WO2018078112A1 - Glp-1 agonist (eg liraglutide) for use in the treatment of multiple sclerosis - Google Patents

Abstract

A method is provided for treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis by providing a GLP-1 receptor agonist. Kits are also provided, which comprise a GLP-1 receptor agonist and one or more further agents.

Description

GLP-1 AGONIST (EG LIRAGLUTIDE) FOR USE IN THE TREATMENT OF

MULTIPLE SCLEROSIS

Technical field

The present invention relates to treatment of multiple sclerosis by modulation of the GLP-1 axis.

Background

Current multiple sclerosis (MS) treatments are non-curative, side-effect prone and expensive, highlighting the need for expanded treatment options for patients. Newly diagnosed MS patients exhibit hyperinsulinemia and decreased insulin sensitivity suggesting that obesity is a potential risk factor for MS. Treating underlying metabolic syndrome with classic anti-diabetic drugs such as metformin and pioglitazone ameliorates metabolic disturbances, reduces MRI-evident lesion frequency and dampens T-cell pro-inflammatory response in MS patients. Metformin also reduces disease severity and pro-inflammatory response in an experimental model of multiple sclerosis. Obese multiple sclerosis patients have been shown to have a less pronounced response to interferon treatment underlining the need for further investigation of metabolic disturbances and pharmacological targets for treating MS through improved metabolic control.

Summary

The present disclosure provides methods of treating multiple sclerosis and/or cognitive impairment associated with multiple sclerosis. The methods are based on

pharmacological modulation of the GLP-1 axis.

In a first aspect, a method is provided for treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis by administering a GLP-1 receptor agonist. In particular, a method is provided, where liraglutide or a functional derivative thereof is administered. The methods may also comprise administration of a further drug, such as another drug, which stimulates the modulation of GLP-1 axis, such as a DPP-IV inhibitor.

In another aspect, a GLP-1 receptor agonist is provided for use in treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis. A kit-of-parts is also provided in a further aspect, which kit-of-parts comprise a GLP-1 receptor agonist, such as liraglutide, and a further agent, such as a DPP-IV inhibitor.

The same kit-of-parts is also provided for use in treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis.

Description of Drawings

Figure 1. EAE-emulsion induces weight loss in all animals. EAE was induced with EAE emulsion at day 0. Animals were randomly selected for vehicle (Veh) and liraglutide (Lira) treatment arms and treated by blinded investigators twice-daily with saline (n=15) or 20C^g/kg of Lira (n=15) (s.c). Healthy animals were treated equally without EAE emulsion (n=Veh:7, Lira:6). (a) Weight of EAE (closed line) and healthy (dotted line) animals for each treatment arm (Veh:black; Lira:blue). Lira treatment induces weight loss at the initial phase of the experiment. All animals receiving EAE- emulsion experienced a weight loss, even in animals with clinical score of 0 at day 1 1. This reflects a full penetrance of the induction. Arrows denote the interval when mean weight loss began for Veh (black) and Lira (blue) and is described as EAE-associated weight loss phase. . Statistics are derived from: normality test (Shapiro-Wilk), thereafter two-way ANOVA and Holm-Sidak multiple comparisons test.

Figure 2. Liraglutide treatment delays clinical presentation and reduces clinical score in EAE. EAE was induced with EAE emulsion at day 0. Animals were randomly selected for vehicle (Veh) and liraglutide (Lira) treatment arms and treated by blinded investigators twice-daily with saline (n=15) or 20C^g/kg of Lira (n=15) (s.c). Healthy animals were treated equally without EAE emulsion (n=Veh:7, Lira:6). Clinical scores were conducted twice-daily (a) and plotted as Veh (black) and Lira (blue) median ± interquartile range. Disease debut: a groupwise clinical score that was significantly higher than 0 is denoted in (a) with arrows for Veh (black) and Lira (blue). Asterisks represent a significant difference in animals with EAE treated with Veh vs. Lira, (b) Median clinical score at termination (via humane endpoint or day 1 1 ) is significantly lower in Lira animals than Veh with EAE. Statistics are derived from: (a, b) non- parametric analysis of clinical scoring. Statistical significance is reported as p<^*0.05, ^**0.01 , ^***0.001 . Figure 3. Liraglutide treatment significantly increases antioxidant capacity and reduces neurodegenerative precursor APP in the EAE brain. Brains were isolated from EAE animals and the brainstem and right hemisphere of the cerebrum were homogenized for immunoblotting. Manganese superoxide dismutase (MnSOD) levels were significantly increased -1 .6- and -2.6-fold of EAE animals treated with Lira in the brainstem and cerebrum, respectively. Levels of the marker of axonal damage, amyloid-precursor protein (APP), were reduced by 30% in the cerebrum of Lira-treated animals. There was no difference in APP levels in the brainstem of Lira-treated rats. APP was significantly higher in the brainstem than in the cerebrum. Values are reported as mean ± SEM of MnSOD, and APP levels relative to housekeeping protein GAPDH and significance was tested with parametric analysis after normality was tested (Shapiro-Wilk) (b-f); (a) data was log-transformed, re-tested for normality and tested with parametric analysis. Statisical significance is reported as p<*0.05, **0.01 , ^•0.001 ; n=6-7.

Figure 4. Liraglutide treatment does not affect astroglial GFAP levels in EAE brain. Brains were isolated from EAE animals and the brainstem and right hemisphere of the cerebrum were homogenized for immunoblotting. Astroglial marker glial fibrillary acidic protein (GFAP) levels (e, f) were significantly higher in the cerebrum than the brainstem but were not affected by liraglutide treatment. Values are reported as mean ± SEM of GFAP levels relative to housekeeping protein GAPDH and significance was tested with parametric analysis after normality was tested (Shapirop-Wilk); n=6-7.

Figure 5. EAE-emulsion induces weight loss in all rats. EAE was induced with EAE emulsion at day 0. Lewis rats were randomly selected for vehicle (Veh) and liraglutide (Lira) treatment arms and treated by blinded investigators twice-daily with saline (n = 14) or 200 μg kg of Lira s.c. from day 0 (n=13) or at first-sign-of-disease (LateLira) (n=12). Healthy animals were treated equally without EAE emulsion (n = Veh: 10, Lira:6). Weight of EAE (closed line) and healthy (dotted line) animals for each treatment arm (Veh: 10, black; Lira: 10, red). Lira treatment induces weight loss at the initial phase of the experiment. All rats receiving EAE-emulsion experienced a weight loss, including rats with clinical score of 0 at day 1 1 . This reflects a 100% penetrance of the EAE induction. Figure 6. Early and Late onset of liraglutide therapy improves clinical score in EAE. EAE was induced with EAE emulsion at day 0. Lewis rats were randomly selected for vehicle (Veh) and liraglutide (Lira) treatment arms and treated by blinded investigators twice-daily with saline (n = 14) or 200 μg kg of Lira s.c. from day 0 (n=13) or at first-sign-of-disease (LateLira) (n=12). Clinical scores were conducted twice-daily and plotted as Veh (black), Lira (red), and LateLira (green) median ± interquartile range. Theta marks the time point when the clinical score for Veh and Lira treatment arms were statistically above 0. LateLira did not statistically rise above 0. Statistics are derived from non-parametric analysis of clinical scoring and Wilcoxon Signed Rank Test for the hypothetical value of zero. Statistical significance is reported as ^*, ^**, ^***p < 0.05, 0.01 , 0.001 relative to Veh.

Figure 7. Liraglutide treatment activates CREB in the brainstem of EAE rats.

Brains were isolated from EAE rats and the brainstem was homogenized for immunoblotting with preservation of phosphorylation state. Brains were probed for

CREB and phosphorylated CREBSer133 and are reported as relative to housekeeping protein GAPDH. Statistics are reported at p=0.016 from Student's t-test after testing for normality (Shapiro-Wilk). Figure 8. Albumin enters the brain during EAE and liraglutide significantly reduces the infiltration of peripheral albumin. Brains were isolated from EAE rats and the brainstem was homogenized for immunoblotting with preservation of phosphorylation state. Brains were probed for albumin and are reported as relative to housekeeping protein GAPDH. Statistics are reported as ^**, ^*** p<0.01 , 0.001 from One-Way ANOVA after testing for normality (Shapiro-Wilk).

Detailed description

In general, methods and agents are provided herein for treating multiple sclerosis and/or cognitive impairment associated with multiple sclerosis based on a

pharmacological modulation of the GLP-1 axis. The activity of the GLP-1 axis can be modulated by provision of GLP-1 receptor agonists.

Thus, in a main aspect, a method is provided of treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis by administering a GLP-1 receptor agonist. GLP-1 receptor agonist

Any agent, which is capable of modulating the GLP-1 axis by inhibiting activation of the GLP-1 receptor, can be applied in the methods, treatments and kits-of-parts provided herein. In particular, any GLP-1 receptor agonist is applicable.

Non-limiting examples of GLP-1 receptor agonist include a natural GLP-1 , a GLP-1 analogue or a GLP-1 derivative. In its broadest sense, the term "natural GLP-1 " refers to a naturally occurring molecule of the glucagon family of peptides or of the family of exendins. The glucagon family of peptides are encoded by the pre-proglucagon gene and encompasses three small peptides with a high degree of homology, i.e. glucagon (1 -29), GLP-1 (1 -37) and GLP-2 (1 -33). The term "natural GLP-1 " also refers to the human GLP-1 (7- 37), the sequence of which is disclosed as SEQ ID NO:1 in WO 2006097537 and included herein by reference, and to the human GLP-1 (7-36)NH2. Exendins are peptides expressed in lizards. Examples of naturally occurring exendins are exendin-3 and exendin-4.

The term "analogue" as used herein referring to a peptide means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. In its broadest sense, the term "GLP-1 analogue" or "analogue of GLP-1 " as used herein refers to an analogue of a natural GLP-1 . It does not include a natural GLP-1 as such. In a particular embodiment, the term "GLP-1 analogue" or "analogue of GLP-1 " as used herein refers to an analogue of human GLP-1 (7-37) or GLP-1 (7-36)NH2. Non-limiting examples of GLP-1 analogues comprise exenatide and taspoglutide. In a particular embodiment, the "GLP-1 analogues" comprise analogues with a maximum of 17 amino acid modifications (i.e. up to 17 amino acids have been modified in total, where the changes can be amino acid substitutions, additions and/or deletions) compared to a natural GLP-1 of reference or, in particular, compared to human GLP-1 - (7- 36)NH2 or GLP-1 (7-37). All amino acids for which the optical isomer is not stated is to be understood to mean the L-isomer. In embodiments of the invention a maximum of 17 amino acids have been modified (substituted, deleted, added or any combination thereof) relative to a natural GLP-1 of reference or, in particular, relative to human GLP-1 -(7-36)NH2 or GLP-1 (7-37). In embodiments of the invention a maximum of 15 amino acids have been modified I. In embodiments of the invention a maximum of 10 amino acids have been modified I. In embodiments of the invention a maximum of 8 amino acids have been modified. In embodiments of the invention a maximum of 7 amino acids have been modified. In embodiments of the invention a maximum of 6 amino acids have been modified. In embodiments of the invention a maximum of 5 amino acids have been modified. In embodiments of the invention a maximum of 4 amino acids have been modified. In embodiments of the invention a maximum of 3 amino acids have been modified. In embodiments of the invention a maximum of 2 amino acids have been modified. In embodiments of the invention 1 amino acid has been modified relative to a natural GLP-1 of reference or, in particular, relative to human GLP-1 -(7-36)NH2 or GLP-1 (7- 37). In a particular embodiment, the amino acid modifications of this paragraph are relative to human GLP-1 (7- 37).

The term "derivative" as used herein in relation to a peptide means a chemically modified peptide or an analogue thereof, wherein at least one substituent has been attached to the unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently modified. The substituent may also be referred to as a "side chain". The peptide to which the substituent(s) is attached may also be referred to as the "parent" peptide. In its broadest sense, the term "GLP-1 derivative" or "derivative of GLP-1 " as used herein refers to a derivative of a parent peptide selected from a natural GLP-1 or an analogue thereof. It does not include a natural GLP-1 as such as defined herein. In particular, the term "GLP-1 derivative" does not include glucagon (1 -29), GLP-1 (1 -37) and GLP-2 (1 -33), the human GLP-1 (7-37)), the human GLP-1 (7- 36)NH2, exendin-3 and exendin-4. In a particular embodiment, the term "GLP-1 derivative" or "derivative of GLP-1 " refers to a derivative of a parent peptide selected from human GLP-1 (7-37) or GLP-1 (7- 36)NH2 or an analogue thereof. In a particular embodiment, the term "GLP-1 derivative" or "derivative of GLP-1 " as used herein refers to a derivative of a parent peptide selected from a GLP-1 analogue, where said analogue comprises a maximum of 17 amino acid modifications compared to a natural GLP-1 of reference or, in particular, compared to human GLP-1 -(7-36)NH2 or GLP-1 (7-37), or, in particular, compared to human GLP-1 (7-37).

Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters, polyethylene glycol (PEG) groups, sialylation groups, glycosylation groups and the like of a parent peptide. In one embodiment, the parent peptide is a GLP-1 analogue as defined above.

In particular embodiments, the side chain has at least 10 carbon atoms, or at least 15, 20, 25, 30, 35, or at least 40 carbon atoms. In further particular embodiments, the side chain may further include at least 5 hetero atoms, in particular O and N, for example at least 7, 9, 10, 12, 15, 17, or at least 20 hetero atoms, such as at least 1 , 2, or 3 N- atoms, and/or at least 3, 6, 9, 12, or 15 O-atoms.

In one embodiment, the term "GLP-1 derivative" refers to acylated GLP-1 parent peptide. In a particular embodiment, the term "GLP-1 derivative" refers to acylated GLP-1 parent peptide where the parent peptide is selected from a GLP-1 analogue comprising a maximum of 17 amino acid modifications compared to a natural GLP-1 of reference or, in particular, compared to human GLP-1 -(7-36)NH2 or GLP-1 (7-37).

The side chain may be covalently attached to a lysine residue of the GLP-1 parent peptide by acylation. Additional or alternative conjugation chemistry includes alkylation, ester formation, or amide formation, or coupling to a cysteine residue, such as by maleimide or haloacetamide (such as bromo-/fluoro-/iodo-) coupling. For the preparation, an active ester of the side chain is covalently linked to an amino group of a lysine residue, preferably the epsilon amino group thereof, under formation of an amide bond (this process being referred to as acylation). Preferred side chains include, for example, fatty acids and fatty diacids. The term fatty acid refers to aliphatic monocarboxylic acids having from 4 to 28 carbon atoms. The fatty acid may be branched or unbranched. The fatty acid is preferably even numbered. The fatty acid may be saturated or unsaturated. The term fatty diacid refers to fatty acids as defined above but with an additional carboxylic acid group in the omega position. Thus, fatty diacids are dicarboxylic acids.

In a particular embodiment, the side chain(s) is a fatty acid having 10 to 20 carbon atoms, and preferably 14 to 20 or 16 to 18 carbon atoms, optionally with a spacer.

Non-limiting examples of GLP-1 derivatives also include:

• NE37-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino) methyl]cyclohexanecarbonyl]amino]butanoyl]

amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ace tyl]- [lmp⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]-GLP-1 -(7-37)-peptide;

• N^E26-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino) butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg³⁴]- GLP-1 -(7-37)-peptide, also called semaglutide;

• N^E26-[(4S)-4-carboxy-4-(hexadecanoylamino)butanoyl]-[Arg³⁴]-GLP-1 -(7-37)- peptide, also called liraglutide;

• N^E26-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4- carboxyphenoxy)decanoylamino] butanoyl]amino]ethoxy]ethoxy]acetyl]amino] ethoxy]ethoxy]acetyl],N^E37-[2-[2-[2-[[2-[2-[2-[[(4S)-4- carboxy-4-[10-(4- carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy] ethoxy]acetyl] amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Arg³⁴,Lys³⁷]-GLP-1 -(7-37)-peptide;

• N^E26-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(3-carboxyphenoxy)

dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]ac etyl],N^E37-[2-[2-[2-[[2-[2-[2-[[(4S)-4- carboxy-4-[12-(3- carboxyphenoxy) dodecanoylamino] butanoyl]amino]ethoxy]ethoxy]acetyl]amino]

ethoxy]ethoxy]acetyl]- [Aib⁸, Arg³⁴, Lys³⁷] - GLP-1 -(7-37)-peptide;

• lixisenatide;

• albiglutide; and

• dulaglutide.

In a particular embodiment, the GLP-1 derivative is liraglutide or is semaglutide. The chemically modified derivatives of natural GLP-1 can be prepared for example as described in patent US 6,451 ,762 or in Knudsen et. al. (2000) J Med Chem 43, 1664- 1669.

Non-limiting examples of divalent metal include zinc (Zn), calcium (Ca), manganese (Mn) or magnesium (Mg). As non-limiting examples, the source of zinc may be zinc chloride, zinc acetate, zinc sulphate or zinc oxide. Amongst these, at least, zinc acetate allows an easy preparation of solutions. The divalent metal stabilizes the composition during storage. It helps minimizing the burst release and associated side effects. Non-limiting examples of polycationic compound include protamine, chitosan, a chitosan derivative, polylysine or polyarginine. As non-limiting examples, protamine can come from protamine chloride, protamine acetate, protamine sulphate. The polycationic compound helps controlling the physical properties of the composition. It also helps improving the sustained release.

The various features of the composition contribute in the optimisation of the

composition properties and advantages.

In one embodiment, the GLP-1 :divalent metal molar ratio in the composition is 1 :>2. Such ratios are associated with a reduction of the burst release and related side effects such as injection site reaction. It also increases the chemical and physical stability of the composition and of the GLP-1 molecule itself. It also helps controlling and increasing the sustained-release of the GLP-1 compound after injection into the body and the associated protraction action.

The GLP-1 receptor agonist is for example a GLP-1 analogue. The GLP-1 receptor agonist applied in the methods, treatments and kits-of-parts provided herein could be selected from the group consisting of liraglutide, exenatid, dulaglutid, lixisenatid, albiglutide, semaglutid and functional derivatives thereof. The drugs are marketed under trade names indicated in table 1 below.

Table 1. Preferred GLP-1 receptor agonists

Trade name compound

Victoza liraglutide

Bydureon exenatid Byetta

Trulicity dulaglutid

Lyxumia lixisenatid

Eperzan/Tanzeum albiglutide

N/A semaglutid

In a preferred embodiment, the GLP-1 receptor agonist is liraglutide, exenatid and/or functional derivatives thereof. In a more preferred embodiment, the GLP-1 receptor agonist is liraglutide or a functional derivative thereof. Liraglutide, also known as NN221 1 , is a long-acting glucagon-like peptide-1 receptor agonist, which is marketed as Victoza for the treatment of type 2 diabetes. Liraglutide is also marketed under the brand name Saxenda as a treatment for adults, who are obese or overweight.

Liraglutide is an injectable drug, and as such, this is also the preferred route of administration according to the present invention. As a long-acting agent, liraglutide can be administered once daily.

The methods, treatments and kits-of-parts provided herein may include provision of a liraglutide derivative. In one such embodiment, the liraglutide derivative is semaglutide.

Treatment

The methods, treatments and kits-of-parts disclosed herein are generally provided for treatment of multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis. One aspect of the present disclosure also relates to a GLP-1 receptor agonist for use in treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal injury.

According to one feature of the invention, the GLP-1 receptor agonist used herein is capable of delaying disease onset and disease progression in an experimental autoimmune encephalitis disease model. The GLP-1 receptor agonist may also be effective in reducing leukocyte invasion into tissues of the CNS and reducing the destruction of the underlying neuronal tissues of the CNS, and thus ameliorating the clinical manifestations of multiple sclerosis. In particular, the GLP-1 receptor agonist could reduce the migration of monocytes and T cells, which are active in presenting myelin antigens and administering cytotoxic effects on the cells producing them.

The present invention is based on observations using a well-accepted animal model for MS, the experimental autoimmune encephalomyelitis (EAE) mouse. EAE is an experimental disease state that shares many clinical and pathological features with MS in humans. Many FDA-approved MS therapies were first discovered and developed based on the EAE models in mice and rats (reviewed by Steinman and Zamvil, 2006). Active immunization of EAE mice with several different protein components of myelin, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin

oligodendrocyte glycoprotein (MOG), induces the production of autoimmune antibodies and the clinical symptoms of ascending paralysis. For example, EAE can be induced in C57BL/6 mice following immunization with MOG peptide amino acids 35-55 (Aharoni, R., et al.) or PLP peptide amino acids 139-151 (PLPp (139-151 )). Immunization with MOG or PLP induces myelin-specific autoimmune reactions, which cause

demyelination and morbidity similar to that of MS. The clinical features of EAE include inflammation and demyelination of the CNS by large numbers of infiltrating

lymphocytes, monocytes and macrophages.

In experiments carried out in support of the present invention, administration of a the GLP-1 receptor agonist, liraglutide, to EAE mice were found to delays disease onset and disease progression in EAE; cf. example 1. Moreover, it was found that liraglutide treatment increases anti-oxidant MnSOD levels and reduces APP.

Multiple sclerosis can be accompanied by cognitive impairments, and in one aspect, the methods, treatments and kits-of-parts disclosed herein are provided for treatment of cognitive impairment in patients with multiple sclerosis. Cognitive changes are a common symptom of MS - approximately half of all people with MS will develop problems with cognition. Cognitive impairments include the ability to learn and remember information, organize, plan and problem-solve, focus, maintain and shift attention, understand and use language, accurately perceive the environment, and perform calculations.

In MS, the following functions are more likely to be affected:

Memory (acquiring, retaining and retrieving new information)

- Attention and concentration (particularly divided attention)

Information processing (dealing with information gathered by the five senses) Executive functions (planning and prioritizing)

Visuospatial functions (visual perception and constructional abilities)

Verbal fluency (word-finding)

Thus, in a preferred embodiment, cognitive impairment in patients with multiple sclerosis includes impairments in memory, attention and concentration, information processing, executive functions, visuospatial functions and/or verbal fluency. In one specific embodiment, such cognitive impairments is memory loss/amnesia and/or depression.

Further drug

The methods and treatments provided herein may in addition to a GLP-1 receptor agonist comprise one or more further drug agents. Such agents are preferably intended to enhance or support the effect of the GLP-1 receptor agonist and/or reduce undesired side-effects. The kits-of-parts disclosed herein below also comprise in addition to the GLP-1 receptor agonist one or more further drug agents. In one embodiment, the further drug agent is a conventional agent for treatment of multiple sclerosis and related disorders. Conventional drugs for the treatment and management of multiple sclerosis include but are not limited to: ABC (i.e., Avonex- Betaseron/Betaferon-Copaxone) treatments (e.g., interferon beta 1 a (AVONEX^®, REBIF^®), interferon beta 1 b (BETASERON^®, BETAFERON^®), and glatiramer acetate (COPAXONE^®); chemotherapeutic agents (e.g., mitoxantrone (NOVANTRON E^®), azathioprine (IMURAN^®), cyclophosphamide (CYTOXAN^®, NEOSAR^®), cyclosporine (SANDIMMUNE^®), methotrexate, and cladribine (LEUSTATIN^®); corticosteroids and adreno-corticotrophic hormone (ACTH) (e.g., methylprednisolone (DEPO-MEDROL^®, SOLU-MEDROL^®), prednisone (DELTASONE^®), prednisolone (DELTA-CORTEF^®), dexamethasone (MEDROL^®, DECADRON^®), adreno-corticotrophic hormone (ACTH^®), and corticotrophin (ACTHAR^®); pain mediation (dysaesthesia) (e.g., carbamazepine (TEGRETOL^®, EPITOL^®, ATRETOL^®, CARBATROL^®), gabapentin (NEURONTIN^®), topiramate (TOP AM AX^®), zonisamide (ZONEGRAN^®), phenytoin (DILANTIN^®), desipramine (NORPRAMIN^®), amitriptyline (ELAVIL^®), imipramine (TOFRANIL^®, IMAVATE^®, JANIMINE^®), doxepin (SINEQUAN^®, ADAPIN^®, TRIADAPIN^®,

ZONALON^®), protriptyline (VIVACTIL^®), cannabis and synthetic cannabinoids

(MARINOL^®), pentoxifylline (TRENTAL^®), ibuprofen (NEUROFEN^®), aspirin, acetaminophen, and hydroxyzine (ATARAX^®); and other treatments (e.g., natalizumab (ANTEGREN^®), alemtuzumab (CAMPATH-1 H^®), 4-aminopyhdine (FAMPRIDINE^®), 3,4 diaminopyridine, eliprodil, IV immunoglobin (GAMMAGARD^®, GAMMAR-IV^®,

GAMIMUNE N^®, IVEEGAM^®, PANGLOBULIN^®, SANDOGLOBULIN^®,

VENOGLOBULIN^®).

In one particular embodiment, the one or more further drug agent is a DPP-IV inhibitor. The further drug is for example selected from the group consisting of sitagliptin, teriflunomid, dimethylfumarat, interferon beta-1 a, glatirameracetat, natalizumab, fingolimod, alemtuzumab, Mitoxantron, Daclizumab and Ocrelizumab. The drugs are marketed under trade names indicated in table 2 below.

Table 2. Preferred DPP-IV inhibitor

Trade name compound

Aubagio: teriflunomid

Tecfidera dimethylfumarat

Rebif interferon beta-1 a

Copaxone glatirameracetat

Tysabri natalizumab

Gilenya fingolimod

Lemtrada alemtuzumab

Mitoxantron Mitoxantron

Zinbryta Daclizumab

Ocrevus Ocrelizumab In another embodiment, the further drug agent is an anti-inflammatory drug. A range of anti-inflammatory drugs are available in the art. Such drugs can be Nonsteroidal Antiinflammatory Drugs (NSAIDs) or Steroidal Anti-inflammatory Drugs (SAIDs). Examples of anti-inflammatory drugs, any of which may be used as a further drug according to the present invention are

aspirin

celecoxib (Celebrex)

diclofenac (Cambia, Cataflam, Voltaren-XR, Zipsor, Zorvolex)

diflunisal (Dolobid - discontinued brand)

etodolac (Lodine - discontinued brand)

ibuprofen (Motrin, Advil)

indomethacin (Indocin)

ketoprofen (Active-Ketoprofen [Orudis - discontinued brand])

ketorolac (Toradol - discontinued brand)

nabumetone (Relafen - discontinued brand)

naproxen (Aleve, Anaprox, Naprelan, Naprosyn)

oxaprozin (Davpro)

piroxicam (Feldene)

salsalate (Disalsate [Amiqesic - discontinued brand])

sulindac (Clinoril - discontinued brand)

tolmetin (Tolectin - discontinued brand)

Adminstration

Administration routed of the GLP-1 receptor agonist employed in the methods, treatments and kits-of-parts depends on the properties and characteristics of the specific agonist. Generally, it is preferred that the GLP-1 receptor agonist is provided by oral, subcutaneous, intravenous or intramuscular administration. The GLP-1 receptor agonist can be provided in specific tools adapted for self-administration, for example in a pen or the agonist can be prepared for pen administration. Thus, in one embodiment, the GLP-1 receptor agonist is administered using a pen.

The same applies to the further drug agent, which may be used together with the GLP- 1 receptor agonist in certain embodiments of the methods and treatments as well as the kits-of-parts provided herein. Generally, clinically effective and tolerable administration forms and dosage regimes are known for the preferred compounds mentioned in table 1 and table 2, above.

The GLP-1 recepto agonist is in one embodiment administered in a daily amount of at east 0.5 mg, such as at least 0.6, such as at least 0.7, such as at least 0.8, such as at least 0 9, such as at least 1 0, such as at least 1 1 , such as at least 1 2, such as at least 1 3, such as at least 1 4, such as at least 1 5, such as at least 1 6, such as at least 1 7, such as at least 1 8, such as at least 1 9, such as at least 2 0, such as at least 2 1 , such as at least 2 2, such as at least 2 3, such as at least 2 4, such as at least 2 5, such as at least 2 6, such as at least 2 7, such as at least 2 8, such as at least 2 9, such as at least 3 0, such as at least 3 1 , such as at least 3 2, such as at least 3 3, such as at least 3 4, such as at least 3 5, such as at least 3 6, such as at least 3 7, such as at least 3 8, such as at least 3 9, such as at least 4 0, such as at least 4 1 , such as at least 4 2, such as at least 4 3, such as at least 4 4, such as at least 4 5, such as at least 4 6, such as at least 4 7, such as at least 4 8, such as at least 4 9, such as at least 5 0, such as at least 5 1 , such as at least 5 2, such as at least 5 3, such as at least 5 4, such as at least 5 5, such as at least 5 6, such as at least 5 7, such as at least 5 8, such as at least 5 9, such as at least 6 0, such as at least 6 1 , such as at least 6 2, such as at least 6 3, such as at least 6 4, such as at least 6 5, such as at least 6 6, such as at least 6 7, such as at least 6 8, such as at least 6 9, such as at least 7 0, such as at least 7 1 , such as at least 7 2, such as at least 7 3, such as at least 7 4, such as at least 7 5, such as at least 7 6, such as at least 7 7, such as at least 7 8, such as at least 7 9, such as at least 8 0, such as at least 8 1 , such as at least 8 2, such as at least 8 3, such as at least 8 4, such as at least 8 5, such as at least 8 6, such as at least 8 7, such as at least 8 8, such as at least 8 9, such as at least 9 0, such as at least 9 0, such as at least 9 2, such as at least 9 3, such as at least 9 4, such as at least 9 5, such as at least 9 6, such as at least 9 7, such as at least 9 8, such as at least 9 9, such as at least 10.0 mg per day.

The GLP-1 receptor agonist is in one embodiment administered in a daily amount of less than 10.0 mg, such as less than 9.9, such as less than 9.8, such as less than 9.7, such as less than 9.6, such as less than 9.5, such as less than 9.4, such as less than 9.3, such as less than 9.2, such as less than 9.1 , such as less than 9.0, such as less than 8.9, such as less than 8.8, such as less than 8.7, such as less than 8.6, such as less than 8.5, such as less than 8.4, such as less than 8.3, such as less than 8.2, such as less than 8.1 , such as less than 8.0, such as less than 7.9, such as less than 7.8, such as less than 7.7, such as less than 7.6, such as less than 7.5, such as less than

7.4, such as less than 7.3, such as less than 7.2, such as less than 7.1 , such as less than 7.0, such as less than 6.9, such as less than 6.8, such as less than 6.7, such as less than 6.6, such as less than 6.5, such as less than 6.4, such as less than 6.3, such as less than 6.2, such as less than 6.1 , such as less than 6.0, such as less than 5.9, such as less than 5.8, such as less than 5.7, such as less than 5.6, such as less than

5.5, such as less than 5.4, such as less than 5.3, such as less than 5.2, such as less than 5.1 , such as less than 5.0, such as less than 4.9, such as less than 4.8, such as less than 4.7, such as less than 4.6, such as less than 4.5, such as less than 4.4, such as less than 4.3, such as less than 4.2, such as less than 4.1 , such as less than 4.0, such as less than 3.9, such as less than 3.8, such as less than 3.7, such as less than

3.6, such as less than 3.5, such as less than 3.4, such as less than 3.3, such as less than 3.2, such as less than 3.1 , such as less than 3.0, such as less than 2.9, such as less than 2.8, such as less than 2.7, such as less than 2.6, such as less than 2.5, such as less than 2.4, such as less than 2.3, such as less than 2.2, such as less than 2.1 , such as less than 2.0 mg.

In one embodiment, the GLP-1 receptor agonist is administered in a daily amount of 0.5-5.0 mg per day. More preferred the GLP-1 receptor agonist is administered in a daily amount of 0.8-4.0 mg, such as 0.8-3.5, such as 0.8-3.3, such as 0.8-3.1 , such as 0.8-3.0, such as 1.0-3.0, such as 1.1 -3.0, such as 1.2-3.0, such as 1.3-3.0, such as 1 .4-3.0, such as 1.5-3.0, such as 1.6-3.0, such as 1.7-3.0, such as 1.8-3.0, such as 1 .9-3.0, such as 2.0-3.0 mg. In a preferred embodiment, the GLP-1 receptor agonist is administered in a daily amount of 1.8-3.0 mg.

The GLP-1 receptor agonist is preferably administered subcutaneously.

In one specific embodiment, the GLP-1 receptor agonist is liraglutide or a functional derivative thereof, and for example, the GLP-1 receptor agonist is liraglutide or a functional derivative thereof and is administered in a daily amount of 0.5-5.0 mg per day. More preferred the GLP-1 receptor agonist is liraglutide or a functional derivative thereof and is administered in a daily amount of 0.8-4.0 mg, such as 0.8-3.5, such as 0.8-3.3, such as 0.8-3.1 , such as 0.8-3.0, such as 1.0-3.0, such as 1.1 -3.0, such as 1 .2-3.0, such as 1.3-3.0, such as 1.4-3.0, such as 1.5-3.0, such as 1.6-3.0, such as 1 .7-3.0, such as 1.8-3.0, such as 1.9-3.0, such as 2.0-3.0 mg. In a preferred embodiment, liraglutide or a functional derivative thereof is administered in a daily amount of 1.8-3.0 mg, preferably by subcutaneous administration.

In one embodiment, the GLP-1 receptor agonist is liraglutide or a functional derivative thereof and is administered in a daily amount of 0.5 mg, or 0.6, or 0.7, or 0.8, or 0.9, or 1 .0, or 1.1 , or 1.2, or 1 .3, or 1 .4, or 1.5, or 1.6, or 1 .7, or 1 .8, or 1.9, or 2.0, or 2.1 , or 2.2, or 2.3, or 2.4, or 2.5, or 2.6, or 2.7, or 2.8, or 2.9, or 3.0, or 3.1 , or 3.2, or 3.3, or 3.4, or 3.5, or 3.6, or 3.7, or 3.8, or 3.9, or 4.0, or 4.1 , or 4.2, or 4.3, or 4.4, or 4.5, or 4.6, or 4.7, or 4.8, or 4.9, or 5.0, or 5.1 , or 5.2, or 5.3, or 5.4, or 5.5, or 5.6, or 5.7, or 5.8, or 5.9, or 6.0, or 6.1 , or 6.2, or 6.3, or 6.4, or 6.5, or 6.6, or 6.7, or 6.8, or 6.9, or 7.0, or 7.1 , or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0, or 8.1 , or 8.2, or 8.3, or 8.4, or 8.5, or 8.6, or 8.7, or 8.8, or 8.9, or 9.0, or 9.0, or 9.2, or 9.3, or 9.4, or 9.5, or 9.6, or 9.7, or 9.8, or 9.9, or 10.0 mg per day. In a preferred embodiment, the GLP-1 receptor agonist is liraglutide or a functional derivative thereof and is administered in a daily amount of 1 .8 mg, preferably by subcutaneous administration.

Kit

One aspect of the present disclosure relates to a kit-of-parts, wherein a GLP-1 receptor agonist is combined with one or more further agents, which confer a synergistic effect on the GLP-1 receptor agonist by enhancing or supporting the effect of the GLP-1 receptor agonist and/or reduce undesired side-effects. Thus, one aspect relates to a kit-of-parts comprising a GLP-1 receptor agonist and one or more further agents. The GLP-1 receptor agonist can be selected from those described herein above, and thus be selected from the group consisting of liraglutide, exenatid, dulaglutid, lixisenatid, albiglutide, semaglutid and functional derivatives thereof. In a preferred embodiment the GLP-1 receptor agonist is liraglutide or a functional derivative thereof as described above.

The further agent is preferably a drug for efficient in treatment of multiple sclerosis, as defined herein above. The further agent is for example a DPP-IV inhibitor, and the agent can be a drug selected from the group consisting of sitagliptin, teriflunomid, dimethylfumarat, interferon beta-1 a, glatirameracetat, natalizumab, fingolimod, alemtuzumab, Mitoxantron, Daclizumab and Ocrelizumab. The kits-of-parts for practicing the methods of the invention may include one or more containers comprising a GLP-1 receptor agonist and one or more further agents as described herein and instructions for use in accordance with any of the methods provided herein. Generally, these instructions comprise a description of how to administer the GLP-1 receptor agonist. The kit may further comprise instructions for identifying when to administer the drug agents and/or measuring the effectiveness of treatment. The instructions generally include information relating to dosage, dosage scheduling (frequency of administration), and route of administration. The instructions supplied in the kits may be written or machine/computer readable as in the form of a data file or spreadsheet. The containers may be unit doses, bulk packages (e.g., multi- dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

The kits may also comprise tools for administering GLP-1 receptor agonists, including pens, syringes, needles, catheters, inhalers, pumps, alcohol swaps, gauze, CNS biopsy apparatus, histological antibodies and stains, etc. In a preferred embodiment, the kit comprises one or more pens for easy end-user administration. The components of the kit are sterilized as needed. Kits may include date stamps, tamper-proof packaging, and radio frequency identification (RFID) tags or other inventory control features. The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a GLP-1 R agonist. The container may further comprise a second pharmaceutically active agent. Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

The kit may comprise one or more compositions, e.g. compositions comprising the GLP-1 receptor agonist and further agents in separate compositions, or comprising the active agents in the same composition. In one embodiment, the composition is an aqueous composition. In one embodiment, the composition is in the form of particles, not yet in suspension. In one embodiment, the composition is in the form of a suspension of particles. In one embodiment, it is a suspension of particles into an aqueous vehicle.

In one embodiment, the pharmaceutical composition of the invention is in the form of a non-aqueous suspension of particles. The non-aqueous medium can be, as a non- limiting example, an oil, such as MCT (medium chain triglyceride). The composition can be in a form that is ready-to-use, for examples where particles are pre-mixed into a suspension, or the composition can be stored in a form that needs to be mixed before use, i.e. in the form of particles only, not yet in suspension.

In another embodiment, the pharmaceutical composition of the invention is in the form of a suspension of particles wherein the particles can be further incorporated into at least one biodegradable polymer, such as PLGA (poly(lactic-co-glycolic acid), the resulting combination being either present as spheres or rods. The spheres, consisting of both the particles and at least one biodegradable polymer, can either be premixed into an oil such as MCT, and thereby ready to use, or separately stored i.e. in the form of spheres only, not yet in suspension. In the latter case, mixing of the spheres and medium has to take place before use. In addition, the obtained spheres can be stored separately from an aqueous medium. In this case, mixing of the spheres and aqueous medium has to take place shortly before use, in order to avoid the biodegradable polymer to degrade prior to dosing.

In further embodiments, the composition may also comprise one or several of the followings: a tonifier or isotonic agent, such as sodium chloride, glycerol, propylene glycol, mannitol, sucrose, trehalose;

a buffer, such as TRIS (tris(hydroxymethyl)aminomethane), HEPES (4-(2- hydroxyethyl)-1 - piperazineethanesulfonic acid), GlyGly etc, possibly with a pH adjusting agent such as hydrochloric acid, sodium hydroxide, acetic acid etc; a preservative agent, such as phenol, m-cresol, benzyl alcohol etc, and mixtures thereof;

an additional stabilizer, such as amino acids, surfactants etc. These additional components, however, are especially appropriate with aqueous compositions.

The kits-of -of-parts disclosed herein are also provided for use in treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis, as described herein above.

Examples

Example 1

Glucagon-like peptide-1 analogue, liraglutide, delays onset of experimental autoimmune encephalitis in Lewis rats

The glucagon-like peptide-1 (GLP-1 ) class of anti-diabetic drugs improves metabolic control and has a neuroprotective potential in humans. This example investigates the effects of the GLP-1 receptor agonist, liraglutide: a long-acting GLP-1 analogue designed to extend the half-life of GLP-1 receptor activation that can cross the blood- brain barrier. In particular, the potential of liraglutide as a candidate multiple sclerosis therapy was investigated by assessing the clinical efficacy in an active, monophasic rat model of experimental autoimmune encephalitis (EAE). This model is characterized by an aggressive onset and we were primarily interested in the effect of liraglutide on the induction phase of experimental autoimmune encephalitis.

Methods

Female Lewis rats (Charles River, Germany) aged 1 1 -12 weeks, weighing -21 Og were housed under standard conditions. Studies were conducted to minimize suffering and were approved by the Danish Animal Inspectorate (2015-15-0201 -00647). Weight was monitored daily throughout the experiment.

EAE Induction

EAE Emulsion: 100 μΙ_ complete Freund's adjuvant (CFA; BD, 2631 10, Denmark (DK)), 200 g M. Tuberculosis H37Ra (MT; BD, 231 141 , DK), 100 μg guinea pig myelin basic protein (MBP; Sigma-Aldrich, DK, M2295), and 100 μΙ_ 0.9% saline). 200 μΙ_ of EAE- emulsion was administered intra-dermally under isoflurane anesthesia at three sites at the base of the tail. Animals were randomized directly thereafter and blindly treated with vehicle (saline, n=15) or liraglutide (200μg kg (n=15) ) s.c. twice-daily. This dose is neuroprotective in mice and clinically relevant to the anti-diabetic effect in humans. Healthy controls were treated similarly without EAE emulsion (vehicle, n=7; and liraglutide, n=6). Clinical Scoring and predefined endpoints

Clinical scoring was performed blinded by two observers twice-daily using the following scale relating to progressive degrees of paralysis: 0- No clinical signs of EAE; 1 - Abolished tail tone; 2- Mild paresis of one or both hind legs; 3- Moderate paresis of one or both hind legs; 4- Severe paresis of one or both hind legs; 5- Paresis of one of both hind legs and incipient paresis of one or both forelegs; 6- Moribund. Animals were deemed terminally ill according to predefined humane endpoints designed in consultation with the Danish Animal Inspectorate: animals registering a clinical score of ≥4, a≥20% loss of initial body weight or when animal caretakers deemed an animal to be moribund before clinical score of 4. The study was designed to terminate on the peak of disease severity to assess the effect of liraglutide on the acute phase (day 1 1 ) before remission. Animals reaching predefined humane endpoints before day 1 1 were terminated (clinical score of≥4 or a≥20% loss of initial body weight).

Immunoblotting

Brains were removed and the right cerebrum and brainstem were isolated and stored at -80°C (vehicle, n=6; liraglutide, n=7) for immunoblotting. In this model, the brainstem shows marked pathological changes in gene expression at day 9 with increased proinflammatory and reduced anti-inflammatory cytokines. Brain tissue was homogenized with protease+phosphatase inhibitors (Roche, complete mini; Phosphosafe; Millpore; DK), protein content quantified, aliquoted and stored at -22°C. 30 μg of protein was run on 12% bis-tris gels in MES buffer, transferred to PVDF membranes and blocked in 5% tris-buffered saline + skim milk powder + 0.05% Tween. Primary antibodies were applied in blocking solution: anti-manganese superoxide dismutase (MnSOD), Millipore 06-984, 1 :1000; anti-amyloid precursor protein (APP), Abeam 32136, UK, 1 :1000; anti- glial fibrillary acidic protein (GFAP), DAKO, IS52430, DK; anti-glyceraldehyde 3- phosphate dehydrogenase (GAPDH), Millipore MAB 374, DK; 1 :10000. Secondary antibodies- anti-rabbit/anti-mouse secondary antibodies (Dako, DK)- were applied 1 :2000 and 1 :3000, respectively and visualized with SuperSignal Femto substrate (Thermo Scientific, Denmark) and CCD camera (Bio-Rad Chemidoc XRS imager, Denmark). Images were quantified with ImageJ and reported relative to housekeeping protein GAPDH.

Data analysis

Clinical scores: Mann-Whitney for individual time points. Cumulative survival: Log-Rank test. Weight: normality (Shapiro-Wilk), thereafter two-way ANOVA and Holm-Sidak multiple comparisons test. Immunoblotting: normality, and Student's t-test for parametric data (APP, GFAP, MnSOD_cerebrum) and log-transformation, normality test and thereafter Student's t-test for non-parametric data (MnSOD_brainstem)- Results

The penetrance of the EAE induction (defined as a clinical score>0 or EAE-induced weight-loss) was 100% for all rats (Figure 1 and 2) as expected. Liraglutide induced weight loss in all animals in the initial days of the study (Fig. 1 ). Weight of healthy liraglutide-treated animals was indistinguishable from healthy-vehicle weight by day 7 (p>0.05).

Liraglutide delays disease onset and disease progression in EAE

EAE-associated weight loss - described as a downward slope in mean weight - began at day 7 in EAE-vehicle rats and day 9 in EAE-liraglutide rats, pre-empting the presence of clinical symptoms (Fig. 1 ). The disease onset (i.e. first median clinical score statistically significantly > 0, Fig. 2a) was delayed by liraglutide treatment: day 8 for vehicle- (p<0.05) and day 10 for liraglutide-treated rats (p<0.05). Moreover, EAE- vehicle rats were significantly more impaired than EAE-liraglutide rats at day 8, 9 (p<0.05), and 10, 1 1 (p<0.0001 ) (Fig 2a). The clinical score at study termination (via humane endpoint or day 1 1 ) was significantly lower for liraglutide-treated animals: median of 2 vs. 5 (p=0.0003, Fig.1 b), where 14 of 15 EAE-vehicle rats achieved the humane endpoint compared to 5 of 15 EAE-liraglutide rats (93% vs. 33%, p=0.0004).

Liraglutide treatment increases anti-oxidant MnSOD levels and reduces APP

Liraglutide increased the mitochondrial anti-oxidant protein MnSOD by -1 .6-

(brainstem, p=0.003) and -2.6-fold (cerebrum, p<0.0001 ) in liraglutide-treated animals relative to the EAE-vehicle group (Figure 3a, b). Liraglutide decreased the

neurodegenerative precursor APP (Fig. 3c, d) in the cerebrum by 30% (p=0.036) relative to EAE-vehicle rats. APP was significantly higher in the brainstem than the cerebrum (p<0.001 ) however, was not affected by treatment (p=0.82). GFAP levels were not changed by liraglutide treatment in the respective brain regions (Fig. 4a, b). GFAP levels were significantly higher in the cerebrum than the brainstem (p<0.001 , Fig. 4). Discussion

This is the first example of the effects of GLP-1 -class agents in an experimental model of multiple sclerosis. Liraglutide reduces clinical debut and severity in this aggressive monophasic model. The antidiabetic drug, metformin, is effective in EAE and in multiple sclerosis patients suggesting that the positive results in this EAE model could be translated into a therapeutic option for multiple sclerosis (cf. Negrotto L, Farez MF, Correale J.

Immunologic Effects of Metformin and Pioglitazone Treatment on Metabolic Syndrome and Multiple Sclerosis. JAMA neurology (2016). doi: 10.1001/jamaneurol.2015.4807. PubMed PMID: 26953870 and Nath N, Khan M, Paintlia MK, Singh I, Hoda MN, Giri S. Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis. Journal of immunology (2009) 182(12):8005-14. doi: 10.4049/jimmunol.0803563. PubMed PMID: 19494326; PubMed Central PMCID: PMC2965405).

Liraglutide treatment is known to elicit different metabolic effects, which could contribute to the protective effect of liraglutide in multiple sclerosis, however, liraglutide may also activate neuroprotective pathways such as the MnSOD-regulating CREB pathway. Indeed, the increased MnSOD levels (Fig. 2) observed in this study supports an improved mitochondrial antioxidant capacity that may play a role in buffering oxidative stress. Previous work in this model has shown that gene expression of the MnSOD-encoding gene, Sod-2, is increased in the brainstem in Lewis rats with EAE (cf. Pedersen DS, Tran TP, Smidt K, Bibby BM, Rungby J, Larsen A. Metallic gold beads in hyaluronic acid: a novel form of gold-based immunosuppression?

Investigations of the immunosuppressive effects of metallic gold on cultured J774 macrophages and on neuronal gene expression in experimental autoimmune encephalomyelitis. Biometals (2013) 26(3):369-85. doi: 10.1007/s 10534-013-9616-4. PubMed PMID: 23653168). The present data suggests that this increase is potentiated with liraglutide treatment.

The pathophysiological role of APP in MS is complex but APP is a known marker of cerebral lesions and a biomarker of disease progression and axonal damage in MS and EAE. Recent genomic work suggests that APP may primarily be a marker of early neuronal stress in EAE. Thus, the reduction in APP observed in the cerebrum (where frank pathology is diffuse) may reflect a protective mechanism engaged by liraglutide treatment as the neuropathology develops and/or a reduction in peripheral and central nervous system inflammation in liraglutide-treated animals. Increased MnSOD levels may play a role in controlling buffering these stress signals. Liraglutide did not however affect APP in the brainstem, where - as expected based on the caudal-rostral nature of this model - the APP levels were significantly higher than the cerebrum. This likely reflects increased neuronal stress in the brainstem. This is supported by previous work in the brainstem of this model describing increased gene expression of proinflammatory cytokines, reduced anti-inflammatory cytokines yet no signs of cell death signaling.

Liraglutide treatment did not change GFAP expression in the brain although there tended to be an increase in the brainstem of treated animals. Astroglial up-regulation of GLP-1 receptors may be an important mechanism of action for GLP-1 agonism in the brain as GLP-1 receptors are up-regulated in reactive astroglia after cortical lesion and protect astrocytes in culture.

In summary, the present example demonstrates a strong positive effect of liraglutide, which is approved for human use and has a well-described safety profile and good tolerance, apart from initial gastrointestinal side effects. It is shown for the first time that liraglutide delays clinical disease progression in EAE. Moreover, this is associated with improved antioxidant capacity and reduced neuronal damage. Taken together with the promising results in Alzheimer's patients, and the prevalence of metabolic disturbances in patients with MS, these data strongly support GLP-1 -based therapy as a future contributor to the MS treatment paradigm and specifically confirms the potential of liraglutide in such a treatment strategy.

Example 2

In this second set of experiments we designed a study to further expand upon and support the high-powered study from our first experiment shown in Example 1 regarding liraglutide as a long-term treatment and moreover, add a treatment group that is initiated at the first sign of disease. This second group represents a clinical scenario where treatment is initiated during an MS attack. Furthermore, based on our previous findings establishing CREB as an important pathway for GLP-1 -based neuroprotection, we believed that the CREB pathway was activated in the brain of EAE rats treated with liraglutide. The CREB pathway is intimately involved in improving the protective protein milieu of neurons, improving mitochondrial function, promoting neuronal proliferation important for hippocampal function, and importantly, cognitive improvement. Increased CREB activation may therefore, protect against both short- term and long-term damage such as lesions and neurodegeneration and also support cognitive function.

Patho-physiologically, EAE and MS are autoimmune diseases, in which an important disease initiating feature is the process in which autoreactive cells of the immune system infiltrate the brain causing lesions and damage. We wished to evaluate whether liraglutide treatment would reduce infiltration of the peripheral blood system into the brain, suggesting that the blood-brain barrier (BBB) is more intact with liraglutide treatment. We therefore assessed the albumin content of the brain with vehicle and liraglutide treatment to determine the leakage of periphery into the brain proper. Methods

Female Lewis rats (Charles River, Germany), weighing -218 g, were housed under standard conditions. Studies were conducted to minimize suffering and were approved by the Danish Animal Inspectorate (2015-15-0201-00647). Weight was monitored daily throughout the experiment. EAE Induction

EAE Emulsion: 100 μΙ_ complete Freund's adjuvant (CFA; BD 263810, Denmark (DK)), 200 Mycobacterium tuberculosis H37Ra (MT; BD, 231 141 , DK), 100 guinea pig myelin basic protein (MBP; Sigma-Aldrich, DK, M2295), and 100 μΙ_ 0.9% saline.

EAE-emulsion was administered intra-dermally under isoflurane anesthesia at three sites at the base of the tail, totalling two hundred microliters in volume. Animals were randomized directly thereafter and blindly treated with vehicle (saline, n = 14) or liraglutide (200 μg kg; n = 13) s.c. twice-daily similar to our previous experiment. This dose is neuroprotective in mice and clinically relevant to the anti-diabetic effect in humans. Furthermore, a third group was added where treatment of liraglutide (twice- daily, 200 μg kg s.c.) was initiated at the first sign of disease (n =12). This was defined as the first day of weight loss. Healthy controls were treated similarly without EAE emulsion (vehicle, n = 10; and liraglutide, n = 10). Clinical Scoring and predefined study endpoints

Clinical scoring was performed blinded by two observers twice-daily using the following scale relating to progressive degrees of paralysis: 0, No clinical signs of EAE; 1 , Abolished tail tone; 2, Mild paresis of one or both hind legs; 3, Moderate paresis of one or both hind legs; 4, Severe paresis of one or both hind legs; 5, Paresis of one of both hind legs and incipient paresis of one or both forelegs; 6, Moribund. Animals were deemed terminally ill according to predefined humane endpoints designed in consultation with the Danish Animal Inspectorate: animals registering a clinical score of ≥4, a≥20% loss of initial body weight or when animal caretakers deemed an animal to be moribund before clinical score of 4.

The study design was to follow disease until 'peak disease severity' to assess the effect of liraglutide treatment and late-liraglutide treatment (LateLira) on the acute phase of EAE, before remission. From our first study, we expected that this time point would be day 1 1 post-induction of EAE. Animals reaching predefined humane endpoints before day 1 1 were terminated (clinical score of≥4 or a≥20% loss of initial body weight).

Tissue processing

At day 1 1 (or the first time a rat reached a clinical score of 4 is such disease severity occurred before day 1 1 ), rats were anesthisized with hypnorm/dormicum relative to body weight, blood was extracted into citrate anticoagulant from cardiac puncture and subsequently, perfused transcardially with heparinized saline. Tissues were extracted thereafter and preserved in either RNALater, formaldehyde or flash-frozen in liquid nitrogen. Whole blood was spun at 4°C, plasma extracted and flash-frozen in liquid nitrogen.

Immunoblotting

Flash-frozen brainstem region was homogenized with protease + phosphatase inhibitors, protein content quantified, aliquoted and stored at -22°C. Thirty micrograms of protein was run on 12% bis-tris gels in MES buffer, transferred to PVDF membranes and blocked in 5% tris-buffered saline + skim milk powder + 0.05% Tween. Primary antibodies were applied in blocking solution: anti-albumin (1 :1000, DAKO, A0001 ); anti- CREB (1 :1000, Cell Signaling, 9197); anti-pCREB (1 :1000, Millipore, 06-519; blocking solution was 5% tris-buffered saline + bovine albumin serum + 0.05% Tween); and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 :10000, Millipore MAB 374). Secondary antibodies- anti-rabbit/anti-mouse secondary antibodies (Dako, DK) were applied 1 :2000 and 1 :3000, respectively, and visualized with SuperSignal Femto substrate (Thermo Scientific, Denmark) and CCD camera (Bio-Rad Chemidoc XRS imager, Denmark). Images were quantified with ImageJ and reported relative to housekeeping protein GAPDH.

Data Analysis

Clinical scores: Mann-Whitney for individual time points and Wilcoxon Signed Rank Test for determining when clinical scores were significantly above zero. Weight:

normality (Shapiro-Wilk), thereafter two-way ANOVA and Holm-Sidak multiple comparisons test. Immunoblotting: normality, and Student's i-test or One-Way ANOVA. Level of statistical significance was set at 0.05.

Immunoprecipitation

Preclearing

A mixture of antibody buffer containing 1 % BSA, 0.1 % 2M NaN3 in PBS-T and A/G magnetic beads (Pierce protein, Thermo-Scientific) was prepared. The mixture was put into a magnetic rack, where the liquid was discarded and the magnetic beads were saved. Antibody buffer was added to the magnetic beads mixture and vortexed. Magnetic beads mixture was added to the brain homogenate obtained from cerebellum of a three week old Sprague-Dawley rat and incubated on ice for one hour. The solution of magnetic beads mixture and brain homogenate was put into a magnetic rack. The beads were saved and washed three times in antibody buffer and

subsequently stored at 4 °C. The liquid was saved and magnetic beads mixture was added and incubated on ice for 30 minutes. This step was repeated three times and antibody buffer was added to the liquid

Immunoprecipitation

Serum from healthy rats and treated rats receiving either placebo, early or late liraglutide treatment, were applied for immunoprecipitation. The precleared brain homogenate along with serum from the EAE animals was mixed and incubated on ice for one hour. The magnetic beads mixture was added to each of the samples and incubated on ice for one hour. After incubation the samples were placed in a magnet rack to isolate the beads which were washed consecutively three times in antibody buffer followed by elution incubation in 120 mM Sodium deoxycholate (SDC) in 50 mM triethylammonium bicarbonate (TEAB) (pH 8.5) for 10 min at 95°C.

Proteomics Sample Preparation

The proteomic sample preparation was performed according to the FASP digestion protocol by Bennike et al. regarding sample preparation with ethyl acetate phase inversion to facilitate surfactant removal 80. The elute was transferred to individual YM- 10 kDa spin filters for digestion (Millipore, Billerica, MA, USA) and centrifuged at 14,000 g for 15 min at room temperature, which apply to all future centrifugation steps performed. Protein disulfide bonds were reduced with 12 mM tris(2- carboxyethyl)phosphine (Thermo Scientific, Waltham, MA, USA) and alkylated with 50 mM chloroacetamide (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C followed by centrifugation. The reducing and alkylating agents were dissolved in 120 mM SDC, 50 mM triethylammonium bicarbonate (TEAB) (pH 8.5). In preparation for digestion, 10mM CaCI and 50 mM TEAB buffer was added to the spin filter and centrifuged. A 1 :25 (w/w) Chymotrypsin:protein ratio dissolved in digestion buffer was added to the spin filter, and the samples were digested overnight at 37°C. The flow-through containing the peptides was retrieved by addition of 50 mM TEAB buffer and centrifugation. To facilitate SDC removal, a phase separation was performed with 3:1 (v/v) ethyl acetate:sample and acidified by addition of Trifluoroacetic acid (TFA) to a final concentration of 0.5%. Total phase separation was achieved by 2 min agitation followed by centrifugation. The aqueous phase was collected and vacuum centrifuged overnight and the dry peptide product was stored at -80 °C until time of analysis. Mass Spectrometry Analysis

The peptides were resuspended in 2% acetonitrile (ACN), 0.1 % formic acid (FA) 0.1 % TFA, whereafter they were briefly sonicated. Five μg total peptide material was analyzed per liquid chromatography-mass spectrometry analysis. The samples were analyzed using a UPLC-nanoESI MS/MS setup with a NanoRSLC system (Dionex, Sunnyvale, CA, USA). The system was coupled online with an emitter for nanospray ionization (New objective picotip 360-20-10) to a Q Exactive Plus mass spectrometer (Thermo Scientific, Waltham, USA). The peptide material was loaded onto a 2 cm trapping reversed phase Acclaim PepMap RSLC C18 column (Dionex), and separated using an analytical 75 cm reversed phase Acclaim PepMap RSLC C18 column (Dionex). Both columns were kept at 60 °C. The sample was eluted with a gradient of 96% solvent A (0.1 % FA 0.1 % TFA) and 4% solvent B (0.1 % FA 0.1 % TFA in ACN), which was increased to 10% solvent B on a 1 min. ramp gradient at a constant flow rate of 300 nL/min. Subsequently, the gradient was raised to 30% solvent B on a 45 min ramp gradient. The mass spectrometer was operated in positive mode selecting up to 20 precursor precursor ions with a mass window of m/z 1 .6 based on highest intensity for HCD fragmenting at normalized collision energy of 27. Selected precursors were dynamically excluded for fragmentation for 30 sec.

Protein Identification and Quantitation Data Analysis

A label-free relative quantitation analysis was performed in MaxQuant 1.5.7.4. The raw files were searched against the Rattus norvegicus Uniprot database UP000002494 81 , 82. All standard settings were employed with carbamidomethyl (C) as a static peptide modification, and deamidation (NQ), oxidation (M), formylation (N-terminal and K), and protein acetylation (N-terminal) as variable modifications. The output contains a list of proteins identified below 1 % false discovery rate and their abundances was further filtered and processed in Perseus v1 .5.8.5. All reverse hits identified proteins were removed from further analysis and the data was log2-transformed in order to approximate normal distribution. Two unique peptides or more was a requirement for protein quantitation. Additionally, a non-zero quantitation value in at least 50% the samples in one of the groups was required for the quantifiable proteins. Applied Statistics

The identified quantifiable proteins in the different groups were investigated for differences in the expression of the proteins between the groups by unpaired two-tailed t-tests were performed. P values of 0.05 were used for truncation and only proteins found in at least 50% the samples in one of the groups was considered for the t-test.

Results Development of EAE

Clinical symptoms began to appear at day 9 in EAE-Veh (4 of 14) and EAE-Lira (2 of 13) and at day 10 in EAE-LateLira (1 of 12). Clinical scoring was determined to be statistically above zero at day 10 and 1 1 in EAE-Veh and EAE-Lira; EAE-LateLira remained statistically not above zero at Day 1 1 (p=0.06). EAE-Veh rats had

significantly higher clinical scores from EAE-LateLira from all days where clinical scores were present (day 9, 10 and 1 1 ) and significantly higher than EAE-Lira on day 10, and 1 1 . First-sign-of-disease treatment with liraglutide (EAE-LateLira) improved the clinical score at day 10 and 1 1 from liraglutide treatment being at the start of the study (EAE-Lira). This represents both an improvement on the treatment regime, clinically, and a more realistic (and thus potentially translatable) scenario for a patient in a clinical setting.

Increased CREB activation and reduced BBB leakage in rats receiving short-term liraglutide

In our first set of experiments, we showed that liraglutide increases antioxidant MnSOD and reduces neurodegenerative protein APP in the brain tissue proper. In the second set of experiments we show that liraglutide treatment significantly activates the CREB system in the brainstem of EAE-LateLira rats. 'Significant activation' is presented as an ~2-fold increase in phosphorylated CREB relative to unphosphorylated CREB (i.e. pCREB/CREB). Furthermore, as expected, the BBB is compromised in the brainstem of EAE-Veh rats when compared to healthy controls as shown by a marked ~3-fold increase in albumin content in the perfused brainstem. Moreover, EAE-LateLira rats have significantly less albumin leakage into the brain (p<0.01 ). Treatment effect on auto-antibodies A multitude of mechanisms are believed to be involved in MS pathophysiology. One possibility is that an autoimmune response is initiated when T cells become activated outside the CNS through molecular mimicry or by-stander activation then transcending to the central nervous system alongside activated B cells and

monocytes/macrophages. As potential marker of disease and target of treatment the involvement of B cells and autoantibodies in MS has been indicated as the humoral immune defence could likely play a central role in the pathogenesis of MS. In fact, a correlation has been reported between the degree of disease progression and the intrathecal antibody. This corresponds with the hypothesis that autoantibodies stimulated by altered peptide ligand response and epitope spreading could induce a loss of tolerance for self-proteins. The involvement of autoantibodies in the

pathogenesis of MS is thus suggested to participate or perhaps initiate immune activity. Supporting a role of the B cell compartment in particular in MS pathology, recent drug developments like Ocrelizumab, reduce the presence of CD20-expressing plasma B cells, hereby ameliorating the disease cause in primary progressive MS. Targeting the autoantigen responds may be a potential target for treatment and/or indicative of the underlying processes affected by specific treatment modalities.

To this end, the effects of glp-1 (liraglutide) treatment on MS-related autoantibodies were investigated in the Lewis rat model (EAE) of MS

In short, the blood-borne autoantibodies against neuronally degenerated

autoantibodies were investigated following immunoprecipitation with brain tissue. A Mass spectrometry approach was used for characterizing the auto-antibodies detected. Antibodies

Investigating autoantibodies statistically significantly up-regulated with EAE disease we found that such upregulation was statistically significantly reduced increase upon treatment (early or late) with regards to antibodies directed at:

• Myelin proteolipid protein

· Q4KLZ0 protein ( encoded by the Vnn1 gene)

Investigating autoantibodies significantly down-regulated with EAE disease we found that a treatment modulated this response i.e. a statistically significantly increased level of auto-antibodies (compared to EAE disease) upon treatment (early and late) with regards to antibodies directed at the following proteins: • Hemoglobin subunit alpha-1/2

• Ig kappa chain C region, B allele

• Actin, cytoplasmic 1 ;Actin, cytoplasmic 1 , N-terminally processed Blood samples from EAE versus control also revealed a statiscally significantly increased amount of auto-antibodies towards

• Glutathione peroxidase 3

• Olfactory receptor These findings of increased levels of auto-antibody presences were statically significantly reduced with early liraglutide treatment with a similar trend in the late liraglutide treated animals

Trending towards an increase in the EAE group were the auto-antibodies towards

• Fructose-bisphosphate aldolase C

· Actin

Indicating a treatment response, the level of Fructose-bisphosphate aldolase C autoantibodies was significantly lower in the early treated-liraglutide treated group compared to the EAE group with a similar trend in the late-treated liraglutide group. Trending towards an decrease in the EAE group (compared to controls) were the autoantibodies towards

• 78 kDA glucose-regulated protein

• Acyl-CoA-binding protein Indicating a treatment response, the level of 78 kDA glucose-regulated protein and Acryl-CoA binding protein auto-antibodies were significantly higher in the early liraglutide treated group compared to the EAE group with a similar trend in the late- treated liraglutide group. Discussion

Based on the first study, we believed that not only would our well-powered study be reproducible, but also that there was a case for a similar or improved clinical outcome with a first-sign-of-disease treatment regime. Furthermore, we believed that the neuroprotective pathway CREB would be activated by liraglutide treatment in the EAE brain and infiltration of the periphery into the brain would be reduced. This second set of experiments has shown that both long-term and first-sign-of-disease treatment is favourable in terms of clinical scoring. Interestingly, manifestation of EAE was significantly reduced in the late-initiated treatment. Moreover, this regime activates a well-described and potent protective pathway, CREB. This suggests that the brain tissue in EAE rats receiving liraglutide may have a more protective protein expression profile (as seen for example, with increased MnSOD) and therefore may be less likely to suffer cellular damage or develop long-term degeneration of the brain. CREB activation is also intimately connected to improved cognition, through increased BDNF expression and increased neurogenesis in the hippocampal and subventricular stem cell niches in the brain. Furthermore, EAE rats with this treatment regime have less infiltration of peripheral blood and thus less leakage of BBB in the brainstem. This is potentially a result of both reduced T-cell inflammation in the blood and a less permeable endothelial BBB, since GLP-1 treatment on T-cells is known to reduce their pro-inflammatory state (especially at low concentrations), and GLP-1 reduces cell adhesion molecules and protects against pro-inflammatory stress in endothelial cells.

Autoantibodies in brain related to disease

As described above, we conducted a large-scale analysis of autoantibodies for associations with EAE disease and changes directly related to both early and late treatment with liraglutide. One major finding from this analysis is that autoantibodies to myelin proteolipid protein (PLP) are significantly increased in EAE disease, and are significantly decreased when rats with EAE are treated with early and late liraglutide (alongside Q4KLZ0 protein ( encoded by the Vnn1 gene)). Autoantibodies in the brain to PLP show that EAE drives the production of self-antigens towards a component of the myelin sheath. These autoantibodies will likely result in more intense immune response to myelin sheath and therefore demyelination, which is classical in both MS and EAE. Indeed, autoantibodies towards PLP have been shown to be increased in MS patient cohorts and are associated with different disease courses, especially those with extension demyelination. In this context, it is an important finding that autoantibodies towards PLP is decreased in liraglutide treated animals in both chronic and acute treatment settings. This suggests that liraglutide treatment is both reduces

inflammatory response and reduces an important signal that amplifies demyelination. Clinically one would expect that this would result in less demyelination and less intense inflammation around the myelin sheath itself. EAE disease results in the reduction of autoantibodies to Hemoglobin subunit alpha- 1/2; Ig kappa chain C region, B allele; Actin, cytoplasmic 1 ; and Actin, cytoplasmic 1 , N-terminally processed. Both early and late treatment with liraglutide reverses this decline. This finding further supports the claim that liralgutide is delaying disease processes. Interestingly, hemoglobin is produced in the brain and the hemoglobin subunits are not organized into tetramers as they are in classically in the erythrocyte. Cerebral hemoglobin is associated with protection against oxidative stress in neurons and in astroglia and is down-regulated in Parkinson's and Alzheimer's diseases. With this background, we suggest that liraglutide treatment may increase cerebral hemoglobin alpha 1/2 contributing protective effects to the brain, and this increased is reflected in an increase in autoantibodies. Moreover, this increase in autoantibody response under neurorprotective treatment of GLP-1 agonists may be associated with secretion of hemoglobin within the brain for neuroprotective purposes, as is seen in the retina. Indeed, if this was the case in the brain, a reduction of hemoglobin secretion for protective purposes in EAE would result in a brain that is more susceptible to oxidative damage, and that liralglutide treatment restores this hemoglobin-based protection.

Consequences of improved clinical scoring

With these experiments, we are able to show, with high power, that liraglutide treatment improves clinical outcome when treated long-term and when initiated at the time of symptoms. With the low side-effect profile of GLP-1 -based therapies, this suggests that GLP-1 receptor agonism may be beneficial for MS patients as a long- term treatment, similar to type II diabetes, and also suitable for a time-of-attack therapy. These data expand the applicably of this therapy to the heterogeneity that is present in MS patient subgroup, expanding the treatment options for medical professionals.

Potential consequences of increased CREB activation

Furthermore, our data shows that GLP-1 agonism activates the CREB pathway in the EAE brain. The CREB pathway is intimately involved in neurogenesis, and cognition, and increasing the cellular capacity to withstand oxidative stress, pro-inflammatory and pro-apoptotic environments. Interestingly, we have previously shown that GLP-1 treatment only activates the CREB system under conditions of cellular stress. This suggests that GLP-1 treatment will activate a protective, pro-cognition system in specific 'at-risk' sites of the brain during times of attack. This further suggests that GLP- 1 receptor activation is safe for long-term, chronic treatment.

In the adult brain, the CREB system is involved in the production of many pro-survival proteins and proteins involved in preserving the mitochondria, promoting proliferation of neural stem cells, increasing the genesis of synapses and improving cognition. BDNF is an important mediator of improved cognition, neurogenesis, cell survival and we have previously shown that liraglutide treatment increases CREB and BDNF in the diseased brain. Moreover, we have also shown liraglutide to increase pro-survival protein neuroglobin, and pro-mitochondrial biogenesis signalling, CREB-regulated proteins associated with protection against oxidative stress in short-term and neurodegeneration in the long-term.

An important aspect of autoimmune pathogenesis is the access of autoreactive T-cells inside the brain proper. This can be achieved, to the determent of the patient, through increased chemokine activity and/or increased permeability of the BBB due to unrelated circumstances. We propose that GLP-1 agonism results in reduced infiltration of the periphery into the brain, of which we have shown through reduced albumin leakage. The potential consequences of this result would be reduced access to lesion sites and potential new sites to cause lesions, reduced edema and reduced inflammation inside the brain, a process that is induced when peripheral proteins are detected by the cerebral immune system, and a process that exacerbates damage at the lesion site. This data suggests that autoreactive attacks may be reduced in frequency and in the extent of damage caused leading to less relapses, more manageable symptoms upon attack and less long-term degeneration at lesion sites.

Glutathione peroxidase autoantibodies are increased in EAE. This finding suggests that EAE disease results in increased oxidative stress in the brain and is supported by previous work in EAE. Importantly, liraglutide treatment reduces autoantibodies to this enzyme intimately involved in the redox cycle of the cell suggesting a reduction of oxidative stress. Indeed, we have shown above that the antioxidant MnSOD is increased in the brain and that the CREB system, which has a strong association to protection against oxidative stress, is increased with liraglutide treatment. Additionally, liraglutide treatment in other brain pathologies shows increased CREB and reduced oxidative stress. This autoantibodiy finding appears to be related a protection against oxidative stress with liraglutide therapy.

Vanin-1 (VNN1 ), also known as pantetheinase, is a cell-membrane anchored or secreted enzyme that hydrolyzes D-pantetheine into cysteamine and pantothenate (vitamin-B5) [19]. Pantothenate is part of a metabolism pathway that is the precursor to acetyl-CoA for production of energy and cysteamine is involved in redox control of oxidative stress similar to glutathione, discussed above. Autoantibodies to VNN1 are, similar to glutathione, increased in EAE, a disease effect that is not present in liraglutide treated EAE rats. This suggests that the EAE rat brain is under significantly more distress than liraglutide treated EAE brain. This is consistent with our data showing increased antioxidants in the brain and activation of the antioxidant pathway CREB in the brain of liraglutide-treated EAE rats. 78kDa glucose-related protein (GRP78) is a stress-related heat shock protein with potent anti-inflammatory effects. This protein has previously been shown to act directly on lymphocytes and macrophages to reduce their pro-inflammatory state. Therefore, the finding that GRP78 is reduced in EAE is consistent with a pro-inflammatory mileu that is lacking anti-inflammatory input leading to disease. Liraglutide treatment increases or retains these levels during EAE and shows a tendency to increase GRP78 autoantibodies. We suggest liraglutide may exert a potent anti-inflammatory mechanism through this immunomodulatory protein. Indeed, treatment with GRP78 in a model of arthritis reduces the autoimmune reaction. Fructose-biphosphate aldolase C is an enzyme involved in glycolysis in the

hippocampus and cerebellum. In late treatment with liraglutide, autoantibodies to this protein are increased when compared to EAE. This may represent an increase in glycolysis in these two regions. Indeed, the hippocampus is a region involved in CREB activation, BDNF production and cognition where we claim liraglutide to have an important effect. Moreover, the cerebellum is a region involved motor coordination. As the EAE pathology progresses, there may be more activity in this region of the brain involved in preservation of coordination movement. Indeed, despite losing a similar amount of weight due to EAE, LateLira rats maintained their ability to locomote, which may be due, in part to changes in in the cerebellum reflected here. Autoantibodies to olfactory receptor 1069 were increased in EAE disease. The olfactory areas of the brain are well-described to be highly vulnerable to disease, especially from immune infiltrating disease. Indeed, in EAE, olfactory regions are inflamed before clinical symptoms are detected. Liraglutide treated rats had control level autoantibodies to this olfactory receptor. These autoantibodies likely represent an increase in damage to the olfactory regions with EAE resulting in immune cell recognition and antibody production. Since this an early event in the disease progress, this data suggests that liraglutide treatment protects the brain from this early phase damage, as represented by normal autoantibody levels for this olfactory protein.

Acyl-CoA binding protein, also known as diazepam-binding inhibitor, is a neuropeptide GABA receptor modulator with reduced autoantibodies in EAE disease and significantly increased autoantibodies in liraglutide treated rats. This suggests that GABAergic signals are modified with disease, and preserved and/or restored with liraglutide.

Furthermore, Diazepam-binding inhibitor is found to be modified into

triakontatetraneuropeptide and octadecaneuropeptide, and both of these

neuropeptides have reduced autoantibodies in EAE and increased with liraglutide treatment. These neuropeptides act on GABA receptors of astroglia modulating binding and controlling intracellular pathways promoting calcium influx and, in particular for octadecaneuropeptide, promote antioxidant production in astrocytes. Indeed, octadecaneuropeptide treatment induces production of superoxide dismutase and catalase and increases their activity under oxidative stress, thereby protecting astroglia from damage. This further suggests that redox capacity in EAE is reduced and liraglutide protects astroglia from oxidative stress in EAE.

Claims

Claims

1 . A method of treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis by administering a GLP-1 receptor agonist.

2. The method according to any of the preceding claims, wherein said GLP-1

receptor agonist is selected from the group consisting of liraglutide, exenatid, dulaglutid, lixisenatid, albiglutide, semaglutid and functional derivatives thereof.

3. The method according to any of the preceding claims, wherein said GLP-1

receptor agonist is liraglutide or a functional derivative thereof.

4. The method according to any of the preceding claims, wherein cognitive

impairment includes impairments in memory, attention and concentration, information processing, executive functions, visuospatial functions and/or verbal fluency.

5. The method according to any of the preceding claims, said method further

comprising treating said multiple sclerosis with a further drug agent.

6. The method according to any of the preceding claims, wherein said further drug agent is a DPP-IV inhibitor.

7. The method according to any of the preceding claims, wherein said further drug agent is selected from the group consisting of sitagliptin, teriflunomid, dimethylfumarat, interferon beta-1 a, glatirameracetat, natalizumab, fingolimod, alemtuzumab, Mitoxantron, Daclizumab and Ocrelizumab.

8. The method according to any of the preceding claims, wherein said GLP-1

receptor agonist is administered by oral, subcutaneous, intravenous or intramuscular administration, and/or wherein said GLP-1 receptor agonist is administered using a pen.

9. The method according to any of the preceding claims, wherein said GLP-1

receptor agonist is liraglutide or a functional derivative thereof and is

administered in a daily amount of 1 .8 mg by subcutaneous administration.

10. A GLP-1 receptor agonist for use in treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis.

1 1 . A kit-of-parts comprising a GLP-1 receptor agonist, such as liraglutide, and one or more further agents.

12. The kit-of-part according to claim 1 1 , wherein said further agent is a DPP-IV inhibitor.

13. The kit-of-part according to claim 1 1 , wherein said further agent is a drug for treatment of multiple sclerosis, such as agent is selected from the group consisting of sitagliptin, teriflunomid, dimethylfumarat, interferon beta-1 a, glatirameracetat, natalizumab, fingolimod, alemtuzumab, Mitoxantron,

Daclizumab and Ocrelizumab.

14. A kit-of-parts as defined in claims above for use in treating multiple sclerosis and/or cognitive impairment in patients with multiple sclerosis.

15. The kit-of-part according to claim 14, wherein said wherein cognitive impairment includes impairments in memory, attention and concentration, information processing, executive functions, visuospatial functions and/or verbal fluency.