`including bone marrow
`(Monaco et al., 1998). During immunosuppression, latent
`infection can be reactivated, and JCV disseminates to the
`CNS (Tornatore et al., 1992). That might have resulted either
`from compromised T-cell surveillance of the CNS or from
`mobilization of stem cells and JCV from the bone marrow
`(Papayannopoulou and Nakamoto, 1993; Ransohoff, 2005),
`where a4b1 integrin serves as a retaining signal (Simmons
`et al., 1992). Since JCV is not found in rodents, this adverse
`event could not have been anticipated from pre-clinical inves-
`tigations. Therefore, this drug cannot be called a failure of
`prediction, especially as many thousand patients needed to be
`treated to unravel potential adverse effects. In addition, the
`recently published two-year phase III trials underline its com-
`pelling effects on relapse rate and clinical progression
`(Polman et al., 2006; Rudick et al., 2006). In March 2006,
`The Peripheral and CNS Drugs Advisory Committee, under
`The Food and Drug Administration, voted unanimously to
`recommend the return of natalizumab for the treatment of
`RR multiple sclerosis in a subset group of patients.
`
`Co-stimulatory molecules
`Despite its promise in EAE, anti-CD40 ligand (CD154)
`(Howard et al., 1999) was not developed because of its throm-
`boembolic complications in man (Kawai et al., 2000), which
`result from its expression on human but not murine platelets.
`Anti-CD4 therapy was effective in EAE (Waldor et al., 1985),
`but not
`in human studies (van Oosten et al., 1997).
`Anti-CD52, which depletes both CD8+ and CD4+ T-cells
`(Coles et al., 1999b), was never evaluated in EAE, but is
`very effective against new lesions in multiple sclerosis, though
`30% of treated multiple sclerosis patients develop auto-
`immune hyperthyroidism (Coles et al., 1999a). On the
`other hand, IL-2 receptor blockade with the humanized
`anti-CD25 antibody (daclizumab) caused impressive reduc-
`tions in MRI lesions and improvements in some clinical
`measures (Bielekova et al., 2004). In this case, the theoretical
`role of CD25 in promoting T regulatory cells, and equivocal
`EAE data (Engelhardt et al., 1989; Reddy et al., 2004), might
`have argued against its use in multiple sclerosis. Interestingly,
`there is little evidence that it perturbs T regulatory or TH
`function; indeed it may act by expanding immunoregulatory
`
`NK cells (Bielekova et al., in review). CTLA-4-Ig interferes
`with co-stimulation from CD80/CD86 molecules on antigen-
`presenting cells (APCs) to the stimulatory or inhibitory
`ligands CD28 and CTLA-4 (Alegre et al., 2001). Data in
`EAE indicate that CTLA-4-Ig is much more effective as a
`preventive pre-treatment (Cross et al., 1995) than in therapy
`of ongoing disease (Cross et al., 1999). Treatment with
`CTLA-4-Ig is also effective in other autoimmune diseases
`such as rheumatoid arthritis (Kremer et al., 2003), and is
`currently being tested in a phase III trial in multiple sclerosis.
`
`Cytokines
`Cytokines have different effects at different stages of patho-
`genesis, for example, in the induction phase and the chronic/
`relapsing phase in EAE. These differences suggest a pleiotro-
`pic role in CNS inflammation and might explain some of the
`below-described discrepancies between EAE and multiple
`sclerosis.
`Interferon-b ( IFN-b), the first drug approved for multiple
`sclerosis, had not been previously tested in EAE. It exerts a
`wide variety of effects on the immune system: it inhibits both
`leukocyte proliferation and antigen presentation; it biases
`towards production of anti-inflammatory cytokines and it
`inhibits T-cell migration across the blood-brain barrier
`(Billiau et al., 2004). Although widely used in multiple sclero-
`sis, its long-term effectiveness and side-effects are still uncer-
`tain (Filippini et al., 2003). With other cytokines, effects have
`seemed contradictory in EAE vis a` vis multiple sclerosis. In
`it was found that IFN-g knockout mice
`the mid-1990s,
`develop lethal EAE (Ferber et al., 1996), and IFN-g admin-
`istration in EAE showed a protective effect on disease severity
`(Krakowski and Owens, 1996). By then, its use in multiple
`sclerosis patients had already led to a modest increase in
`disease exacerbations (Panitch et al., 1987). Although this
`study is limited, it is unlikely that IFN-g will ever be tested
`again in multiple sclerosis.
`In contrast, tumour necrosis factor-a (TNF-a) has long
`been considered a key mediator of multiple sclerosis patho-
`genesis (Sharief and Hentges, 1991), and its blockade by
`antibodies or soluble TNF receptors prevents or reverses dis-
`ease in EAE models (Ruddle et al., 1990; Selmaj et al., 1991,
`1995). Paradoxically, this approach worsens disease in multi-
`ple sclerosis patients and had to be discontinued (The Lener-
`cept Multiple Sclerosis Study Group and The University of
`British Columbia Multiple Sclerosis/MRI Analysis Group,
`1999; van Oosten et al., 1996). Indeed, a substantial number
`of cases developed their first demyelinating event while being
`treated with anti-TNF-a agents for other diseases such as
`rheumatoid arthritis or Crohn’s disease (Hyrich et al.,
`2004). Despite the data that TNF-a is an important compo-
`nent in the pathogenesis in EAE, a precise role for TNF-a in
`multiple sclerosis remains unclear. However, subsequent EAE
`/
`experiments using TNF-a gene deleted mice (TNF-a
`)
`surprisingly showed that TNF-a/
`mice displayed profound
`neurological impairment and high mortality with extensive
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`demyelination and monocytic cell infiltration in comparison
`with control mice (Liu et al., 1998). Conversely, treatment of
`the TNF-a/
`mice but also wild-type mice with recombi-
`nant TNF-a reduced disease severity in afflicted mice and
`prevented development of EAE in pre-treated mice. These
`studies suggested that TNF-a may be protective in the
`CNS during the development of demyelinating disease also
`in mice and may serve to limit the extent of immune-
`mediated inflammation, as was implicated by the study in
`multiple sclerosis patients. Further, we can only speculate on
`the reasons for the adverse outcome of TNF-blocking
`approaches in multiple sclerosis, but it has been demon-
`strated that TNF signalling is also important for remyelina-
`tion (Arnett et al., 2001; Diemel et al., 2004).
`Transforming growth factor (TGF)-b2 ameliorates EAE
`and is a very potent immunosuppressive cytokine. However,
`it has never been approved as a treatment in multiple sclero-
`sis, as it is associated with nephrotoxicity in multiple sclerosis
`patients, which was not checked in EAE (Calabresi et al.,
`1998). Interleukin (IL)-10 is another important suppressive
`cytokine, produced mainly by regulatory CD4+ T cells
`(O’Garra et al., 2004). Its selective upregulation in the
`CNS during the recovery phase of EAE prompted the evalua-
`tion of IL-10 treatment in a Lewis rat EAE model. Systemic
`administration during the initiation phase suppressed EAE
`disease (Rott et al., 1994). Again IL-10 treatment in a phase II
`clinical trial in multiple sclerosis patients had to be stopped,
`owing to lack of efficacy (Wiendl et al., 2000).
`
`Neurotrophic factors
`Apart from cytokines, several neurotrophic factors have been
`proposed as a novel therapeutic in multiple sclerosis. These
`proteins regulate survival and differentiation of neurons by
`binding to specific neurotrophin receptors (Thoenen and
`Sendtner, 2002). They are involved in skewing the cytokine
`balance in the CNS from TH1 to TH2 responses (Villoslada
`et al., 2000), and neuroprotective effects have been proposed
`(Hohlfeld et al., 2005). The clinical potential of one such
`factor, insulin-like growth factor-1 (IGF-1), was first studied
`in a rat EAE model, in which myelin regeneration and clinical
`recovery was enhanced (Yao et al., 1995). In contrast, there
`was no clear effect in one phase-I/II study in seven patients
`(Frank et al., 2002).
`
`Anti-inflammatory substances,
`immunosuppression and
`immunomodulation
`Cytokine secretion and proliferation of T cells are regulated
`by intracellular cyclic AMP (cAMP) levels, which are reduced
`by phosphodiesterases (PDEs). The type 4 PDE (PDE4) is a
`cAMP-specific phosphodiesterase expressed in cells of the
`immune system and the CNS (Engels et al., 1994). Inhibition
`of PDE4 activity by rolipram ameliorates EAE severity
`(Sommer et al., 1995). It was originally developed and
`
`evaluated in clinical studies as an anti-depressant (Zeller
`et al., 1984). Since rolipram and other PDE4 inhibitors
`(Dinter et al., 2000) demonstrated efficacy in various EAE
`models, it seemed a promising candidate for clinical use in
`multiple sclerosis patients. However, a phase II clinical trial
`was halted owing to lack of efficacy on the primary outcome
`measure, that is, the reduction of gadolinium-enhancing
`inflammatory CNS lesions by MRI (Martin, R., Bielekova, B.,
`Stu¨rzebecher, C.S., Richert, N., Frank, J.A., Ohayon, J.,
`McCartin, J., McFarland, H.F., unpublished data).
`Peroxisome proliferator-activated receptors (PPARs) are
`members of the nuclear hormone receptor superfamily of
`ligand-activated transcriptional factors that include receptors
`for steroids, thyroid hormone, vitamin D and retinoic acid
`(Mangelsdorf et al., 1995). PPAR-g is expressed in adipose
`tissue, on macrophages, T cells, and endothelial and vascular
`smooth muscle cells. The natural 15-deoxy-D12,14-PGJ2
`(15d-PGJ2) and the synthetic anti-diabetic thiazolidinedione
`are PPAR-g ligands; their administration before and at the
`onset of clinical signs of EAE significantly reduced its severity
`(Diab et al., 2002). Another orally administered PPAR-g
`agonist pioglitazone reduced the incidence and severity in
`C57BL/6 EAE and B10.Pl murine models. Pioglitazone also
`reduced clinical signs when given after disease onset (Feinstein
`et al., 2002). So far there is only casuistic information about
`PPAR-g agonists in multiple sclerosis (Pershadsingh et al.,
`2004), but clinical trials are being planned.
`Statins block the enzyme 3-hydroxy-3-methylglutaryl-
`coenzyme A reductase; they are widely used to lower choles-
`terol
`levels and prevent cardiovascular disease. They are
`highly
`effective
`in reversing EAE,
`shifting the pro-
`inflammatory TH1-type cytokine profile to a TH2-type
`pattern and also interfering with antigen presentation
`(Youssef et al., 2002). In this case, exploratory trials in multi-
`ple sclerosis patients have shown promising results with up to
`44% reduction in the number of contrast-enhancing MRI
`lesions (Vollmer et al., 2004), so they are a solitary success.
`Mitoxantrone is a potent anti-inflammatory cytostatic
`anthracenedione, and suppresses both B and T lymphocytes
`and macrophages, resulting in down-modulation of the
`inflammatory cascade. Since it had already been approved
`for the therapy of malignancies, this immunosuppressant
`could logically have been assessed in multiple sclerosis with-
`out studying EAE. Not surprisingly, mitoxantrone has potent
`effects in EAE. It suppressed paralysis in acute EAE, prevented
`its development when administered during the induction
`period and still showed some effects when given after clinical
`signs and symptoms appeared (Ridge et al., 1985; Levine and
`Saltzman, 1986). These results parallelled recent proof of
`efficacy in multiple sclerosis (Hartung et al., 2002). However,
`cardiotoxicity was a reported adverse effect in humans;
`though not observed in the early EAE studies (Ridge et al.,
`1985; Levine and Saltzman, 1986), it was also detected in
`careful retrospective analysis in treated mice. Meanwhile,
`the more serious problem of inducing haematopoietic malig-
`nancies is predictable for a cytotoxic agent, and has already
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`1946
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`Brain (2006), 129, 1940–1952
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`M. A.