Myasthenia Gravis - Therapeutic Aspects

**39**

**Chapter 4**

**Abstract**

antibodies, will be discussed.

the human immune system [1].

nAChR-specific B cells

**1. Introduction**

Structure-Based Approaches

Myasthenia Gravis

*Jiang Xu, Kaori Noridomi and Lin Chen*

to Antigen-Specific Therapy of

A majority of Myasthenia Gravis (MG) cases (~85%) are caused by pathological autoimmune antibodies to muscle nicotinic acetylcholine receptors (nAChRs). An attractive approach to treating MG is therefore blocking the binding of autoimmune antibodies to nAChR, or removing specifically nAChR antibodies, or selectively inhibiting and eliminating nAChR-specific B cells. This chapter will review highresolution structural studies of muscle nAChR and its complexes with antibodies derived from experimental autoimmune Myasthenia Gravis (EAMG). Based on these structural analyses, various strategies, including using small molecules to block the binding of MG autoimmune antibodies, and engineered chimeric nAChR antigen to specifically target and eliminate B cells that produce nAChR-specific

**Keywords:** crystal structure, nicotinic acetylcholine receptor, antigen-specific therapy,

Myasthenia Gravis (MG) is an autoimmune disease that afflicts a significant human population. MG patients suffer from a variable degree of skeletal muscle weakness. The symptoms range from mere lack of muscle strength to life-threatening respiratory failure. MG is a chronic disease that can last many years and negatively impact the quality of living and life expectancy of afflicted individuals. Although MG rate is reported to be 7–20 out 100,000 [1] and the diagnosed MG cases are increasing, probably due to increased awareness of this debilitating disease, the aging population and other intrinsic and extrinsic factors that disturb

The majority of MG cases (~85%) are caused by pathological autoantibodies to muscle nicotinic acetylcholine receptors (nAChRs), a ligand-gated ion channel that mediates rapid signal communication between spinal motor neurons and the muscle cells. Autoantibodies against other neuromuscular junction (NMJ) proteins, including muscle-specific kinase (MuSK) and lipoprotein-related protein 4 (LRP4), can also cause muscle weakness in a small fraction of patient [2, 3]. The heterogeneous nature of MG autoantibody presents a challenge to both diagnosis and treatment of the disease. Current treatment regimens for MG include anticholinesterase inhibitors, thymectomy, immunosuppressants, plasmapheresis, or intravenous immunoglobulins [4].

Myasthenia Gravis, autoimmune antibodies, chimeric nAChR antigen,

#### **Chapter 4**

## Structure-Based Approaches to Antigen-Specific Therapy of Myasthenia Gravis

*Jiang Xu, Kaori Noridomi and Lin Chen* 

#### **Abstract**

 A majority of Myasthenia Gravis (MG) cases (~85%) are caused by pathological autoimmune antibodies to muscle nicotinic acetylcholine receptors (nAChRs). An attractive approach to treating MG is therefore blocking the binding of autoimmune antibodies to nAChR, or removing specifically nAChR antibodies, or selectively inhibiting and eliminating nAChR-specific B cells. This chapter will review highresolution structural studies of muscle nAChR and its complexes with antibodies derived from experimental autoimmune Myasthenia Gravis (EAMG). Based on these structural analyses, various strategies, including using small molecules to block the binding of MG autoimmune antibodies, and engineered chimeric nAChR antigen to specifically target and eliminate B cells that produce nAChR-specific antibodies, will be discussed.

**Keywords:** crystal structure, nicotinic acetylcholine receptor, antigen-specific therapy, Myasthenia Gravis, autoimmune antibodies, chimeric nAChR antigen, nAChR-specific B cells

#### **1. Introduction**

Myasthenia Gravis (MG) is an autoimmune disease that afflicts a significant human population. MG patients suffer from a variable degree of skeletal muscle weakness. The symptoms range from mere lack of muscle strength to life-threatening respiratory failure. MG is a chronic disease that can last many years and negatively impact the quality of living and life expectancy of afflicted individuals. Although MG rate is reported to be 7–20 out 100,000 [1] and the diagnosed MG cases are increasing, probably due to increased awareness of this debilitating disease, the aging population and other intrinsic and extrinsic factors that disturb the human immune system [1].

The majority of MG cases (~85%) are caused by pathological autoantibodies to muscle nicotinic acetylcholine receptors (nAChRs), a ligand-gated ion channel that mediates rapid signal communication between spinal motor neurons and the muscle cells. Autoantibodies against other neuromuscular junction (NMJ) proteins, including muscle-specific kinase (MuSK) and lipoprotein-related protein 4 (LRP4), can also cause muscle weakness in a small fraction of patient [2, 3]. The heterogeneous nature of MG autoantibody presents a challenge to both diagnosis and treatment of the disease.

Current treatment regimens for MG include anticholinesterase inhibitors, thymectomy, immunosuppressants, plasmapheresis, or intravenous immunoglobulins [4].

Most MG patients respond favorably to these treatment options to achieve effective symptom relief, and in some cases even clinical remission. Cholinesterase inhibiting drugs can temporarily enhance neuromuscular transmission by delaying the breakdown of acetylcholine (ACh) to compensate for the loss of NMJ nAChRs, but this treatment option only works in a fraction of patients and does not alter the autoimmune response. The more broadly used nonspecific immunosuppressive drugs work by inhibiting lymphocyte activation and proliferation but have little effect on long-lived plasma cells that are terminally differentiated and continue producing pathogenic antibodies [5, 6]. This may explain why treatment with nonspecific immunosuppressive drugs takes long time to show clinical improvement.

There are two major limitations in the current MG treatment. First, up to 10% of MG patients do not tolerate or are resistant to the available treatments [7]. Second, all immunosuppressant drugs, which are often used in the long-term control of chronic MG, inevitably carry the serious risks of infection and cancer. As such continued efforts have been put into searching for better MG treatment, as evident by the long list of clinical trials (ClinicalTrials.gov) testing well known immunosuppressive drugs such as methotrexate and azathioprine, as well as new biologics agents such as the anti-CD20 monoclonal antibody rituximab (which depletes B cells) and the anticomplement C5 monoclonal antibody eculizumab.

 An ideal therapeutic approach to MG would be to inhibit the pathogenic autoimmune response to nAChR specifically without disrupting other functions of the immune system. Because nAChR is a dominant autoantigen in MG, it has served as the primary target for a wide range of studies attempting to develop antigenspecific therapy to induce immune tolerance to nAChR [8–14]. While some of these approaches showed promising results in animal model of experimental autoimmune MG (EAMG), translation to human MG treatment is uncertain. Furthermore, introducing an autoantigen like nAChR or its derivative peptides risks to inadvertently enhance the pathogenic autoimmune response.

Here, we will first review structural and molecular features of nAChR and its complexes with autoantibodies. Based on insights derived from structural studies, we will discuss several strategies to specifically inhibit the binding of pathological autoantibodies to nAChR or specifically eliminate nAChR-specific B cells.

#### **2. Structural study of nAChR**

 As the first isolated neurotransmitter receptor and ion channel, nicotinic acetylcholine receptors (nAChRs) have been the focus of extensive studies to understand the basic mechanisms of neuronal signaling. These receptors are also being targeted for drug development against a variety of diseases, including addiction, depression, attention-deficit/hyperactivity disorder (ADHD), schizophrenia, Alzheimer's disease, pain and inflammation [15]. nAChRs have been analyzed by a variety of biochemical, biophysical and electrophysiological experiments [16]. Tremendous efforts have been put into pursuing the atomic structure of nAChR. Electron microscopic analyses of nAChR from *Torpedo marmorata* by Unwin and colleagues have led to a 4 Å resolution model of the intact channel [17, 18], providing one of the most comprehensive structural model for nAChR. The structural details, however, are limited by the relatively low resolution. In this regard, the high-resolution structure of the acetylcholine binding protein (AChBP) published by Sixma and colleagues in 2001 was a major breakthrough [19]. AChBP shares ~24% sequence identity with nAChRs and has the same pentameric assembly. Its structures in different bound states have provided detailed information on the binding of a variety of agonists and

*Structure-Based Approaches to Antigen-Specific Therapy of Myasthenia Gravis DOI: http://dx.doi.org/10.5772/intechopen.84715* 

 antagonists [20]. But AChBP does not function as an ion channel and may lack necessary structural features required for transmitting the ligand-binding signal across the protein body [21, 22]. The crystal structures of several prokaryotic homologues of nAChR have also been determined from different species and in different states [23–25]. These structures together with detailed biochemical and biophysical characterization have provided a great deal of insight into the fundamental mechanisms of ligand-dependent channel gating (reviewed in Corringer et al [26]). More recently, the structure of the anionic glutamate receptor (GluCl) from *C. elegans* [27], and human α4β2 neuronal nicotinic receptor have also been determined [28]. However, direct structural information of mammalian muscle nAChRs at high resolution will be needed for further dissecting the mechanisms of neuromuscular junction signal transmission and for drug development against MG [29].

#### **3. High-resolution structural analysis enabled by stabilizing nAChR mutants**

 Although large quantities of nAChR were available from *Torpedo* electric ray organ, crystallization was not successful, probably due to the heterogeneity of the protein samples prepared from the natural source. Heterologous expression in bacterial results in insoluble protein is due to the lack of proper post translation modifications such as glycosylation. Yeast *Pichia pastoris* has been a favorable expression system for overexpressing nAChR because of its mammalian-like glycosylation system. However, the expressed nAChR protein or extracellular domain (ECD) is often unstable, leading to aggregation and low yield [30, 31]. We have employed a number of strategies to overcome this difficulty, including expressing different family members of nAChR or its sub-domain (mostly ECD), constructing AChBPnAChR chimera, and introducing specific mutations to enhance expression and stability [32]. Using the nAChR α1 as an example, we screened a PCR-generated mutant library of mouse nAChR α1 ECD for variants with increased expression and stability which led to the isolation of a triple mutant (V8E/W149R/V155A) that has much improved expression and stability than the wild type protein, and ultimately the determination of the crystal structure of nAChR α1 ECD bound to a-bungarotoxin at 1.94 Å resolution [22]. Structure comparison with the 4 Å electron microscopic model of nAChR and AChBP reveals that the isolated ECD is very similar to its counterpart in the intact channel and that the stabilizing mutations do not appear to alter the overall structure of the ECD.

All of the three mutations map to the surface of the protein (**Figure 1a**), with one (V8E) located on the N-terminal helix and the other two (W149R and V155A) located on loop B. The V8E mutation introduces a salt bridge with Lys84 (**Figure 1b**), whereas the W149R mutation introduces a salt bridge with Asp89 (**Figure 1c**). These salt bridges apparently contribute to protein stability as evident by the well-defined electron density of these exposed residues with long and charged side chains. Thus, the mutations seem to enhance the protein stability through at least two mechanisms. One is to remove surface exposed hydrophobic residues, including V155A (**Figure 1d**); the other is to introduce salt bridges on the protein surface. These observations suggest that the ECD of nAChR may be rationally engineered to improve solubility and stability. In principle, one can use homology models to guide the selection of exposed hydrophobic residues and to engineer surface salt bridges, which can increase the stability of recombinant mammalian nAChRs. This insight will be important for the design of stable chimeric nAChR antigen for specific targeting and elimination of nAChR-specific B cells (discussed further below).

#### **Figure 1.**

 *Mutations that stabilize nAChR α1 ECD. (a) The three mutations (boxed and indicated by arrow) are mapped on the surface of nAChR α1 ECD (dark green) and away from the binding site of α-bungarotoxin (orange) and the glycan (magenta); (b) the mutation Val8Glu establishes a salt bridge with Lys84. The surrounding structure is well ordered, showing well-defined electron density; (c) the mutation Trp149Arg establishes a salt bridge with Asp89. The side chains of both residues show well-defined electron density; (d) the mutation of Val155Ala removes an exposed hydrophobic residue. The surrounding structure is well ordered (Adapted from Chen [33]).* 

#### **4. Functionally instrinsic instability of nAChR ECD**

Most proteins have a densely packed hydrophobic core that is important for stable folding in aqueous solution. However, a hydration pocket was found inside the beta sandwich core of the nAChR α1 ECD [22]. This hydration pocket consists of two buried hydrophilic residues, Thr52 and Ser126, two ordered water molecules, and a few cavities, creating a packing defect near the disulfide that connects the two beta sheets. Both Thr52 and Ser126 are highly conserved in nAChRs but are substituted by large hydrophobic residues (Phe, Leu or Val) in the non-channel homologue AChBPs. This observation suggests that the nAChR ECD has evolved with a nonoptimally packed core, hence predisposed to undergo conformational change during ligand-induced gating. Replacing Thr52 and Ser126 with their hydrophobic counterparts in AChBP significantly impaired the gating function of nAChR without affecting the folding of the protein structure [22]. This role of the hydration pocket on the conformation flexibility/dynamics of the nAChR ECD is supported by recent molecular dynamics studies [34]. This model also suggests that the specific location of the hydration cavity is important for a particular class of pentameric LGICs [35]. A practical implication of these observations is that one can design stabilization mutants of LGICs, including nAChR ECD, by structure-guided modifications of such packing defects, which are evolved for intrinsic ion channel functions but may be detrimental to recombinant production of proteins as therapeutic antigen.

#### **5. Structural studies of the complexes between nAChR ECD and EAMG antibodies**

 Antibodies generated by the immune system may bind various epitopes on nAChR. It is therefore important to know if MG autoantibodies are randomly distributed to various epitopes and if they contribute equally or differently to the disease phenotype. This question is also therapeutically relevant if one wishes to use small molecules or single valent antibody [36] to block the binding of most

#### *Structure-Based Approaches to Antigen-Specific Therapy of Myasthenia Gravis DOI: http://dx.doi.org/10.5772/intechopen.84715*

 pathologically relevant autoantibodies to nAChR. Mammalian muscle nAChR has a pentameric structure composed of two α1, one β1, one δ, and one ε (adult form) or γ (fetal form) subunit(s) [18]. Extensive studies suggest that autoantibodies to α1 play a major role in MG pathology [37–40]. Furthermore, more than half of all autoantibodies in MG and EAMG bind an overlapping region on the nAChR α1 subunit, known as the main immunogenic region (MIR) [41]. The MIR is defined by the ability of a single rat monoclonal antibody (mAb), mAb35, to inhibit the binding of about 65% autoantibodies from MG patients or rats with EAMG [42–44]. Subsequent studies have mapped MIR to a peptide region that spans residues 67–76 on nAChR α1 [45, 46]. Monoclonal antibodies directed to the MIR can passively transfer EAMG and possess all the key pathological functions of serum autoantibodies from MG patients [37]. Moreover, a recent study showed that titer levels of MIR-competing autoantibodies from MG patients, rather than the total amount of nAChR autoantibodies, correlate with disease severity [47]. These observations suggest that autoantibodies directed to the MIR on nAChR α1 play a major role in the pathogenesis of MG [41]. However, autoantibodies classified as MIR-directed by competition assay may not necessarily have the same binding mechanisms to nAChR: two MIR-competing autoantibodies may share common or overlapping epitopes or may bind different epitopes but compete through steric effect [14].

 Given their established myasthenogenic role, extensive efforts have been put into characterizing the interactions between MG autoantibodies and nAChR using biochemical [45, 46, 48–53], structural [22, 54–56], and modeling approaches [57]. More recently, the first crystal structures of human (pdb code: 5HBT) and mouse (pdb code: 5HBV) nAChR ECD bound by the Fab fragment of an EAMG autoantibody, Fab35 were determined [58]. Both crystal structures are very similar, so the discussion here will focus mainly on the human complex (pdb code: 5HBT). The crystal structure, which also contains α-Btx that binds and stabilizes nAChR ECD to facilitate crystallization, shows that Fab35 binds to nAChR α1 in an upright orientation, away from the α-Btx (**Figure 2**). The Fab35 binding sites on nAChR α1 include the MIR and the N-terminal helix. Fab35 has the canonical IgG antibody structure where the complementarity determining regions (CDRs) from the heavy chain, CDR-H2 and CDR-H3, and the light chain, CDR-L3, form the binding site for nAChR α1. Contacting residues from Fab35 and nAChR α1 (defined as being closer than 4.5 Å) can be mapped using the crystal structure. Such contacting analysis revealed several "hotspots" on nAChR α1 that make numerous contacts to Fab35, including Asn68 and Asp71 from the MIR loop and Arg6 and Lys10 from the N-terminal helix. As shown in **Figure 3**, each of these four "hotspots" anchors an extensive network of interactions that display remarkable chemical complementarities. The importance of these hotspots are supported by extensive mutagenesis studies [50, 51, 53, 59], which showed that Asn68 and Asp71 of the MIR are essential for MG autoantibody binding, while the surrounding Pro69 and Tyr72, when mutated, also affect the interaction between the antibody and the receptor. Mutation of N68D and D71K in the intact receptor also suggested ASn68 and Asp71 are of vital importance for the interaction [49]. On the N-terminal helix of *Torpedo* nAChR α1, two exposed residues, Arg6 and Asn10, which correspond to Arg6 and Lys10 in human nAChR α1, respectively, are found to be critical to MG antibody binding by mutational analyses [53]. Many nAChR residues found to be important for antibody binding by mutagenesis studies, including Asn68 and Asp71of the MIR and Arg6 and Lys10 of the N-terminal helix, were indeed found to be interaction"hotspots" at the Fab35/nAChR α1 interface. More recent studies using natively folded nAChR α1α7 chimera proteins [52] or GFP-fused protein fragments [53] showed that the N-terminal helix (residues 1–14) and the nearby loop region (residues 15–32) are also important for high affinity MG antibody binding. These biochemical observations are in excellent agreement with the binding interface structure observed in the crystals (**Figure 2**).

#### **Figure 2.**

*Crystal structure of the ternary complex of nAChR α1 ECD bound by Fab35 and α-Btx. (a) Ribbon representation of nAChR α1 ECD (α1: cyan) in complex with α-Btx (green) and Fab35 (heavy chain (H, yellow) and light chain (L, magenta)). The variable domains (VH and VL) and the constant domains (CH and CL) of the antibody are indicated accordingly. (b) Surface representation of the ternary complex. (c) Zoomed-in view of the binding interface. The complementarity determining regions of the heavy chain and light chain are indicated as H1, H2, H3, L1, L2, and L3, respectively (Adapted from Noridomi et al. [58]).* 

#### **Figure 3.**

*Detailed interactions between Fab35 and nAChR α1 ECD at the binding interface. (a) Binding interactions at the vicinity of Asp71 of α1 (located at the MIR). (b) Interactions at the vicinity of Asn68 of α1 (located at the MIR). (c) Interactions involving Arg6 and Lys10 of α1 (located at the N-terminus of α1). (d) Interactions mediated by His3 of α1 (located at the N-terminus of α1) (Adapted from Noridomi et al. [58]).* 

 Although biochemical mapping of antibody-binding residues on nAChR α1 were performed with different antibodies (e.g., mAb210 and mAb132A) [45, 46, 48–53], it is remarkable that these biochemical data agree so well with the crystal structure. The fact that many MIR residues at the center of the antibody-receptor interface are important for the high affinity binding of a variety of MG antibodies suggests that many MIR-directed autoantibodies share similar binding mechanisms to the

core MIR/N-helix region. This is a rather surprising finding given the potential heterogeneity of nAChR antibodies mentioned above. An important implication of this finding is that it may be possible to find small molecule inhibitors to block the binding of a large fraction of pathological MG autoantibodies to nAChR.

### **6. Structural comparison of Fab35 with other MG autoantibodies**

 To see how various MG/EAMG mAbs may bind nAChR through similar or different mechanisms, we compared the structure of Fab35 with that of two other MG mAbs (Fab198: pdb code 1FN4 and Fab192: pdb code, 1C5D) that have been determined previously [55, 56]. Superposition of the structure of Fab198 and Fab35 from the ternary complex shows that these two Fabs share a similar antigen-binding site (**Figure 4a**). As such, the MIR loop fits snugly into the pocket formed by the CDR-H2, CDR-H3 and CDR-L3 loops of Fab198, as predicated by previous modeling studies [57]. The CDR-H2 loop of Fab198 is also in a position to interact with the N-terminal α-helix adjacent to the MIR (**Figure 4b**). Even more remarkably, many key α1-binding residues in Fab35 are also conserved in Fab198 and they appear to have similar contacts to nAChR α1 in the modeled Fab198/nAChR α1 binding interface (**Figure 4b**). These residues include Trp47 from CDR-H2, Arg50 from CDR-H2, and Tyr95 from CDR-L3 at the center of the MIR-binding pocket, and Trp52 and Asp54 (both from CDR-H2) which interact with the N-terminal α-helix. In contrast to the structural similarities shown above, the CDR-H3 loops between Fab198 and Fab35 differ significantly in length and sequence. The CDR-H3 loop of Fab198 is too short to interact with the surface pocket of nAChR α1, which is occupied by the corresponding CDR-H3 loop of Fab35 in the complex crystal structure (**Figure 4b**). These structural analyses suggest that mAb35 and mAb198 share a high degree of similarity in binding mechanism to the core MIR/N-terminal helix region but differ in the periphery of the binding interface. On the other hand, superposition of the structure of Fab192 onto that of Fab35 in the ternary complex reveals substantial differences (not shown here). The variable domains (VH and VL) have a significant rotational twist, such that the MIR loop does not fit into the antigen-binding site of Fab192. What is more, the key α1-binding residues of Fab35, like Arg50 and Trp52 of CDR-H2, are not conserved in Fab192. These structural differences suggest that Fab192 may differ significantly from Fab35 in terms of binding mechanisms to nAChR α1, confirming and extending the differences previously recognized between the two [52].

#### **Figure 4.**

*Structural comparisons among MG mAbs. (a) Superposition of Fab198 [55] (heavy chain: purple and light chain: dark green) onto Fab35 in the Fab35/nAChR α1/α-Btx ternary complex using the Cα backbone. (b) Detailed comparison of the binding interface. The residues are colored according to their protein subunits.* 

#### **7. MG autoantibody repertoire and MIR-directed autoantibodies**

A number of studies showed that the total amount of nAChR antibodies in the serum of MG patients does not seem to correlate with disease severity, suggesting that various nAChR antibodies that bind different regions on nAChR may contribute differently to this disease [41, 60–62]. As discussed above, the total amount of autoantibody from MG patients directed to the MIR of nAChR α1 subunit did show significant correlation with disease severity [47]. These observations suggest that autoantibodies directed to nAChR α1 MIR play a major role in the pathogenesis of MG [41]. It is now clear that many MIR-directed autoantibodies bind a composite epitope consisting of the original MIR (α1, 67–76) and the N-terminal helix (α1, 2–14) (N-helix) and surrounding regions (α1, 15–32). The structural analyses above and published biochemical data suggest that some MIR-directed autoantibodies (e.g., mAb35 and mAb198) bind epitopes centered around the MIR/N-helix core region while others (e.g., mAb192) seems to require epitopes outside the MIR/N-helix core. Nevertheless, based on crystallography studies and structure-guided analyses of existing biochemical data, it can be concluded that despite the heterogeneity of MG autoantibody repertoire a large fraction of MG autoantibodies share a highly-conserved binding mechanism to a core region on the nAChR, suggesting that it is possible to use a single or a limited set of small molecules to block the binding of a large fraction of MG autoantibodies. Because MG autoantibodies directed to the MIR region on nAChR are most relevant to the MG disease, MIR and its surrounding region are therefore an attractive target site for developing small molecules to block the binding of MG autoantibodies. Blocking the binding of MG autoantibodies to nAChR will likely have a direct impact on the antibody-mediated pathologies and may even alter the long-term immune response to nAChR in MG patient.

#### **8. Small molecules blocking the binding of MIR-directed autoantibody to nAChR**

Targeting protein-protein interface for drug development is generally more challenging than the enzyme active sites [63]. This is especially true for flat protein interfaces lacking features for small molecule binding. However, successes have been achieved with a number of well-known targets, including the p53/MDM2 complex [64], the Bcl-xL/Bak complex [65] and the IL2/IL2R complex [66, 67]. A common feature of these complexes is that the protein-protein binding interfaces contain concave pockets lined with hydrophobic residues, which may provide favorable anchoring points for small molecules to bind and compete with protein-protein interactions. The crystal structure of the Fab35/nAChR α1 complex revealed that their binding interface is characterized by mutual insertions of loops into the pockets of binding partners. On the receptor side (**Figure 5**), the MIR loop inserts deeply into a surface pocket between VH and VL, and the N-terminal α-helix sits into a groove on the surface of VH. On the antibody side (**Figure 6**), the CDR-H3 protrudes into a surface pocket formed by the N-terminal α-helix, the loop following the N-terminal α-helix, the MIR and the loop preceding the MIR (referred to as the CDRH3 pocket here after). Based on these structural features, two MG inhibitor design strategies can be envisioned. One is to find small molecules that bind the surface pockets on Fab35 (**Figure 5**). But this approach faces the potential issue of antibody heterogeneity in sera of human MG patients because small molecule inhibitors may bind some but not other pathological autoantibodies, as it is highly possible antibodies binding to the same epitope may have subtle differences in their antigen-binding site structures. Another approach is to find small molecules to bind the CDRH3 pocket on nAChR (**Figure 6**). Small molecules bound to this site will directly interfere with the binding

*Structure-Based Approaches to Antigen-Specific Therapy of Myasthenia Gravis DOI: http://dx.doi.org/10.5772/intechopen.84715* 

#### **Figure 5.**

*Surface pockets on Fab35 bound by the nAChR MIR loop (white dashed circle) and the N-terminal helix (black dashed circle).* 

#### **Figure 6.**

*The surface pocket (green dashed circle) on nAChR α1 bound by the CDR-H3 loop from Fab35 (indicated as H3 in the figure).* 

of mAB35 by competing with its CDR-H3. Even for other mAbs with short CDR-H3, such as mAb198, the compounds may also block the binding of CDR-H3 through steric hindrances. Moreover, since the CDRH3 pocket is immediate adjacent (about 6–8 Å) (**Figure 6**) to the MIR/N-helix core region critical for the binding of a large group of MG autoantibodies, compounds bound to CDRH3 could sterically and/or allosterically inhibit the binding of most pathological MG autoantibodies efficiently. Because of its concaved structure, CDRH3 pocket could serve as the anchoring point to design and/or screen small molecules that bind nAChR α1 and complete with MG autoantibodies directed to MIR and its nearby regions.

#### **9. nAChR-specific B cell inhibition and depletion with engineered antigen chimera**

 The fact that pathogenic B cell clones can populate for a long time in patients' body may explain why MG is usually a chronic disease. Ectopic germinal centers are found

 in the thymus of many MG patients who are diagnosed with thymoma or thymus hyperplasia, where nAChR-specific B lymphocyte are constantly activated, selected and matured to produce the antibody, leading to the disease [68]. This disease model underlies the rationale of thymectomy as a widely adopted treatment of MG, but the result varies depending on the subtype of the disease, with a complete remission rate of 25–53% [69]. These results suggest there are possibly other unknown sites where nAChR specific B cells are activated, selected and matured [13].

 Using B cell surface marker CD20 [70–72] or possibly CD19 [73] as the target, disease-causing B cells can be depleted at the cost of killing normal B cells. For example, an ongoing clinical trial, NCT02110706, is testing if rituximab, which targets CD20 on B cells, can be a safe and beneficial therapeutics for MG. In general, treatment with B cell depletion agent often requires a long recovery time before B cells return to normal level again [71]. Moreover, the treatment has been reported to have a short effective duration time for MuSK-positive MG [74]. Long-term usage of such agent may compromise immunological function with increased risk of infection such as Progressive multifocal leukoencephalopathy (PML) and malignancy [72]. As such, strategies targeting nAChR specific B cells seem to be attractive. Since each B cell expresses B cell Receptors of the same idiotype as its secreted antibody on its surface, one can use such property to specifically target autoreactive B cell as long as the antigenicity of the autoimmune disease is clear. The idea was borrowed from immunotoxins [75] in which an antigen-toxin chimera was constructed. The antigen moiety is used to target the B cells that express the BCR of the same idiotype as the antibody and the toxin moiety is responsible for conveying death signal to the target B cells. In a pioneering study in 1983 the author fused thymoglobulin with ricin to treat an autoimmune disorder-Hashimoto's thyroiditis [76]. Another attempt was tried a decade later in another autoimmune disease-Pemphigus Vulgaris, in which the authors constructed antigen-toxin fusion protein that can specifically target Dsg3-specific hybridoma cells [77]. Similar strategies have also been attempted in the treatment of MG. In a study of 2006, the author fused the nAChR α1 ECD to a plant toxin and showed its effectiveness in specifically killing of α1-specific B cells [78]. More recently, researchers have developed a variant of such strategy in which nAChR α1 ECD was fused with Fc domain of antibody, which was used to convey the negative signal, since B cells express and only express one kind of Fc receptor, namely FcRγIIB, which transduce negative signal for B cell activation. Consequently, such chimeric protein will specifically target the nAChR α1 specific B cell via the binding to the BCR and deliver negative signal to inhibit α1 specific B cells [79, 80].

The idea of antigen-chimera in the treatment of MG seems attractive but will not be practical unless the chimeric protein is stable enough to be used as a therapeutic agent. As mentioned above, nAChR α1 is just one subunit of the nAChR pentamer and is intrinsically unstable, making the expression of wild type nAChR α1 ECD in stable soluble form very challenging. However, as discussed earlier in this chapter, crystallography studies of nAChR α1 ECD in recent years have accumulated extensive experience and knowledge in designing strategic mutations to improve the stability and expression level of nAChR α1 ECD protein while preserve the binding of MIR-directed MG autoantibodies [22, 31, 58] These progresses will greatly facilitate the approach to using engineered antigen chimera to specially inhibit and eliminate nAChR-specific B cells for MG treatment.

#### **10. Outlook**

Insights from structural studies and molecular biology/biochemical analyses may ultimately lead to precision medicine and personalized treatment of MG by *Structure-Based Approaches to Antigen-Specific Therapy of Myasthenia Gravis DOI: http://dx.doi.org/10.5772/intechopen.84715* 

 antigen profiling of patient and the use of corresponding molecular missiles to eliminate antigen specific antibodies or B-cells, induce antigen specific tolerance, or blocking nAChR-autoantibody binding by small molecules. These approaches, once established in the treatment of MG, could be expanded to other autoimmune diseases with well-defined antigen targets.

### **Author details**

Jiang Xu, Kaori Noridomi and Lin Chen\* Molecular and Computational Biology, Departments of Biological Sciences and Chemistry, University of Southern California, Los Angeles, CA, USA

\*Address all correspondence to: linchen@usc.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Plasmapheresis in Treatment of Myasthenia Gravis

*Valerii Voinov*

#### **Abstract**

Treatment of myasthenia gravis is still a rather difficult task, since there is no single tactic to use different drugs (corticosteroids, rituximab, immunoglobulins), especially since it is associated with a number of side effects. They are not able to remove the accumulating autoantibodies and immune complexes, the large size of which does not allow them to be excreted by the kidneys as well. Special problems of treatment arise when myasthenic crises develop associated with respiratory failure requiring artificial lungs ventilation. Plasmapheresis can help to solve this for it is possible to remove antibodies and other pathological metabolites. In addition, regular plasmapheresis is able not only to prevent exacerbations but also to reduce doses of the maintenance therapy with less risk of their side effects, which is confirmed by our own experience.

**Keywords:** myasthenia gravis, autoimmunity, autoantibodies, drug therapy, plasmapheresis

#### **1. Introduction**

 Myasthenia gravis (MG) is a relatively rare disease, affecting about 140 people per million [1, 2]; however, its frequency has been increasing in the recent years, especially in the elderly population with mortality rate of 0.27/100,000 people, and in intensive care units, mortality of such patients reaches 5.3% [3, 4]. However, MG also affects children, manifesting in three forms: transient neonatal myasthenia, congenital myasthenic syndrome, and juvenile MG [5]. In the latter case, the disease onset can be from 11 months to 17 years [6]. Although the disease has been known for decades, a single tactic of its treatment has not yet been developed. In many respects, it depends on the variety of forms and their etiopathogenetic features. In particular, the main focus is on the use of drug therapy, and too little attention is paid to plasmapheresis. Therefore, the main objective of this study is to justify the need for plasmapheresis in the treatment of MG.

#### **2. Etiology and pathogenesis**

MG is a long-term neuromuscular disease that leads to various degrees of the skeletal muscles weakness. The most commonly affected muscles are those of the eyes, face, and swallowing [7]. In this case, IgG antibodies appear to nicotine acetylcholine (ACh) receptors of the postsynaptic membrane, which leads to the muscle weakness increase [8]. In some cases, antibodies can also emerge to the muscle-specific kinase (MuSK) [9]. In this case, antibodies against MuSK can produce plasmoblasts, and in such cases, removal of B-lymphocytes does not exclude recurrence of MG [10]. It also does not exclude autoantibodies presence to other postsynaptic proteins (anti-titin, anti-integrin antibodies) in small amounts [11–13].

#### **3. Drug therapy**

#### **3.1 Cholinesterase inhibitors**

Cholinesterase inhibitors (pyridostigmine bromide) delay the disease progression and increase the availability of ACh on the motor end membranes and lead to their strength increase [14]. Cholinergic side effects, including hyperactivation of the smooth muscles of the urinary bladder and intestines causing diarrhea, abdominal cramps, increased salivation, sweating, and bradycardia, are dose limiting and lead to noncompliance to the treatment plan [15].

#### **3.2 Corticosteroids**

The most common tactic for MG treatment is based on corticosteroids therapy [16]. However, such therapy is not deprived of a large number of adverse reactions. They lead to *Cushingoid syndrome*. Glucocorticoids, in particular, are *diabetogenic*  hormones for they suppress glucose consumption by the tissues, and its production by the liver becomes increased. Besides, they can also directly suppress the release of insulin, thus showing that β-cells of pancreatic islets are one of their targets. Other complication of long glucocorticoid therapy is *osteoporosis*. It is considered that these hormones inhibit proliferation and differentiation of osteoblasts and stimulate their apoptosis. There is also an indirect mechanism of bones resorption caused by secondary hyperparathyreosis due to intestinal calcium adsorption decrease. Glucocorticoids effect on hypothalamus and gonads causes *hypogonadism* [17]. Development of chronic inflammatory polyneuropathy is also described [18]. There are evidences about correlation between such intensive and prolonged immunosuppressive therapy and the onset of tumors [19, 20].

#### **3.3 Immunoglobulins**

Administration of large doses of immunoglobulins does lead to such serious complications as aseptic meningitis, hemolytic anemia, cardiac rhythm disorders, and neurologic frustration in children with thrombotic thrombocytopenic purpura. Arthritis, thromboembolic complications, vasculitis, and a systemic lupus erythematosus are the side effects of autoantibodies and circulating immune complexes. Besides, there are other complications such as lethal hypersensitive (allergic) myocarditis and refractory heart failure, rash and skin itch, a leucopenia, a neutropenia, fever, etc. [21–24]. Presence of immune complexes may be the cause of it [25]; however, the main cause that must be recognized is the technology of immunoglobulins preparation from thousands (!) of donors having different blood types with full set of anti-A and anti-B isohemagglutinins (α and β), which lead to destruction of the corresponding erythrocytes [26]. At the same time, plasma exchange was necessary to relieve such hemolytic complication [27].

#### **3.4 Rituximab**

In the recent years, treatment of autoimmune diseases with rituximab—chimeric monoclonal antibody to CD20 antigen of B-lymphocytes—has become rather widespread, which should reduce the production of autoantibodies [28]. Rituximab is believed to be the first choice therapy [7]. Nevertheless, there are also complications of such treatment described leading even to *fulminant hepatitis* and *multiple organ failure* development [28–30]. Rituximab, cetuximab, and panitumumab have direct nephrotoxic effect [31]. *Rituximab* and *alemtuzumab* are reported to cause interstitial pneumonia development [32]. Prospectively, after rituximab treatment, neutropenia with pneumonia and other infectious complications may develop in up to 17% [33, 34]. There are reports about development of male infertility due to either gonadal dysfunction or antisperm autoantibodies production [35]. In addition, as noted above, removal of B-cells is not always accompanied by decrease in the autoantibodies reproduction [10].

### **4. Plasmapheresis**

Considering the disease autoimmune nature, direct removal of antibodies by plasmapheresis is more effective [9, 36–38]. It causes normalization of immunoglobulin levels and reduction of the circulating immune complexes (CICs) in 1.7–2 times. The overall subjective improvement is observed in 94% of patients after a primary set of five plasma exchange procedures with their addition if necessary [39]. In severe cases, patients can be quickly disconnected from the artificial lung ventilation, but it is a relatively short-term effect and requires repeated sets of procedures [40].

Nevertheless, along with plasmapheresis, the same results are obtained by intensive intravenous immunoglobulin administration at a dose of 0.4 g/kg daily for 3 or 5 days [41, 42]. Though, using intensive plasmapheresis, we can achieve better results in the treatment of myasthenic crises, rather than by intravenous administration of immunoglobulins, the course of which costs \$78.814 [43–47]. Immunoadsorption methods are also used; however, the best results are achieved in combination with plasma exchange [16].

 It is advisable to carry out three to five procedures of plasmapheresis with removal of plasma up to 2.0–2.5 ml/kg of the body weight [48]. It is also possible to carry out daily procedures of plasma exchange removing smaller amounts of plasma, instead of the abovementioned plasma exchange, being carried out every other day [49]. Similarly, plasma exchange provides faster positive effect (already after the first procedure) in patients resistant to rituximab [50]. Nevertheless, carrying out plasma exchange along with rituximab treatment appeared more effective [51].

 Plasmapheresis before thymectomy greatly facilitates the postoperative period [52–55]. Moreover, in cases when thymoma recurs postoperatively after a course of a plasma exchange, its involution is observed [56].

In juvenile forms of MG, plasmapheresis with immunoglobulins appears successful [57, 58], and it was noted that *plasma exchange yields more stable results* than IVIG therapy [44].

It should be noted that in the earliest symptoms of MG such as weakness of the cervical paraspinal muscles (*dropped head syndrome*), plasma exchange and immunoadsorption are justified [59].

 The use of specific IgG-immunoadsorption to remove antibodies to ACh receptors [60] seems prospective as well as new systems for cascade plasmapheresis [53, 61].

At a cascade plasma exchange, the level of soluble molecules of intercellular adhesion decreases more effectively and the quantity of the T-regulating cells increases [62]. After a cascade plasma exchange, they observe increase in the SatO2 levels associated with decrease in pCO2 [63].

 Nevertheless, in the comparable groups of patients with MG, there were no significant differences noted in the effectiveness of immunoadsorption or cascade plasmapheresis [64, 65]. On the other hand, there were no benefits found of immunoglobulin transfusions before cascade plasmapheresis or immunoadsorption [66]. After a cascade plasma exchange, they also noted a decrease in cytotoxic activity of the natural killer cells that even more improves the effectiveness of such treatment [67].

MG development is also possible in infants due to "graft-versus-host" disease (GVHD) following bone marrow transplantation. The course of plasmapheresis with subsequent administration of immunoglobulins was quite effective [68].

 Our own experience shows that there are two possible applications of plasmapheresis. In myasthenic crises accompanied by swallowing and breathing disorders when patients need artificial lung ventilation, it is really necessary to urgently conduct a massive plasma exchange, removing 1–1.5 of the total plasma volume (TPV) with compensation with albumin and fresh frozen donor plasma for four to five procedures every day or every other day [69, 70]. The same tactic is described in the American Society for Apheresis Guidelines on the Use of Therapeutic Apheresis in Clinical Practice [71].

Then, to achieve a more stable remission, it is necessary to repeat procedures of less massive plasmapheresis at intervals of 2–4 weeks, removing only 0.3–0.5 TPV. The same tactic is used in less severe degrees of the disease, when the removed plasma volume can be compensated only by crystalloid solutions. In this case, the primary course also consists of four such plasmapheresis procedures, followed by one procedure every 1–2 months. Given the fact that MG can be observed in young children up to the development of myasthenic crises, it is desirable to use equipment with a small volume of filling. In our practice, we use a device for membrane plasmapheresis called "Hemophenix" ("Trackpore Technology," Russia) with an internal filling volume up to 70 ml, which can be used even in unstable hemodynamics, including in children. The advantage is a single-needle access using any peripheral vein.

 Our practice includes 15 patients with MG. Two of them were in acute stage of the myasthenic crisis with respiratory failure, requiring connection to artificial lungs ventilation. One of them was a girl of 8 years old, who had complication of GVHD on the background of lymphocytic leukemia. She had already been on artificial lung ventilation for 10 days without visible effect (**Figure 1**). After two procedures of plasma exchange in a volume of 1.2 TPV, she was already able to breathe herself. In total, five such procedures were performed with a good effect of restoring the motor activity except for some left eyelid ptosis, which persisted after a month (**Figure 2**). The second patient had been on the artificial lung ventilation for 2 weeks in one of the clinics in Sofia, Bulgaria (**Figure 3**). Also, after two plasma exchange procedures, it was possible to switch him off the artificial lung ventilation (**Figure 4**), and after the last fourth procedure, he was already able to move without assistance and was discharged from the clinic.

 The other patients were in different degrees of MG severity, and they performed a conventional plasmapheresis in the volume of 0.3–0.5 TPV with replacement of the removed plasma with an isotonic solution of sodium chloride. The course of treatment consisted of four such procedures, conducted every other day. Most of the procedures were performed in outpatient settings. The main task was to stabilize the condition and prevent the disease recurrence. One of them was in

*Plasmapheresis in Treatment of Myasthenia Gravis DOI: http://dx.doi.org/10.5772/intechopen.81354* 

#### **Figure 1.**

*Girl M of 8 years old and 18 kg body weight. Myasthenic crisis with artificial ventilation for 10 days. Plasma exchange using the "Hemophenix" device.* 

#### **Figure 2.**

*The same girl a month after the course of plasma exchange.* 

#### **Figure 3.**

*Patient T of 28 years old. The first session of plasma exchange on the device "Hemophenix" on the background of artificial lung ventilation, carried out for 2 weeks.* 

**Figure 4.**  *The same patient after two sessions of plasma exchange. Disconnected from the ventilator.* 

quite serious condition and was able to move only with someone's assistance. After the primary course of plasmapheresis, we followed the tactics of a "programmed" plasmapheresis once per month, which enabled him to return to his physical work of an auto mechanic. The follow-up period is 6 years.

#### **5. Conclusion**

The autoimmune nature of the disease undoubtedly is an indication for plasmapheresis since it is the only way to remove large-molecule pathological products (autoantibodies, immune complexes) that cannot be excreted by the kidneys. Our experience shows that after such courses of plasmapheresis, conducted twice a year, it is possible to practically reduce the doses of corticosteroids and other medicines by half and, thereby, avoid the toxic consequences of their use.

#### **Author details**

Valerii Voinov First I.P. Pavlov State Medical University of Saint Petersburg, Russia

\*Address all correspondence to: voinof@mail.ru

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Plasmapheresis in Treatment of Myasthenia Gravis DOI: http://dx.doi.org/10.5772/intechopen.81354* 

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**69**

**Chapter 6**

**Abstract**

the endogenous ACh.

myasthenia gravis

**1. Introduction**

Anticholinesterases

*Zeynep Özdemir and Mehmet Abdullah Alagöz*

**Keywords:** acetylcholine, acetylcholinesterase, butyrylcholinesterase, anticholinesterases, neostigmine, pyridostigmine, distigmine, ambenonium,

The autonomic nervous system (ANS) works out of our request, and it differs from the somatic system with this feature. Autonomic afferent and efferent fibers enter and exit the central nervous system through the spinal and cranial nerves. It connects with the medulla spinalis and intermediate neurons, which mediate autonomic reflexes in the brain stem [1, 2]. Changes in the internal and external environment and emotional factors affect autonomic activity through fibers, which descend from the hypothalamus. ANS shows its effect through neuromediators. Acetylcholine (ACh) and noradrenaline (NA) are the main neurotransmitters in the autonomic nervous system. ACh is released from all preganglionic endings. ACh is secreted from all postganglionic parasympathetic fibers, and it acts through muscarinic receptors [3, 4]. Autonomic nervous system disorders may occur with an abnormally high parasympathetic activity or abnormally low parasympathetic activity and/or abnormally high sympathetic activity or abnormally low sympathetic activity. MG, a neuromuscular junction disease that occurs due to ACh receptor deficiency in the postsynaptic membrane at the neuromuscular junction, is one of these disorders. The origin of the disease is thymus, because myoid cells form the source of receptor antigens. ACh release in the normal muscular junction leads to a localized end-plate potential, resulting in muscle contraction [5]. Although MG patients have normal nerve anatomy and function, there is a decrease in the number

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known serine hydrolase enzymes responsible for the hydrolysis of acetylcholine (ACh). Although the role of AChE in cholinergic transmission is well known, the role of BChE has not been elucidated sufficiently. The hydrolysis of acetylcholine in the synaptic healthy brain cells is mainly carried out by AChE; it is accepted that the contribution to the hydrolysis of BChE is very low, but both AChE and BChE are known to play an active role in neuronal development and cholinergic transmission. Myasthenia gravis (MG) is a muscle disease characterized by weakness in skeletal muscles and rapid fatigue. Anticholinesterases, which are not only related to the immune origin of the disease but also have only symptomatic benefit, have an indispensable role in the treatment of MG. Pyridostigmine, distigmine, neostigmine, and ambenonium are the standard anticholinesterase drugs used in the symptomatic treatment of MG. All of these compounds may increase the response of the myasthenic muscle to recurrent nerve impulses, primarily by protecting

## **Chapter 6**  Anticholinesterases

*Zeynep Özdemir and Mehmet Abdullah Alagöz* 

### **Abstract**

 Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known serine hydrolase enzymes responsible for the hydrolysis of acetylcholine (ACh). Although the role of AChE in cholinergic transmission is well known, the role of BChE has not been elucidated sufficiently. The hydrolysis of acetylcholine in the synaptic healthy brain cells is mainly carried out by AChE; it is accepted that the contribution to the hydrolysis of BChE is very low, but both AChE and BChE are known to play an active role in neuronal development and cholinergic transmission. Myasthenia gravis (MG) is a muscle disease characterized by weakness in skeletal muscles and rapid fatigue. Anticholinesterases, which are not only related to the immune origin of the disease but also have only symptomatic benefit, have an indispensable role in the treatment of MG. Pyridostigmine, distigmine, neostigmine, and ambenonium are the standard anticholinesterase drugs used in the symptomatic treatment of MG. All of these compounds may increase the response of the myasthenic muscle to recurrent nerve impulses, primarily by protecting the endogenous ACh.

**Keywords:** acetylcholine, acetylcholinesterase, butyrylcholinesterase, anticholinesterases, neostigmine, pyridostigmine, distigmine, ambenonium, myasthenia gravis

#### **1. Introduction**

 The autonomic nervous system (ANS) works out of our request, and it differs from the somatic system with this feature. Autonomic afferent and efferent fibers enter and exit the central nervous system through the spinal and cranial nerves. It connects with the medulla spinalis and intermediate neurons, which mediate autonomic reflexes in the brain stem [1, 2]. Changes in the internal and external environment and emotional factors affect autonomic activity through fibers, which descend from the hypothalamus. ANS shows its effect through neuromediators. Acetylcholine (ACh) and noradrenaline (NA) are the main neurotransmitters in the autonomic nervous system. ACh is released from all preganglionic endings. ACh is secreted from all postganglionic parasympathetic fibers, and it acts through muscarinic receptors [3, 4]. Autonomic nervous system disorders may occur with an abnormally high parasympathetic activity or abnormally low parasympathetic activity and/or abnormally high sympathetic activity or abnormally low sympathetic activity. MG, a neuromuscular junction disease that occurs due to ACh receptor deficiency in the postsynaptic membrane at the neuromuscular junction, is one of these disorders. The origin of the disease is thymus, because myoid cells form the source of receptor antigens. ACh release in the normal muscular junction leads to a localized end-plate potential, resulting in muscle contraction [5]. Although MG patients have normal nerve anatomy and function, there is a decrease in the number

of postsynaptic ACh receptors. During a muscle contraction under normal conditions, the release of ACh, which is caused by impulses along the axon, decreases gradually in each impulse. This decrease does not cause problems in the postsynaptic membrane when there is no pathology. However, in addition to the reduced number of receptors in MG, the ACh-receptor complex is also decreasing gradually; therefore, rapid fatigue is observed [6, 7].

#### **2. Acetylcholine and cholinergic receptors**

The neurotransmitter is ACh in all of the preganglionic autonomic fibers constituting the peripheral parts of the autonomic nervous system, in all postganglionic parasympathetic fibers and in several postganglionic sympathetic nerve fibers, and these ACh release fibers are called cholinergic fibers [8]. ACh is synthesized in the cytosol at the end of the nerve fibers, and then it is transported into the vesicles from the membrane of the vesicles (**Figure 1**). ACh is stored here in a very dense manner, with about 10,000 molecules in each vesicle. When an action potential reaches the nerve end, a large number of calcium channels are opened on the nerve end, since it has a large number of voltage-gated calcium channels.

**Figure 1.**  *Biosynthesis, transmission, and inactivation of ACh [3].* 

#### *Anticholinesterases DOI: http://dx.doi.org/10.5772/intechopen.81994*

 As a result, the calcium concentration in the nerve end increases by 100 times; this increases the speed of incorporation of ACh vesicles with the nerve end membrane by 10,000 times. This incorporation allows the exocytosis of acetylcholine to the synaptic range by causing rupture of many vesicles. About 125 vesicles are usually ruptured with each action potential. The ACh is then broken up with AChE in a few milliseconds to the acetate ion and choline. Choline is taken back to the nerve end to be used in the formation of ACh again [9].

Studies have shown that many tissues respond to stimulation and inhibition are generated by compounds, which mimic the action of neuronal release of ACh or the neurotransmitter administered externally. Peripheral cholinergic receptors interacting with ACh are found in the parasympathetic postganglionic nerve endings in the smooth muscles and neuromuscular junction in the skeletal muscles. Cholinergic receptors are divided into two groups, namely nicotinic and muscarinic. The distribution of muscarinic receptors in the brain adapts to the distribution of ACh [10]. Receptors are mostly found in the striatum, neocortex, hippocampus, superior colliculus, locus coeruleus, and pons nuclei; whereas, the quantity of them in the hypothalamus, spinal cord, and cortex is low. These receptors in neuromuscular motor ends and ganglia are the first neurotransmitter receptors that have been isolated and purified in active form. It is contemplated that the receptor is comprised of two polypeptide chain monomers, which are connected to each other by a disulfide bond and have five subunits. When ACh is bound to these receptors, it allows the passage of the small cations such as Ca++, Na+ , and K+ by leading to an increase in membrane permeability. The physiological effect of this condition is the formation of depolarization at the motor ends and consequently the muscular contraction or the continuation of nerve stimulation at the neuromuscular junction. Muscarinic receptors play an important role in regulating the functions of organs stimulated by the autonomic nervous system. The effect of ACh on these receptors in parasympathetic synapses may be stimulating or inhibiting. ACh both stimulates the secretion by activating the salivary glands and leads to the contraction of the respiratory system. The compound also inhibits cardiac contractions and relaxes the smooth muscles in the blood vessels. Recent studies have shown that there are five subtypes of muscarinic receptors (M1, M2, M3, M4, and M5). M1 is found in neuronal structures such as the central nervous system and ganglions, M2 is found in the heart, M3 is found in the smooth muscles in the glands, and M4 is also found in the striatum and lungs [8–10].

#### **2.1 Cholinesterase enzymes and cholinesterase inhibitors**

AChE is a hydrolytic enzyme of the class of the serine hydrolase enzyme, which plays a major role in the hydrolysis of ACh in cholinergic synapses of the autonomic nervous system and central nervous system [11]. Electron microscopy studies using histochemical techniques have shown that this enzyme is located on both the nerve endings and the postjunction or postsynaptic membrane at the cholinergic synapses or junctions. This enzyme, which is also called main cholinesterase, hydrolyzes ACh most rapidly among choline esters. It can also hydrolyze methacholine, but it is ineffective against benzoylcholine. A second type of cholinesterase, which is called pseudocholinesterase, breaks down acetylcholine more slowly. Because this enzyme is the most rapidly broken choline ester butyrylcholine, pseudocholinesterase is called butyrylcholinesterase (BChE). Butyrylcholinesterase is not found in synapses and does not contribute to the hydrolysis of acetylcholine [12]. Both AChE and BChE are polymorphic and exist as homomeric and heteromeric molecular forms characterized by subunit relationships and hydrodynamic properties. Heteromeric molecular forms contain catalytic subunits linked to the lipid or triple helix collagen

tail and are often referred to as asymmetric or A forms of AChE. The G1, G2, and G4 forms, which are homomeric hydrophilic globular forms of AChE, contain one, two, and four identical subunits, respectively. The G4 form is secreted by neurons and secretory cells. An amphiphilic glycophospholipid-bound form is a dimer of G2, which has two subunits having a glycophospholipid link to the cell membrane. Less polymorphism is observed in BChE, and only hydrophilic and asymmetric forms have been defined for this enzyme [11].

The structure of AChE reveals an active site containing a catalytic triadglutamate (E327), histidine (H440), and serine (S200) at the base of a narrow valley about 20 A depth. This general amino acid arrangement represents the serine hydrolase enzyme family. The gorge in AChE is coated with 14 aromatic amino acids consisting of phenylalanine, tyrosine or tryptophan, and the base of the gorge contains a number of anionic residues, which are collectively responsible for the interaction of ACh with the positively charged trimethylammonium group and the acceleration of binding of the cationic ligands. BChE contains six less aromatic amino acids than AChE in the gorge. These structural studies help to elucidate the molecular basis of the specificity between the active center and the ligand. In particular, the main substrate differences between AChE and BChE can be determined by the presence of two phenylalanines (F288 and F290), providing a rigid acyl binding pocket in AChE. In addition, replacement of these amino acids with leucine and valine to provide a less structurally restricted pocket in BChE can also determine the main substrate differences between AChE and BChE. In addition, AChE contains a peripheral anionic region responsible for allosteric inhibition by cationic ligand interactions in the catalytic region. This peripheral anionic region proposed by Changeux in 1966 links agents such as propidium to residues around the edge of the gorge. This peripheral anionic region may play a role in the catalytic process by mediating substrate inhibition [13–17].

The effects of ACh released from autonomic and somatic motor nerves are terminated by enzymatic degradation of the molecule by AChE that is present in high concentrations in cholinergic synapses and synthesized in both nerve and muscle tissues [18]. The drugs, which inhibit AChE, are called anticholinesterase agents. The characteristic pharmacological effects of anticholinesterases occur primarily by inhibiting the hydrolysis of AChE by the AChE enzyme in cholinergic pathways. Inhibition of cholinesterases induces ACh receptors by leading increased ACh in nerve synapses and neuromuscular junction. Continuous stimulation of ACh receptors results in cholinergic synaptic paralysis and central and peripheral clinical symptoms in the central nervous system, autonomic ganglia, parasympathetic and sympathetic nerve endings, and somatic nerves due to the accumulation of ACh in the motor end plates. Muscarinic effects due to parasympathetic activity and nicotinic effects due to sympathetic activity are seen. The main symptoms and signs depend on the balance between muscarinic and nicotinic receptors [8, 18].

Inhibition of the AChE prolongs the life of the neurotransmitter at the junction, thus resulting in pharmacological effects similar to those observed when acetylcholine is administered. AChE is the primary target of these drugs, but BChE is also inhibited. Anti-AChEs are used in the treatment of diseases such as myasthenia gravis, atony in the gastrointestinal tract, glaucoma, and Alzheimer's disease. These compounds are also used as nerve gases and insecticides. Anti-AChE agents can be divided into three groups based on their mechanism of action: competitive agonists, short-acting inhibitors, and long-acting inhibitors [19]. Before World War II, only reversible anti-ChE agents were known and their prototype is physostigmine. Organophosphates as highly toxic chemicals, which were first developed as agricultural insecticides, were also developed as a potential chemical warfare agent shortly before World War II. It is known that

#### *Anticholinesterases DOI: http://dx.doi.org/10.5772/intechopen.81994*

the excessive toxicity of these compounds is due to the irreversible inactivation of AChE. Thus, organophosphate inhibitors are sometimes referred to as "irreversible cholinesterase inhibitors." Strong nucleophiles such as pralidoxime can break phosphorus enzyme binding [9, 19].

Anti-AChEs, which are currently used for treatment in the postoperative period of intestinal system and atony of the smooth muscles of bladder, glaucoma, myasthenia gravis, and termination of the effects of competitive neuromuscular muscle relaxants produce nonselectively both muscarinic and nicotinic effects as indirect effects by increasing the ACh concentration. Longacting and hydrophobic ChE inhibitors are also the only inhibitors with limited, well-documented efficacy in the treatment of dementia symptoms of Alzheimer's disease [8, 20].

MG is a muscle disease characterized by weakness in skeletal muscles and rapid fatigue. Anti-AChEs, which are not only related to the immune origin of the disease but have only symptomatic benefit, have an indispensable role in the treatment of MG. Pyridostigmine, distigmin, neostigmine, and ambenonium are the standard anticholinesterase drugs used in the symptomatic treatment of MG. All of these compounds may increase the response of the myasthenic muscle to recurrent nerve impulses, primarily by protecting the endogenous ACh [8, 21, 22].

#### *2.1.1 Pyridostigmine*

 The most commonly used anti-ChE in daily treatment is pyridostigmine bromide (**Figure 2**). The effect of the drug starts in 15–30 min, reaches maximum in 1–2 hours, and lasts 3–4 hours or longer. Pyridostigmine bromide, used for the treatment of MG and for protection against exposure to nerve agents, is a carbamate-derived reversible AChE inhibitor [23–25]. Due to the quaternary amine structure, it is relatively weakly absorbed from the gastrointestinal system. The elimination of half-life of pyridostigmine bromide after a single dose of 60 mg in healthy volunteers was found to be 200 min. This requires frequent usage. Ninetynine percent of AChE inhibitors, including pyridostigmine bromide, are administered orally [26–28].

#### *2.1.2 Distigmine*

Distigmine is a carbamate-derived reversible ChE inhibitor. The compound synthesized chemically by Schmid has a chemical structure consisting of two molecules of pyridostigmine bonded together by hexamethylene bonds (**Figure 3**). Distigmine is clinically used in some Asian and European countries, including Japan and Germany, and the main clinical indication for distigmine is myasthenia gravis.

**Figure 2.**  *Structure of pyridostigmine bromide.* 

However, in Japan, distigmin was also used for glaucoma and underactive bladder [29, 30].

#### *2.1.3 Neostigmine*

Neostigmine (**Figure 4**) is commonly used to reverse nondepolarizing neuromuscular blocking agents. The drug increases the rate of recovery from moderate nondepolarizing neuromuscular blockade and reduces the incidence of residual blockade. However, doses of neostigmine used in clinical practice may cause muscle weakness when administered after complete recovery from neuromuscular blockade. Since the first studies investigating the effects of neostigmine were performed in anesthetized patients, the results may be mixed with the presence of anesthetic agents, which are known to be in the neuromuscular blockade. Later, the effect of neostigmine was supported by the studies that the volunteers did not receive anesthetic agents. However, the effects of neostigmine on maximum voluntary muscle strength have not been previously investigated [31, 32].

#### *2.1.4 Ambenonium*

In addition, compounds, which are structurally different from the above-mentioned carbamates for the treatment of MG, are also used. One of them, bisquaternary inhibitor ambenonium dichloride (**Figure 5**), is known to be one of the compounds with the highest inhibition ability against AChE in sub-nM range [33].

 The superior effect potential is unique for the compound, which does not form any covalent bonds with the active site of the enzyme. Binding studies on the AChEambenonium complex have shown that the compound is capable of making very

*Structure of neostigmine.* 

#### *Anticholinesterases DOI: http://dx.doi.org/10.5772/intechopen.81994*

convenient contacts with the amino acids of the catalytic and peripheral AChE sites. Ambenonium produces less muscarinic side effects than carbamates. Unlike shortacting anti-AChE compounds, it is advantageous because it produces a larger and longer-lasting therapeutic effect during the night and waking period. In addition, the bisquaternary structure inhibits the passage from blood-brain barrier (BBB) after a conventional oral or intravenous route of administration. Another anti-AChE compound, edrophonium chloride (**Figure 6**), is used as a diagnostic tool for MG. It has a rapid onset and short pharmacological effect, so it cannot be used for therapeutic purposes [33, 34].

The optimal single oral dose of the anti-ChE agents can be determined empirically, when the MG is diagnosed. Basic records are made for a range of signs and symptoms, which reflect comprehension strength, vital capacity, and the strength of various muscle groups. The patient is given an oral dose of pyridostigmine at 30–60 mg, neostigmine at 7.5–15 mg, or ambenonium at 2.5–5 mg. Improvement in muscle strength and changes in other symptoms are recorded at frequent intervals until they return to the baseline state. After a baseline hour or longer, the drug is reintroduced, the dose is increased to one and a half times the initial amount, and the same observations are repeated. These repeats continue with increments of half the initial dose until the optimal dose is achieved. The duration of action between the oral doses is required to maintain the muscle strength of these drugs, which is usually 2–4 h for neostigmine, 3–6 h for pyridostigmine, or 3–8 h for ambenonium. However, the required dose may vary from day to day. Physical or emotional stress, intercurrent infections, and menstruation usually require an increase in the frequency or size of the dose. Unpredictable exacerbations and remissions of the myasthenic condition may require adjustment of the dosage. Pyridostigmine has sustained release tablets containing a total of 180 mg, 60 mg of this is released immediately and the drug concentration is 120 mg for several hours. This preparation is valuable in maintaining patients in periods of 6–8 h, but it should be limited to use before bedtime [5, 8, 35].

 There is always a risk of cholinergic crisis, if the effects of anti-ChE drugs are weak and there is no any improvement in the symptoms of the disease even with high doses of AChE. In cholinergic crisis, nausea, vomiting, sweating, salivation, colic, diarrhea, miosis, bradycardia, etc., are observed and myasthenic weakness increases [7]. In addition, many drugs, including curariform agents,

**Figure 5.**  *Structure of ambenonium.* 

**Figure 6.**  *Structure of edrophonium.* 

certain antibiotics, and general anesthetics prevent neuromuscular transmission. Therefore, the application of these drugs to patients with MG requires an appropriate adjustment of the anti-ChE dose and other measures [8].

#### **2.2 Newly developed cholinesterase inhibitors for MG**

Muscarinic cardiovascular and gastrointestinal side effects of anti-ChE agents can usually be controlled by atropine or other anticholinergic drugs. However, these anticholinergic drugs mask many side effects of an excessive amount of anti-ChE agents. Tolerance in most patients may eventually lead to muscarinic effects [5, 7, 35, 36]. For these reasons, it is aimed to investigate more effective drugs for the treatment of MG and to prevent the hepatotoxicity and known gastrointestinal side effects, while creating the targeted pharmacological effect with the synthetic analogues at the development stage.

 Musilek et al. performed the synthesis of 20 new bis-isoquinolinium inhibitors (**Figure 7**) in a study and determined whether the compounds would be effective in the treatment of MG. They evaluated the newly prepared compounds *in vitro* on human recombinant AChE and human plasmatic BChE and compared the inhibitory capabilities of the compounds expressed as IC50 with ambenonium dichloride, edrophonium chloride, BW284c51, and ethopropazine hydrochloride, which have been selected as standard. In three of the compounds they have obtained, they had promising results in which their compounds inhibited both enzymes better than or similar to edrophonium and BW284c51, however, worse than ambenonium *in vitro.*  The kinetic assays are confirmed noncompetitive inhibition of human-recombinant AChE (hAChE) with two promising new compounds selected [37].

In a study in which neostigmine, pyridostigmine, and physostigmine quaternary phenylcarbamates were synthesized and evaluated their activity, N-monophenylcarbamate analogues together with their precursors of neostigmine methyl sulfate and pyridostigmine bromide and the N-methylammonium analogues of phenserine, tolserine, cymserine, and phenylethylcymserine were synthesized as long-acting peripheral inhibitors of AChE or BChE (**Figure 8**). According to the results of the study, only N-phenylcarbamate of 3-dimethylamiophenol to N-phenylcarbamate of 3-hydroxy-1-methylpyridinium bromide compounds had marginal ChE inhibitor of activity and compound N(1)-methylammonium bromide of (−)-phenserine, (−)-tolserine, (−)-cymserine, and (−)-phenylethylcymserine were strong anti-ChEs [38].

Monarsen (EN101) is an antisense oligodeoxynucleotide, which acts at the level of mRNA and selectively reduces the production of The enzymatic isoform of readthrough AChE (AChE-R) by the destruction of the AChE-R mRNA. This compound selectively lowers the AChE-R levels in both blood and muscle, but, does not affect the synaptic variant of synaptic AChE (AChE-S). It was tested in experimental

**Figure 7.**  *Structure of bis-isoquinolinium derivatives.* 

*Anticholinesterases DOI: http://dx.doi.org/10.5772/intechopen.81994* 

#### **Figure 8.**

*Structure of neostigmine N-phenylcarbamate of 3-dimethylamiophenol and 3-hydroxy-1-methylpyridinium and N(1)-methylammonium bromide of (-)-phenserine, (-)-tolserine, (-)-cymserine, and (-)-phenylethylcymserine.* 

autoimmune MG rats that oral or intravenous administration of EN101 reduced AChE in blood and muscle and increased survival, muscle strength, and disease severity. The stabilization of the reduction of the compound motor action potential (CMAP) on the responsive neurostimulation system and muscles was also observed during the entire treatment. This effect has been found to be comparable to that of pyridostigmine, which is worn out for hours and causes significant fluctuations in muscle strength. It was also found that clinical and electrophysiological improvement was associated with a decrease in autoimmune responses [39–41].

#### **3. Conclusions**

Anti-ChEs have an indispensable role in the symptomatic treatment of the MG, which is not directed against the immune origin. AChE inhibitors improve neuromuscular conduction by preventing disruption of circulating ACh in the neuromuscular junction. The compounds used in the treatment of MG have a positive charge in the molecule to provide the peripheral effect of the action and minimal blood-brain barrier penetration. However, the most prescribed carbamate inhibitors may cause many serious side effects, such as carbamylation of AChE. As a result, it is important to individually arrange treatment for each MG patient. The effect of treatment should be optimized for vital muscles such as respiratory and swallowing; because, different muscles are affected by varying levels. Since the nonselective AChE inhibitors are the most effective compounds at the beginning of MG, the dosage of AChE inhibitors is ideally reduced as they develop strength by immunosuppressive therapy. Decrease in activity over time may be related to increased levels of the AChE-R isoform, which may cause morphological and physiological abnormalities in the neuromuscular junction. Myasthenia gravis (MG) is usually caused by antibodies either to the acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase (MuSK) or at the neuromuscular junction. Patients with MuSK antibodies generally do not respond to the treatment; whereas, patients with AChR antibodies respond specifically to the treatment. For these reasons, EN101, a selective AChE inhibitor that specifically targets the isoform of AChE (AChE-R), has been developed recently and the AChR-antibody may be important for symptomatic relief in seropositive MG.

#### **Conflict of interest**

None to declare.

*Selected Topics in Myasthenia Gravis* 

### **Author details**

Zeynep Özdemir and Mehmet Abdullah Alagöz Faculty of Pharmacy, Department of Pharmaceutical Chemistry, İnönü University, Malatya, Turkey

\*Address all correspondence to: zeynep.bulut@inonu.edu.tr

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Isam Jaber Al-Zwaini and Ali AL-Mayahi*

Myasthenia gravis is a rare potentially fatal chronic autoimmune disorder. Circulating autoantibodies directed against components of the neuromuscular junction of skeletal muscles, most commonly nicotinic acetylcholine receptor and associated protein in the postsynaptic membrane, block neuromuscular transmission resulting in muscle weakness. Tis muscle weakness typically worsens with continued activity, improves on rest, and is of variable severity ranging from mild ocular muscle weakness to severe generalized muscle weakness, involving the respiratory muscle with impending respiratory failure. Te worldwide prevalence of myasthenia gravis is 100–200 per million population, afecting more than 700,000 people all over the world. Te prevalence rate has increased since the 1950s due to improved diagnostic precision and decreased mortality rate.

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