Neuroinflammation

Italy

Preface

Alireza Minagar, MD, MBA, Shreveport, LA, United States

Since the publication of the 2011 edition of Neuroinflammation, tremendous scientific progress has been made in explorations on the molecular mechanisms, etiopathogenesis, and treatment of many diverse neuroinflammatory conditions. Neuroinflammation remains at the center of many neurological conditions and the role of nervous system interactions with the immune system and inflammatory phenomena is becoming widely apparent as to have always been with us. Indeed the continuing progress in neuroinflammation has driven the rapid proliferation of drug targets and drugs now being used to treat these conditions and the novel diagnostic and imaging modalities which have been developed and refined since the last edition.

The first edition of the book Neuroinflammation was a critical accomplishment because of the high caliber of the scientific content contributed by my colleagues and the depth to which they delved into their reports, which were communicated by a panel of nationally and internationally recognized scientists and clinicians. Due to current intense focus on expanding our knowledge about the neuroinflammatory processes, we have continued to collect and update our previous chapters and to add new ones that build on these new findings.

This, our second edition, summarizes the most current scientific findings in the swiftly advancing and burgeoning field of neuroinflammation and, again, argues that neuroinflammation deserves its own distinctive area of neuroscience. The diverse chapters of the present book also highlight the pragmatic and practical applications of these new developments toward treating neuroinflammatory diseases by facilitating the development of innovative biologic immunomodulators and immunosuppressants. This book, in its entirety, is dedicated to the eager scientists around the world who devotedly and unreservedly spend their life to research the various aspects of the field of neuroinflammation. I truly hope that the contents of this book will enhance the scientific curiosity of the younger colleagues so they can proceed with their research.

As the editor of this book, I must acknowledge the labors of the many authors who contributed to our book, and thank them for taking some much time away from their very many research and clinical duties to condense and summarize in review form the invaluable contributions submitted here. Without their perseverance in communicating their opinions and summarizing the contemporary knowledge in this field, this book would have never arrived and their valuable impressions would not have been shared. I cannot begin to fully express how well the many chapters complement one another and extend from our last edition.

I also would like to appreciate the editorial and production team and its members at Elsevier, Inc. for their tireless efforts to collect and produce these chapters. Much appreciation is due to Ms. Kathy Padilla, Ms. Melanie Tucker, Ms. Kristi Anderson, Ms. Natalie Farra, Ms. April Farr, and many of the editorial staff at Elsevier who were stalwart colleagues, advisors, and coaches providing thoughtful and outstanding support that helped tremendously with the creation of this current edition. In conclusion, this book is dedicated to the patients affected by these conditions, their families and physicians. We hope this edition provides valuable insights to the readers of this volume. My patients have taught me so much—I hope that the intense pace of study in neuroinflammation will improve the quality of life for all of us whose lives have been touched by forms of these disorders.

Chapter 1

Multiple Sclerosis

Clinical Features, Pathophysiology, Diagnosis, and Management

Reza Rahmanzadeh¹, Abdureza N. Moghadasi¹, Samira Navardi¹, Alireza Minagar² and Mohammad A. Sahraian¹,    ¹Tehran University of Medical Science, Tehran, Iran,    ²Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, United States

Abstract

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that usually affects young adults. The disease affects about 2.5 million people worldwide and is more frequently seen in females. The prevalence of MS appears to be different in various parts of the world, ranging from less than 5/100,000 in some African countries to more than 200/100,000 in Western Europe and North America (Kingwell et al., 2013). MS was first described by the French neurologist Jean-Martin Charcot who, in 1868, assimilated these findings into a single syndrome he called sclerose en plaques, and characterized it as a distinct neurological disease whilst clarifying some clinical and pathological features of the disease. The etiology of this potentially debilitating disease is still unknown but both environmental and genetic factors interact in disease development and progression. Many environmental factors such as vitamin D deficiency, viral infections such as Epstein Barr Virus (EBV), and smoking have been mentioned as triggering factors in genetically susceptible people. To date no single gene or locus has been found to be the gene for MS but several class II major histocompatibility complex (MHC) genes such as HLA DRB1*1501-DQA1*0102-DQB1*0602 alleles have been demonstrated to be associated with MS susceptibility. Over recent decades MS has been presented with its two different aspects of inflammation and degeneration. Although there are several approved and nonapproved medications to overcome or at least control the inflammatory aspect of the disease, at present there is no accepted therapy for controlling the degenerative phase, which may start even at the early stages of the disease.

This chapter covers the clinical manifestations, pathophysiology, diagnosis, and treatment strategies in MS. There are several new disease-modifying therapies that have been approved recently, and there are several options in the pipeline that will be added to the currently approved ones. Recent approved drugs have been associated with rare but serious adverse events that need close monitoring and proper clinical or radiological vigilance in order to reduce catastrophic complications from these drugs.

Keywords

Multiple sclerosis; pathophysiology; autoimmunity; neurodegeneration; diagnosis; management

Clinical Features

Initial Presentation of MS

The first manifestation of MS in most patients is an attack of neurological deficits typical for demyelinating disorders. This first presentation, Clinically Isolated Syndrome (CIS), is monophasic (isolated in time) and usually monofocal (isolated in space).¹ Clinical features that are suggestive of MS are related to the cerebral hemispheres (hemiparesis, hemisensory dysfunctions), the spinal cord (incomplete and lower-than-three-vertebrae transverse myelitis, sphincter incompetency, Lhermitte’s syndrome, asymmetrical limb weakness), brainstem and cerebellar (bilateral internuclear opthalmoplegia, ataxia, nystagmus, sixth nerve palsy, paroxysmal symptoms) and optic nerve involvements (optic neuritis, painful eye movements, reduced visual acuity).²

However, MS may be initiated in a minority of patients with a multifocal relapse, for example a patients with unilateral optic neuritis and concomitant ataxia, or even presentations atypical for demyelinating disorders, e.g., cognitive symptoms, seizures, and encephalopathy.³

Clinical Course of MS

The disease usually starts with a relapsing-remitting course (RRMS) followed by a progressive period in which the patients experience progression and increase in disability without any obvious exacerbations. In 10%–15% of patients the disease starts with progression—primary progressive MS—which is more common in the older population and is predominantly seen in males.⁴ The natural history of MS follows an unpredictable pattern of relapses and progression. Some patients may have few exacerbations and do well for a long period of time, and on the other hand there are patients who have frequent exacerbations and rapid progression despite aggressive treatments. Recently the clinical course of the disease has been redefined as relapsing or progressive according to the nature of the disease. Patients with relapsing or progressive course could be active or nonactive regarding clinical or magnetic resonance imaging (MRI) activities.⁵

MS-Exacerbating Conditions

Several medical conditions influence the frequency and severity of relapses. Firstly, most microbial infections may trigger a new relapse, likely via augmenting Th1-responses, while helminth infections may reduce relapse severity.⁶,⁷ Secondly, immunization, especially to influenza, hepatitis B virus (HBV), and Human Papilloma virus (HPV), may mount disease activity as a result of the molecular similarity of vaccine antigens to myelin components.⁸ Thirdly, pregnancy, likely due to hormonal modulation of the immune response, abolishes MS inflammatory activity.⁹ However, there is an increase in relapse rate in the upcoming months following childbirth. Last but not least, there is the temporary worsening of MS complications upon raising the body temperature, for example following bathing or exercise, and the subsequent improvement after cooling. This feature, named Uhthoff’s phenomenon,¹⁰ is common and sometimes hard to distinguish from a relapse.

Pathophysiology

Pathophysiology of Relapses and Disease Progression

Relapsing-Remitting MS (RRMS) patients experience relapses with differing frequency and severity. During relapses, patients lose some neurologic functions and present various clinical manifestations, e.g., diplopia, ataxia, paresis, and hypesthesia. The underlying causes of relapses are direct/indirect consequences of CNS-targeted inflammation that finally lead to a significant reduction in the velocity of signal transfer, mainly due to demyelination, in the CNS or neuroaxonal demise.¹¹

Inflammatory demyelination is the main pathologic hallmark of MS. Besides the bystander demyelination, as an epi-phenomenon, that can be observed in neuromyelitis optica (NMO),¹² where an autoimmune attack against AQP-4 and ensuing inflammation result in demyelination, and other inflammatory demyelinating diseases, two other processes can produce this pathological feature in MS. First, an inflammatory attack against myelin components leads to an out-to-in destruction of the myelin sheath. Second, a primary oligodendrocyte degeneration, likely due to a disrupted mitochondrial energy production, may show itself with gradual loss of myelin components of the innermost myelin layers from the most distal processes. Accordingly, it has been suggested that the pathophysiology of MS may be driven through two arms: inflammatory processes and degenerative processes.¹³,¹⁴

Inflammation in MS Pathophysiology

The abundance of inflammatory cells infiltrating MS lesions highlights the role of inflammatory processes in MS. It is long believed that T-cells reactive to myelin components initiate inflammatory events that by incorporating and activating macrophages, resulting in myelin destruction.¹⁵ However, recent findings disclosed the detailed roadmap of T-cell trafficking into the CNS.¹⁶,¹⁷ T-cells need to be activated peripherally to be capable to traverse the blood-brain barrier. Although inflammatory cells of the acquired immune system have to pass several checkpoints, eliminating cells expressing receptors with high engagement to self-antigens in a process known as self-tolerance, auto-reactive B- and T-cells may evade the regulatory gates, in the context of a defective central and peripheral tolerance, and reside in peripheral lymphoid organs.¹⁸ In contrast to the previously-believed concept that suggested MS as an absolutely immune-privileged site, it has been shown that CNS antigens (Ags) may be drained into deep cervical lymph nodes via cribriform plate and nasal lymphatics, and also lymphatic vessels of dura and along the cranial nerves.¹⁷ Then, CNS antigens, e.g., myelin-related antigens, inside the deep cervical lymph nodes, are captured by antigen-presenting cells, which present antigens in the context of MHC class 1 or 2 molecules to CD8-positive and CD4-positive T-cells, respectively.¹⁹ Afterward, CNS-Ag-specific T-cells activate and proliferate within the lymph nodes and concomitantly express a considerable level of adhesion molecules, facilitating cell entrance into the CNS. Both CD4 and CD8 T-cells are believed to be important in MS, however, it is suggested that in contrast to the experimental autoimmune encephalitis (EAE) model that is mostly governed by CD4 T-cells, there is a two-step process in MS.²⁰ CD4 T-cells initiate the immunological processes in MS and help B-cell and CD8 T-cells to be activated, however, CD8 T-cells are predominantly involved in tissue destruction during relapses and continuation of the immune processes in chronic lesions. Indeed, CD8 T-cells outnumber CD4 T-cells in lesions, CSF, and blood of MS patients. The greater effectiveness of drugs depleting CD8 T-cells in addition to CD4 T-cells also supports this view. Although the striking efficacy of immunosuppressive therapies confirms the destructive role of CNS-infiltrating inflammatory cells, their accurate target(s) remains elusive. However, to date, several antigens have been reported as possible targets including myelin components such as MBP, PLP, and MOG, axoglial proteins around the Ranvier’s nodes such as neurofascin and contactin-2, and ion channels such as potassium channel KIR 4.1.²¹

Finally, the myelin-directed attack leads to demyelination that considerably influences the velocity of impulse conduction by corresponding axons. In addition, inflammatory cytokines, especially tumor-necrosis factor-a (TNF-a), may pose a similar effect even before establishment of demyelination.

In addition to T-cells, it was recently understood that B-cells play a critical role in MS pathophysiology. The dramatic effect of B-cell depleting therapy on inflammatory activity of the disease and the presence of B-cells and plasma cells in MS type 2 lesions accentuate their prominent role.¹⁴ B-cells are engaged to MS pathophysiology in several ways: antibody production, and potent antigen-presentation to T-cells in lymphoid organs, producing various cytokines in a polarized fashion to augment Th1-mediated response.²² Now it is believed that the pathogenic actions of B-cells are less related to antibodies, and are dependent more on other functions. In addition, it has very recently been shown that B-cells may aggregate within peripheral lymphoid-like organs or follicle-like structures, which provide a favorable place for B-cells to undergo activation and differentiation and also to present antigens to T-cells.²¹ Such structures are more common in MS patients with a more aggressive clinical course and a greater duration of the disease. These structures are believed to be important in the formation of the cortical lesion, especially through increased release of cytokines and inflammatory mediators.²³ In sum, there is an intricate interplay between B- and T-cells in MS that drives the disease pathophysiology through the acute and chronic phases.

Degeneration in MS Pathophysiology

Inflammation-Induced Degenerative Processes

Inflammation may acutely or chronically lead to axonal transaction and degeneration.¹¹ First of all, inflammatory events in a lesion and the accompanying edema may influence axons and neurons by reducing the blood supply. Secondly, several inflammatory cytokines/mediators and an overwhelming amount of free radicals are produced during inflammation, which directly injure the axons.²⁴ Thirdly, during lesion formation increased expression of excitatory neurotransmitters and receptors leads to an elevated intracellular calcium level, which induces apoptotic pathways and results in neural cytotoxicity.²⁵ Lastly, and most importantly, it has been suggested that a chronic inflammation poses several mutations in genes involved in the respiratory chain of neurons and, therefore, leads to mitochondrial failure and axonal degeneration due to energy insufficiency.²⁶ Inflammation may also bring some pathological alterations that later result in degeneration of axons. In more detail, rather than mention events that may continue and lead to axonal degeneration in chronic stages of a lesion, deprivation of myelin-associated supports and oligodendrocyte-produced trophic factors may further augment axonal degeneration and neural apoptosis.²⁷,²⁸ Neuroaxonal degeneration may take place during the MS course in several ways: anterograde wallerian degeneration of transected axons, retrograde degeneration of affected neurons, and migratory loss of neighbor neurons due to loss of synapse-mediated trophic factor transmission.²⁸ Therefore, a huge amount of degenerated still-myelinated axons are buried in the normal-appearing white matter (NAWM). In keeping, the magnetic resonance spectroscopy (MRS) showed that the level of N-acetylaspartate (NAA), a marker of healthy axons and neurons, is lesser in NAWM of MS patients compared to the normal population.²⁹

Primary Degenerative Processes

Neuropathological examinations of samples obtained from MS patients suggest that in a subpopulation of MS patients, oligodendrocyte apoptosis precedes the inflammatory demyelination.¹³ This type of lesion, named type 3, is common in acute and early MS and its prevalence decreases in parallel to the increase in duration of the illness. Type 3 lesions differ in several aspects from type 1 and 2 lesions, which are typical MS lesions reminiscent of lesions detectable in the EAE model. Type 3 lesions, in contrast to other types, are ill-defined and are not centered by vessels and, in addition, they show a preserved layer of myelin around the vessels.¹⁴ Furthermore, there is a preferential loss of myelin-associated glycoprotein (MAG) compared to other myelin components in type 3 lesions, highlighting a primary dying-back distal oligodendrogliopathy. Several underlying mechanisms for primary oligodendrocyte death in MS were proposed, including viral infections, ischemia-hypoxic conditions, glutamate cytotoxicity, secondary to microglial activation, and/or an elevated level of inflammatory mediators and cytokines.³⁰

Pathophysiology of Remissions

Following a relapse in RRMS patients, clinical disabilities disappear over a period of days to weeks. Various mechanisms are responsible for near-complete remission. First, the cytokine profile shows a switch in immune response from Th1 in relapses to Th2 in remissions, which leads to termination of immune attack against CNS. In addition, resolution of inflammation, at the site of lesion, decreases the level of inflammatory mediators and surrounding edema that, respectively, improve impulse conduction and energy supply of neurons. Secondly, although there is a varied loss of oligodendrocytes during lesion formation, oligodendroglial precursor cells migrate to the lesion site and differentiate to re-sheath demyelinated axons.¹³,¹⁴ It has been shown that infiltrating immune cells promote the remyelination process via secreting certain cytokines. Thirdly, CNS has a strong plastic capability that favors adaptive changes following an insult. Neural plasticity in CNS may take place structurally (dendritic spine and axonal sprouting, new synapse formation) and functionally (gaining the lost function of the adjacent diseased brain area).³¹ In this regard, functional MRI and transcranial magnetic resonance studies suggest a considerable functional brain reorganization in various cortices, as motor, sensory, visual cortex, in MS population.³²

Pathophysiology of Cortical Manifestations

Among cortical symptoms of MS, e.g., epilepsy, aphasia, and hemianopsia, cognitive impairment is more frequent and severe.³³ Owing to the development of new imaging techniques, especially the double-inversion recovery (DIR) MRI, it is now well known that the cortex of MS patients is extremely diseased and from early stages contains multiple lesions. Pathologically, cortical lesions based on location are classified into three groups: leukocortical (affecting adjacent white and gray matter), intracortical (totally located in the cortex), and subpial lesions (extending from subpial cortical layers into deeper layers that may occupy multiple gyrus and sulci).²³ It is believed that cortical lesions do not substantially differ from white matter lesions in terms of formation and temporal evolution. Cognitive impairment cannot be well correlated with white matter lesion load or NAWM pathology. However, it is shown that cortical lesions are clinically relevant and may be responsible for physical and cognitive disabilities in MS even more than white matter lesions.³³

Diagnosis

The diagnosis of MS is largely based on a detailed history, neurologic examination, and supportive paraclinical investigations, especially MRI. The first step in diagnosis is finding clinical manifestations typical or atypical for demyelinating disease of the CNS. Diagnosis of MS without any clinical presentations and only with radiological aspects of the disease is not justified yet. Many other conditions may mimic MS both in clinical and radiological aspects.

Several criteria have been proposed and revised since 50 years ago to diagnose the disease accurately and early on. Although these MS criteria have changed a lot, all of them have been proposed based on one general principle of dissemination of lesions in time and space.³⁴

The previous Poser diagnostic criteria for MS was codified for use in clinical trials of MS and included clinically definite MS, and laboratory (cerebrospinal fluid) supported definite, probable, and possible MS. According to these criteria, for definite diagnosis of MS the clinicians should wait for another episode of exacerbation, despite increase in MRI lesions. As MRI was relatively new at that time, it was included as a paraclinical element to support the diagnosis. These criteria may delay the diagnosis, leaving patients unable to start treatment as early as possible in order to prevent future disability.³⁵ In the McDonald criteria, the diagnosis of MS also requires evidence of lesions disseminated in time and space, but MRI findings have a major impact in this regard. Actually MRI could help to demonstrate dissemination in time and space. The first version of McDonald criteria³⁶ was proposed in 2001 and then revised in 2005 and 2010. The most important reason for revision of diagnostic criteria twice in 10 years was the recent data supporting early treatments. Several studies demonstrated that early treatment of MS at the first demyelinating event CIS may delay the later diagnosis of MS and also prevent future disability³⁷ (Fig. 1.1).

Figure 1.1 Axial views of brain T1-weighted sequence showing demyelinating lesions.

In the original McDonald criteria,³⁶ dissemination in space (DIS) can be demonstrated by the Barkhof MRI criteria which requires 3 out of the following 4 elements:

1. At least one Gd-enhancing lesion or nine T2 hyperintense lesions

2. At least one infratentorial lesion

3. At least one juxtacortical lesion

4. At least three periventricular lesions

In the latest revision of McDonald’s criteria, diagnosis of MS is still based on demonstration of DIS and time (DIT). DIS can be demonstrated by one or more lesions in at least 2 of the 4 areas of CNS, which includes periventricular, juxtacortical, infratentorial, and spinal cord.

It should be noted that symptomatic lesions in the brainstem and spinal cord are excluded from the criteria for DIS. In this revision enhancing lesions do not add a score in fulfilling the criteria for DIS. DIT can be demonstrated by the following two elements:

1. A new T2 and/or gadolinium (GD)enhancing lesion on follow-up MRI, with reference to a baseline scan irrespective of the timing of the baseline MRI.

2. Simultaneous presence of asymptomatic GD-enhancing and nonenhancing lesion at any time.

Primary progressive MS (PPMS) can be diagnosed with one year of prospective or retrospective progression plus 2 of the following 3 criteria:

1. Evidence for DIS in the brain based on at least one T2-weighted MRI lesion in at least 1 area characteristic for MS (periventricular, juxtacortical, or infratentorial).

2. Evidence for DIS in the spinal cord based on at least 2 T2-weighted MRI lesions in the cord.

3. Positive CSF (isoelectric focusing evidence of oligoclonal bands and/or elevated IgG index).

It should be noted that symptomatic lesions of brainstem and spinal cord should be excluded.³⁸

Diagnosis of MS in cases with typical clinical and MRI findings are not so sophisticated, but extreme caution needs to be taken before making a diagnosis of MS in patients with vague or atypical presentations and/or imaging findings.

Many diseases such as vasculitis, Lyme disease, and ischemic infarctions may mimic MS and there should be no better explanation for the condition rather than MS to ascertain the diagnosis. Table 1.1 shows the list of MS mimickers.

Table 1.1

Management

The management of MS is complex and includes the use of immunomodulatory agents to modify the course of the disease (Disease-modifying agents), alleviate the symptoms, and treat acute relapses.

Management of Acute Relapses

The acute attacks are unpredictable and vary regarding severity and site of involvements.

Corticosteroids with their antiinflammatory effects have been typically used to treat acute relapses. Steroids seem to hasten the recovery in most patients, especially during the early stages of the disease.³⁹ In patients who do not respond well to steroids or cannot receive them, plasma exchange is the procedure of choice to accelerate recovery of relapses. Intravenous immunoglobulin (IV Ig) is another option in steroid resistant patients and should be considered in special cases. Certain conditions such as fever, infections, and stress may lead to pseudo-exacerbations, in which the patients may experience a worsening of their manifestations. Following resolution of the underlying stress or infections (especially upper respiratory tracts and urinary tract infections), the symptoms resolve and the patients return to their prior status. It is important to differentiate relapses from such pseudo-exacerbations in order to prevent use of steroids in the latter conditions.⁴⁰

Disease-Modifying Therapies

Immunomodulatory agents have been used for more than two decades to prevent clinical exacerbations and possibly delay disability in MS patients. Different preparations of interferon-beta (IFN) and glatiramer acetate (GA) were the only available options for more than one decade.

During the last 10 years, several potent agents with different mechanisms of action, route, and frequency of administration have been approved by the authorities to be used in relapsing forms of the disease. These immunomodulatory and immunosuppressive therapies are further elaborated in this section.

Interferon-Beta

Four preparations of IFN beta have been approved for RRMS and CIS to prevent new exacerbations and worsening of disability. The mechanism of action is not clear and they possibly act with different mechanisms such as stabilizing the blood-brain barrier, reduction of T-cells entry into the CNS, modulating T- and B-cell functions, and altering the expression of cytokines (decreasing pro-inflammatory cytokine release and increasing production of antiinflammatory ones).⁴¹,⁴² These drugs are generally safe and well-tolerated.

IFN beta 1b (Betaseron) was the first disease-modifying therapy approved for RRMS with a dose of 250 µg every other day after demonstrating its clinical and radiological efficacy in reducing the disease activity. The drug could reduce the number of annual relapses and could show a significant decrease in the number of GD enhancing and new T2 lesions on brain MRI.⁴³,⁴⁴ The drug has also been approved for CIS and there are contradictory reports about its effect on secondary progressive MS (SPMS).⁴⁵ Once weekly IFN beta showed its clinical and radiological efficacy in a large randomized trial conducted by Jacobs et al. and has been approved for RRMS and CIS.⁴⁶,⁴⁷ Considering the weekly injection, the patients experienced fewer injection site reactions and flu-like symptoms.⁴⁷ Subcutaneous IFN beta1a is administered three times a week. Like the previous two preparations, Rebif demonstrated clinical and radiological efficacy in randomized trials and was approved for both RRMS and CIS.⁴⁸ Pegylated IFN beta1a demonstrated its efficacy in a large randomized study for relapsing MS and received its approval in 2014. The drug is injected subcutaneously at a dose of 125 µg every two weeks.⁴⁹ Its safety and efficacy seems to be like other preparations, although there is no class I head-to-head trial comparing their efficacies in a randomized trial.

Flu-like symptoms, injection site reactions, and laboratory abnormalities (elevated liver enzymes or leukopenia) are common side effects of IFN beta. The flu-like symptoms are usually seen in the first months of initiation of the drugs and can be managed with early titration and using paracetamol or NSAID drugs. Injection site reaction needs re-evaluation of injection technique and avoidance of cold administration of the drug. Monitoring of liver enzymes and leukocyte count is advised before and following IFN beta administration with regular intervals. Dose reduction or temporary cessation of taking the drug are recommended in case of mild to moderate increase in liver enzymes. The drug should be permanently stopped if the liver enzymes increase significantly (more than 20 times than baseline) or any signs of jaundice appear.⁵⁰

Besides common adverse events, a review of the literature reveals rare complications to which physicians should pay attention, such as induction or aggravation of other immune-mediated diseases. Immune thrombocytopenia, sarcoidosis, and thyroid diseases are a few examples in this respect.⁵⁰–⁵² In summary, IFN beta are generally safe and well-tolerated in the MS population. Due to their valuable safety, they have been considered as first-line therapies in many consensus recommendations and guidelines.⁵³

Glatiramer Acetate

Glatiramer acetate (GA) or Copolymer 1 is a synthetic complex of four amino acids with variable lengths of between 10 and 100 amino acids, which structurally mimic myelin basic protein (MBP), one of the auto-antigens targeted by inflammatory cells. The exact mechanism of action is not clear but it seems to block the formation of myelin reactive T-cells and induce the production of antiinflammatory cytokine.⁵⁴ The efficacy of GA in terms of reducing relapse rate and MRI activity has been demonstrated in different studies, culminating in its approval for RRMS and CIS.⁵⁵ The drug was formerly subcutaneously administered at a dose of 20 mg per day, until 2014 when 40 mg three times a week was approved.⁵⁵

Injection site reaction is the most common side effect of GA, with reactions ranging from mild erythema, redness, pruritus and lumps, to rare cases of skin necrosis. Immediate postinjection reaction presenting with variable severity and symptoms including chest tightness, palpitation, anxiety, sweating, and flushing may occur in about 15% of patients.⁵⁶,⁵⁷ The episodes are self-limiting, lasting from a few to up to 30 min. No laboratory abnormalities have been reported with this drug in most clinical trials. However, there are case reports of the destructive effects of GA on the liver as severe hepatitis.⁵⁶,⁵⁸–⁶⁰ GA is probably the safest DMD approved for MS treatment and is considered as one the first-line therapies. GA is considered to have the least negative effects on pregnancy among all approved DMDs and is widely used in child-bearing females who have a plan for future pregnancy.

Dimethyl Fumarate

The mechanism of action of DMF involves inhibition of proinflammatory pathways via activation of nuclear factor erythroid 2-related factor 2 (Nrf-2) antioxidant response. The drug is taken orally with a starting dose of 120 mg twice a day followed by 240 mg twice daily after one week.⁶¹ The drug was approved for relapsing MS after demonstrating its efficacy in reducing clinical and imaging activity in placebo-controlled trials.⁶²,⁶³ Flushing, gastrointestinal (GI) problems, elevated liver enzymes, and headache are common side effects of DMF. Patients may be recommended to cease medication following the appearance of sever flushing and GI upset (nausea, diarrhea, abdominal pain) especially in the first months of administration. Adverse effects are mostly mild to moderate and resolve with drug continuation.⁶⁴ Decrease in leukocyte count, especially the number of circulating lymphocytes, is an uncommon but important side effect of DMF. A few cases of progressive multifocal leukoencephalopathy (PML) have been reported in MS patients consuming DMF. All cases had low lymphocyte count, except one patient who had a lymphocyte count of more than 500. Lymphocyte count should be monitored every 3 months and if the count remains below 500 during two consecutive tests, the risk-benefit assay for continuation of therapy should be considered.⁶⁵

Teriflunomide

Teriflunomide inhibits the enzyme dihydro-oratate dehydrogenase leading to an inhibition of proliferation of auto-reactive B- and T-cells. The drug was approved in 2013 based on two phase III clinical trials. In TOWER and TEMSO trials Teriflunomide 14 mg per day reduced the annual relapse rate by 36.3% and 31% respectively, compared to placebo.⁶⁶,⁶⁷

The most common side effects of teriflunomide in clinical trials were elevated liver enzymes, hair thinning, and headache. Polyneuropathy is another complication of teriflunomide which is observed in 1.9% of the 14 mg dose users and is mostly mild to moderate.⁶⁸ Teriflunomid is absolutely contraindicated in pregnancy and should not be used in female patients at child-bearing age without effective contraception. In case of adverse events or unwanted pregnancy the drug can be quickly removed by accelerated elimination procedure using oral charcoal or cholestyramine. During therapy with teriflunomide, liver function tests should be performed every 2 weeks in the first 6 months in Europe, while in USA once per month for the first 6 months. The drug should be stopped if liver enzymes increase to more than three-fold the normal limit.⁶⁵ Overall, Teriflunomide is a well-tolerated and effective first-line therapy for RRMS. Both the 7 and 14 mg of Teriflunomide have been approved in USA while only 14 mg is approved in Europe.

Fingolimod

Fingolimod, a sphingosine 1-phsphte receptor modulator which prevents egress of lymphocytes from the lymph nodes, was the first approved oral DMD for the treatment of RRMS. Fingolimod could significantly reduce annual relapse rate and MRI activity in two phase III trials compared to placebo and IFN beta1a. The drug has been approved as first-line therapy in the US but as second-line in Europe. It is administered as a 0.5 mg capsule taken once a day.⁶⁹ Sphingosin 1 phosphte receptor is not specific for lymphocytes and could be seen in other tissues such as heart, macula, and lung. This may explain some of the adverse events related to fingolimod, including bradycardia and macular edema. The patients should be monitored for bradycardia or any cardiac blocks or arrhythmias following the first dose administration. If there is no symptomatic or severe bradycardia the patient can be discharged after six hours.

In the case of bradycardia with a heart rate of 45 beats per minute, or any symptom related to decreased heart rate, or new change in ECG, heart monitoring should be extended until the symptoms or ECG changes are resolved.⁷⁰,⁷¹ Increased risks of infections, macular edema, and elevated liver enzymes are other adverse events that may be seen during treatment with Fingolimod. Patients who are candidates for receiving this medication should be tested for previous exposure to varicella zoster and if the serum antibody is negative, vaccination should be performed for at least two months before starting the drug. Ophthalmologic examination should be performed both before and 3–4 months following treatment with Fingolimod. Liver function tests and lymphocyte counts should be performed regularly and the drug should be temporarily stopped if lymphocyte count is below 200.

Rare cases of PML have been reported with Fingolimod, especially in cases with previous use of Natalizumab. Clinical and radiological vigilance should be performed for opportunistic infections and development of PML. Blood pressure should be monitored in each visit and appropriate management should be performed in the case of increased blood pressure.⁷²,⁷³ In general, Fingolimod is a potent, well-tolerated drug for relapsing MS with special safety concerns such as macular edema or cardiac problems.

Natalizumab

Natalizumab is a humanized mononclonal antibody directed against the α4β1-integrin molecules and inhibits migration of inflammatory cells across the blood-brain barrier to prevent formation of white matter lesions.⁷⁴ Natalizumab was the first monoclonal antibody designed for treatment of MS and was approved following demonstration of its efficacy in reducing annual relapse rate and MRI activity in two randomized trials. Natalizumab is administered intravenously, with 300 mg at four-week intervals, and is considered as one of the most potent drugs in MS treatment.⁷⁵,⁷⁶ The drug was temporarily suspended from the market because of two cases of PML in MS patients. The drug entered the market again as second-line therapy or for patients with aggressive MS with special clinical and radiological monitoring to depict PML at early stages. The risk of PML development in natalizumab-consuming MS patients will increase with duration of therapy, positive serology for JCV, and prior history of immunosuppression use.⁷⁷ So far, more than 650 cases of PML have been reported in association with natalizumab administration, and the treating physicians should closely monitor high-risk patients with clinical and imaging observations. Other side effects, including infusion-related reactions such as headache, dizziness, and chest discomfort, can be treated with premedication and lessening the infusion rate. If the patient develops hypersensitivity reactions the drug should be stopped permanently. A small subset of patients may produce neutralizing antibodies against natalizumab. If the antibodies are persistently positive, the drug should be discontinued. In summary, natalizumab is a very effective and potent treatment for relapsing MS, yielding a profound reduction of the annual relapse rate and of worsening of permanent symptoms. The risk of PML is the most important limiting factor in the use of natalizumab and close monitoring and re-assessing risk-benefit should be considered, especially in JCV seropositive patients.⁷⁸

Alemtuzumab

Alemtuzumab is a humanized monoclonal antibody against CD52 approved for the management of relapsing forms of MS. Efficacy was shown to be high with a relapse reduction rate of about 50% compared with IFN-1βa in two different studies. The patients received two cycles with an interval of 12 months and its efficacy seems to be maintained for years after delivery of the two cycles. The patients received infusion of 12 mg for 5 days in the first cycle and then 3 infusions of 12 mg after one year.⁷⁹,⁸⁰ Infusion-related reactions have been reported to be the most common adverse effects, occurring in 90%–99% of patients. Fever, fatigue, nausea, headache, rash, and pruritus are among the most common infusion-related reactions that can be reduced by premedication with IV steroids and antihistamines. Development of secondary autoimmune disease is a major concern with alemtuzumab treatment. Autoimmune thyroid diseases have been reported in 34% of patients, idiopathic thrombocytopenia purpura in 2% of cases, and Goodpasture syndrome in 0.3%. An increased risk of infections has also been reported with the drug and it is recommended to treat the patient with oral acyclovir 28 days after alemtuzumab infusion in order to prevent herpes infection.⁸¹ There is no reported case of PML in patients with MS treated with alemtuzumab and it could be considered as a proper option in aggressive cases that are high risk for natalizumab-associated PML.

Daclizumab

Daclizumab is a humanized monoclonal antibody targeting CD25, the α subunit of the IL-2 receptor of T-cells. Daclizumab increases the number of other immune cells, particularly regulatory CD56+ natural killer cells, which have a regulatory role in controlling autoimmune cells and their inflammatory consequences. In a randomized placebo-controlled study of subcutaneous injections of daclizumab of 150 or 300 mg every 4 weeks for 52 weeks, the annualized relapse rate was 54% and 50% lower than the placebo, respectively.⁸²

Serious infections were more common in daclizumab-treated patients compared to placebo. Skin reactions including cutaneous including rash, atopic dermatitis, allergic dermatitis, exfoliative dermatitis, and erythema nodosum have also been reported. Given the risk of liver enzyme elevation, monitoring of liver enzymes on a monthly basis is recommended. Due to safety concerns, the drug was approved for patients who failed to respond to at least two other DMDs.⁸³

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Further Reading

1. Kalman B, Lublin FD. The genetics of multiple sclerosis A review. Biomed Pharmacother. 1999;53(8):358–370.

2. Kingwell E, Marriott JJ, Jette N, et al. Incidence and prevalence of multiple sclerosis in Europe: a systematic review. BMC Neurol. 2013;13:128 https://doi.org/10.1186/1471-2377-13-128.

3. Lublin F. History of modern multiple sclerosis therapy. J Neurol. 2005;252(Suppl 3):iii3–iii9 https://doi.org/10.1007/s00415-005-2010-6.

4. Mechelli R, Annibali V, Ristori G, Vittori D, Coarelli G, Salvetti M. Multiple sclerosis etiology: beyond genes and environment. Expert Rev Clin Immunol. 2010;6(3):481–490 https://doi.org/10.1586/eci.10.11.

Chapter 2

Neutralizing Antibodies and Multiple Sclerosis in the Era of Disease Modifying Treatments

Damiano Paolicelli, Antonio Iaffaldano and Alessia Manni,    University of Bari Aldo Moro, Bari, Italy

Abstract

During an era of rapid changes in Multiple Sclerosis (MS), the early identification of nonresponder patients to therapies is crucial in order to provide them an alternative treatment strategy. The need to identify biomarkers of therapeutic response is a challenge in MS. Biopharmaceutical products approved for MS, such as interferon beta (IFNβ), glatiramer acetate (GA), and natalizumab (NTZ), can lead to the development of antidrug antibodies (ADA), with detrimental effects on their efficacy. In this chapter the evidences of the impact of neutralizing antibodies (NAbs) during IFNβ, GA, and NTZ treatment on clinical and MRI outcomes will be reported. The use of NAbs testing in clinical practice remains a powerful tool in the management of MS therapy.

Keywords

Interferon beta; NAbs; immunogenicity; clinical outcomes; MRI outcomes; multiple sclerosis; ADA; natalizumab; glatiramer acetate

Introduction

Biopharmaceuticals (BPs) are drugs produced by means of biotechnology (usually recombinant techniques), not by extraction of a native compound from a biological source. Some examples are insulin (the first biopharmaceutical drug licensed in human medicine), growth factors, interferons alpha and beta, and monoclonal antibodies such as natalizumab. All BPs, which tend to be large protein-based molecules, are potentially able to induce an immunogenetic response characterized by antidrug antibodies (ADA). Immunogenicity of BPs is due both product-specific and host-specific factors. The products can present species-specific epitopes, form aggregates, contain impurities and contaminants, and be modified by oxidation, deamidation, or glycosylation. Host-specific factors include dose, frequency, and duration of treatment, route of administration, immunocompetence, and immune-tolerance and genetic background¹.

ADA may have clinical effects, but the relationship between ADA and clinical effects is not always direct, and possible scenarios range from no demonstrable effects to severe adverse events (SAE). Potential effects of ADA include alteration in the pharmacokinetics,² severe hypersensitivity reactions,³ anaphylactic reactions,⁴ mistargeting of the drug to receptors therefore blocking the desired biological effect,⁵ immune complex formation,⁶ induction of autoimmune reactions,⁷ and crossreactive reactions to endogenous protein counterparts.⁸

During the last 20 years, we have experienced a fast evolution in the therapeutic scenario of Multiple Sclerosis (MS). Currently, more than ten drugs have been approved for the treatment of relapsing remitting (RR) MS: interferon beta (IFNβ) -1a (Avonex™, Rebif™), IFNβ-1b (Betaferon™, Extavia™), glatiramer acetate (Copaxone™), mitoxantrone, natalizumab (Tysabri™), fingolimod (Gilenya™), teriflunomide (Aubagio™), dimethylfumarate (Tecfidera™), pegylated IFNβ-1a (Plegridy™), alemtuzumab (Lemtrada™), and daclizumab (Zinbryta™). In this chapter, we focused attention on the old injectable protein-based treatments, such as IFNβ and glatiramer acetate, and on the first monoclonal antibody approved for MS: natalizumab (NTZ).

Interferon Beta

Immunogenicity

There are two forms of IFNβ approved for MS treatment: -1a, produced in Chinese hamster ovary cells and identical to the human form of IFNβ, and -1b, produced in Escherichia coli and consisting of 165 amino acids because it lacks the N-terminal methionine, while cysteine is substituted with serine at position 17. Currently, four different IFNβ formulations are available: -1a intramuscular (IM) once weekly (Avonex™, Biogen Idec), -1a subcutaneous (SC) three times weekly (Rebif™, Merck Serono), -1b SC every other day (Betaferon™, Bayer—also marketed as Extavia™, Novartis) and pegylated IFNβ-1a SC once every two weeks (Plegridy™, Biogen-Idec).

IFN-β is the most commonly prescribed first-line disease-modifying treatment (DMT) for RRMS; it reduces the annualized relapse rate (ARR) and the brain magnetic resonance imaging (MRI) activity, and decreases disease progression.⁹ However, up to 45% of patients may develop antiIFNβ antibodies during the treatment, with a reduction of its efficacy.¹⁰

With respect to their methods of detection, anti-IFNβ antibodies are classified as binding antibodies (BAbs) or neutralizing antibodies (NAbs).¹¹ BAbs are quantified by enzyme-linked immunosorbent assay (ELISA).¹² BAbs may be detected already during first months of therapy, occurring in up to 80% of treated patients.¹³ BAbs do not necessarily inhibit IFNβ biological activity; however, in the long term, a subset may transform into NAbs.¹⁴ A minority of patients present BAb-negative tests yet have neutralizing activity due not to antibodies but to soluble IFNAR receptors.¹⁵

NAbs are considered a subgroup of BAbs that prevent the binding between IFNβ and its receptor IFNAR, abolishing its biological activity.¹⁶ Longitudinal studies on the development of NAbs suggest that they may develop from 4 to 6 months after beginning the therapy and generally within the first 2 years.¹⁷ In most studies, NAbs development has been shown to be independent of sex, age, and duration of the disease at the beginning of IFNβ.¹⁸ Nevertheless, a recent European cohort analysis conducted on 5638 IFNβ-treated MS patients reported as first time differences in anti-IFNβ ADA occurrence by sex and age. In particular, patients older than 50 years at start of therapy developed ADA more frequently than adult patients younger than 30, and men developed ADA more frequently than women.¹⁹

Immunogenicity of IFNβ is due to drug-related, treatment-related or patient-related factors.²⁰ Primary protein structure, absence of glycosylation, posttranslational modifications, and the presence of aggregates have been identified among the drug-related factors that affect immunogenicity the most. IFNβ-1b preparations show a greater immunogenic potential than IFN-β 1a preparations, due to the absence of glycosylation, amino-acid substitution, and the consequent development of aggregates.²¹ Regarding treatment-related factors, the SC route seems more immunogenic than the IM route. A high frequency of injections also seems to increase the probability of developing NAbs, whereas evidence regarding its dosage is unclear.¹⁰

Among patient-related factors, the general immune status of the patient and the genetic profile are most likely determinants; in particular, it has been suggested that HLADRB1*0401 and HLA-DRB1*0408 are strongly associated with the development of antibodies.²² Moreover, it has been demonstrated that the oligoclonal band negativity in Cerebrospinal fluid (CSF) is a protective factor to develop IFNβ ADA.²³

IFNβ-1b immunogenicity is characterized by low ADA titers and low ADA persistency due to lack of a memory response.²⁴ Conversely, IFNβ-1a immunogenicity, although less frequent, generates higher titer and persistent ADA responses, often with a neutralizing activity.¹⁴,²⁵

The frequency of NAb positivity reported in the studies varies between products and assays used,²⁶ however, on average, we can assume an immunogenic potential of 2%–6% for IFNβ-1a IM, 12%–28% for IFNβ-1a SC, and 28%–47% for IFNβ-1b SC.²⁷

An increase in the understanding of the factors that lead to the enhanced immunogenicity of IFNβ has led to the reformulation of subcutaneous IFNβ-1a. In the new IFNβ-1a SC formulation, human albumin and fetal calf serum have been removed to prevent the formation of mixed albumin-IFNβ-1a aggregates, while the pH has been adjusted to preserve the molecule's stability in the absence of albumin.²⁸ This new formulation has been shown to be less immunogenic compared to the old one (14.8% and 24%, respectively).²⁹,³⁰

In the last few years Peginterferon-β1a SC, a pegylated form of IFNβ-1a developed by attaching a polyethylene glycol (PEG) chain to the parent IFN molecule, has become available.³¹ This process, known as pegylation, has been shown to increase the half-life and reduce the immunogenicity of certain drugs with either increased or sustained efficacy.³² The phase III ADVANCE trial showed a low rate of peginterferon-β1a immunogenicity over 2 years with less than 1% of patients developing NAbs, 6% developing BAbs and ~7% developing anti-PEG antibodies.³³

Neutralizing Antibody Assays

Quantifying NAbs requires the measurement of the loss of the biological activity of IFNβ. IFNβ uses the same metabolic pathway as natural IFNβ, binding the IFN cell-surface receptor (IFNAR). Formation of the IFNβ-IFNAR complex leads to activation of the JAK/STAT signaling pathway.³⁴ At a nuclear level, this activation leads to a change in the expression levels of many genes, including those which have antiviral, antiproliferative, and immunological properties such as the genes of Myxovirus resistance protein A (MxA),³⁵ β2-microglobulin,³⁶ and neopterin.³⁷ In the presence of NAbs these changes are inhibited.

Four assays are described below which make use of these properties of IFNβ or of the IFNβ-induced expression of specific genes.

The Cytopathic Effect (CPE) Assay

The CPE assay is recommended by the World Health Organization³⁸ for the measurement of NAbs and is considered the gold standard method against which all other methods should be compared.¹⁷ In the assay, a virus-susceptible cell line is treated with patient serum and IFNβ prior to treatment with the virus. Cells that are stimulated by the IFNβ to produce antiviral factors remain viable and are quantified. NAb titers are calculated using the Kawade formula, which expresses titer in 10-fold reduction units (TRU) that represent the ability of sera to induce a 10-fold reduction in IFNβ bioactivity.³⁹ The Kawade method calculates titer (t) using the equation t=f (n−1)/9, where f is the reciprocal of the antibody dilution achieving the endpoint, and n is the IFN concentration measured in that day's titration in laboratory units (LU)/mL. NAb titer has also been expressed as neutralization unit per milliliter (NU/mL), which may be equivalent to