`
`www.elsevier.com/locate/jns
`
`Pharmacokinetics and pharmacodynamics of the interferon-betas,
`glatiramer acetate, and mitoxantrone in multiple sclerosis
`Oliver Neuhaus ⁎, Bernd C. Kieseier, Hans-Peter Hartung
`
`Department of Neurology, Heinrich Heine University, Moorenstraße 5, D-40225 Düsseldorf, Germany
`
`Received 2 March 2006; received in revised form 25 April 2006; accepted 1 May 2006
`Available online 27 March 2007
`
`Abstract
`
`Five disease-modifying agents are currently approved for long-term treatment of multiple sclerosis (MS), namely three interferon-beta
`preparations, glatiramer acetate, and mitoxantrone1. Pharmacokinetics describes the fate of drugs in the human body by studying their
`absorption, distribution, metabolism and excretion. Pharmacodynamics is dedicated to the mechanisms of action of drugs. The understanding
`of the pharmacokinetics and pharmacodynamics of the approved disease-modifying agents against MS is of importance as it might contribute
`to the development of future derivatives with a potentially higher efficacy and a more favourable safety profile. This article reviews data thus
`far present both on the pharmacokinetics as well as on the putative mechanisms of action of the interferon-betas, glatiramer acetate, and
`mitoxantrone in the immunopathogenesis of MS.
`© 2007 Elsevier B.V. All rights reserved.
`
`Keywords: Multiple sclerosis; Immunotherapy; Immunomodulation; Immunosuppression; Interferon-beta; Glatiramer acetate; Mitoxantrone
`
`1. Introduction
`
`Multiple sclerosis (MS) is the most common chronic
`central nervous system (CNS) disorder of younger adults and
`a major cause of lasting neurological disability [1].
`Pharmacological therapeutic approaches in MS are based
`on three principles. Apart from (i) treatment of relapses by
`corticosteroids and, in some cases, plasma exchange, and (ii)
`symptomatic treatment, (iii) immunomodulatory or immu-
`nosuppressive long-term treatment with the aim of modify-
`ing the disease course represents the mainstay. However, as
`MS is a chronic disorder requiring chronic therapy, the
`optimal treatment choice for individual patients is often
`difficult to make and in many cases is discussed controver-
`sially [2].
`
`⁎ Corresponding author. Tel.: +49 211 81 17880; fax: +49 211 81 18469.
`E-mail address: oliver.neuhaus@uni-duesseldorf.de (O. Neuhaus).
`1 A sixth disease-modifying agent, natalizumab, has been approved for
`treatment of multiple sclerosis after acceptance of this manuscript. Its
`pharmacokinetics and pharmacodynamics are not subject of this review.
`
`0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
`doi:10.1016/j.jns.2006.05.071
`
`The rationale to influence immunological activity in MS
`is to suppress ongoing inflammation with an aim to prevent
`myelin and axonal damage and thus to prevent clinical
`disability. For years, a number of immunosuppressive agents
`(i.e. inhibitors of crucial components of the immune system
`causing generalized immune dysfunction) were used off
`label, the potential beneficial effects of which in MS were
`limited by systemic adverse effects, such as increased risk of
`cancer or infection. Examples are azathioprine (licensed for
`MS treatment in some countries) or cyclophosphamide. In
`the 1990s, two classes of immunomodulatory agents were
`approved for the treatment of relapsing–remitting MS,
`namely interferon (IFN)-beta (IFN-β1a or IFN-β1b) as
`well as glatiramer acetate (GA) [3–5]. Immunomodulators –
`without generally suppressing immunological properties –
`shift immune responses from pro-inflammatory autoimmune
`conditions (mediated by TH-1 cytokines released from
`autoreactive T cells) towards a more beneficial anti-inflam-
`matory environment (mediated through TH-2 cytokines
`secreted by regulatory T cells) [6]. Both IFN-β and GA have
`been proven at least partially effective in relapsing–remitting
`MS as assessed in pivotal and subsequent trials [7–9]. In
`
`Page 1 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`28
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`secondary progressive MS, treatment options are limited [10].
`All IFN-β preparations have been investigated in phase 3
`clinical trials, but only IFN-β1b, as administered in a European
`Trial [11], in which many of the patients included exhibited
`clinical relapses, significantly delayed time to onset of
`sustained progression of disability [12] as measured by the
`Expanded Disability Status Scale (EDSS) [13]. The more
`recent approval of the immunosuppressant mitoxantrone in
`MS gave clinicians another therapeutical tool for treatment
`of active forms of relapsing–remitting and secondary pro-
`gressive MS [14].
`
`2. Pharmacokinetics
`
`Pharmacokinetics describes absorption, distribution, me-
`tabolism and elimination of a given drug. The action and fate
`of this drug in the body over a period of time is investigated,
`including the processes of permeation through barriers
`between compartments, localization in tissues and biotrans-
`formation. The pharmacokinetics of the drug is directly
`related to its pharmacodynamics, i.e. its interaction with the
`body.
`
`3. Pharmacokinetics of interferon-beta 1a and
`interferon-beta 1b
`
`Human IFN-β is a polypeptide naturally produced by
`human fibroblasts (predominantly but not exclusively, as
`there are reports of other sources of natural IFN-β, such as
`dendritic cells or
`retinal glial cells; upon stimulation,
`
`virtually all mammalian cells can produce IFN-β). IFN-β
`is highly species-specific, i.e. it is effective only in the
`human organism. It consists of 166 amino acids forming five
`α helices (Fig. 1). IFN-β1a is produced in Chinese hamster
`ovary cells and is pharmacologically identical with the natu-
`ral form, i.e. it is glycosylated by oligosaccharides at Asn 80;
`IFN-β1b is produced in E. coli and has slight differences
`compared to the natural form: it is not glycosylated, Met 1 is
`lacking (i.e. IFN-β1b has 165 amino acids), and Cys 17 is
`replaced by Ser. In both IFN-β forms, a disulphide bridge
`extends between Cys 31 and Cys 141. The optimal dosage
`and route of administration (subcutaneously vs. intramus-
`cularly) and the resulting pharmacodynamic properties of
`IFN-β are subjects of controversial discussion [15–19].
`IFN-β1a is a lyophilized glycoprotein produced in
`mammalian cells using the natural human gene sequence
`[20]. Two preparations are licensed for treatment of MS,
`30 μg once a week administered by intramuscular injection,
`and 22 or 44 μg administered three times a week
`subcutaneously. In a comparative study of three routes of
`administration of 60 μg IFN-β1a in healthy donors, the
`intramuscular route was observed to induce the highest area
`under curve (AUC) for serum IFN activity as compared to
`subcutaneous and intravenous routes [20]. This putative
`disadvantage of the subcutaneous route is thought to be
`circumvented by the higher weekly dose. However,
`the
`results of direct dosing comparison studies are contradictory:
`in the pivotal trial of IFN-β1a administered subcutaneously,
`the 44 μg three times a week group was shown to exhibit
`more beneficial effects as compared to the 22 μg three times
`
`Fig. 1. Structure of interferon-beta. Two recombinant interferon (IFN)-β preparations are approved for MS treatment. IFN-β1a is pharmacologically identical
`with the natural form, i.e. it is glycosylated by oligosaccharides at Asn 80; IFN-β1b has slight differences compared to the natural form: it is not glycosylated,
`Met 1 is lacking (i.e. IFN-β1b has 165 amino acids), and Cys 17 is replaced by Ser. In both IFN-β forms, a disulphide bridge extends between Cys 31 and Cys
`141. Structurally important amino acids are highlighted in red.
`
`Page 2 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`29
`
`Fig. 2. Structure of glatiramer acetate. Glatiramer acetate (GA) is the acetate salt of a standardized, randomized mixture of synthetic polypeptides consisting of
`L-glutamic acid, L-lysine, L-alanine, and L-tyrosine with a defined molar residue ratio of 0.14 : 0.34 : 0.43 : 0.09 and an average length of 45 to 100 amino acids.
`One possible example sequence is shown here.
`
`a week group [9]. A similar dose-dependent effect was
`observed in a study comparing 44 μg vs. 22 μg subcutane-
`ously once a week (a dose that is not licensed for treatment of
`MS) [21]. In contrast, a study comparing 30 μg vs. 60 μg
`(double dose) once a week intramuscularly did not reveal
`any differences [22]. Taken together, the optimal route, dose
`and frequency of IFN-β1a in MS remains elusive.
`IFN-β1b is a lyophilized protein produced by DNA
`recombinant technology using E. coli and has a molecular
`weight of 18.5 kDa. It is combined with mannitol and human
`albumin in order to have a neutral pH of 7.2. The approved
`dose is 250 μg every other day administered by subcutane-
`ous injection [23]. A comparative phase III trial assessing the
`double dose of 500 μg every other day subcutaneously is
`currently underway.
`
`4. Neutralizing antibodies to interferon-beta
`
`A major issue of the pharmacokinetics of IFN-β (and also
`affecting its pharmacodynamics)
`is the relevance of
`neutralizing antibodies [15,24–27]. Although it is estab-
`lished that the induction of neutralizing antibodies in patients
`treated with IFN-β reduces its clinical effects and may
`accelerate disease progression [27], the mode of measuring
`the presence of neutralizing antibodies as well as the optimal
`time point (after a given treatment duration vs. when clinical
`non-responsiveness to IFN-β occurs) and the clinical
`consequences are still a matter of debate [26,28].
`
`5. Pharmacokinetics of glatiramer acetate
`
`GA is the acetate salt of a standardized, randomized
`mixture of synthetic polypeptides consisting of L-glutamic
`acid, L-lysine, L-alanine, and L-tyrosine with a defined molar
`residue ratio of 0.14:0.34:0.43:0.09 and an average length of
`45 to 100 amino acids (Fig. 2). The approved dose is 20 mg
`applied subcutaneously every day. After subcutaneous
`administration, GA is quickly degraded to free amino acids
`and small oligopeptides with only 10% remaining at the
`injection site after 1 h [29]. No systemic plasma concentra-
`tions nor any urinary or faecal excretion are detectable. Due
`to its high polarity and hydrophilic nature, the penetration of
`GA through the blood–brain barrier is impeded [29]. Thus,
`GA is most unlikely to reach the central nervous system and
`most probably initiates its major immunological effects in
`the periphery [30].
`
`6. Pharmacokinetics of mitoxantrone
`
`Mitoxantrone has a molecular mass of 517.4 Da and is a
`synthetic anthracenedione derivative related to the anthracy-
`clins doxorubicin and daunorubicin (Fig. 3). The agent
`interacts with topoisomerase-2 and causes single and double
`strand breaks by intercalating the DNA. Due to the rapid
`diffusion of mitoxantrone into different tissue compartments
`followed by a relatively slow elimination phase, its report-
`ed maximum plasma half-life varies between 25 and 215 h
`
`Fig. 3. Structure of mitoxantrone. Mitoxantrone is an anthracenedione derivative related to the anthracyclins doxorubicin and daunorubicin.
`
`Page 3 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`30
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`[31–36]. Mitoxantrone is eliminated both by the kidney and
`the liver, although less than 11% of mitoxantrone was shown to
`be recovered in urine and less than 25% in faeces within 5 days
`after administration [31]. Furthermore, it has been reported to
`persist in the body for up to 9 months [37]. Reports on the
`ability of mitoxantrone to cross the blood–brain barrier are
`contradictory [37–39]. After intravenous administration, there
`is a linear relationship between the dose and the AUC.
`Mitoxantrone is 78% bound to plasma proteins [40].
`
`7. Pharmacodynamics
`
`Pharmacodynamics assesses the action of a given drug in
`an organism, i.e. the interaction between a drug and the
`body. It is the study of the biochemical and physiological
`effects and the mechanisms of drug action as well as the
`relationship between drug concentration and effect.
`On the molecular level, the binding to and activation or
`deactivation of receptors, signal transduction, induction of
`biosynthesis, impact on transport processes contribute to the
`pharmacodynamics of a drug.
`
`8. Current view on the immunopathogenesis of multiple
`sclerosis
`
`In order to understand the pharmacodynamics of disease-
`modifying treatments in MS,
`the current concept of its
`pathogenetic background needs to be highlighted. Deviations
`of immune responses play a central role in the pathogenesis
`of MS [41] by contributing to the formation and perturbation
`of the MS lesion [42]. The initial inflammatory phase is
`characterized by selective demyelination, and eventually
`subsides to a neurodegenerative phase with axonal loss and
`gliosis [41,43]. With ever novel scientific information
`emerging, concepts regarding the pathogenesis of MS are in
`constant flux and shown in Fig. 4 [44]. In genetically
`susceptible individuals an activation of antigen-specific,
`encephalitogenic T cells occurs in response to a yet
`unidentified trigger, probably in the periphery. Once activated,
`T lymphocytes are able to migrate across the blood–brain
`barrier and invade the CNS . Reactivated there, these T cells
`release pro-inflammatory cytokines and orchestrate destruc-
`tion of the myelin sheath by various immune cell types.
`Pathologically, four different pathologic patterns have been
`described [42]: (i) T-cell and macrophage-mediated demye-
`lination; (ii) antibody-mediated demyelination involving
`complement activation; (iii) distal oligodendrogliopathy and
`oligodendrocyte apoptosis; (iv) primary oligodendrocyte
`degeneration. Interestingly,
`the presence of pattern (iii),
`oligodendrocyte apoptosis, has recently been demonstrated
`in very early MS lesions [45]. In parallel to the autoaggressive
`inflammatory phase causing demyelination, recent evidence
`suggests that axonal loss responsible for irreversible disability
`occurs already early in the disease course and – as the disease
`evolves – predominates in the underlying pathogenetic
`mechanisms [41,46,47].
`
`9. Pharmacodynamics of interferon-beta 1a and
`interferon-beta 1b
`
`IFN-β exerts its immunological effects antigen-indepen-
`dently through reducing the secretion of proteolytic matrix
`metalloproteinases that mediate the migration of T cells
`across biological barriers [48,49], as well as possibly
`downregulating MHC class II on various antigen presenting
`cells (APC) [50,51]. IFN-β has immunomodulatory proper-
`ties, antiviral and antiproliferative effects and promotes cell
`differentiation [52]. Although the mechanisms of action of
`IFN-β are not yet finally understood, there is agreement that the
`major effects are mediated by a transmembranous IFN receptor
`leading to upregulation or downregulation of target genes
`[53,54]. IFN-β shares the receptor with IFN-α as so-called
`“type I interferons” (in contrast to the “type II interferon”, I FN-
`γ). The receptor consists of two chains, IFN-αR1 and IFN-
`αR2, or several subvariants. Ligand binding of IFN-β to the
`extracellular domain of the receptor induces an intracellular
`signal transduction cascade by (i) recruitment and activation of
`the cytoplasmic tyrosine kinase (Tyk)-2 (by IFN-αR1) and
`Janus kinase (Jak)-1 (by IFN-αR12); (ii) subsequent phos-
`phorylation and recruitment of “signal
`transducers and
`activators of transcription” (Stat)-1 and Stat-2 forming a Stat-
`1/Stat-2 heterodimer; (iii) migration of the Stat-1/Stat-2
`heterodimer to the nucleus; (iv) association with the p48
`protein forming the active “IFN-stimulated gene factor-3”; (v)
`binding to promoter elements and initiation of the transcription
`of target genes. The variations of this rough scheme of signal
`transduction – that are based on the wide complexity in the
`single interaction steps – are still not completely identified [53].
`IFN-β exerts a wide and ever broadening range of
`immune effects [52,55]. Among other effects, it
`
`• suppresses T-cell proliferation [56,57];
`• diminishes IFN-γ-induced upregulation of MHC class II
`expression [58,59];
`• downregulates matrix metalloproteinases (MMP) [48,49,
`60]; decreases surface-expressed adhesion molecules [61]
`and increases release of soluble adhesion molecules [60]
`(the latter three actions in concert reduce the T-cell
`migratory potential);
`• induces the production of TH2 cytokines and conversely
`reduces synthesis of TH1 cytokines [62,63];
`• inhibits activation of monocytes [64].
`
`Comparing the three interferon preparations, apart from
`clinical parameters as assessed in the pivotal and subsequent
`trials [7–9], some pharmacodynamical differences have been
`demonstrated. In an open trial comparing the cytokine profiles of
`short-term peripheral blood lymphocyte cultures in patients
`treated with IFN-β1a i.m. and IFN-β1b, respectively, IFN-β1a
`has been shown to increase the concentrations of the TH2
`cytokines IL-4 and IL-10 whereas IFN-β1b reduced the
`concentration of the TH1 cytokine IFN-γ [65]. However, the
`TH net effect of both treatments putatively remained comparable.
`
`Page 4 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`31
`
`Fig. 4. Hypothetical pathogenesis of MS. Via their T-cell receptor (TCR), pro-inflammatory T cells are activated in the periphery by foreign or self antigens (Ag) presented on major histocompatibility complex class II
`(MHC-II) by antigen-presenting cells (APC), including dendritic cells and B cells. Activated T cells migrate to, adhere at and penetrate through the blood–brain barrier, a step mediated by adhesion molecules,
`proteases and chemokines. Inside the central nervous system (CNS), T cells are reactivated in the context of MHC-II on resident APC, predominantly microglia cells. These reactivated T cells secrete pro-
`inflammatory cytokines, such as interferon (IFN)-γ or interleukin (IL)-2 and induce CNS inflammation by subsequent activation of macrophages, other T cells and B cells as effector cells. Macrophages and T cells
`attack the oligodendrocytic myelin sheath by cytotoxic mediators, mainly tumor necrosis factor (TNF)-α, oxygen (O2) radicals and nitric oxide (NO). B cells differentiate to plasma cells that secrete demyelinating
`antibodies. They can guide and activate macrophages or ignite the complement cascade with assembly of the membrane attack complex which causes pore formation in myelin membranes. Modified from Ref. [44],
`with permission from Elsevier.
`
`Page 5 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`32
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`10. Pharmacodynamics of glatiramer acetate
`
`GA acts on “signal one” of T cell activation by binding to
`MHC class II molecules irrespective of haplotype [66], and
`possibly crossreacts with several CNS antigens. It also serves
`as an altered peptide ligand (APL) [67] and induces anti-
`inflammatory regulatory GA-reactive T cells capable of a
`“bystander suppression” once reactivated inside the CNS
`[68].
`In contrast to the multiple immunological effects of IFN-β
`that are antigen-nonspecific, the immunomodulatory potential
`of GA is based on immune cells specific for myelin basic
`protein (MBP) and probably other myelin antigens [52,68,69].
`Four major mechanisms of GA have been identified, namely
`
`• competition of GA with MBP for binding to MHC
`molecules [66];
`• competition of the GA/MHC complex with MBP/MHC
`for binding to the T-cell receptor (TCR) [70];
`• activation and tolerance induction of MBP-specific T
`cells through an altered peptide ligand [67];
`• induction of GA-reactive TH2-like regulatory cells
`mediating local bystander suppression [71,72].
`
`Whereas the first two effects are supposed to take place
`only in vitro, the latter two mechanisms are most likely to
`occur also in vivo and could contribute to the anti-
`inflammatory effects of GA [68].
`More recently, a series of hints have emerged that GA may
`also exert neuroprotective effects, although the final proof of this
`hypothesis is still pending:
`
`• GA significantly reduces the number of T1 black holes in
`magnetic resonance imaging (MRI) in MS [73];
`• MR spectroscopy revealed a higher N-acetyl-aspartate
`(NAA)-creatine-ratio in GA-treated than in untreated
`patients [74];
`• GA-treated patients have been observed to exhibit lower
`brain atrophy than patients treated with placebo [75];
`• Human activated GA-reactive T lymphocytes secrete
`brain derived neurotrophic factor (BDNF), that exerts
`neuroprotective properties [76]. T cells with other
`specificity secrete BDNF as well, although at
`lower
`concentrations. In addition, GA-reactive anti-inflamma-
`tory TH2 cells have been shown to secrete higher
`amounts of BDNF than pro-inflammatory TH1 cells [77].
`• In an adoptive transfer mouse model, GA-specific T
`lymphocytes have been demonstrated to migrate to the
`CNS and express BDNF in situ [78]. In a transgenic
`experimental autoimmune encephalomyelitis (EAE)
`model, MBP-specific T cells secreting the neurotrophic
`factor nerve growth factor (NGF) induced a significantly
`milder course of EAE than MBP-specific cells not
`secreting NGF [79]. In addition, other neurotrophic
`factors have been demonstrated to be beneficial in EAE,
`both clinically and pathologically [80,81].
`
`• In another EAE model the burden of axonal damage has
`been observed to be diminished by GA treatment [82].
`• In non-inflammatory animal models for motoneuron
`diseases or trauma, beneficial effects of GA (plus complete
`Freund's adjuvant) have been demonstrated [83].
`
`11. Pharmacodynamics of mitoxantrone
`
`Mitoxantrone interacts with topoisomerase-2 and causes
`both crosslinking and single and double strand breaks by
`intercalating the DNA [84]. Apart
`from the cytotoxic
`efficacy of mitoxantrone, immunosuppressive effects and
`even antiviral and antibiotic effects have been observed
`[85,86]. More recently, immunomodulatory properties have
`been suggested, as a number of distinct
`immunological
`effects have been described [40,87,88]. Still, more research
`is warranted to illuminate the immunological effects of
`mitoxantrone in MS, as its specific mechanisms of action
`targeting the immune system still widely remain unclear.
`
`11.1. Immunosuppressive properties
`
`Mitoxantrone is a potent immunosuppressive agent target-
`ing proliferating immune cells [89–92]. It inhibits proliferation
`of macrophages, B lymphocytes and T lymphocytes [89,91].
`
`11.2. Effects on helper and suppressor T cells
`
`In an in vitro system testing an anti-sheep red blood cell
`response, mitoxantrone was observed to inhibit T helper
`activity and – conversely – to enhance T suppressor functions
`[90]. In contrast, in an in vivo mouse model, the induction of
`suppressor T cells was also abrogated by mitoxantrone [90]. T
`helper cells were indirectly inhibited by mitoxantrone-induced
`macrophages.
`
`11.3. Induction of cell death
`
`Mitoxantrone was shown to induce apoptosis of B
`lymphocytes [93] and other types of antigen-presenting cells
`[94]. Comparison of peripheral blood mononuclear cells
`(PBMC) obtained from MS patients before and immediately
`after application of mitoxantrone exhibited a decreased
`proliferation of PBMC based on necrotic cell death, predom-
`inantly in B cells [95]. Thus, there is apparently a bimodal
`mechanism of cell death induced by mitoxantrone: apoptosis at
`lower concentrations and cell lysis at higher concentrations.
`Previous pharmacokinetic studies in oncology revealed maxi-
`mum serum concentrations of mitoxantrone between 308 and
`839 ng/ml and terminal half-lifes between 38.4 and 71.5 h [33–
`36]. Thus, in the first approximately 10 days after infusion of
`12 mg/m2 body surface, maximum serum concentrations
`exceed 20 ng/ml (a putative threshold between induction of
`necrosis and apoptosis [94]), whereas the latter 80 days of a
`three-month dosage regime will have concentrations below
`20 pg/ml. Thus, mitoxantrone apparently may act via short-term
`
`Page 6 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`33
`
`Fig. 5. Putative action sites of interferon-beta, glatiramer acetate, and mitoxantrone in the pathogenesis of MS. The action sites of the currently approved disease-modifying drugs are highlighted (see Fig. 4).
`(a) Antigen presentation by antigen-presenting cells is inhibited. (b) Proliferation of autoreactive T cells is blocked or tolerance is induced. (c) T-cell migration, adhesion and penetration are abrogated. (d) T-cell
`reactivation inside the central nervous system (CNS) is blocked. (e) Release of pro-inflammatory cytokines by autoreactive T cells is diminished. (f ) A cytokine shift from pro-inflammatory T-helper (TH)-1 to anti-
`inflammatory TH2 cytokines is induced. (g) A special mechanism of action of glatiramer acetate (GA) is the induction of T cells cross-reactive with both GA and CNS antigens (CNS Ag) which after reactivation
`inside the CNS exert a local bystander suppression effect through the release of TH2 cytokines. (h) Activation of macrophages or secretion of pro-inflammatory mediators are curtailed. (i) Apoptosis of autoreactive T
`cells is induced. ( j) Activation of B cells and their differentiation to plasma cells is blocked. (k) Axonal damage may be reduced. Further abbreviations: IFN, interferon; IL, interleukin; MHC, major histocompatibility
`complex; TCR, T- cell receptor. Modified from Ref. [44], with permission from Elsevier.
`
`Page 7 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`34
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`immunosuppressive effects by the induction of cell lysis leading
`to blood leukocyte reduction postinfusion and to inhibition of
`proliferation of all types of immune cells demonstrated in vitro
`[89,90,95]. In addition, a long-term immunological impact of
`mitoxantrone is considered to occur at lower concentrations by
`the induction of programmed cell death in antigen-presenting
`cells [94]. Consistent with this hypothesis, the clinical effects of
`mitoxantrone in MS have been suggested to last up to 1 year
`posttreatment [96].
`
`11.4. Effects on the cytokine network
`
`Recent ex vivo analysis of the cytokine profile of im-
`mune cells obtained from patients before and during treat-
`ment with mitoxantrone revealed a decrease of monocytes
`expressing the anti-inflammatory cytokine IL-10 and of
`T lymphocytes expressing IL-2R-β1 after 6 months of
`treatment [97].
`An overview of the immunological effects of IFN-β, GA,
`and mitoxantrone is given in Fig. 5 and Table 1.
`
`12. What do we learn from pharmacokinetics and
`pharmacodynamics?
`
`In recent years there have been major advances in
`understanding how cellular and humoral immune responses
`
`contribute to the pathogenesis of MS. In parallel, the knowledge
`on the mechanisms of action of agents aimed to modify the
`disease course has been growing continuously. As the efficacy
`of the currently available drugs is often unsatisfactory, new
`developments of therapeutic strategies against MS are highly
`warranted. Principally, this can be pursued by five ways:
`
`• the optimization of dose and route of administration of the
`currents drugs by learning from their pharmacokinetics
`(e.g. higher doses such as IFN-β1b 500 μg vs. 250 μg or
`GA 40 mg vs. 20 mg; new galenics (lipid micelles; oral or
`intranasal applications [98]);
`• the development of derivatives of the current drugs by
`learning from their pharmacodynamics (e.g. pixantrone
`instead of mitoxantrone with the aim of reducing drug-
`related cardiotoxicity [99]);
`• the evaluation of agents with other
`indications but
`reasonable for treatment of MS based on their known
`pharmacodynamics including immunomodulatory prop-
`erties (e.g. statins [100], or hormones [101,102]);
`• the design of fully new agents based on pathogenetical
`concepts (natalizumab as a selective adhesion molecule
`inhibitor is an example [103,104]);
`• the combination of available substances by learning from
`the single agents' pharmacokinetics and pharmacody-
`namics [105].
`
`Table 1
`Immunological effects of IFN-β, glatiramer acetate, and mitoxantrone [52,55,68,88]
`IFN-β
`
`Immune mechanism
`
`Glatiramer acetate
`↓ Proliferation of MBP-specific T cells
`
`No known effect
`No known effect
`
`Mitoxantrone
`
`Antigen-independent
`suppression of T-cell proliferation
`↓ Activation of B lymphocytes
`No known effect
`
`Direct and promiscuous binding to HLA-DR molecules No known effect
`↓ T-cell migration
`↓ Migration of PBL from GA-treated patients (unknown
`mechanism)
`No known effect
`
`No known effect
`
`No known effect
`No known effect
`
`No known effect
`No known effect
`
`↑ IL-10 in serum and of mRNA for TGF-β and IL-4; ↓
`mRNA for TNF-α in PBL; shift of GA-reactive T cells
`from TH1 towards TH2 during GA treatment
`Induction of anergy in MBP-specific T-cell clones
`
`↓ IL-10 in monocytes; ↓ IL-2R-β1
`in T lymphocytes during
`mitoxantrone treatment
`No known effect
`
`Proliferation of T cells
`in vitro
`B-cell activation
`Regulation of MHC
`expression
`MHC binding
`T-cell migration
`
`Adhesion molecule
`expression
`
`MMP expression
`Chemokine and chemokine
`receptor expression
`Cytokine shift in PBL
`
`APL effect on CNS-
`specific T cells
`Effects on APC
`
`Antigen-independent suppression
`of T-cell proliferation
`No known effect
`↓ IFN-γ-induced upregulation of
`MHC class II expression
`No known effect
`↓ T-cell migration
`
`↑ Soluble adhesion molecules
`(sICAM-1, sVCAM-1), ↓ surface-
`expressed adhesion molecules
`(VLA-4)
`↓ Expression of MMP-9
`↓ Secretion of chemokines, ↓
`expression of chemokine receptors
`Induction of TH2 cytokines and
`reduction of TH1 cytokines in
`PBL
`No known effect
`
`↓ IFN-γ induced FcγRI
`expression in monocytes
`
`↓ TNF-α and cathepsin-B production in a monocytic cell
`line, ↓ monocyte and dendritic cell functions
`
`↓ Proliferation of monocytes and
`macrophages; induction of
`apoptosis
`No known effect
`
`Neuroprotection
`
`Secretion of BDNF by GA-reactive T cells;
`Positive clinical and MRI effects
`neuroprotection in EAE
`on disease progression
`↑, increase; ↓, inhibitory effect; APC, antigen presenting cells; APL, altered peptide ligand; BDNF, brain-derived neurotrophic factor; CNS, central nervous
`system; EAE, experimental autoimmune encephalomyelitis; GA, glatiramer acetate; HLA, human leukocyte antigen; ICAM, intercellular adhesion molecule;
`IFN, interferon; IL, interleukin; MBP, myelin-basic protein; MHC, major histocompatibility complex; MRI, magnetic resonance imaging; MMP, matrix
`metalloproteinase; PBL, peripheral blood lymphocytes; sICAM, soluble intercellular cell adhesion molecule; sVCAM, soluble vascular cell adhesion molecule;
`TGF, transforming growth factor; TH, T helper function; TNF, tumor necrosis factor; VLA, very late antigen.
`
`Page 8 of 11
`
`YEDA EXHIBIT NO. 2012
`MYLAN PHARM. v YEDA
`IPR2014-00643
`
`
`
`O. Neuhaus et al. / Journal of the Neurological Sciences 259 (2007) 27–37
`
`35
`
`An overview on future developments in MS is given in Ref.
`[106]. The primary goal remains targeting of key elements of
`the immunological cascade that culminates in neural and glial
`tissue damage through disease-modifying agents.
`One step behind the horizon is the development of further
`therapeutic strategies against the demyelination as well as the
`axonal damage in MS, the latter of which is known to be
`associated with disability progression [46,47]. And further steps
`behind, i.e. neuroregenerative strategies, are still speculative.
`
`References
`
`[1] Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG.
`Medical progress: multiple sclerosis. N Engl J Med 2000;343:
`938–52.
`[2] Multiple Sclerosis Therapy Consensus Group. Escalating immu-
`notherapy of multiple sclerosis. New aspects and practical applica-
`tion. J Neurol 2004;251:1329–39.
`[3] Paty DW. Interferon beta-1b. In: Hawkins CP, Wolinsky JS, editors.
`Principles of treatments in multiple sclerosis. Oxford, Woburn:
`Butterworth-Heinemann; 2000. p. 24–37.
`[4] Wolinsky JS. Glatiramer acetate. In: Hawkins CP, Wolinsky JS,
`editors. Principles of
`treatments in multiple sclerosis. Oxford,
`Woburn: Butterworth-Heinemann; 2000. p. 71–94.
`[5] Goodin DS. Therapeutic developments in multiple sclerosis. Expert
`Opin Investig Drugs 2000;9:655–70.
`[6] Hartung HP, Bar-Or A, Zoukos Y. What do we know about the
`mechanism of action of disease-modifying treatments in MS? J Neurol
`2004;251(Supplement 5):V/12–29.
`[7] The IFNB Multiple Sclerosis Study Group. Interferon beta-1b is
`effective in relapsing–remitting multiple sclerosis: I. Clinical results
`of a