GLATIRAMER ACETATE: MECHANISMS OF ACTION IN
`MULTIPLE SCLEROSIS
`
`Tjalf Ziemssen and Wiebke Schrempf
`
`Multiple Sclerosis Center Dresden, Neurological University Clinic
`Dresden University of Technology, Dresden 01307, Germany
`
`I. Introduction
`II. Pathology and Immunology
`III. MS as Neurodegenerative Disease
`IV. The Janus Face of CNS-Directed Autoimmune Inflammation
`V. Neurotrophic Factors Are Released by DiVerent Immune Cells
`VI. Glatiramer Acetate: Historical Remarks
`VII. GA: Overview of Clinical Studies
`VIII. GA: Imaging Studies
`IX. GA: Animal Models
`X. GA in MS: Mechanisms of Action
`A. Effects of GA by Binding to MHC Class II Molecules
`B. Effects of GA on the APC Level
`XI. EVects of GA on the B-Cell Level
`XII. EVects of GA on the T-Cell Level
`A. Th1–Th2 Shift
`B. GA as Altered Peptide Ligand
`C. Secretion of Neurotrophic Factors
`
`D. Induction of GA-Specific CD8þ Suppressor T Cells
`E. Induction of Regulatory CD4þCD25þ T Cells
`
`XIII. Conclusions
`References
`
`Glatiramer acetate (GA), formerly known as copolymer 1, is a mixture of
`synthetic polypeptides composed of four amino acids resembling the myelin basic
`protein (MSP). GA has been shown to be highly eVective in preventing and
`suppressing experimental autoimmune encephalomyelitis (EAE),
`the animal
`model of multiple sclerosis (MS). Therefore, it was tested in several clinical studies
`and so approved for the immunomodulatory treatment of relapsing-type MS.
`In contrast to other immunomodulatory MS therapies, GA has a distinct
`mechanism of action: GA demonstrates an initial strong promiscuous binding to major
`histocompatibility complex molecules and consequent competition with various
`(myelin) antigens for their presentation to T cells. In addition, antigen-based therapy
`generating a GA-specific immune response seems to be the prerequisite for GA
`
`INTERNATIONAL REVIEW OF
`NEUROBIOLOGY, VOL. 79
`DOI: 10.1016/S0074-7742(07)79024-4
`
`537
`
`Copyright 2007, Elsevier Inc.
`All rights reserved.
`0074-7742/07 $35.00
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`therapy. GA treatment induces an in vivo change of the frequency, cytokine secretion
`
`pattern and the eVector function of GA-specific CD4þ and CD8þ T cells, probably
`by aVecting the properties of antigen-presenting cells such as monocytes and dendritic
`cells. As demonstrated extensively in animal experiments, GA-specific, mostly,
`T helper 2 cells migrate to the brain and lead to in situ bystander suppression of the
`inflammatory process in the brain. Furthermore, GA-specific cells in the brain express
`neurotrophic factors like the brain-derived neurotrophic factor (BDNF) in addition
`to anti-inflammatory T helper 2-like cytokines. This might help tip the balance in favor
`of more beneficial influences because there is a complex interplay between detrimental
`and beneficial factors and mediators in the inflammatory milieu of MS lesions.
`
`I. Introduction
`
`Multiple sclerosis (MS) is the most common inflammatory demyelinating disease
`of the central nervous system (CNS). It is believed to be a multifocal immune-
`mediated disorder in which the myelin sheath or the oligodendrocyte is targeted
`by the immune system in genetically susceptible people. There is a considerable
`heterogeneity in terms of clinical, radiological, and pathological changes, mirrored
`by a high intrapatient and interpatient variability in the clinical course and its
`manifestations. The disease aVects approximately 0.1% of the population in tem-
`perate climates. It is a disease of young people with a lifelong and often disabling
`course. MS is manifested in physical symptoms (relapses and disability progression),
`CNS inflammation, brain atrophy and cognitive dysfunction.
`About 85% of the patients begin with a relapsing-remitting disease, where
`neurological symptoms and signs develop over several days, plateau, and then
`usually improve over days to weeks. Approximately two-thirds of patients with
`relapsing-remitting MS (RRMS) undergo a conversion to a secondary progressive
`disease course (SPMS), where relapse frequency lessens over time and progressive
`neurological dysfunction emerges. The remaining 15% of patients begin the
`disease course with a gradually progressive neurological dysfunction, typically a
`slowly worsening myelopathy [primary progressive MS (PPMS)].
`
`II. Pathology and Immunology
`
`The pathological hallmark of MS is the demyelinating plaque which consists
`of infiltrating lymphocytes and macrophages, damage to the blood–brain barrier,
`and loss of myelin.
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`GLATIRAMER ACETATE: MECHANISMS OF ACTION IN MULTIPLE SCLEROSIS 539
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`Periphery
`
`CNS
`
`APC
`
`Blood−brain barrier
`
`6
`
`1
`
`3
`
`4
`
`5
`
`TMyelin
`
`2
`
`BMyelin
`
`BMyelin
`
`APC
`
`TMyelin
`
`FIG. 1. Hypothetical pathophysiological cascade in MS. Details (inclusive numbers) in the text.
`
`Oligodendrocytes synthesize and maintain the axonal myelin sheath of up to
`40 neighboring nerve axons in the CNS. Compact myelin consists of a condensed
`membrane, spiraled around axons to form the insulating segmented sheath
`needed for saltatory axonal conduction; voltage-gated sodium channels cluster
`at the unmyelinated nodes of Ranvier between myelin segments, from where the
`action potential is propagated and spreads down the myelinated nerve segment to
`trigger another action potential at the next node.
`In MS, the composition of the inflammatory infiltrate varies depending on the
`stage of demyelinating activity. Early symptoms of MS are widely believed to
`result from this inflammatory demyelination which leads to slowing or blockade
`of axonal conduction. The regression of symptoms has been attributed to the
`resolution of inflammatory edema and to partial remyelination.
`Current concepts assume that MS occurs as a consequence of immune toler-
`ance breakdown in genetically susceptible individuals (Hafler, 2004). The major
`contributing factors thus include genetics, environment, and immune dysregula-
`tion. T helper (Th) cells are considered to play a pivotal role in the whole self-
`reactive immune response of the CNS, primarily characterized by inflammatory
`demyelination. Putting experimental data from human studies and animal experi-
`ments together, the following pathophysiological cascade seems quite attractive
`which is shown and numbered in Fig. 1:
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`1. Autoreactive T cells, known to exist in any individual, are activated in the
`periphery, probably by molecular mimicry (i.e., recognition of epitopes
`that are common to autoantigens and microbial structures as exogenous
`triggers) or by self-antigens.
`2. T cells may trigger B-cell activation and antibody formation, the latter
`potentially exerting detrimental eVector functions at the myelin sheath
`(Ziemssen and Ziemssen, 2005).
`3. T-cell activation enables transmigration through the blood–brain barrier
`to the sites of inflammation, a cascade of events influenced by adhesion
`molecules, chemotactic factors and migration promoters.
`4. Recognizing their antigens presented by microglia, the local antigen-
`presenting cells (APCs), the autoreactive T cells are reactivated.
`
`5. T cells of both CD4þ and CD8þ cytotoxic phenotypes release proinflam-
`
`matory cytokines. An inflammatory cascade is initiated followed by the
`further recruitment of inflammatory cells like monocytes.
`6. Underlying immunoregulatory defects, such as decrease of regulatory
`T cells in the circulation of patients with MS, allow the further pathological
`activation of autoreactive T cells.
`
`III. MS as Neurodegenerative Disease
`
`Although MS seems to be primarily an inflammatory autoimmune disease, it has
`become evident that axonal loss plays an important role in the pathogenesis of
`disability in patients with MS. While axonal pathology was elegantly and precisely
`described in classic MS neuropathologic studies more than a century ago (Charcot,
`1877), it has reemerged as a major focus of research (Trapp et al., 1998). The
`important question to be addressed is not whether there is axonal loss in MS but
`when and to what extent does the axonal loss occur. The timing and degree of axonal
`loss is of importance not only in its relationship to the etiology of the disease but may
`be central to the appearance of clinical symptoms and the progressive deterioration
`associated with the disease. The fact that axonal loss is irreversible has important
`implications for when and which therapeutic intervention should be used.
`It is likely that various mechanisms contribute to axonal damage during diVerent
`stages of disease. In active lesions, the extent of axonal transection correlates with
`inflammatory activity while even there seems to be an inflammation-independent
`axonal loss (Perry and Anthony, 1999). Hence, axonal loss may be caused by
`inflammatory products of activated immune and glial cells, including proteolytic
`enzymes, cytokines, oxidative products, and free radicals, although the precise
`molecular mechanisms of axonal damage are poorly understood. In addition, the
`magnitude of axonal loss in chronic MS lesions without pronounced inflammatory
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`infiltrates suggests that mechanisms other than inflammatory demyelination con-
`tribute to the degeneration of axons. Several conditions interfere with attempts of
`axonal regrowth after lesions develop. These include the lack of neurotrophic factors
`that support growth, the presence of a glial scar (depending on the site of lesion) or
`the presence of inhibitory molecules that impede axonal growth. Evidence shows
`that axon degeneration following injury has similarities with the cellular mechanisms
`underlying programmed cell death (Perry and Anthony, 1999).
`The biochemical events underlying the inflammatory and neurodegenerative
`phases of the disease are not known in detail and may be quite diVerent (Steinman,
`2001). Inflammation involves activation of T and B cells in the periphery, crossing
`the blood–brain barrier and homing to the lesion site. In the lesion, T cells are
`reactivated by myelin antigens, release cytokines that attract macrophages and
`activate microglia which start to destroy the myelin sheath. Anti-myelin antibodies
`bind complement, attract macrophages, and stimulate opsonization of myelin.
`Demyelination leads to reversible and to some extent irreversible impairment of
`functionality of the axon whose conduction properties are deteriorated, thus
`accounting for the clinical symptoms associated with relapses.
`Neurodegeneration is likely to be a complex process (Chitnis et al., 2005;
`Grigoriadis et al., 2004), especially when it takes place in inflammatory disorders
`like MS. To a significant extent, axonal loss seems to be a major consequence of
`
`demyelination and inflammation, for example, by binding of CD8þ T cells to
`
`exposed axons and secretion of toxic factors. However, other mechanisms not
`directly related to demyelination and inflammation are also likely to be important.
`An example is excitotoxicity: glutamic acid can bind to excitatory amino acid
`receptors on the cell bodies, dendrites, or axon terminals of neurons and initiate a
`process of neuronal cell death (Steinman, 2001).
`Theoretically, there are several possible relationships between inflammation
`and neurodegeneration in MS. Three exclusive hypotheses can be envisaged:
`(a) that neurodegeneration is entirely secondary to inflammation, (b) that inflam-
`mation is entirely secondary to neurodegeneration, or (c) that inflammation and
`neurodegeneration are entirely independent. On the other hand, nonexclusive
`hypotheses stating that neurodegeneration is partially dependent and partially
`independent of inflammation or vice versa can also be put forward, and these
`seem intuitively more likely (Ziemssen, 2005).
`There are a number of clinical arguments in favor of some independence
`between the inflammatory and neurodegenerative processes. For example, in a
`clinical trial of alemtuzumab (Campath-1H), a monoclonal antibody directed
`against CD52 which leads to T-cell depletion, in secondary progressive MS (SPMS),
`a gradual extinction of exacerbations and lesion activity visible on magnetic reso-
`nance imaging (MRI) was demonstrated (Coles et al., 1999). However, disability
`continued to progress in about half the patients in whom progressive brain atrophy
`and axonal degeneration could be observed using MRI and magnetic resonance
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`spectroscopy (MRS). The investigators concluded firstly that inflammation and
`demyelination were responsible for relapses of MS and could be prevented
`by alemtuzumab treatment and secondly that ongoing axonal degeneration
`accounted for the progressive phase of disability. Even though axonal injury may
`have been conditioned by prior inflammation, this process can continue despite
`complete suppression of inflammatory activity.
`There is also neuropathologic evidence for a dissociation between inflamma-
`tory demyelination and axonal injury from a series of 42 biopsy samples obtained
`from patients with MS (Bitsch et al., 2000). Acute axonal injury was visualized by
`amyloid precursor protein (APP) staining. There was no relationship between the
`expression of APP or axonal density and the extent of demyelination, with axonal
`injury being observed even in lesions that were successfully remyelinating. Simi-
`larly, there was no association between the extent of axonal injury and markers of
`acute inflammation such as tumor necrosis factor (TNF)- or inducible nitric
`oxide synthase. However, axonal injury was correlated to some extent with the
`extent of infiltration by CD8þ T lymphocytes and macrophages. A dissociation
`
`between neurodegeneration and inflammatory demyelination is also observed in
`lesions within the cortex. These lesions are characterized by a significant degree of
`axonal transection and apoptosis of neuronal cell bodies. However, the extent of
`infiltration by T lymphocytes and macrophages and the expression of inflamma-
`tory markers are low (Bo et al., 2003; Peterson et al., 2001).
`MRI studies in very early disease also suggest that inflammation and neuronal
`injury are not strictly related. A study of 31 subjects presenting with a clinically
`isolated syndrome evaluated inflammatory lesion activity with classical T2- and
`T1-weighted images after gadolinium enhancement and measured a surrogate
`marker of axonal injury, the size of the N-acetylaspartate peak (NAA) determined
`in the whole brain (Filippi et al., 2003). In these patients, the mean size of the NAA
`peak was some 20% lower than that observed in matched controls. No correlation
`was observed between the size of the NAA peak and lesion volume on either T1 or
`T2 images. The investigators concluded that significant axonal injury occurs early
`in the disease and that this is only indirectly linked to inflammatory activity.
`Studies such as these suggest that treatment strategies for MS need to address
`both the inflammatory and neurodegenerative components of the disease, and that
`anti-inflammatory therapies may only be able to control the inflammation-related
`neurodegenerative process adequately (Chitnis et al., 2005).
`
`IV. The Janus Face of CNS-Directed Autoimmune Inflammation
`
`Inflammation is considered to be a key feature in MS pathogenesis. The
`neurotoxic eVects of inflammation are well established and thought to be at least
`partially responsible for the observed axonal damage. Recently, an increasing
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`body of experimental evidence supports the view of a dual role of the immune
`system in CNS-directed autoimmune inflammation. A number of studies have
`proposed that autoimmune inflammation may have neuroprotective eVects in the
`CNS (Ziemssen, 2005).
`On the one hand, MS and its animal models, representing neuroimmunolo-
`gical diseases which arise when immune cells attack the nervous system, provide
`the paradigm for the deleterious interaction between cells of the immune and
`nervous systems. EAE, the MS animal model, can be induced by active immuni-
`zation with CNS autoantigens MBP or by the transfer of autoantigen-specific
`T cells into naive syngenic recipients (Gold et al., 2006). On the other hand, it was
`demonstrated that MBP-specific, encephalitogenic T cells may have seemingly
`neuroprotective (side) eVects. The neuroprotective and regenerative potential of
`immune cells was first coined by Schwartz et al. (1999).
`They demonstrated that autoimmune T cells could protect neurons in an
`animal model of secondary degeneration after a partial crush injury of the optic
`nerve (Moalem et al., 1999). In several experiments, T cells with diVerent specifi-
`cities [specific for MBP, control antigen ovalbumin (OVA), or a heat-shock
`protein (hsp) peptide] were activated by restimulation with their respective anti-
`gens in vitro, and then injected into rats immediately after a unilateral optic nerve
`injury. Seven days after injury, the optic nerves were analyzed by immunohisto-
`chemistry for the presence of T cells. Small numbers of T cells could be found in
`the intact (uninjured) optic nerves of rats injected with anti-MBP T cells, which is
`consistent with previous observations that activated MBP-specific T cells home to
`intact CNS white matter.
`A much more pronounced accumulation of T cells, however, was observed in
`the crushed optic nerves of the rats injected with T cells specific for MBP, hsp peptide,
`or OVA. The degree of primary and secondary damage to the optic nerve axons
`and their attached retinal ganglion cells was measured by injecting a neurotracer
`distal to the site of the optic nerve lesion immediately after the injury, and again
`after 2 weeks. The number of labeled retinal ganglion cells as marker for viable
`axons was significantly greater in the retinae of the rats injected with anti-MBP
`T cells than with anti-OVA or anti-hsp peptide T cells. Thus, although all three
`T-cell lines (TCLs) accumulated at the site of injury, only the MBP-specific,
`autoimmune T cells had a substantial eVect in limiting the extent of secondary
`degeneration. This neuroprotective eVect was confirmed by electrophysiological
`studies.
`The results demonstrate that T-cell autoimmunity can mediate significant
`neuroprotection after CNS injury. The authors speculate that after injury, ‘‘cryptic’’
`epitopes might become available and might be recognized by endogenous non-
`encephalitogenic (benign) T cells. After local stimulation, these protective autoreac-
`tive T cells could exert their neuroprotective eVect. The findings further substantiate
`the idea that ‘‘natural autoimmunity’’ can be benign and may even function as a
`protective mechanism (Cohen, 1992).
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`Macrophages seem to represent another type of immune cell which is capable
`of mediating neuroprotection and/or stimulating recovery of CNS lesions. The
`injection of activated macrophages into transected rat spinal cord stimulated
`tissue repair and partial recovery of motor function (Rapalino et al., 1998). The
`neuroprotective activity of immune cells is not restricted to the CNS. After
`experimental axotomy of the facial nerve of immunodeficient SCID mice, the
`survival of facial motor neurons was severely impaired compared to immunocom-
`petent wild-type mice. Reconstitution of SCID mice with wild-type splenocytes
`containing T and B cells restored the survival of facial motor neurons in these
`mice to the level of the wild-type controls (Serpe et al., 1999).
`It is equally evident that a large number of neurotoxic and proinflammatory
`mediators are produced and released by immune cells (Kerschensteiner et al.,
`2003). The neutralization of toxic inflammatory mediators may improve the
`outcome of MS and experimental models of CNS damage. On the other hand,
`we know that immune cells can supply a number of neuroprotective mediators,
`including neurotrophic factors, anti-inflammatory cytokines, and prostaglandins
`which may reduce tissue damage (Hohlfeld et al., 2006).
`The idea that inflammatory reactions may not always be harmful under
`certain conditions even confers neuroprotection and repair, has important con-
`sequences for the design of immunomodulatory therapies for MS (Hohlfeld et al.,
`2006). Undebatably, there is convincing rationale for immunosuppressive treat-
`ment when the noxious eVects of the inflammatory reaction prevail. Because
`nonselective immunosuppressive treatments will suppress both destructive and
`beneficial components of inflammation, therapy is likely to fail when the benefi-
`cial eVects of CNS inflammation outweigh its negative consequences. It seems, for
`example, possible that the lack of beneficial (side) eVects of inflammation during
`the late phase of MS with little inflammation but ongoing axonal loss contributes
`to the pathogenesis of neurodegeneration. In MS, it is unfortunately unclear
`whether there is a stage of the disease when the inflammatory reaction is more
`beneficial than harmful.
`The concept of the neuroprotective role of inflammation can be extended to
`neurodegenerative, ischemic, and traumatic lesions of the CNS, considering that
`inflammation is a universal tissue reaction crucial for defense and repair (Ziemssen
`and Ziemssen, 2005).
`
`V. Neurotrophic Factors Are Released by Different Immune Cells
`
`The precise mechanisms involved in immune-mediated neuroprotection re-
`main to be clarified. A number of recent studies have shown that several types of
`immune cells and hematogenic progenitor cells express one or more neurotrophic
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`factors. For example, nerve growth factor (NGF) is produced by B cells, which
`also express the trkA receptor and p75 NGF receptor (Torcia et al., 1996). Because
`neutralization of endogenous NGF caused apoptosis of memory B cells, it was
`concluded that NGF is an autocrine growth factor for memory B cells.
`More recently, another neurotrophin, brain-derived neurotrophic factor
`(BDNF), was found to be expressed in immune cells. BDNF was originally cloned
`in 1989 as the second member of the neurotrophin family which includes NGF
`and neurotrophins (NT)-3, -4/5, -6, and -7 (Lewin and Barde, 1996). Since then,
`the important role of BDNF in regulating the survival and diVerentiation of
`various neuronal populations, including sensory neurons, cerebellar neurons,
`and spinal motor neurons, has been firmly established. Neurons are the major
`source of BDNF in the nervous system. BDNF binds to diVerent types of recep-
`tors: the tyrosine kinase receptor B (trkB) which exists in two isoforms—the full-
`length receptor (gp145trkB) and the truncated receptor (gp95trkB) lacking the
`tyrosine kinase domain—and the p75 neurotrophin receptor (Klein et al., 1991). It
`is thought that BDNF and NT-4/5 exert their biological function via the full-
`length form of trkB receptor which expression seems to be restricted to neuronal
`cell populations.
`Immune cells can be a potent source of the neuroprotective factor BDNF
`in neuroinflammatory disease (Hohlfeld et al., 2006). Activated human T cells,
`B cells, and monocytes are able to secrete bioactive BDNF after in vitro activation
`(Kerschensteiner et al., 1999). The BDNF secreted by immune cells is bioactive as
`it supports neuronal survival in vitro. In histology, BDNF immunoreactivity was
`found in T cells and macrophages in active and inactive MS lesions.
`Similar observations were recently reported by several other groups of in-
`vestigators. After experimental injury of the striatum, activated macrophages and
`microglia cells transcribe mRNA for glial cell line-derived neurotrophic factor
`(GDNF) and BDNF (Batchelor et al., 1999). This could help to explain the
`sprouting of dopaminergic neurons observed after experimental injury. Tran-
`scripts for BDNF and NT-3 and their receptors trkB and trkC were found in
`subpopulations of human peripheral blood cells (Besser and Wank, 1999). BDNF
`protein was secreted by cultured T-cell clones.
`Besides BDNF, there is a robust expression of the full-length BDNF receptor
`gp145trkB in neurons in the vicinity of MS plaques (Stadelmann et al., 2002).
`Single neurons with clearly pronounced trkB immunoreactivity close to MS
`lesions can be observed, suggesting an upregulation of trkB in a proportion of
`damaged neurons. Additionally, full-length trkB immunoreactivity is present in
`reactive astrocytes within the lesions. The restriction of trkB expression to neural
`cell types in MS underscores the possibility that there may be BDNF signaling
`from infiltrating cells to neurons in neuroinflammatory lesions, as would be
`necessary for immune cells to support neuronal survival or provide axonal
`protection.
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`Within the MS lesion, immune cells seem to be the major source of BDNF
`(Stadelmann et al., 2002). They are likely to release this substance in the immedi-
`ate vicinity of nerve cell processes, which—according to the observed trkB
`expression—are likely to be responsive to the neuroprotective eVects of BDNF.
`This neurotrophin-mediated neuroimmune signaling network could be a major
`factor that helps to preserve axons in a microenvironment that is clearly capable
`of exerting significant neurotoxicity. Thus, it should be considered as a beneficial
`aspect of neuroinflammation that could be worth preserving therapeutically, or
`even reinforcing using tailored immunomodulatory treatment strategies.
`
`VI. Glatiramer Acetate: Historical Remarks
`
`w
`
`), formerly known as copolymer 1, is the
`Glatiramer acetate (GA, Copaxone
`acetate salt of a standardized mixture of synthetic polypeptides containing the
`four amino acids L-alanine, L-glutamic acid, L-lysine, and L-tyrosine with a
`defined molar ratio of 0.14:0.34:0.43:0.09 and an average molecular mass of
`4.7–11.0 kDa, that is, an average length of 45–100 amino acids (Arnon et al.,
`1996; Teitelbaum et al., 1997a,b).
`In the 1960s, Drs. Sela, Arnon, and their colleagues at the Weizmann
`Institute, Israel were involved in studies on the immunologic properties of a series
`of polymers and copolymers which were developed to resemble MBP, a myelin
`protein. MBP in Freund’s complete adjuvant induces EAE, the best animal model
`of MS. They were interested in evaluating whether these polypeptides could
`simulate the ability of MBP and fragments and regions of the MBP molecule to
`induce EAE (Teitelbaum et al., 1971, 1973, 1974a,b). None of these series was
`capable of inducing EAE, but several polypeptides were able to suppress EAE in
`guinea pigs. Copolymer 1, later known as GA, was shown to be the most eVective
`polymer in preventing or decreasing the severity of EAE. The suppressive eVect is
`a general phenomenon and not restricted to a particular species, disease type, or
`encephalitogen used for EAE induction.
`Abramsky et al. (1977) were the first who treated a group of severe RRMS
`patients with intramuscular GA 2–3 mg every 2–3 days for 3 weeks, then weekly for
`2–5 months. No conclusions could be drawn regarding drug eYcacy but there were
`no significant undesirable side eVects. Three clinical trials in the 1980s were
`performed showing some evidence of eYcacy that was adequate for support of the
`Food and Drug Administration (FDA) approval and a good safety profile (Bornstein
`et al., 1982, 1984, 1987). However, the results of these studies must be interpreted
`with caution because before 1991 the production of the drug was not standardized
`( Johnson, 1996). DiVerent batches had variable suppressive eVects on EAE which
`could also imply variable eVects in MS patients.
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`In 1991, a phase III multicenter trial with a daily 20-mg dose of an s.c.
`administered, highly standardized GA preparation was started in the United
`States. This double-blind, placebo-controlled study demonstrated that GA signif-
`icantly reduced the relapse rate without significant side eVects ( Johnson et al.,
`1995).
`In 1996, GA was approved by the US FDA as a treatment of ambulatory
`patients with active RRMS. Since then GA has been licensed for approval in
`many other countries (Ziemssen et al., 2002b).
`
`VII. GA: Overview of Clinical Studies
`
`Four early exploratory open studies were performed in the late 1970s and
`early 1980s in order to obtain indications of dosing and safety (Abramsky et al.,
`1977; Bornstein et al., 1982, 1984, 1987). In total, 41 patients with relapsing-
`remitting or SPMS were enrolled in these studies. The treatment schedules, doses
`of GA, and treatment duration were quite variable from study to study. The
`maximum dose of 20 mg/day was well tolerated and no severe adverse eVects
`were detected in these studies.
`To assess more comprehensively the eYcacy of GA in patients with RRMS, a
`definitive phase III trial was conducted at 11 US medical centers with a total of
`251 RRMS patients who received GA at a dosage of 20 mg or placebo by daily
`s.c. injection for 2 years ( Johnson et al., 1995). As the primary end point, the mean
`annualized relapse rates were 0.59 for the GA-treated group and 0.84 for the
`
`placebo group, a 29% reduction was statistically significant ( p ¼ 0.007). Trends in
`
`the proportion of relapse-free patients and median time to first relapse favored
`GA treatment. Patients in both groups with higher disability at entry, measured by
`the Expanded Disability Status Scale (EDSS), had a higher relapse rate, while the
`largest reduction in relapse rate between groups occurred in patients with a
`baseline EDSS of 0–2 (33% vs a reduction of 22% in patients with an EDSS
`score at entry >2). When the proportion of patients who improved, were un-
`changed, or worsened by 1 EDSS step from baseline to end of study (2 years)
`worsened ( p ¼ 0.037). The eVect of treatment was constant throughout the entire
`
`was evaluated, significantly more patients on GA improved and more on placebo
`
`study duration. Patient withdrawals were 19 (15.2%) from the copolymer 1 group
`and 17 (13.5%) from the placebo group at approximately the same intervals. The
`treatment was well tolerated. The most common adverse experience was the local
`injection-site reaction. Rarely, the transient self-limiting immediate post-injection
`reaction followed the injection in 15.2% of those treated with GA and 3.2% of
`those treated with placebo.
`
`Page 11 of 34
`
`YEDA EXHIBIT NO. 2083
`MYLAN PHARM. v YEDA
`IPR2015-00644
`
`

`
`548
`
`ZIEMSSEN AND SCHREMPF
`
`In an extension of this study up to 35 months with unchanged blinding and
`study conditions, the clinical benefit of GA for both the relapse rate and for
`neurological disability was sustained ( Johnson et al., 1998). Thus, the reduction of
`
`annual relapse rate was 32% in favor of GA ( p ¼ 0.002). The results of this
`
`extension study confirmed the excellent tolerability and safety profile of GA. This
`study was further on extended as open-label study with all patients receiving
`active drug. The published data from approximately 10 years of ongoing trial
`showed a persistent reduced relapse rate of about 1 relapse per 5 years (Ford et al.,
`2006). After more than 10 years of observation, 108 out of 232 patients (47%)
`were still participating. Sixty-two percent of the group who stayed on GA without
`interruption were neurologically unchanged or improved from baseline by at least
`one step of the EDSS scale in contrast to 28% of the withdrawn patients. This is
`the first time that the benefits of an immunomodulatory treatment over this
`clinically highly relevant long-term time period are demonstrated in a planned
`neurological follow-up study.
`Studies using an oral formulation of GA (CORAL) (Filippi et al., 2006) and
`applying GA to PPMS patients (PROMISE) (Wolinsky and PROMiSe Trial
`Study Group, 2004) failed to demonstrate significant treatment eVects. There
`are promising new data on using a higher GA dose (40 mg) per injection which
`are studied in a multicenter, controlled trial (FORTE).
`
`VIII. GA: Imaging Studies
`
`Several early MRI studies indicated a trend towards benefit with GA, but
`were limited by the small number of evaluated patients. Wolinsky et al. were able
`to demonstrate a definite, but modest eVect of GA on MRI enhancements of MS
`patients in the US open-label GA extension MRI trial for relap