Autoimmunity Reviews 6 (2007) 469 – 475
`
`www.elsevier.com/locate/autrev
`
`Glatiramer acetate: Mechanisms of action in multiple sclerosis
`Wiebke Schrempf, Tjalf Ziemssen ⁎
`
`Multiple Sclerosis Center, Department of Neurology, Dresden University of Technology, Fetscherstraße 74, 01307 Dresden, Germany
`
`Received 2 January 2007; accepted 6 February 2007
`Available online 6 March 2007
`
`Abstract
`
`Glatiramer acetate (GA) is a mixture of synthetic polypeptides composed of four amino acids resembling myelin basic
`protein (MBP). GA has been shown to be effective in preventing and suppressing experimental autoimmune encephalomyelitis
`(EAE), the animal model of multiple sclerosis. It was tested in several clinical studies and approved for the immunomodulatory
`treatment of
`relapsing-type MS in 1996. Glatiramer acetate demonstrates a strong promiscuous binding to major
`histocompatibility complex molecules and inhibits the T cell response to several myelin antigens. In addition, it was shown
`to act as a T cell receptor antagonist for the 82–100 MBP epitope. Glatiramer acetate treatment causes in vivo changes of the
`frequency, cytokine secretion pattern and effector function of GA-specific T cells. It was shown to induce GA-specific
`regulatory CD4+ and CD8+ T cells and a TH1–TH2 shift with consecutively increased secretion of antiinflammatory cytokines.
`GA-specific TH2 cells are able to migrate across the blood–brain barrier and cause in situ bystander suppression of
`autoaggressive TH1 T cells. In addition glatiramer acetate was demonstrated to influence antigen presenting cells (APC) such as
`monocytes and dendritic cells. Furthermore secretion of neurotrophic factors with potential neuroprotective effects was shown.
`© 2007 Elsevier B.V. All rights reserved.
`
`Keywords: Glatiramer acetate; Multiple sclerosis; Experimental Autoimmune Encephalomyelitis (EAE); Neuroprotection; Myelin Basic Protein
`(MBP)
`
`Contents
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`1. Multiple sclerosis.
`2. Drug development and clinical studies .
`3. GA as antigen-based therapy .
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`3.1.
`Effects of GA on APC .
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`3.2.
`Effects of GA on T cells .
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`3.3.
`Effects of GA on B cells .
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`3.4. Neuroprotection .
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`3.5.
`Biomarkers .
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`Take-home messages .
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`Take-home messages
`References .
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`⁎ Corresponding author. Tel.: +49 351 458 3859; fax: +49 351 458 5873.
`E-mail address: Ziemssen@web.de (T. Ziemssen).
`
`1568-9972/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
`doi:10.1016/j.autrev.2007.02.003
`
`Page 1 of 7
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`YEDA EXHIBIT NO. 2009
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`470
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`W. Schrempf, T. Ziemssen / Autoimmunity Reviews 6 (2007) 469–475
`
`1. Multiple sclerosis
`
`Multiple sclerosis (MS) is a chronic, inflammatory
`demyelinating disease affecting about 0.1% of the
`population in temperate climates. It varies in terms of
`clinical, radiological and pathological features and is
`characterized by physical and neuropsychological
`symptoms. Pathological findings include axonal dam-
`age, brain atrophy and demyelinating plaques consisting
`of inflammatory cells [1]. Different disease entities are
`known leading from relapsing–remitting (RRMS) to
`primary (PPMS) and secondary progressive forms
`(SPMS). Current concepts assume that MS occurs as a
`consequence of immune tolerance breakdown in genet-
`ically susceptible individuals. The major contributing
`factors include environmental factors and immune
`dysregulation.
`The mechanisms of CNS inflammation involve
`activation of autoreactive, myelin specific T helper (TH)
`cells in the periphery possibly by molecular mimicry.
`These activated lymphocytes are able to cross the blood–
`brain barrier and are reactivated by recognising their
`specific target antigen presented by antigen presenting
`cells (APC) e.g. microglia. Reactivation of T cells results
`in increased secretion of proinflammatory cytokines and
`chemokines. An inflammatory cascade is initiated which
`causes activation and recruitment of macrophages and
`other inflammatory cells to the site of inflammation.
`Activation of B cells leads to augmented release of anti-
`myelin antibodies which bind complement and stimulate
`opsonization of myelin peptides. This ongoing destruc-
`tion of the myelin sheath causes reversible and to some
`extent irreversible impairment of functionality of the axon
`by deterioration of its conduction properties. As a major
`consequence of inflammation, axonal loss by binding of
`cytotoxic CD8+ T cells to demyelinated axons, release of
`cytotoxic factors and cytokines is proposed. In addition
`mechanisms not directly related to demyelination and
`inflammation i.e. excitotoxicity caused by glutamic acid is
`shown to initiate a process of neuronal cell death [1].
`
`2. Drug development and clinical studies
`
`Glatiramer acetate is a random mixture of synthetic
`polypeptides composed of four amino acids (L-glutamic
`acid, L-lysine, L-alanine and L-tyrosine) in a residue
`molar ratio 4.2 : 3.4 : 1.4 : 1.0 with an average molecular
`mass of 4700–11,000 Da. Myelin basic protein (MBP)
`is a major protein of the myelin sheath and is used as
`encephalitogenic protein for induction of experimental
`autoimmune encephalomyelitis (EAE)
`in animals.
`Glatiramer acetate was discovered in the 1960s when
`
`studies on the immunological properties of a series of
`polymers and copolymers developed to resemble MBP
`were conducted. Instead of EAE induction several of
`these polypeptides were able to decrease the severity or
`prevent EAE [2]. Copolymer 1,
`later known as
`glatiramer acetate, was shown to be the most effective
`polymer. It took several years until first exploratory
`open studies on patients with either relapsing remitting
`or secondary progressive multiple sclerosis were
`performed in the late 1970s and early 1980s. However
`the results of these studies must be interpreted with
`caution as drug production was not standardized before
`1991. In 1991 a phase III multicentre, double-blind,
`placebo-controlled trial with standardized glatiramer
`acetate preparation was conducted at 11 US medical
`centres with 251 RRMS patients receiving treatment for
`two years. Relapse rate decreased about 30% which was
`statistically significant (P = 0.007) [4]. In 1996 GA was
`approved by the US Food and Drug Administration
`(FDA) as a treatment for patients with active relapsing–
`remitting MS. Since then, studies using an oral formu-
`lation of GA (CORAL) and applying GA to primary
`progressive multiple sclerosis patients (PROMISE)
`failed to demonstrate significant
`treatment effects.
`There are promising new data on using a higher GA
`dose (40 mg) which are currently studied in a multi-
`centre, double-blind, controlled trial (FORTE).
`
`3. GA as antigen-based therapy
`
`In contrast to other immunomodulatory MS therapies
`glatiramer acetate seems to preferentially affect immune
`cells in an antigen-specific way. The peptide mixture is
`applied to patients with a putative autoimmune disease
`over many years by daily injection. From a broader
`perspective GA is one of the few practical examples of
`therapeutic vaccination distinct
`from prophylactic
`vaccination against
`infectious diseases. The precise
`mechanism of action of glatiramer acetate is not fully
`understood yet. The mechanisms discussed include
`competition with myelin peptides especially MBP for
`binding to major histocompatibility molecules (MHC)
`on APC, antagonism at the T cell receptor of myelin
`specific T cells and induction of GA-specific suppres-
`sive/regulatory T cells secreting potentially antiinflam-
`matory cytokines upon activation (Table 1). The vast
`majority of studies performed initially in EAE and
`subsequently in MS have focused on evaluating and
`targeting T cell responses especially the role of CD4+ T
`cells. Nevertheless there is increasing evidence for
`immunomodulating effects of GA on CD8+ T cells but
`also on B cells and APC.
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`Table 1
`Immunological effects of glatiramer acetate
`
`MHC II-complex
`
`MBP–MHC-
`complex
`T cell receptor
`
`T cell proliferation in
`vivo after treatment
`T cell proliferation
`in vitro
`
`T cell migration
`T cell features
`
`Effect
`• Promiscuous binding to various
`HLA-DR alleles [5]
`• Displacement of already bound MBP
`from binding site of MHC II-complex
`• T cell receptor antagonism to the
`82–100 MBP epitope [15]
`• Decreased GA specific T cell frequency
`in patients on GA treatment [15,18]
`• Suppression of MBP specific
`T cell lines [6]
`• Reduced proliferation of GA-specific
`CD4+ T cells [23]
`• Reduced, mechanism unclear
`• Shift from TH1 to TH2 cells [6,14,16,17]
`• Bystander suppression [22]
`• Induction of suppressive/regulatory CD4+
`and CD8+ T cells [25–27]
`Effects on monocytes • Reduced activation upon stimulation [7]
`• Increased IL-10, IL-4 secretion [9]
`• Inhibition of TNF-α secretion in vitro [8]
`• Inefficient maturation [12]
`• Reduced ability to stimulate T-cells [10,11]
`• Increased IL-10 secretion [9,10]
`• Controversial findings regarding
`TNF-α [8,9]
`• Decreased IL-12 secretion [10]
`• Increased IL-10 secretion
`• Decreased production of IL-12/TNF-α [11]
`• Enhanced secretion of
`IL-10 and TGF-β [14]
`• Inhibition of transformation to an
`activated microglia form [13]
`• Enhanced secretion of neurotrophic
`factors (BDNF, NT-3/4) [34]
`• Decreasing neuronal damage [35]
`• Increased neuronal proliferation [35]
`
`Effects on dendritic
`cells
`
`Effects on
`macrophages
`Astrocyte/microglia
`
`Neuroprotection
`
`3.1. Effects of GA on APC
`
`In vitro studies have shown that GA competes with
`myelin peptides from binding to MHC molecules and is
`able to displace MBP peptides from APC. GA binds to
`many different alleles of MHC class II molecules without
`antigen processing and without any obvious preference
`(“promiscuous binding”) [5]. Thus GA inhibits MHC II
`assisted activation of myelin specific T cells [6]. GA
`treatment leads to a generalized, antigen-non-specific
`modulation of APC function i.e. monocytes and macro-
`phages resulting in a reduced reactivity towards proin-
`flammatory stimuli [7]. First
`in vitro experiments
`demonstrated that GA blocked the activation of a
`monocytic cell line in the absence of T cells by inhibiting
`the induction of HLA-DR and HLA-DQ proteins leading
`to a reduction of Tumor Necrosis Factor α (TNF-α)
`
`release and decline of cathepsin B activity [8]. GA-treated
`monocytes and macrophages also produced increased
`amounts of Interleukin-10 (IL-10) [9]. GA-treated
`dendritic cells were able to shift the phenotype of naive
`T cells towards TH2 like T cells. In addition GA inhibited
`the secretion of IL-12 and TNF-α by human dendritic
`cells and macrophages [10]. In contrast to these findings
`GA exposed bone-marrow-derived dendritic cells pro-
`duced increased amounts not only of IL-10 but also of
`TNF-α upon stimulation maybe due to a less effective
`autoregulation via IL-10 [9]. Sanna et al. [11] recently
`demonstrated that in MS patients pretreatment with GA
`significantly decreased the proliferative effect of dendritic
`cells on lymphocytes in vitro. Furthermore GA treated
`MS patients showed a significant reduction of GA
`induced proliferation of peripheral blood mononuclear
`cells. Plasmacytoid dendritic cells (pDCs) from multiple
`sclerosis patients were shown to exhibit an inefficient
`maturation process after stimulation compared to controls.
`GA treatment partially restored phenotype and function of
`pDCs in these patients [12]. Chabot et al.
`[13]
`demonstrated a reduced ability of GA-treated T cells to
`interact with microglia. Consecutively the secretion of
`proinflammatory cytokines was reduced in microglia-T
`cell-coculture experiments. Furthermore CNS infiltration
`of GA-specific T cells resulted in increased expression of
`Transforming Growth Factor β (TGF-β) and IL-10 by
`resident astrocytes and microglia [14].
`
`3.2. Effects of GA on T cells
`
`In addition to competition for binding to MHC mole-
`cules and thus inhibiting the T cell response to several
`myelin antigens, GA is proposed to act as an antagonist to
`MBP/MHC at MBP-specific T cell receptors (TCR). It
`was demonstrated to operate as an “altered peptide ligand”
`to the 82–100 epitope of MBP [15]. This effect presum-
`ably occurs in the periphery at the injection sites or in the
`corresponding draining lymph nodes. The immunomod-
`ulatory effect of GA is primarily attributed to its ability to
`induce in vivo changes of the cytokine secretion pattern
`and the effector function of GA-reactive T helper cells.
`Modulation of the immune response with glatiramer
`acetate leads to deviation of cytokine production in
`response to MBP from so-called TH1 cytokines to TH2
`cytokines [6,16,17]. This so called TH1–TH2 shift is
`characterized by increased secretion of antiinflamma-
`tory cytokines like IL-4 and decreased TNF-α and
`Interferon-γ (IFN-γ) production [18]. After prolonged
`GA treatment T cell response of MS patients remains
`TH2 biased [19]. In EAE experiments GA-reactive TH2
`cells could be detected in the CNS [20]. Furthermore,
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`human TH1 and TH2 GA-reactive T cells were shown
`to migrate across an artificial blood–brain barrier in vitro
`[21]. So it can be concluded that activated TH2 cells are
`able to cross the blood–brain barrier (BBB), accumulate in
`the CNS [20] and express in situ antiinflammatory
`cytokines like IL-4, IL-10, TGF-β and IL-5, which not
`only modulate the local milieu but are able to suppress the
`activity of encephalitogenic TH1 cells [16–18]. This so
`called bystander suppression is regarded as essential part
`of the mechanisms of action of GA [22] (see Fig. 1). It
`seems very unlikely that sufficient amounts of GA reach
`the CNS to compete with locally degraded myelin
`antigens. So it might be speculated that GA-specific TH2
`cells are reactivated in the CNS when crossreacting with
`locally presented myelin autoantigens.
`In the setting of autoimmunity, one of the goals of
`successful therapeutic immune modulation is the induc-
`tion of peripheral tolerance, a large part of which is
`mediated by regulatory T cells. GA treatment gradually
`reduces the proliferative reactivity of GA-specific
`CD4+ T cells [23] probably by inducing apoptosis in
`some T cells [24]. Karandikar et al. [25] demonstrated
`
`that during GA treatment proliferative CD4+ T cell
`responses were comparable in healthy individuals and
`MS patients, whereas untreated MS patients showed a
`deficit in CD8+ T cell-mediated proliferation towards
`GA compared with healthy subjects. GA-specific treat-
`ment enhanced the frequency and suppressive ability of
`GA specific CD8+ T cells to those seen in healthy
`individuals. CD8+ T cells from GA treated patients and
`healthy subjects, but not those from untreated patients
`with MS, exhibited potent, HLA class I-restricted, GA-
`specific cytotoxicity [26]. In addition to these regula-
`tory CD8+ T cells Hong et al. [27] demonstrated that
`expression of Foxp3 in CD4+ T cells was significantly
`increased in MS patients treated with GA. This
`induction of regulatory CD4+ T cells was mediated by
`IFN-γ and to a lesser degree TGF-β1. Adoptive transfer
`of GA-specific T cells suppresses induction of EAE by
`different encephalitogens, but cannot cure ongoing
`EAE, which shows that T cells alone have only a limited
`role when disease is established [15,22]. By contrast
`GA injections not only prevent but also suppress
`established EAE [2].
`
`Fig. 1. Mechanisms of action of glatiramer acetate in multiple sclerosis. 1. GA exhibits competitive binding at the MHC-II complex and TCR-
`antagonism. In addition GA is able to displace MBP from the binding site on MHC-II molecules. Treatment with GA leads to the induction of antigen
`specific TH2 T cells in the periphery. 2. In addition CD8+ and CD4+CD25+ regulatory T cells are induced by GA therapy. 3. The constant activation
`seems to have an important impact on the induction and maintenance of the regulatory/suppressive immune cells. 4. Because of the daily activation,
`GA T cells are believed to be able to cross the blood–brain barrier. 5. Inside the CNS, some GA-specific T cells cross-react with products of local
`myelin turnover presented by local APCs. 6. In response anti-inflammatory cytokines are secreted which dampen the local inflammatory process
`(bystander suppression). 7. Furthermore GA specific T cells secrete neurotrophic factors which might favor remyelination and axonal protection.
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`3.3. Effects of GA on B cells
`
`A report including data from three different therapeutic
`trials demonstrated that all GA treated patients developed
`anti-GA antibodies which declined 6 months after
`treatment initiation but were still detectable. Anti-MBP-
`antibody titers were low and remained unchanged during
`treatment. Antibodies were of the IgG class with IgG1
`isotype levels two- to threefold higher than those of IgG2
`[3]. Farina et al. showed GA-specific IgG4 antibodies in
`GA-treated MS patients in a cross-sectional study. In
`addition they showed the occurrence of GA antibodies of
`the IgM, IgG1 and IgG2 class in some untreated patients
`[28]. Also in GA-treated primary progressive MS patients
`GA-reactive antibodies of the IgG1 subclass predomi-
`nated [29]. IgG1 subsequently decreased while anti-GA
`antibodies of the IgG4 subclass increased and remained
`high for the 3 years of follow-up. This isotype switch to
`IgG4 seems to reflect the interaction of B cells with TH2
`lymphocytes [30]. The presence of GA-reactive antibodies
`in some untreated control subjects suggests that such
`antibodies, which are mainly of the IgM isotype, are present
`in the common B cell immune repertoire. One might
`speculate that they are T cell independent, polyspecific
`“natural” antibodies produced by so called B1 cells.
`Teitelbaum et al. did not observe any neutralizing
`activity in sera of GA-treated patients. GA antibodies did
`not interfere with GA action neither in terms of MHC
`binding and T cell stimulation nor regarding suppression
`of EAE [31]. However in vitro experiments by Salama
`et al. demonstrated inhibition of GA-specific T cell lines
`by purified GA antibodies [32]. No clear evidence has yet
`been found that GA antibodies inhibit clinical efficacy in
`vivo. Moreover relapse-free patients seem to develop
`higher antibody titers, which would be consistent with a
`beneficial rather than neutralizing activity of the anti-
`bodies [3]. In a murine model of demyelinating disease
`induced by Theiler's virus remyelination of spinal cord
`axons was enhanced by GA antibodies [33]. These results
`support the hypothesis that the antibody response in GA
`treated patients may be beneficial by facilitating repair of
`demyelinated lesions.
`
`3.4. Neuroprotection
`
`Human GA-specific TH1-, TH2-, and TH0 cells all
`showed low levels of basal secretion of Brain Derived
`Neurotrophic Factor (BDNF) and an increase of BDNF
`production after stimulation [34]. This ability of GA-
`specific cell could also be demonstrated in situ in the
`mouse model of MS (EAE) resulting not only in de-
`creased neuronal damage, but also increased neuronal
`
`proliferation [35]. Gilgun-Sherki et al. demonstrated a
`significant reduction of axonal loss and neuronal damage
`in myelin oligodendrocyte glycoprotein (MOG)-induced
`EAE, a model characterized by a chronic disease course
`[36]. Kipnis et al. showed that in a model of traumatic
`injury of the optic nerve the posttraumatic spread of
`degeneration could be attenuated by either active im-
`munization with GA on the day of injury or by adoptive
`transfer of GA-specific T cells [37]. After restimulation
`by cross-reactive myelin antigens in the CNS, GA-
`reactive T cells seem to secrete not only immunomod-
`ulatory cytokines but also neurotrophic factors. There-
`fore these T cells might confer neuroprotection in
`addition to bystander suppression.
`
`3.5. Biomarkers
`
`Similar to effects seen in patients treated with other
`immunomodulatory drugs some GA-treated patients do
`not respond to therapy. Early identification of such non-
`responders would be very useful for adjusting therapy
`regimens. For this reason there has been much interest in
`the development of appropriate biomarkers. We are just
`running a study (COPIMMUNONET) which aims at
`finding a link between clinical outcome and immuno-
`logical parameters specific for GA.
`
`Take-home messages
`
`• Glatiramer acetate is an approved drug for the
`treatment of relapsing–remitting multiple sclerosis, a
`chronic inflammatory disease of the central nervous
`system.
`• Treatment with glatiramer acetate results in reduction
`of clinical exacerbations and progression rate in
`multiple sclerosis patients as well as a decrease of
`burden lesion shown on MRI scans.
`• Its proposed mechanisms of action include competi-
`tion with myelin antigens for binding to APC,
`antagonism at specific T cell receptors, induction of
`GA-specific regulatory CD4+ and CD8+ T cells which
`downregulate inflammation by secretion of antiin-
`flammatory cytokines and cause bystander suppres-
`sion of proinflammatory TH1 cells in the CNS.
`• In addition, neuroprotective effects as well as reg-
`ulatory effects on B cells, monocytes and dendritic
`cells have been shown.
`
`References
`
`[1] Steinman L. Multiple sclerosis: a two-stage disease. Nat
`Immunol 2001;2:762–4.
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`MYLAN PHARM. v YEDA
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`Prevalence of autoantibodies (ANA, anti ds-DNA, ENA, IMF) and rheumatic syndromes in patients with
`lymphoproliferative diseases.
`
`In the development of rheumatic syndromes as well as of lymphoproliferative disorders it is probable that there are
`common genetic, environmental and immunoregulatory pathogenetic mechanisms. The purpose of this study,
`(Chloraki-Bobota A. et. al. (J BUON 2006; 11: 485-9) was to determine the frequency of simultaneous
`presentation of both lymphoma or other lymphoproliferative diseases, and rheumatic syndromes. All patients with
`lymphoproliferative diseases (1920 patients) followed at Metaxa Cancer Hospital, Piraeus, Greece, during the last
`5 years, were included in this study. 312/1920 (16.2%) patients presented with non-Hodgkin's lymphoma (NHL),
`645/1920 (33.5%) had myeloma, 558/1920 (29%) had leukemia and miscellaneous other hematological
`malignancies. Anti nuclear antibodies (ANA), ribosomal P and intermediate filament antibodies (IMF), Anti-
`dsDNA and extractable nuclear antigens (ENA: Sm, RNP, SSA), were analyzed in all. 388/1920 (4.6%) patients
`were ANA positive. On the other hand, clinical symptoms attributed to autoimmune diseases (arthralgias, morning
`stiffness etc) plus autoantibodies other than ANA were present only in 8/312 (2.5%) patients with NHL, among
`them one with anti-cardiolipin antibodies. It is interesting that from these 8 patients, 3 had MALT lymphoma and 3
`diffuse B-cell large lymphoma. It was concluded that the simultaneous presence of lymphoproliferative diseases
`and rheumatic syndromes are more frequent among lymphoma patients than in other lymphoproliferative diseases.
`Therefore, the screening of antibodies in MHL patients may be useful for the discovery and the treatment of an
`underlying autoimmune disease.
`
`Proinflammatory mediator-induced reversal of CD4+CD25+ regulatory T cell-mediated suppression in rheumatoid
`arthritis.
`
`CD4+CD25+ T regulatory cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis
`(RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB)
`counterparts. Despite the presence of these immunoregulatory cells in RA, c