Novel diagnostic tools and solutions for multiple sclerosis treatment: a patent review (2009 - 2014).

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Review

Novel diagnostic tools and solutions for multiple sclerosis treatment: a patent review (2009 -- 2014)

1.

Introduction

2.

Diagnosis of multiple sclerosis

3.

Treatment of MS

Anna Maria Papini† & Enrico K€onig

4.

Miscellaneous patents

†

5.

Expert opinion

Universit a degli Studi di Firenze, Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology (www.peptlab.eu), Dipartimento di Chimica ‘ Ugo Schiff’ , Sesto Fiorentino, Italy

Introduction: With > 2 million people affected by multiple sclerosis (MS) worldwide, the elucidation of its etiopathogenesis is of highest interest. Ongoing research in medicine, molecular biology, chemistry and physics aims to improve the life of MS patients by increasing efficacy and decreasing adverse side effects of presently available drugs. A precise diagnosis of this complex disease, which can take different courses, is fundamental to finding an efficient treatment strategy. Areas covered: We present a summary of diagnostic and therapeutic patents granted between 2009 and 2014. Diagnostic inventions use both genetic and proteomic approaches or measure cerebral venous hemodynamics. Instead, new treatments rely on small molecules and/or the active manipulation of proteins that are involved in the pathogenesis of MS. Expert opinion: There are some promising approaches among recently published patents. In particular, genetic profiling for diagnosis, combination of novel drugs with FDA-approved drugs to reduce side effects, and the personalisation of MS treatments according to a more defined diagnosis are considered as important. In the light of the latest developments, we discuss the complex picture of MS, which we assume to be different events connected by a causal chain consisting of circulatory abnormalities, altered redox processes in CNS immune cells, oligodendropathy, inflammation and finally autoimmunity. Keywords: autoimmunity, circulatory abnormality, inflammation, oligodendropathy, redox processes Expert Opin. Ther. Patents [Early Online]

1.

Introduction

Myelin covers most axons of vertebrate nerve cells in both brain and spinal cord as well as sensory and motor axons of the nervous system to provide a fast signal transduction in a saltatory manner. The latter, as the main function of myelin, is seriously impeded when the lipid rich, insulating myelin sheath is damaged through pathological processes, resulting in the entire disruption of communicating nerve cells and thereby leading to severe life-complicating symptoms. Multiple sclerosis (MS) is such a demyelinating disease and it is accepted to be characterized by inflammation at the onset, particularly causing damage of the white matter in the brain and blood--brain barrier (BBB) disruption. The inflammatory stage possibly plays a role in triggering an autoimmune response since ‘novel’ antigens can become exposed to the circulating immune cells beyond the BBB. It is considered by far the most common human disorder of the CNS, which currently affects > 2 million people globally (as of 2008, see [1]). In most patients, MS will 10.1517/13543776.2015.1043267 © 2015 Informa UK, Ltd. ISSN 1354-3776, e-ISSN 1744-7674 All rights reserved: reproduction in whole or in part not permitted

1

€ nig A. M. Papini & E. Ko

Article highlights. . .

.

.

We present an update on important patents for multiple sclerosis (MS) therapeutics and diagnostics. Further, we discuss this complex neurological disorder trying to explain why some treatments are not sufficient in all patients. We assume that a chain of different events lead to MS, that is circulatory abnormalities -- death of oligodendrocytes -- inflammation -- autoimmunity. However, inflammation itself may potentially cause MS alone.


This box summarizes key points contained in the article.

leave the individuals with severe neurological disabilities that are life-defining [2] for the patients and their families, implicating high financial costs for medical care and treatment, loosing work/unemployment and reorganizing everyday life. Although being first described by Jean-Martin Charcot almost 150 years ago [3], the etiopathogenesis of MS is not yet understood. This being said, an entire cure of MS is still a distant prospect despite the remarkable milestones that have been achieved during decades of intensive studies to understand this complex neurological disorder. The results from the Weizmann Institute of Science prominently contributed to the development of the most used and effective disease-modifying therapies (DMT) approved by the American FDA, in particular, IFN-b1a (REBIF from Merck Serono, Darmstadt, Germany and AVONEX from Biogen Idec, Cambridge, USA) [4], IFN-b1b (BETASERON from Bayer, Leverkusen, Germany) [5] and the polypeptide mixture glatiramer acetate (COPAXONE from Teva Pharmaceutical Industries, Israel) [6]. Only a few months ago, a long-lasting patent dispute between Teva Pharmaceutical and rival companies has been reconciled to the favor of the inventing company, whose patent for the bestselling MS drug Copaxone expires in September 2015 [7]. The finding that the peptide mixture of Copaxone stimulates anti-inflammatory Th2 cells through cytokine secretion [8] can probably be considered as a key event in MS drug discovery. The monoclonal antibody natalizumab (TYSABRI from Biogen Idec, Cambridge, USA) [9] and the cytotoxic/chemotherapeutic agent mitoxantrone (NOVANTRONE from Immunex, now Amgen, Thousand Oaks, USA) [10] complete the list of FDA-approved DMTs. Other commercial drugs available are corticosteroids, methotrexate, cyclophosphamide, azathioprine and immunoglobulins for intravenous injections. Altogether, these agents can be generally grouped into immunomodulatory drugs (IFN-b, glatiramer acetate), immunosuppressive drugs (mitoxantrone, azathioprime, cyclophosphamide) and monoclonal antibodies (natalizumab). It is noteworthy that natalizumab is also used in selective immunosuppressive treatments. However, this treatment remains the only one for MS based on an antibody and approved by the FDA. 2

However, treatment depends on the MS patients’ disease course. Today, four different MS patterns with clinical relevance are recognized: i) the relapsing-remitting MS (RRMS, with 80 -- 85% of cases at onset); ii) primary progressive MS (PPMS, with 10 -- 15% at onset); iii) progressive relapsing MS (PRMS, with 5% at onset); and iv) secondary progressive MS (SPMS) [11-13]. The relapsing forms are characterized by isolated attacks followed up by complete or partial recovery, whereas the progressive course is characterized by a constant increase of neurological disabilities over time. Patients with RRMS may convert to SPMS, in terms of accumulating disabilities without proper recovery, at a relatively constant rate of approximately 2.5% year with a median time span of 19 years after the onset of RRMS [14]. Women are more prone to get MS than men and commonly the disease appears between the age of 15 and 45, with an average age of 29 at the onset. Apparently, populations with Caucasian background have a higher probability to develop MS compared to Asian and African populations, raising the question of a genetic predisposition [2]. Most of the drugs stated above are for treatment of RRMS, whereas no drugs for treating PPMS have been developed yet [15]. However, using MRI together with clinical and para-clinical diagnostic methods allow clinicians to recognize the four different MS subtypes and cases of clinically isolated syndrome, which is a disease form with single episodes of neurological attacks that do not progress into one of the previously stated MS courses. The diagnostic criteria established by the International Panel on Diagnosis of MS, also referred to as the McDonald Criteria [16], have been regularly revised on a 5-year basis [17,18]. Needless to say that a clear defined picture and understanding on how the onset of MS is triggered will help to find more promising ways for a complete cure of patients. Therefore, most therapeutic agents aim to improve patient’s life via DMTs, whose development in the past was based on two different hypotheses of possible etiopathogenic mechanisms that are currently under debate: autoimmunity versus oligodendrogliopathy [19]. Without any doubt, both aspects do contribute to the pathophysiology of MS. Nevertheless, at the cellular level redox changes and reactive oxygen and nitrogen species have been previously underestimated. Their involvement in MS pathogenesis has been recently discussed and an important role in the early events correlated with first clinical symptoms is emerging [20]. The first hypothesis is based on an autoimmune response against the proteins involved in myelin formation, later leading to its degradation. Therefore, many research efforts have been focused on the major myelin proteins, that is, myelin basic protein (MBP); myelin oligodendrocyte glycoprotein (MOG); proteolipid protein that totally make up to 15 -- 30% of the myelin sheath, whereas the glycolipid family of galactocerebrosides are the most abundant myelin lipids (Figure 1) [21]. Demyelination likely is a consequence of inflammation, as a combination of previously reported

Expert Opin. Ther. Patents (2015) ()

Novel diagnostic tools and solutions for multiple sclerosis treatment

B.

A.

MAG Oligodendrocyte MOG


Myelin sheath

Axon

PLP

Node of ranvier

MBP

CNP

C.

Extracellular domain Symbol: Outer leaflet

PLP

MBP

Proteins Inner leaflet PLP MBP PLP

Phospholipid

Cytoplasmic domain Inner leaflet Glycolipid

Outer leaflet

Cholesterol

Extracellular domain

Figure 1. The architecture and molecular composition of myelin in the CNS. Oligodendrocytes form multiple layers of myelin sheath (see cutaway) around the neuronal axon insulating the latter for a fast saltatory signal transduction along the nodes of ranvier that remain unsheated (A). The enlarged box (B) gives a three-dimensional view of the molecular composition of myelin consisting of lipids and 15 -- 30% proteins. An hypothetical arrangement of complex lipids (cholesterol, phospholipids, and glycolipids) and the most abundant proteins (PLP, MBP) in CNS myelin illustrates the distribution and relative constant molar proportion of the three lipid classes cholesterol : phospholipids : galactocerebrosides of 2:2:1 (C). CNP: 2¢3¢-cyclic-nucleotide 3¢-phospodiesterase; MAG: Myelin-associated glycoprotein; MBP: Myelin basic protein; MOG: Myelin oligodendrocyte glycoprotein; PLP: Proteolipid protein. Adapted from [21].


3



observations on CD4+ T lymphocyte activation, impeded regulation on TH1/TH2 lymphocytes and production of autoantibodies (Abs) against myelin proteins by B lymphocytes, as well as inhibition of CD8+ suppressor lymphocytes that can be observed [8,22-25]. Regarding the presence of different Ab families, research on the development of MS biomarkers gives some more insights into both diagnosis and prognosis of RRMS. Briefly, it was demonstrated that post-translationally modified peptides, which represent suspected epitopes incorporated into synthetic antigenic peptide probes, are promising tools to detect auto-Abs in autoimmune diseases [26-31]. Using a ‘chemical reverse approach’ [26,30], it turned out that post-translational modifications (PTMs), and in terms of MS this is an aberrant N-glucosylation in human MOG peptide epitope [32], are strongly correlated with the course/pathogenesis of the disease suggesting to play a role in triggering the autoimmune response. The elevated levels of IgG and/or IgM in sera of a patients population compared to healthy controls can eventually be used as biomarkers [33,34]. In a similar way, citrullination in MBP has been found to be a likewise important PTM contributing to MS pathogenesis [35-39]. The second hypothesis trying to explain MS initiation, suspects a disordered mechanism in myelin producing glia cells, particularly oligodendrocytes, in the CNS causing dysmyelination (note the distinction from demyelination). As a consequence of dysmyelination, macrophages are activated to remove myelin debris. However, the amount of myelin often exceeds the capacity of activated macrophages, which in turn results in residual debris in the lesion. It is noteworthy that both scenarios may not be mutually exclusive as the inflammatory response appearing ‘autoimmune-like’ may be a mere epiphenomenon due to the incomplete macrophagemediated removal of myelin debris. The potential correlation between circulatory abnormalities and an increased level of the vasoactive agents NO and H2O2 altering the natural redox cycle in astrocytes and macrophages that subsequently become the center of oxidative/nitrosative stress might contribute to the formation of the very first lesions [20]. The negative impact of this process is the degeneration of oligodendrocytes and an eventual acceleration of MS disease course (Figure 2) accompanied by subsequent BBB breakdown, inflammation and autoimmunity [20]. Nonetheless, infection and genetic predisposition are also considered to contribute to MS pathogenesis. Most likely, a combination of all the above-mentioned pathophysiological factors plays a role in the process that leads to the clinical picture of MS. Many scientists are still trying to get a better understanding of this neuropathological disorder. As a result a large number of new patents both for diagnosis and treatments of MS patients appeared over the last 5 years. A search on the website of the European patent office (www.worldwide.espacenet. com) for ‘MS AND 2014’ resulted in 300 hits, 86 of which explicitly entitle MS in the head of their claims. In this review, we want to summarize only those new patents concerning 4

1) the diagnosis and 2) treatment of MS that have been published in the period between 2009 and 2014. For convenience and to give a direct overview, these patent claims were subdivided into three different categories according to the approach and concept applied (genetics, and/or proteomics, miscellaneous).

2.

Diagnosis of multiple sclerosis

As stated above, there are many factors that contribute to the pathogenesis of MS, some of which are used to develop diagnostics. All diagnostic patents that will be discussed here are presented in Table 1. One feature that coincides strongly with the disease onset is the obstruction of venous flow at the extracranial level. When Zamboni et al. [40] made the observation to determine the rate of reflux as a function of an increased resistance in the cerebral veins they proposed to use this value as a diagnostic feature for MS patients [41]. However, recent studies demonstrated that a modified venous blood flow is not causally associated with MS [42,43]. In another claim related to blood flow, a procedure to overcome the blockage of internal jugular or azygous veins causing, or at least contributing to the symptoms of MS, was presented where stenosis is being opened by surgery [44].

MS diagnosis based on proteomics A promising discovery appears to be the use of an intracellular actin-binding and calcium-dependent regulatory protein with a key role in macrophage motility [45]. This protein, namely gelsolin, has been found to be implicated in injured tissue and inflammation when depletion of plasma gelsolin is observed [46]. The mechanism behind gelsolin depletion is an increasing level of binding to actin in cells that are exposed following tissue breakdown. Amongst others, gelsolin also binds to inflammatory mediators and to the cell proliferation stimulator lyophosphatidic acid [47]. However, the invention of Stossel et al. [48] is based on the discovery that plasma gelsolin level is reduced in animal models of MS (experimental autoimmune encephalomyelitis) and that reduced levels of this protein precede manifestation of MS. Therefore, gelsolin levels in plasma appear to be a very useful tool for diagnosis and monitoring of MS. Moreover, gelsolin administration can suppress demyelinating attacks and therefore this protein can be a promising novel treatment for MS as well. Cytoskeleton proteins as autoantigens of MS involved in neurodegenerative processes have been investigated by some groups [28,49]. For example, following a proteomic approach, a significant cross-reaction between anti-N-Glc Abs [33,34] and anti-a actinin 1 Abs in MS patients has been reported suggesting that intracellular anti-a actinin 1 gets exposed upon tissue breakdown triggering an autoimmune response [28]. 2.1



(1) Fe2+ + H2O2 (2) (3)

*O2*O2-

Fe3+ + OH- + *OH ONOOH ONOO- + H+

+ NO +

*OH + NO2

*OOH

+H

(4) NO or ONOO- + protein

nitrosylation

Neuron


Myelin

(1–4) Astrocyte

(1,2,4) ODC

oxHDL PARP

–

DNA

NAD+ +

+ ONOO-

+

Caspase-3 ONOO-+

NO

+ COX2

+

iNOS

+

DNA Condensation

NF-κB

+ H2O2

+ *OH or *OOH + Lipid

Lipid peroxidation

HNE MDA

+

NADH/NAD

– NO –

H2O2 +

+ ETC

Mitochondria H2O2 –

CuZnSOD

NO

*O2-

Perivascular space

Macrophage (1)

*O2-

NO Smooth muscle cells

NOX2

TJ

NOX1

eNOS TJ Endothelium

Blood

+

+

TJ

NO

Figure 2. Redox mechanisms that hypothetically contribute to the onset of MS. Through shear stress in the capillary the . production of NO and O2-- is induced in endothelial cells, and subsequently H2O2 is formed by CuZnSOD. Both NO and H2O2 enter astrocytes, where two different redox loops are activated, which result in the activation of key enzymes (e.g., NF-kB, iNOS, COX2) and a feedback-enhanced inhibition of the ETC (inner loop). The second loop is characterized by an increased NADH/NAD+ ratio, PARP activation and DNA damages provoked by ONOO-. NO-activated macrophage/microglia activation release H2O2 and NO, while lipid peroxidation and the production of HNE, MDA and oxHDL are a result of prooxidative conditions. ODC death is provoked by NO, HNE, and H2O2 that leak from astrocytes and macrophages. An increased level of NO provokes caspase-3 inhibition and ONOO- formation in ODC. HNE provokes the activation of NOX2 in macrophages. The box shows side-reactions (1--4) that take place in astrocytes, ODC and macrophages. Symbols represent positive (+) and negative (-) effects on concentration, gene expression or activity. Adapted from [20]. COX2: Cyclooxygenase 2; CuZnSOD: Copper, zinc superoxide dismutase; ETC: Electron transport chain; H2O2: Hydrogen peroxide; HNE: 4-hydroxy-2-nonenal; iNOS: Inducible nitric oxide synthase; MDA: Malondialdehyde; ODC: Oligodendrocytes; oxHDL: Oxidized high-density lipoproteins; PARP: Poly (ADP-ribose) polymerase. Expert Opin. Ther. Patents (2015) ()

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Table 1. Patents for MS diagnosis. Name of agent/molecular classification

Mechanism of action

Patent number

Ref.

Venous flow

Measurement of obstruction of venous flow at the extracranial level Opening of stenosis by surgery Regulator of macrophage motility Gene expression profiling Serological antigen selection combined with cDNA phage display library Single-nucleotide polymorphism Codon signature with mutation

WO2009107152

[41]

WO2012092444 US8440662 EP2069533 EP2579038

[44] [48] [50] [51]

WO2011101466 WO2010011894

[52] [53]

Unblock jugular or azygous veins Gelsolin MS-associated genes Biomarkers


Gene ID INPP4b VH4-34 cDNA: Complementary DNA.

2.2

MS diagnosis based on genetics

The rapidly evolving developments in genetics and genomics offer some application for MS diagnosis. For example, the expression profiling of a panel of 72 MS-associated genes allows the identification of patients with an accuracy of 75%, while only two of these genes are being analyzed [50]. Most of the MS-associated genes encode immune response-related molecules (i.e., cytokines etc. - e.g., ITGAL, CASP9, NFKBIB, STAT3, NFKB1, ITGAM, BCL2, CD4, IL1B, HSPA1A, ICAM1, IL18R1, CD14, IFI16, PI3, TGFBR2 or CD8A). Similarly, a panel of eight biomarkers has been provided from two inventors through the application of a technique called Serological Antigen Selection [51]. The method identifies antigens binding MS-specific auto-Abs present in the cerebrospinal fluid. These antigens were obtained from a cDNA phage display library derived from MS brain plaques and thus represent protein/peptide epitopes involved in the disease. The biomarker panel enables subtyping of MS patients, discriminating them from healthy controls with a specificity of 86% and sensitivity of 45%. A simpler approach, in terms of identifying single mutations, was suggested by analyzing single nucleotide polymorphism in human chromosome 4 encoding the inositol-polyphosphate-4-phosphatase type II (gene ID INPP4b) [52] or sequencing B cells associated with MS expressing VH4-34 to spot codon signatures comprising mutations at codon 31B, 56 and/or 81 [53]. The gene underlying the second invention often causes B cells to be autoreactive towards sugars exposed on blood cells and either undergo negative selection in adults, or class switch to the rare IgD isotype rather than IgG, presumably to dampen the response in healthy individuals [54]. 3.

Treatment of MS

The patents listed in the following propose new potential MS treatment strategies and are presented in Table 2. MS is a multi-factorial disease. Not only genetic predisposition but also environmental factors associated with immune dysregulation can contribute to the disease onset 6

and progression. This is possibly the reason why genetic approaches have not yet provided efficient solutions for MS treatment. The only exception in this regard is an invention that proposes the use of an antisense oligonucleotide indirectly reducing the blood levels of integrin VLA4. VLA4 is also a target of natalizumab therapy, because it plays an important role in leukocyte activation, trafficking and signaling [55]. The effect of reduced protein translation appears to be more specific as the antisense oligonucleotide acts on a single gene. In contrast, small molecules bind protein domains that are often common to a whole protein family. Virtually, all treatments rely on manipulation of biochemical pathways strongly involved in MS pathophysiology, merely targeting key proteins or enzymes responsible for signaling and metabolism. One such suspected protein is MBP, from which several immune-dominant antigenic peptides have been previously identified [8,36-39,56]. Apitope Technology (Bristol, UK) identified a series of MBP antigenic peptides showing a significant capacity to bind either MHC class I or MHC class II molecules on antigen-presenting cell and then presented to T-cell receptor without additional antigen processing [57]. In fact, the name of the company refers to this characteristic (Apitope: Antigen Processing-Independent epiTOPE), while the subject of the claim is the capacity of the ‘apitopes’ to induce tolerance in vivo and an effective treatment of MS using a mix of four of such apitopes. Another invention claims an approach of treating MS from the other side of the immune reaction, namely the receptors of antibody heavy chains (Fc), in particular Fcg receptors. Fcg receptors bind to immunocomplexes (immunoglobulins and antigens). The invention is based on the use of these receptors that are produced via recombinant technologies and with selective binding affinity to the MS-associated antigens that need to be purified from the patients’ plasma [58]. Fundamental to this invention is the assumption that MS is a B-cellmediated disease, a crucial point as we will discuss later in detail. Important for the proposed therapy are defined conditions that need to be fulfilled before a possible Fcg receptors therapy. Briefly, a positive response to treatment with antiCD20 antibody or IGIV should be observed as well as the




Table 2. Patents for MS treatment. Name of agent/molecular classification

Mechanism of action

Patent number

Ref.

VLA4 MBP antigens Fcg receptors Daclizumab in combination with IFN-b Angiopoetin-2 inhibitor H4L4 antibody Daptomycin Muramyl dipeptide Idebenone (2,3-dimethoxy-5-methyl-6(10-hydroxydecyl)-1,4-benzoquinone) Nrf2 (transcription factor)

Antisense oligonucleotide Apitopes, antigen processing independent epitopes Recombinant receptors mAB against IL-2 (CD25) Anti-inflammatory Lipopeptide antibiotic Activation of inflammasomes Regulation of redox chain in ETC

US8759314 EP2211892 EP2331114 EP2425250 WO2009158432 US8664209 WO2010147484 WO2010124713

[55] [57] [58] [60,61] [62] [65] [66] [71]

Neuroprotective character through fine-tuning of antioxidant enzymes Sphingosine 1-phosphate receptor agonist Sphingosine 1-phosphate receptor agonist Inhibition of autoimmune activity Combination with interferon beta Immunosuppression

WO2008097596

[72]

EP2183224 US8741923 WO2014003605 WO2011005907 WO2009042892

[74] [75] [76] [81] [83]

Pyrimidine derivates Oxadiazole fused heterocyclic derivatives ex vivo-expanded CD4+ T reg cells Teriflunomide Cyclosporine A ETC: Electron transport chain.

presence of MS-associated Abs (e.g., anti-MOG, anti-MBP, anti-aquaporin 4) in order to ascertain the proposed therapy to be efficient. As discussed below, anti-aquaporin 4 Abs are characteristic for a different disease form, in particular neuromyelitis optica (NMO). In fact, only in 2004 anti-aquaporin 4 Abs have been demonstrated to be diagnostics of NMO [59] and then introduced as diagnostic criteria in 2006 (see Section 5). However, the Fcg receptors approach appears to be of high diagnostic value with respect to the identification of different MS courses and possibly discriminate other demyelinating diseases. To the best of our knowledge, however, clinical trials for the administration of individually designed Fcg receptors have not been reported yet. In contrast, a novel immunosuppressive agent, a monoclonal antibody against the IL-2 receptor (CD25) named daclizumab [60], was tested in a Phase II clinical trial in combination with IFN-b (CHOICE study) [61] showing a considerable effect in terms of a reduced number of new or enlarged lesions in the white matter of the brain via MRI as well as a reduction in annualized relapse rate. To monitor the response to the daclizumab therapy, they also showed that a change in HLA-DR+CD4+ T-cell counts is a useful biomarker to follow the disease course. An agent that was shown to ameliorate the course of MS using the angiopoetin-2 (ANG-2) inhibitor is the H4L4 antibody [62]. The invention aims to reduce the number of perivascular macrophages that develop from circulating monocytes through ANG-2 treatment, thereby preventing the negative inflammatory conditions that are characteristic for some MS patients. Daptomycin, also known as LY146032, is a cyclic lipopeptide antibiotic obtained from fermentation of Streptomyces roseosporus. It has been found to act against Gram-positive strains responsible for skin or soft tissue infection [63] and the FDA finally approved this agent for treatment in such cases. The preparation of daptomycin has been described in

a separate patent [64]. Although the effect of daptomycin on MS patients had not been reported yet, the inventor reported anecdotally the improvement of a 53-year-old woman with SPMS, who showed no improvement with other treatments. Thereafter, 30 different MS patients with progressive and relapsing forms similarly unresponsive to treatments were tested and 47% of them showed significant positive results [65]. This result even if demonstrated on a limited number of patients may confirm the involvement of infection at the onset of disease. More studies should be performed to investigate the effects of this promising agent. The invention of Gelder and Webster [66] proposes the use of the peptidoglycan constituent muramyl dipeptide (MurNAc-l-Ala-d-isoGln), which is also responsible for a specific protein interaction known for its involvement in Crohn’s disease [67]. Basically, muramyl dipeptide acts like a pathogenassociated antigen and is recognized by the immune system, which leads to the activation of inflammasomes in a direct way rather than through toll-like receptor-mediated pathway, thereby inducing the secretion of anti-inflammatory cytokines (e.g., IL-1a and IL-1b) [68]. Thus, its application in treating MS patients was intended and the inventors claim that treatment of PPMS, but also RR form, with this agent is possible showing no negative side effects. Additionally, the invention embodies muramyl dipeptide to be cross-linked with a microparticle containing DNA fragments thereby simultaneously modulating the immune system in a desired fashion to improve the MS disease course. However, clinical studies still remain to be performed. 4.

Miscellaneous patents

The following series of patents cannot be classified unambiguously. Nevertheless, some of them appear to have a potential application both in diagnosis and treatment of MS.


7



As stated previously, redox processes can have a crucial effect in maintaining immune cells in physiological conditions [20]. Idebenone (2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone) is a small molecule analogue of the antioxidant coenzyme Q10 from the electron transport chain. Due to its important role in regulation of redox equilibrium, it has been applied to treat several diseases associated with iron overload [69]. The property of idebenone to protect the cell membrane from oxidative damage [70] makes it a promising candidate to treat MS patients. A recent patent claim includes a possible way to treat PPMS patients using this quinone derivative [71]. This is of particular importance considering that the course of PPMS cannot be slowed down with the existing therapies proven to be efficient for RRMS [15]. Another key molecule in the regard of redox balance is the nuclear factor E2-related factor 2 (Nrf2), a transcription factor inducing the expression of a set of essential antioxidant enzymes. Upon enzyme activity, the resulting antioxidative compounds seem to be neuroprotective avoiding metabolic and toxic stress of immune cells, particularly in the CNS. Therefore, a patent claims for a set of methods to identify the precise candidate, an enzyme and/or a compound to facilitate the selection of an appropriate drug candidate with neuroprotective properties. In a nutshell, this would allow highly personalized treatment of MS patients through a fine-tuned and controlled regulation of the Nrf2 pathway [72]. Fingolimod (Gilenya form Novartis) represents a small molecule analogue of sphingosine 1-phosphate (S1P), which promotes immunosuppressive activity by modulating leukocyte trafficking, lymphocytes sequestering and other processes important for the immune response. However, treating MS patients with this agent aims to prevent lymphocytes being directed to the CNS, thereby avoiding the undesired autoimmune response. The therapeutic effects of fingolimod have been already tested in a placebo-controlled trial [73], while similar compounds with the same target, the S1P receptor, are subject to novel patent claims. Good agonist candidates, sharing the mimicking property of fingolimod, are for example, pyrimidine derivatives [74] and oxadiazole-fused heterocyclic derivatives [75], which seem to have the potential for a combined treatment with FDA-approved immunomodulating MS drugs. In a different patent claim, the inverse correlation of the number of regulatory T cells (T reg) and the degree of disability during relapse are subject of interest. As a reduced number of T reg cells appears to coincide with progression of the disease, the inventors propose a so-called ‘immune correction therapy’ by the administration of autologous ex vivo-expanded CD4+CD25+Foxp3+CD127low T reg cells [76]. This therapeutic approach aims to reduce or even inhibit the autoimmune activity provoked by autologous cytotoxic T and B cells, thereby ameliorating the symptoms in the relapsing phases of MS. 8

Cladribine [77], which was evaluated in Phase II [78,79] and Phase III clinical studies [80], provokes undesired effects when applied alone. An increased number of infections related to compromised immune function or bone marrow suppression led the inventors to combine cladribine with IFN-b. The latter has also been proposed with the agent teriflunomide [81,82], whereas a combination with any other FDAapproved MS drug was proposed, but not yet investigated, for the immunosuppressant 11-mer peptide cyclosporine A [83]. 5.

Expert opinion

In the last years, combinations of different drugs appeared to be a key focus when newly discovered agents for MS treatment have been presented in patent claims. Many of such new compounds were proposed to be combined with the FDA-approved MS drugs. However, the latter show adverse side effects and often need a prolonged use (up to 6 months daily injections). Flu-like symptoms (IFNs), headaches, dizziness, itching (particularly with natalizumab) or dysfunction in hematopoiesis (corticosteroids) can appear, and immunosuppressive effects are known to lead to an increased susceptibility to infections. Besides, immunomodulatory agents, like altered peptide ligands, seem to exacerbate rather than improve the course of MS [56] and even the production of anti-IFN-Ab has been reported upon treatment [84]. Tysabri (natalizumab) shows the most severe side effects, provoking progressive multifocal leukoencephalopathy [85,86]. Nonetheless, level of efficacy on relapse frequency is higher in patients treated with natalizumab than with copaxone, one of the most used MS drug [2]. However, copaxone is not equally effective in all patients, probably because of the different disease forms of MS. Apparently, the latter observation is due to emerging opinion that MS is a multi-faceted disease, which is now underscored with recent clinical studies. For example, the presence of anti-aquaporin-4 Abs is diagnostic of patients affected by NMO, previously included as a sub-group of MS patients [59]. The ongoing debate on the causal reason for the onset of MS may simply reflect the need for stratification of patients sub-populations suffering from demyelinating processes, although the McDonald criteria seem to be reliable to some extent [16-18]. At this stage, however, it cannot be ruled out to find other obscure and yet uncategorized conditions amongst the four established MS forms that will define them as distinct diseases. Genetic profiling of patients, as introduced in some innovative patents [50-53], may provide some help in this regard. Doubtless, a key factor in terms of MS onset is to understand the ‘true’ nature of the disease. Again, the complex picture and different components that appear to vary between individuals having MS might just reflect various factors that fuse together once the typical symptoms become evident. The nature of the onset per se can be physiological (oligendendropathy), infective/inflammatory [19] or also metabolic




(redox changes) [20]. The detailed insights gained from redox processes may even explain why inflammation occurs, and later contributes to neurodegeneration and autoimmunity triggered by neo-antigens getting exposed upon tissue disruption and breakdown of the BBB [20]. Nevertheless, it is noteworthy that inflammation alone can be a singular reason for the development of MS, although the precise pathogen(s) has never been identified until today. However, if all these different conditions were indeed part of such a causal chain, it could be assumed to place the obstruction of cerebral circulation [20] at the beginning of the pathologic sequence leading to MS. Support for this comes from the work of Zamboni et al. [40], who is among the inventors for a diagnostic patent claim measuring cerebral venous hemodynamics [41]. Although the hypothesis called chronic cerebrospinal venous insufficiency (CCSVI) is not commonly accepted [42,43], Zamboni et al. [87] use it to explain the pathophysiological observations in the inflammatory phase of some MS patients. Again, the argument that several MS sub-populations exist might also help here to explain the opposite opinions regarding CCSVI. Regarding autoimmunity, there is a long list of myelinassociated antigens (proteins, sulfatide, glycolipids), which all have been subject to numerous studies [19,27,32,35-39,88]. Some results suggest that aberrant PTMs, in particular, N-glycosylation in myelin proteins can be responsible for triggering an auto-Ab response. However, no patents claim for citrullinated myelin proteins yet, although this PTM has been described for many years as potentially involved in MS pathophysiology [35-38]. In our experience we did not detect any ACPA (anti-citrullinated peptide antibodies) in MS patients (unpublished results). However, it is now accepted that glycosylation plays a crucial role in cell--cell communication and molecular interactions in general. Interestingly, the intervention proposing the administration of Fcy receptor emphasizes several times that these receptors need to be glycolsylated in certain positions to perform specific antigen binding [58]. More important for the scope

of this review is the fundamental assumption within that patent claim that MS is a B-cell-mediated and/or an autoantibody driven form [58] rather than a T-cell-mediated pathology as it is commonly believed [39,89-92]. This could also be in agreement with the hypothesis that MS is a group of disease forms more than a single disease [93]. In conclusion, we believe that genetic profiling together with refined diagnostic tools will help to identify MS subpopulations. For these different groups, different treatments might be necessary. Once the right treatment strategy is found, patients may get a more personalized therapy (e.g., Fcg receptor therapy [58], regulation of redox processes through fine-tuning of the Nrf2 pathway [72], or ex-vivo expanded T reg cell administration [76]), thereby reducing the adverse side effects and hopefully increasing their quality of life. In contrast, for the purely inflammatory, and probably initial MS course, an immediate therapy with appropriate anti-inflammatory agents may be advisable. For such cases, the administration of daptomycin [65] and muramyl dipeptide [66] ought to be considered for an alternative therapy, although some more studies need to be done to prove efficacy. Preferably at the early stage of inflammation, the demyelinating processes should be treated to prevent the progression into autoimmune phases, because once the immune system is activated against self proteins (or glycolipids etc.), treatment becomes more difficult and is merely limited to immunosuppressive therapies with their undesired side effects.

Declaration of interest Erico K€onig was supported by the Marie Curie Fellowship (PITN-GA-2012-317297). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


9


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Affiliation

Anna Maria Papini†1,2 & Enrico K€onig1,3,4 † Author for correspondence 1 Universita degli Studi di Firenze, Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology (www.peptlab. eu), Dipartimento di Chimica ‘Ugo Schiff’, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy Tel: +39 055 4573561; Fax: +39 055 4574912; E-mail: [email protected] 2 University of Florence, Department of Chemistry ‘Ugo Schiff’, Via della Lastruccia 3-13, I-50019 Sesto Fiorentino, Italy 3 University of Florence, Drug Research and Child Health, Section of Pharmaceutical Sciences and Nutraceutics, Department of Neurosciences, Psychology, Via Ugo Schiff 6, I-50019 Sesto Fiorentino, Italy 4 Toscana Biomarkers SRL, Via Fiorentina 1, I-53100 Siena, Italy

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