COMBINATION THERAPY WITH MINOCYCLINE AND HYDROXYCHLOROQUINE FOR THE TREATMENT OF MULTIPLE SCLEROSIS fMSl

CROSS REFERENCE TO RELATED APPLICAITON

[0001] This application claim priority to US 62/412,555, filed October 25, 2016, the entire contents of which are incorporated by reference in their entirety.

FIELD

[0002] The present disclosure relates generally to compound(s), composition(s), and method(s) for the treatment of multiple sclerosis in a subject.

BACKGROUND

[0003] Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), characterized by demyelination, axonal and neuronal degeneration ¹ . The majority of patients present with a relapsing-remitting phenotype (RRMS) in early adulthood and progress after 15-20 years to a progressive disease course (e.g., secondary progressive MS or SPMS). About 15% of the patients present with a primary progressive phenotype. Whereas the number of therapeutics for RRMS has been growing over the last decade, treatment options are not yet available for the progressive phase of the disease. Reasons for that are manifold.

SUMMARY

[0004] In one aspect there is provided a method of treating multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline and a therapeutically effective amount of hydroxychloroquine, of a functional derivative thereof.

[0005] In one example, said multiple sclerosis is relapsing remitting multiple sclerosis.

[0006] The method of claim 1 , wherein said multiple sclerosis is progressive multiple sclerosis. [0007] In one example said multiple sclerosis is primary progressive multiple sclerosis.

[0008] In one example said multiple sclerosis is secondary progressive multiple sclerosis.

[0009] In one example wherein said multiple sclerosis is progressive relapsing multiple sclerosis.

[0010] In one example said treatment further comprises administering a

therapeutically effective amount of Laquinimod, Fingolimod, Masitinib, Ocrelizumab, Ibudilast, Anti-LINGO-1 , MD1003 (high concentration Biotin), Natalizumab, Siponimod, Tcelna (imilecleucel-T), Simvastatin, Dimethyl fumarate, Autologous haematopoietic stem cell transplantation, Amiloride, Riluzole, Fluoxetine, or a functional derivative thereof.

[0011] In one example said treatment further comprises administering a

therapeutically effective amount of teriflunomide (Aubagio), interferon beta-1 a (Avonex; Rebif), pegylated interferon beta-1a (Plegridy), interferon beta-1 b (Betaseron; Extavia), glatiramer acetate (Copaxone), alemtuzumab (Lemtrada), mitoxantrone (Novantrone), natalizumab (Tysabri), or functional derivative thereof.

[0012] In one example said subject is a human.

[0013] In another aspect there is provided a use of minocycline, or a functional derivative thereof, and a use of hydroxychloroquine, of a functional derivative thereof, for treating multiple sclerosis in subject in need thereof.

[0014] In another aspect there is provided a use of minocycline, or a functional derivative thereof, and a use of hydroxychloroquine, or a functional derivative thereof, in the manufacture of a medicament for treating multiple sclerosis in subject in need thereof.

[0015] In one example said multiple sclerosis is relapsing remitting multiple sclerosis.

[0016] In one example said multiple sclerosis is progressive multiple sclerosis. In one example said multiple sclerosis is primary progressive multiple sclerosis.

[0017] In one example said multiple sclerosis is secondary progressive multiple sclerosis.

[0018] In one example said multiple sclerosis is progressive relapsing multiple sclerosis. [0019] In one example further comprising a use of a therapeutically effective amount of Laquinimod, Fingolimod, Masitinib, Ocrelizumab, Ibudilast, Anti-LINGO-1 , MD1003 (high concentration Biotin), Natalizumab, Siponimod, Tcelna (imilecleucel-T), Simvastatin, Dimethyl fumarate, Autologous haematopoietic stem cell transplantation, Amiloride, Riluzole, Fluoxetine, or a functional derivative thereof, for the treatment of multiple sclerosis, relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, secondary multiple sclerosis, or progressive relapsing multiple sclerosis.

[0020] In one example further comprising a use of a therapeutically effective amount of Laquinimod, Fingolimod, Masitinib, Ocrelizumab, Ibudilast, Anti-LINGO-1 , MD1003 (high concentration Biotin), Natalizumab, Siponimod, Tcelna (imilecleucel-T), Simvastatin, Dimethyl fumarate, Autologous haematopoietic stem cell transplantation, Amiloride, Riluzole, Fluoxetine, or a functional derivative thereof, in the manufacture of a medicament for the treatment of multiple sclerosis, relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, , secondary multiple sclerosis, or progressive relapsing multiple sclerosis.

[0021] In one example further comprising a use of a therapeutically effective amount of tenflunomide (Aubagio), interferon beta-1 a (Avonex; Rebif), pegylated interferon beta-1 a (Plegridy), interferon beta-1 b (Betaseron; Extavia), glatiramer acetate (Copaxone), alemtuzumab (Lemtrada), mitoxantrone (Novantrone), natalizumab (Tysabri), or functional derivative thereof, for the treatment of multiple sclerosis, relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, secondary multiple sclerosis, or progressive relapsing multiple sclerosis.

[0022] In one example further comprising a use of a therapeutically effective amount of teriflunomide (Aubagio), interferon beta-1 a (Avonex; Rebif), pegylated interferon beta-1 a (Plegridy), interferon beta-1 b (Betaseron; Extavia), glatiramer acetate (Copaxone), alemtuzumab (Lemtrada), mitoxantrone (Novantrone), natalizumab (Tysabri), or functional derivative thereof, in the manufacture of a medicament for the treatment of multiple sclerosis, relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, , secondary multiple sclerosis, or progressive relapsing multiple sclerosis.

[0023] In one example the subject is a human. [0024] In another aspect there is provided a kit for the treatment of multiple sclerosis, relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, or progressive relapsing multiple sclerosis comprising: 1. minocycline or a functional derivative thereof; 2. hydroxychloroquine or a functional derivative thereof; and Instructions for the use thereof.

[0025] In one example said multiple sclerosis is relapsing remitting multiple sclerosis.

[0026] In one example said multiple sclerosis is progressive multiple sclerosis.

[0027] In one example said multiple sclerosis is primary progressive multiple sclerosis.

[0028] In one example said multiple sclerosis is secondary progressive multiple sclerosis.

[0029] In one example said multiple sclerosis is progressive relapsing multiple sclerosis.

[0030] In one example further comprising one or more of Laquinimod, Fingolimod,

Masitinib, Ocrelizumab, Ibudilast, Anti-LINGO-1 , MD1003 (high concentration Biotin), Natalizumab, Siponimod, Tcelna (imilecleucel-T), Simvastatin, Dimethyl fumarate,

Autologous haematopoietic stem cell transplantation, Amiloride, Riluzole, Fluoxetine, or a functional derivative thereof.

[0031] In another aspect there is provided a pharmaceutical composition comprising minocycline or a functional derivative thereof and hydroxychloroquine or a functional derivative thereof, for treating relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, or progressive relapsing multiple sclerosis.

[0032] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures. [0034] Fig. 1 - Minocycline and hydroxychloroquine attenuate iron mediated neurotoxicity. FeS0 ₄ induced neuronal cell death as shown by the reduced number of MAP2- positive neurons (A, C) after 24h compared to the untreated control (B). Minocycline (D) and hydroxychloroquine (HCQ, E) prevented iron-mediated neurotoxicity individually. A combination effect of minocycline and hydroxychloroquine at 1 , 2 or 5 μΜ (F) each did not elevate surviving neurons compared to both treatments applied individually (A); note that we do not consider there to be a combination effect if the impact of the combination is significant only to one drug. Representative immunofluorescent images are shown (bar represents 100 μηι). Data (mean ± SEM) are normalized to the respective control condition across 3 independent experiments of quadruplicate wells per test condition per experiment (controls 4-16 wells per experiment). One-way analysis of variance (ANOVA) with Dunnett ^' s multiple comparisons test as post-hoc analysis compared to FeS04. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001.

[0035] Fig. 2 - Minocycline but not hydroxychloroquine has anti-oxidative properties in the HORAC assay. In the HORAC assay, the degradation of fluorescein by hydroxyl radicals results in loss of fluorescence that can be measured over 60 min. The presence of gallic acid as a strong anti-oxidant control causes an upward shift in the fluorescence degradation curve compared to the blank reaction (no anti-oxidant) (A). Minocycline but not hydroxychloroquine alters the fluorescence degradation curve (B). HORAC gallic acid equivalents (GAEs) were calculated by the integration of the area under the curve in comparison to 12.5 μΜ gallic acid (C). Five μΜ of minocycline doubled the GAE

demonstrating twice elevated anti-oxidative potency compared to gallic acid. The

combination of minocycline and hydroxychloroquine did not further elevate GAE beyond either drug, and indeed showed a level that is intermediate between the 2 medications. Data of 3 independent experiments ± SEM are shown, where each experiment consisted of quadruplicate samples. RFU: Relative fluorescent units.

[0036] Fig. 3 - Low concentrations of minocycline and hydroxychloroquine had a combination effect in reducing the proliferation of T-lymphocytes. Minocycline reduced T- lymphocyte proliferation from 1 μΜ, whereas hydroxychloroquine reduced proliferation only at 5 μΜ (A); data were normalized to counts per minute (cpm) of activated T-cells. The combination at 1 and 2 μΜ each reduced proliferation further when compared to both drugs in isolation, with a ceiling effect leading to 51 % reduced proliferation at a concentration of 5 μΜ each (A). TNF-a levels were reduced upon treatment with minocycline but increased with hydroxychloroquine (B); the combination reflected the minocycline outcome. IFN-γ altered using the combination but not upon treatment with either drugs alone (B). Reduced proliferation particularly for minocycline was reflected in a cell cycle arrest with increased percentage of cells in the G1 phase and a lower amount in the S-phase (C); the combination effect reflected that of minocycline. Shown are data of n=3 independent experiments performed in quadruplicates (A), quadruplicate wells of one experiment (B) and one representative experiment performed twice, depicted as mean ± SEM of quadruplicate wells (repeat experiment in duplicates) (C). Data are depicted as mean ± SEM. One-way analysis of variance (ANOVA) with Dunnett ^' s multiple comparisons test (A) or Sidak ^' s multiple comparisons test (B, C) as post-hoc analysis compared to activated T-lymphocytes. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001.

[0037] Fig. 4 - Reduced T-lymphocyte proliferation is mediated via m-TOR, AKT and

ERK1/2 signaling. Minocycline (p<0.01) and hydroxychloroquine (p<0.05) reduced the percentage of p-m-TOR (A), p-AKT (B) and p-ERK1/2 (C) positive T-cells, reflected in a shift of respective histograms. The combination did not exhibit additional effects over both individual medications. The upper panels show % of total cells, the lower panels depict the median fluorescence intensity of the gated population. Shown are data of quadruplicate wells (mean ± SEM) of one individual experiment that was conducted twice. One-way analysis of variance (ANOVA) with Sidak ^' s multiple comparisons test as post-hoc analysis compared to activated T-lymphocytes. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001.

[0038] Fig. 5 - Minocycline and hydroxychloroquine both reduced the proliferation of

B-cells. B-cells were isolated by positive selection achieving a purity of 97% (A). Anti- CD40L/BCR/IL-4 activated B-cells increased their proliferation compared to control, as assessed with 3H-thymidine incorporation (counts per minute, cpm) (B). Proliferation was reduced individually by minocycline and hydroxychloroquine but the combination was not statistically significant from either alone. TNF-a release was unchanged by drug treatment (C), with data normalized to activated B-cells. Values are mean ± SEM of triplicate wells of one experiment that was corroborated across three independent experiments. One-way analysis of variance (ANOVA) with Sidak ^' s multiple comparisons test as post-hoc analysis compared to activated B-cells. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001.

[0039] Fig. 6 - The combination of minocycline and hydroxychloroquine attenuates acute EAE. Mice were treated with suboptimal doses of minocycline (25 mg/kg) or hydroxychloroquine (50 mg/kg) from the day of induction of MOG-EAE (day 0). While vehicle treated mice were at peak EAE clinical severity at days 18 and 19, the individual treatments resulted in lower clinical scores while the combination virtually prevented manifestation of disease (A). Histological scores evaluated by a previously described method ⁴¹ showed the lowest amount of inflammation in the combination group (B; *p<0.05 against vehicle) on average (C: hematoxylin/eosin and luxol fast blue); arrows depict inflammatory lesions, arrowheads show meningeal inflammation. TNF-a levels were decreased in the blood in combination treated mice, concomitant with increased IL-5 levels (G, H). Two-way ANOVA with Sidak ^' s multiple-comparisons test as post-hoc analysis (A). Significant changes in the post-hoc analysis against the combination group are presented as * for the vehicle group and + for the minocycline group. B, G and H were analyzed with one-way ANOVA and Tukey ^' s multiple comparisons test as post-hoc analysis. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001.

[0040] Fig. 7 - Long-term outcomes in chronic EAE. Mice were treated with minocycline (25 mg/kg) or hydroxychloroquine (50 mg/kg) from the day of induction of MOG- EAE (day 0). Whereas minocycline and hydroxychloroquine delayed onset of clinical disease by 5 days, the combination therapy suppressed EAE completely until day 22 (A). Treatment was stopped at day 22 whereupon the combination group developed clinical signs. The sum of scores showed a trend towards lower disease burden in the combination group over the entire 30 days of observation (B), or during the 22 days of treatment (C). N=6 animals per group for vehicle, minocycline or hydroxychloroquine, and n=4 for combination. Two-way ANOVA with Sidak ^' s multiple-comparisons test as post-hoc analysis (A). Significant changes in the post-hoc analysis against the combination group are presented as * for the vehicle group, + for the minocycline group and # for the hydroxychloroquine group. B, C one-way ANOVA and Tukey ^' s multiple comparisons test as post-hoc analysis. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001. DETAILED DESCRIPTION

[0041] In one aspect, there is provided a method of treating, prophylaxis, or amelioration of a neurological disease by administering to a subject in need thereof minocycline, or a functional derivative thereof, and hydroxychloroquine, or a functional derivative thereof. In a specific example, the neurological disease is multiple sclerosis (also referred to as "MS").

[0042] The term "multiple sclerosis" refers to an inflammatory disease of the central nervous system (CNS) in which the insulating covers of nerve cells in the brain and spinal cord are damaged. This damage disrupts the ability of parts of the nervous system to communicate, resulting in a wide range of signs and symptoms, including physical, mental, and psychiatric.

[0043] In one example, as described herein there is provided a treatment for multiple sclerosis in a subject.

[0044] As used herein, "multiple sclerosis" includes multiple sclerosis or a related disease, and optionally refers to all types and stages of multiple sclerosis, including, but not limited to: benign multiple sclerosis, relapsing remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, progressive relapsing multiple sclerosis, chronic progressive multiple sclerosis, transitional/progressive multiple sclerosis, rapidly worsening multiple sclerosis, clinically-definite multiple sclerosis, malignant multiple sclerosis, also known as Marburg's Variant, and acute multiple sclerosis. Optionally, "conditions relating to multiple sclerosis" include, e.g., Devic's disease, also known as Neuromyelitis Optica; acute disseminated encephalomyelitis, acute demyelinating optic neuritis, demyelinative transverse myelitis, Miller-Fisher syndrome,

encephalomyelradiculoneuropathy, acute demyelinative polyneuropathy, tumefactive multiple sclerosis and Balo's concentric sclerosis.

[0045] In one example, the multiple sclerosis is relapsing remitting multiple sclerosis.

[0046] In one example, the multiple sclerosis is progressive multiple sclerosis.

[0047] In one example, the multiple sclerosis is primary progressive multiple sclerosis.

[0048] In one example, the multiple sclerosis is secondary progressive multiple sclerosis. [0049] In one example, the multiple sclerosis is progressive relapsing multiple sclerosis.

[0050] In a specific example, as described herein there is provided a treatment for relapsing remitting multiple sclerosis in a subject.

[0051] In a specific example, as described herein there is provided a treatment for progressive multiple sclerosis in a subject.

[0052] As used herein, "progressive" multiple sclerosis refers to forms of the disease which progress towards an ever-worsening disease state over a period of time. Progressive multiple sclerosis includes, for example, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, and progressive relapsing multiple sclerosis.

[0053] These subtypes may or may not feature episodic flare-ups of the disease, but are each associated with increased symptoms, such as increased demyelination or pain and reduced capacity for movement, over time.

[0054] In a specific example, as described herein there is provided a treatment for primary progressive multiple sclerosis in a subject.

[0055] In a specific example, as described herein there is provided a treatment for secondary progressive multiple sclerosis in a subject.

[0056] In a specific example, as described herein there is provided a treatment for progressive relapsing multiple sclerosis.

[0057] The term "subject", as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human.

[0058] The term "treatment" or "treat" as used herein, refers to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject in the early stage of disease can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent progression.

[0059] In some examples, treatment results in prevention or delay of onset or amelioration of symptoms of a disease in a subject or an attainment of a desired biological outcome, such as reduced neurodegeneration (e.g., demyelination, axonal loss, and neuronal death), reduced inflammation of the cells of the CNS, or reduced tissue injury caused by oxidative stress and/or inflammation in a variety of cells.

[0060] In some examples, treatment methods comprise administering to a subject a therapeutically effective amount of a compound or composition described herein and optionally consists of a single administration or application, or alternatively comprises a series of administrations or applications.

[0061] The term "pharmaceutically effective amount" as used herein refers to the amount of a compound, composition, drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician, for example, the treatment of progressive multiple sclerosis. This amount can be a therapeutically effective amount.

[0062] The compounds and compositions may be provided in a pharmaceutically acceptable form.

[0063] The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions, and/or dosage forms (such as unit dosages) which are suitable for use in contact with the tissues of a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. is also "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0064] In one example, there is provided a method of treating multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline, or functional derivative thereof, and a therapeutically effective amount of hydroxychloroquine, or a functional derivative thereof. [0065] In one example, there is provided a method of treating relapsing remitting multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline, or functional derivative thereof, and a therapeutically effective amount of hydroxychloroquine, or a functional derivative thereof.

[0066] In one example, there is provided a method of treating progressive multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline, or functional derivative thereof, and a therapeutically effective amount of hydroxychloroquine, or a functional derivative thereof.

[0067] In one example, there is provided a method of treating primary progressive multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline, or functional derivative thereof, and a therapeutically effective amount of hydroxychloroquine, or a functional derivative thereof.

[0068] In one example, there is provided a method of treating secondary progressive multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline, or functional derivative thereof, and a therapeutically effective amount of hydroxychloroquine, or a functional derivative thereof.

[0069] In one example, there is provided a method of treating progressive relapsing multiple sclerosis comprising administering to a subject in need thereof, a therapeutically effective amount of minocycline, or functional derivative thereof, and a therapeutically effective amount of hydroxychloroquine, or a functional derivative thereof.

[0070] The term "functional derivative" and "physiologically functional derivative" as used herein means an active compound with equivalent or near equivalent physiological functionality to the named active compound when used and/or administered as described herein. As used herein, the term "physiologically functional derivative" includes any pharmaceutically acceptable salts, solvates, esters, prodrugs derivatives, enantiomers, or polymorphs.

[0071] In some examples the compounds are prodrugs.

[0072] The term "prodrug" used herein refers to compounds which are not pharmaceutically active themselves but which are transformed into their pharmaceutical active form in vivo, for example in the subject to which the compound is administered. [0073] Minocycline, or a functional derivative thereof, and hydroxychloroquine, or a functional derivative thereof, may be administered either simultaneously (or substantially simultaneously) or sequentially, dependent upon the condition to be treated.

[0074] Minocycline, or a functional derivative thereof, and hydroxychloroquine, or a functional derivative thereof, may be administered either simultaneously (or substantially simultaneously) or sequentially, dependent upon the condition to be treated, and may be administered in combination with other treatment(s). The other treatment(s), may be administered either simultaneously (or substantially simultaneously) or sequentially.

[0075] In one example, the other or additional treatment further comprises administering a therapeutically effective amount of Laquinimod, Fingolimod, Masitinib, Ocrelizumab, Ibudilast, Anti-LINGO-1 , MD1003 (high concentration Biotin), Natalizumab, Siponimod, Tcelna (imilecleucel-T), Simvastatin, Dimethyl fumarate, Autologous haematopoietic stem cell transplantation, Amiloride, Riluzole, Fluoxetine, or a functional derivative thereof.

[0076] In one example, the other or additional treatment further comprises administering a therapeutically effective amount of teriflunomide (Aubagio), interferon beta- la (Avonex; Rebif), pegylated interferon beta-1a (Plegridy), interferon beta-1 b (Betaseron; Extavia), glatiramer acetate (Copaxone), alemtuzumab (Lemtrada), mitoxantrone

(Novantrone), or natalizumab (Tysabri), or functional derivative thereof.

[0077] The actual amount(s) administered, and rate and time-course of

administration, will depend on the nature and severity of progressive multiple sclerosis being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

[0078] The formulation(s) may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. [0079] The compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot / for example, subcutaneously or intramuscularly.

[0080] Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in- water liquid emulsion or a water- in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

[0081] Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain antioxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other micro particulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.

[0082] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried

(lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs. [0083] Methods of the invention are conveniently practiced by providing the compounds and/or compositions used in such method in the form of a kit. Such a kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[0084] In one example, there is described a kit for the treatment of multiple sclerosis, relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, or progressive relapsing multiple sclerosis comprising: minocycline or a functional derivative thereof; hydroxychloroquine or a functional derivative thereof; and Instructions for the use thereof.

[0085] In one example, said multiple sclerosis is relapsing remitting multiple sclerosis.

[0086] In one example, said multiple sclerosis is progressive multiple sclerosis.

[0087] In one example, said multiple sclerosis is primary progressive multiple sclerosis.

[0088] In one example, wherein said multiple sclerosis is secondary progressive multiple sclerosis.

[0089] In one example, said multiple sclerosis is progressive relapsing multiple sclerosis.

[0090] In another example, the kit further comprises one or more of Laquinimod,

Fingolimod, Masitinib, Ocrelizumab, Ibudilast, Anti-LINGO-1 , MD1003 (high concentration Biotin), Natalizumab, Siponimod, Tcelna (imilecleucel-T), Simvastatin, Dimethyl fumarate, Autologous haematopoietic stem cell transplantation, Amiloride, Riluzole, Fluoxetine, or a functional derivative thereof; and instructions for use.

[0091] In another example, the kit further comprises one or more of teriflunomide

(Aubagio), interferon beta-1a (Avonex; Rebif), pegylated interferon beta-1 a (Plegridy), interferon beta- 1 b (Betaseron; Extavia), glatiramer acetate (Copaxone), alemtuzumab (Lemtrada), mitoxantrone (Novantrone), natalizumab (Tysabri), or functional derivative thereof.

[0092] In one example there is described a pharmaceutical composition comprising minocycline or a functional derivative thereof and hydroxychloroquine or a functional derivative thereof, for treating relapsing remitting multiple sclerosis, progressive multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, or progressive relapsing multiple sclerosis.

[0093] In one example minocycline or a functional derivative thereof and

hydroxychloroquine or a functional derivative thereof are in a single dosage form.

[0094] In one example minocycline or a functional derivative thereof and

hydroxychloroquine or a functional derivative thereof are in separate dosage forms.

[0095] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these example are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

[0096] Examples

[0097] In the following examples, standard methodologies were employed, as would be appreciated by the skilled worker.

[0098] Methods

[0099] Cell culture

[00100] Human neurons

[00101] Human neurons were isolated from brain tissues of therapeutically aborted 18- 20 week old fetuses, in accordance with ethics approval of the University of Calgary ethics committee. Neurons were isolated as previously described ¹⁷ . Cells were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1 μΜ sodium pyruvate, 10 μΜ glutamine, 1X non-essential amino acids, 0.1 % dextrose and 1 %

penicillin/streptomycin (complete medium; all culture supplements from Invitrogen,

Burlington, Canada). The initial isolates of mixed CNS cell types were plated in poly-L- ornithine coated (10 μg/ml) T75 flasks and cultured for at least two cycles17 in medium containing 25 μΜ cytosine arabinoside (Sigma-Aldrich, Oakville, Canada) to inhibit astrocyte proliferation and to deplete this major contaminating cell type. For experiments, the neuron- enriched cultures were retrypsinized and cells were plated in poly-L-ornithine pre-coated 96- well plates at a density of 100,000 cells/well in 100 μΙ of the complete medium supplemented with cytosine arabinoside. Medium was changed to AIM V ^® Serum Free Medium (Invitrogen) after 24h. Minocycline and/or hydroxychloroquine (Sigma-Aldrich) were added after 1 h in different concentrations, followed by a challenge with 50 μΜ FeS0 ₄ 1 h hence. Twenty-four h thereafter, cultures were fixed using 4% paraformaldehyde and stored until staining in phosphate buffered saline (PBS) (4°C).

[00102] Splenocvtes

[00103] We followed a previously published protocol ¹⁸ . Spleens from female C57/BL6 mice were harvested and after mechanical dissociation, the cell suspension was passed through a 70 μηι cell strainer and separated by Ficoll gradient centrifugation (1800 RPM, 30 min). The cell pellet was resuspended in Roswell Park Memorial Institute 1640 (RPMI 1640) medium, supplemented with 10% fetal bovine serum, 1 % sodium pyruvate, 2 mM L-alanyl-L- glutamine, 1 % penicillin/streptomycin, 1 % HEPES and 0.05 mM 2-mercaptoethanol (all supplements were from Invitrogen). Cells were plated in a concentration of 2.5 x 10 ⁵ cells per well in 100 μΙ media and T-lymphocytes were activated with 1 ,000 ng ml-1 plate-bound anti- CD3 and 1 ,000 ng ml ^"1 anti-CD28 suspended in media. Directly before plating, wells were treated with minocycline, hydroxychloroquine or the combination of both in different concentrations. After 48h, ³ H-thymidine was added at 1 μθϊ per well; cells were harvested 24h later on filter mats and counts per minutes were measured using a liquid scintillation counter.

[00104] B-cells

[00105] After separation of human peripheral blood mononuclear cells (PBMCs) from whole venous blood by Ficoll gradient (1800 RPM, 30 min), B-cells were isolated by positive selection with CD19 directed microbeads (Stemcell Technologies). Purity was assessed by FACS after staining for CD19 (Stemcell Technologies). Cells were plated in a concentration of 2.5 x 10 ⁵ /well in X- VIVO™ medium (Lonza, Mississauga, Canada) supplemented with 1 % penicillin/streptomycin and 1 % Glutamax, and treated with drugs for 1 h. Following that, cells were stimulated with 10 μg/ml IgM BCR cross-linking antibody (XAb) (Jackson

ImmunoResearch), 1 μg/ml anti-CD40L and IL-4 (20 ng/ml) for 24h as previously

described ¹⁹ . After 24h conditioned media were harvested for ELISA. Medium as well as respective drugs were re-added followed by application of ³ H-thymidine in a concentration of 1 μθϊ per well. Cells were harvested after 24h on filter mats and counts per minutes were measured using a liquid scintillation counter.

[00106] Flow cytometry [00107] Splenocytes were harvested 48h after drug treatment and washed with PBS, followed by resuspension in PBS with 2% FBS. Cell cycle analysis was performed using propidium iodide staining (50 μg/ml). Intracellular staining was performed following fixation and permeabilization of T-lymphocytes using the Fixation/Permeabilization Solution Kit (BD Biosciences, Mississauga, Canada). Antibodies were anti-human/mouse phospho-AKT (S473) APC antibody, anti-human/mouse phospho-mTOR (S2448) PE-Cyanine7 antibody and anti-human/mouse phospho-ERK1/2 (T202/Y204) PE antibody (all eBioscience, San Diego, CA). Stained cells were analyzed on a FACSCalibur™ with the software CellQuest™ (BD Biosciences).

[00108] Immunocvtochemistry and microscopy

[00109] Staining was performed at room temperature. A blocking buffer was first applied for 1 h followed by incubation with primary antibody overnight in 4°C. Neurons were identified using anti-microtubule-associated protein-2 (MAP-2) antibody (dilution 1 :1 ,000; Sigma-Aldrich) and Alexa Fluor 546 secondary antibody (dilution 1 :250, Invitrogen). Cell nuclei were stained with Hoechst S769121. Cells were stored in 4 °C in the dark before imaging.

[00110] Images were taken using the automated ImageXpress® imaging system (Molecular Devices, Sunnyvale, CA) through a 10x objective microscope lens, displaying 4 or 9 sites per well. Images were analyzed with the software MetaXpress® (Molecular Devices, Sunnyvale, CA) using the algorithm "multiwavelength cell scoring" ²⁰ . Cells were defined according to fluorescence intensity and size at different wavelengths. Data from all sites per well were averaged to one data point.

[00111] Cytokine analysis

[00112] Splenocyte and/or B-cell conditioned media were analyzed for TNF-a or IFN-γ using single ELISA kits according to the manufacturer ^' s protocol (Invitrogen). Blood from EAE mice was investigated using a multiplex cytokine LUMINEX assay (Eve Technologies, Calgary, Canada).

[00113] Hydroxyl radical antioxidant capacity (HORAC) assay

[00114] The potential anti-oxidant properties of minocycline and hydroxychloroquine were analyzed with the hydroxyl radical antioxidant capacity (HORAC) assay, in accordance with the procedure outlined in Ciz et al. 2010 ²¹ . In this assay, hydroxyl radicals generated by a Co(ll)-mediated Fenton-like reaction oxidize fluorescein causing loss of fluorescence The presence of an anti-oxidant reduces the loss of fluorescence and this can be monitored every 5 min over a period of 60 min with a Spectra Max Gemini XS plate reader (Molecular Devices, Sunnyvale, CA, USA) and the software SoftMax Pro version 5. For monitoring fluorescence, we used an excitation wavelength of A=485nm and an emission wavelength of A=520nm.

[00115] The anti-oxidant gallic acid (GA) is commonly used to generate a standard curve in HORAC assays. The antioxidative capacity of a test agent is then expressed as HORAC gallic acid equivalents (HORAC GAE) by integrating the area under the curves of the test drug ²² . In our experiments, we used 12.5 μΜ gallic acid as reference when calculating the HORAC GAE.

[00116] Induction and treatment of experimental autoimmune encephalomyelitis (EAE)

[00117] Female C57/BL6 mice of age 8 to 10 weeks at the start of the experiment were used. EAE was induced by the subcutaneous application of 50 μΙ of myelin

oligodendrocyte glycoprotein (MOG) peptide (amino acids 35-55, synthesized by the Peptide Facility of the University of Calgary), which was emulsified in 100 μΙ complete Freund ^' s adjuvant (Difco Laboratories, Detroit, Michigan). After anesthesia with ketamine/xylazine, 50 μΙ of the emulsion was injected subcutaneously (sc) on the side of each hind flank. Pertussis toxin (200 μΙ of 0.5 μg/ml solution; List Biological Laboratories, Campbell, CA) was injected intraperitoneally at days 0 and 2. From day 0 animals were treated with minocycline (25 mg/kg; 100 μΙ of 5 mg/ml solution), hydroxychloroquine (50 mg/kg; 100 μΙ of 10 mg/ml solution), 100 μΙ total of both drugs in combination to attain the above mentioned doses, or vehicle by intraperitoneal (IP) injection. All drugs were prepared daily in fresh PBS. Animals were evaluated daily using a 15-point scale as previously described ²³ . EAE clinical disease tends to involve tail dysfunction first, and ascending severity hampers first the hind- and then the forelimbs. The burden of disease was expressed as sum of score of all days of each individual animal over the period of the experiment ²³ .

[00118] We used minocycline and hydroxychloroquine at suboptimal doses so that the potential of additive effects of their combination on EAE disease course could be detected. Thus, we used minocycline at 25 mg/kg and hydroxychloroquine at 50 mg/kg per day;

optimal doses would include 50 mg/kg minocycline twice per day for the first 5 days, and 50 mg/kg once per day thereafter ¹² . For hydroxychloroquine, an optimal dose regimen would include pretreatment prior to MOG immunization, and with a dose of 100 mg/kg providing for best impact ¹⁰ .

[00119] Histological analyses

[00120] Animals were anesthetized with ketamine/xylazine followed by intracardial PBS perfusion. Spinal cords and cerebella were removed. The thoracic cords were fixed in 10% buffered formalin, followed by embedding in paraffin. Tissue was further processed as previously described ²⁴ . Sections were stained with hematoxylin/eosin for general histology, luxol fast blue (LFB) for myelin, Iba1 to visualize microglia and Bielschowsky's silver stain to stain for axons.

[00121] Statistical analysis

[00122] Statistical analysis was performed using the Graphpad Prism software version 7 (La Jolla, CA, USA). In vitro data were analyzed using one-way ANOVA with posthoc analysis as indicated in the figure legends comparing drug treated conditions with controls. EAE data were analyzed using a two-way ANOVA with Sidak ^' s posthoc analysis. Statistical significance was considered as p<0.05 (*), p<0.01 (**), p<0.001 (***) and p<0.0001 (****). All tissue culture experiments were performed in quadruplicates and repeated at least 3 times, if not otherwise specified.

[00123] Results

[00124] Protection against iron neurotoxicity in culture

[00125] Iron deposition and subsequent toxicity to cells is increasingly being implicated as a mediator of injury in MS ^{5, 6} . In culture, FeS0 ₄ is very toxic to neurons, with loss of over 80% of neurons at 50 μΜ over a 24h period (Fig. 1A-C). Minocycline reduced iron neurotoxicity evident from 2 μΜ concentration and above (Fig. 1A,D) while

hydroxychloroquine attenuated neuronal death from 1 μΜ (Fig. 1A,E). On an equimolar basis, hydroxychloroquine is more potent than minocycline in decreasing iron toxicity. We were unable to document additional protective effects when minocycline and

hydroxychloroquine were combined, likely because of the high magnitude of protection already afforded by hydroxychloroquine. Hydroxychloroquine alone, and the combination at 5 μΜ each, prevented neuronal cell death completely (Fig. 1A,F). [00126] Hydroxyl radical scavenging anti-oxidant activity

[00127] We investigated the capacity of the test medications to scavenge free radicals, and particularly the extremely reactive hydroxyl radical, using the HORAC assay. In this assay, the presence of hydroxyl radicals produced a progressive decay of fluorescence that can be detected in real-time in the absence of any anti-oxidants (blank reaction, Fig. 2A). In reactions where the anti-oxidant gallic acid positive control was present, the anti-oxidative effect resulted in an upward shift of the decay. Hydroxychloroquine (5 μΜ) produced a decay in fluorescence very close to that of the blank reaction, suggesting lack of hydroxyl scavenging activity (Fig. 2B). Minocycline (5 μΜ) appeared to have good anti-oxidative effects, as demonstrated by a real-time decay in fluorescence that is elevated above that of blank, and even of the gallic acid control. The combination produced a result very similar to that of minocycline, reflecting the minocycline activity.

[00128] To quantitate the hydroxyl radical scavenging capacity, the area under the curve of test drug was normalized to that of 12.5 μΜ gallic acid to produce the gallic acid equivalent (GAE). Thus, a GAE of one represents anti-oxidative property equivalent to that of the anti-oxidant gallic acid taking into account the molarity of gallic acid and the test drug, whereas a GAE close to zero depicts lack of hydroxyl radical scavenging property. Figure 2C shows that minocycline at 5 μΜ had remarkable hydroxyl radical scavenging property, while lower but still evident effect resulted at higher concentrations. In contrast,

hydroxychloroquine did not manifest hydroxyl radical scavenging property. As a result, the impact of the combination treatment mimicked the response produced by minocycline alone.

[00129] Effects on T-lymphocytes

[00130] Splenocyte cultures were exposed to anti-CD3/anti-CD28 to activate T- lymphocytes. Minocycline from 1 μΜ reduced the proliferation of activated T-cells (Fig. 3A). Hydroxychloroquine was less effective as it did not affect proliferation at 1 and 2 μΜ, and had a marginal (15%, p<0.05) effect at 5 μΜ. The combination of minocycline and

hydroxychloroquine at 1 and 2 μΜ elicited an additional effect in decreasing proliferation when compared to either alone at these respective concentrations (Fig. 3A). Of note, there was a ceiling effect to reducing proliferation, as no additional decrease in mitotic activity resulted from the 5 μΜ in combination (52%), compared to either alone. [00131] We examined levels of pro-inflammatory cytokines in treated cells.

Minocycline reduced the level of TNF-a but not IFN-γ, while hydroxychloroquine did not alter IFN-γ but might even have elevated TNF-a slightly compared to activated T-cell controls (p<0.01) (Fig. 3B). The combination of minocycline and hydroxychloroquine did not reduce TNF-a to level beyond that caused by minocycline alone (Fig. 3B). IFN-γ was lowered by the combination.

[00132] We investigated the phases of the cell cycle to corroborate the reduced [3H]- thymidine incorporation elicited by minocycline and hydroxychloroquine. Propidium iodide cell cycle analyses indicated that minocycline and the combination induced a cell cycle arrest with a higher percentage of cells in the G1 phase (Fig. 3C), whereas hydroxychloroquine did not have an effect. Reduced proliferation was further affirmed by less cells in the S-phase of the minocycline and combination treated conditions. The impact of the combination was driven by the minocycline response.

[00133] We investigated signaling pathways in activated T-cells and their response to medications. We evaluated the phosphorylated (p-, activated) form of mechanistic target of rapamycin (mTOR), protein kinase B (AKT) and extracellular signal-regulated kinases 1/2 (ERK1/2). Both minocycline and hydroxychloroquine reduced p-mTOR, p-AKT and p-ERK1/2 signaling. The combination, however, did not show a clear additive effect (Fig. 4).

[00134] Overall, while minocycline decreased the proliferation of activated T-cells, hydroxychloroquine was largely ineffectual; the consequence of the combination treatment on proliferation appears largely to be due to minocycline. However, hydroxychloroquine does affect T-cells, as indicated by reduction in the activity of signaling pathways.

[00135] Actions on B-lymphocytes

[00136] Due to the growing appreciation of the importance of ectopic B-cell follicular structures for progression ⁷ we next evaluated the effects of minocycline and

hydroxychloroquine on B-cells. By positive selection a purity of 97.0% B-lymphocytes was achieved (Fig. 5A). In anti-CD40L/BCR/IL-4 activated B-cells, proliferation was reduced upon treatment with minocycline (28% reduction), hydroxychloroquine (30%) and the combination (42%); the combination did not differ statistically from either medication alone. Unlike proliferation, none of the medications reduced the production of TNF-a by activated B-cells. [00137] Effects in EAE

[00138] Based on the above results that minocycline and hydroxychloroquine have similar but also differential potency on neuronal survival, hydroxyl radical scavenging capacity, and activity of T- and B-lymphocytes (Table 1), we tested the hypothesis that their combination in EAE would not yield a better outcome than either alone. We employed MOG EAE, which produces an initial peak severity that is followed either by some recovery or lingering disability. Suboptimal doses of drugs were used, so that the potential additive effects of their combination could be discerned. We found that both minocycline and hydroxychloroquine delayed the onset of clinical signs compared to vehicle treated mice by 2 days, and that when vehicle-treated mice were at peak clinical severity (average clinical score of 8.5, with loss of tail and hind limb functions, with involvement of forelimbs), mice treated with minocycline or hydroxychloroquine alone had only involvement of tail and hind limb functions (average clinical disease score of 4) (Fig. 6A). Impressively, the combination of both drugs markedly attenuated EAE severity; indeed, only one mouse out of 5 was sick (clinical score of 7), while the rest had no evidence of clinical impairment.

[00139] Table 1 - Summary of the effects of minocycline and hydroxychloroquine in isolation and in combination.

Functions Minocycline HCQ Combination effect

Mitigating iron neurotoxicity +++ ++++ No

Anti-oxi dative ++++ - No

Inhibiting T cells ++ -/+ Yes, at 1 and 2 μΜ

Inhibiting B cells ++ +++ No

Reducing EAE at suboptimal dose + + Yes; very marked

[00140] Magnitude of response: + 0-25%, ++ 25-50%, +++ 50-75%, ++++ >75%. HCQ: Hydroxychloroquine.

[00141] We sacrificed mice at day 19 for histological analyses. Infiltration of immune cells as assessed by LFB staining was marked in the vehicle group with a score of 4, showing subpial infiltration (Fig. 6B,C). Both medications alone led to reduced infiltration. The combination, however, reduced immune cell infiltration significantly with a score of 1.4 (Fig. 6B,F). Histological findings were corroborated by decreased TNF-a and increased IL-5 levels in the blood (Fig. 6G,H).

[00142] We repeated the above EAE experiment with the intent of prolonging the daily treatment and determining the long term outcomes of EAE mice. Treatment with drugs was initiated at the time of MOG immunization until day 22. Corroborating the first experiment, minocycline and hydroxychloroquine delayed the onset of clinical signs (Fig. 7A). With the longer period of observation, it was evident that while both medications by themselves delayed the ascension to peak clinical severity (by about 5 days), mice eventually succumbed to disease equivalent to that of vehicle-treated mice. Impressively, the combination of minocycline and hydroxychloroquine suppressed clinical signs completely even at 22 days post-MOG immunization, which represent many days post-peak clinical severity in vehicle-treated mice.

[00143] To prove that EAE was indeed induced in the combination treated animals, treatment was stopped after day 22. Thereafter, the combination group developed clinical signs of EAE and finally reached the same peak clinical disease score as the other groups on day 29. The overall burden of disease over the 30 days of experiment, which included days in which the combination-treated mice had succumbed to disease upon drug withdrawal, clearly shows the lowest scores across the mice in the combination group (Fig. 7B). Figure 7C represents the sum of scores in individual mice over the 22 days of treatment, to emphasize the impact of the combination generic drugs in controlling disease activity.

[00144] Overall, these results showcase the combination of minocycline and hydroxychloroquine in effectively reducing EAE severity, even when used at suboptimal doses.

[00145] Discussion

[00146] There is a tremendous need for the development of medications for progressive MS as most attempts to develop therapies for the condition have failed.

Recently, promising results in Phase III trials of ocrelizumab for primary progressive MS ²⁵ and siponimod for secondary progressive MS ²⁶ have been reported; these drugs are likely to be expensive if approved as medications for progressive MS. An alternative for drug development in progressive MS is to repurpose inexpensive generic medications with actions on pathologic features seen in progressive MS. Although the list is incomplete,

characteristics in progressive MS that are thought to promote the neurodegenerative condition include microglia activity, oxidative stress, and iron neurotoxicity that itself produces hydroxyl radical through the Fenton reaction ^{21 , 22} . While the adaptive immune response (T- and B-cells) in the periphery is thought to be less robust in progressive MS compared to the relapsing-remitting form, lymphocytes trapped within the CNS are still evident and diffusely scattered in autopsied progressive MS brains ²⁷ . A recent study of cerebrospinal fluids of T- and B-cell markers in progressive MS found these to be at comparable levels to those found in relapsing-remitting MS ⁴ . With these features as targets, our study focused on minocycline and hydroxychloroquine as potential medications for progressive MS. We therefore tested these two medications in combination, along with the effects of medications in isolation.

[00147] We found that minocycline has greater activity than hydroxychloroquine on an equimolar basis in attenuating the formation of hydroxyl free radical and in inhibiting T-cell proliferation. Conversely, hydroxychloroquine has greater potency in reducing iron neurotoxicity and B-cell proliferation than minocycline. As hydroxychloroquine does not inhibit the elaboration of hydroxyl free radical in the HORAC assay, its benefit in protecting against iron neurotoxicity must be through other mechanisms. Except for a combination effect at low concentrations against T-cell proliferation, the drugs were without clear superiority in combination on the other outcomes measured (Table 1). Surprisingly, without obvious combinational effects in culture, the drugs in combination induced strong synergistic effects in attenuating EAE in mice. While note wishing to be bound by theory, perhaps the medications acted principally on different effector arms of EAE that resulted in the synergistic activity in reducing EAE severity.

[00148] In the EAE experiments, the drug combination was able to suppress clinical signs until the end of treatment on day 22. As first clinical symptoms appeared in the combination group promptly on day 23, one day after the medications were stopped, it is speculative whether clinical disease would have become evident from day 23 if treatment had not been stopped. Given the intensity of the EAE immune responses, it has been our experience that EAE clinical signs ultimately become evident despite the continuation of most immunomodulators.

[00149] While not wishing to be bound by theory, the capacity of the combination therapy of minocycline and hydroxychloroquine to also limit B-cell proliferation might, in addition to other aforementioned properties, therefore be effective in progressive MS.

[00150] The amounts of drugs tested herein are physiologically relevant. Minocycline given at doses of 50 mg/kg in mice leads to serum concentration of 5-10 μg/ml ⁰ which is comparable to serum levels attained in MS patients given 100 mg twice per day (data not shown). Concentrations of 2-3 μΜ hydroxychloroquine in blood can be achieved using standard doses, which could lead to brain concentrations of 4-12 μΜ due to the tendency of the drug to accumulate in tissue ¹⁰ . Thus, the range of concentrations from 1 - 5 μΜ tested herein can be attained in humans.

[00151] References

[00152] 1. Ransohoff RM, Hafler DA, Lucchinetti CF. Multiple sclerosis-a quiet revolution. Nat Rev Neurol. 2015 Mar; 1 1 (3): 134-42.

[00153] 2. Prineas JW, Kwon EE, Cho ES, et al. Immunopathology of secondary- progressive multiple sclerosis. Ann Neurol. 2001 Nov;50(5):646-57.

[00154] 3. Lassmann H, van Horssen J, Mahad D. Progressive multiple sclerosis: pathology and pathogenesis. Nat Rev Neurol. 2012 Nov 5;8(11):647-56.

[00155] 4. Komori M, Blake A, Greenwood M, et al. Cerebrospinal fluid markers reveal intrathecal inflammation in progressive multiple sclerosis. Ann Neurol. 2015

Jul;78(1):3-20.

[00156] 5. Fischer MT, Sharma R, Lim JL, et al. NADPH oxidase expression in active multiple sclerosis lesions in relation to oxidative tissue damage and mitochondrial injury. Brain. 2012 Mar; 135(Pt 3):886-99.

[00157] 6. Hametner S, Wimmer I, Haider L, Pfeifenbring S, Bruck W, Lassmann H. Iron and neurodegeneration in the multiple sclerosis brain. Ann Neurol. 2013

Dec;74(6):848-61.

[00158] 7. Serafini B, Rosicarelli B, Magliozzi R, Stigliano E, Aloisi F. Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis. Brain pathology (Zurich, Switzerland). 2004 Apr; 14(2): 164-74. [00159] 8. Giuliani F, Hader W, Yong VW. Minocycline attenuates T cell and microglia activity to impair cytokine production in T cell-microglia interaction. Journal of leukocyte biology. 2005 Jul;78(1): 135-43.

[00160] 9. Yong VW, Wells J, Giuliani F, Casha S, Power C, Metz LM. The promise of minocycline in neurology. Lancet Neurol. 2004 Dec;3(12):744-51.

[00161] 10. Koch MW, Zabad R, Giuliani F, et al. Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis. Journal of the neurological sciences. 2015 Nov 15;358(1-2): 131-7.

[00162] 11. LM. Metz LML, D.; Traboulsee, A.; Duquette, P.; Yong, V.W.; Eliasziw, M.; Cerchiaro, G.; Greenfield, J.; Riddehough, A.; Yeung, M.; Kremenchutzky, M.;

Vorobeychik, G.; Freedman, M.S.; Bhan, V.; Blevins, G.; Marriott, J.J.; Grand'Maison, F.; Lee, L.; Thibault, M.; Hill, M. . Minocycline reduces the relative risk of multiple sclerosis in people experiencing their first clinical demyelinating event by 44.6%: results of a phase III double-blind placebo controlled Canadian multicentre clinical trial. ECTRIMS Online Library. 2015.

[00163] 12. Brundula V, Rewcastle NB, Metz LM, Bernard CC, Yong VW.

Targeting leukocyte MMPs and transmigration: minocycline as a potential therapy for multiple sclerosis. Brain. 2002 Jun; 125(Pt 6): 1297-308.

[00164] 13. Scholz R, Sobotka M, Caramoy A, Stempfl T, Moehle C, Langmann T. Minocycline counter-regulates pro-inflammatory microglia responses in the retina and protects from degeneration. Journal of neuroinflammation. 2015; 12:209.

[00165] 14. Macdonald H, Kelly RG, Allen ES, Noble JF, Kanegis LA.

Pharmacokinetic studies on minocycline in man. Clinical pharmacology and therapeutics. 1973 Sep-Oct; 14(5):852-61.

[00166] 15. McChesney EW. Animal toxicity and pharmacokinetics of

hydroxychloroquine sulfate. The American journal of medicine. 1983 Jul 18;75(1a): 11-8.

[00167] 16. Wei Y, Nygard GA, Ellertson SL, Khalil SK. Stereoselective disposition of hydroxychloroquine and its metabolite in rats. Chirality. 1995;7(8):598-604.

[00168] 17. Vecil GG, Larsen PH, Corley SM, et al. lnterleukin-1 is a key regulator of matrix metalloproteinase-9 expression in human neurons in culture and following mouse brain trauma in vivo. Journal of neuroscience research. 2000 Jul 15;61 (2):212-24. [00169] 18. Keough MB, Rogers JA, Zhang P, et al. An inhibitor of chondroitin sulfate proteoglycan synthesis promotes central nervous system remyelination. Nature communications. 2016;7:1 1312.

[00170] 19. Li R, Rezk A, Miyazaki Y, et al. Proinflammatory GM-CSF-producing B cells in multiple sclerosis and B cell depletion therapy. Science translational medicine. 2015 Oct 21 ;7(310):310ra166.

[00171] 20. Lau LW, Keough MB, Haylock-Jacobs S, et al. Chondroitin sulfate proteoglycans in demyelinated lesions impair remyelination. Ann Neurol. 2012

Sep;72(3):419-32.

[00172] 21. Ciz MC, H.; Denev, P.; Kratchanova, M.; Slavov, A.; Lojek, A. Different methods for control and comparison of the antioxidant properties of vegetables. FOOD CONTROL. 2010;21 :518-23.

[00173] 22. Ou B, Hampsch-Woodill M, Flanagan J, Deemer EK, Prior RL, Huang D. Novel fluorometric assay for hydroxyl radical prevention capacity using fluorescein as the probe. Journal of agricultural and food chemistry. 2002 May 8;50(10):2772-7.

[00174] 23. Giuliani F, Metz LM, Wilson T, Fan Y, Bar-Or A, Yong VW. Additive effect of the combination of glatiramer acetate and minocycline in a model of MS. Journal of neuroimmunology. 2005 Jan;158(1-2):213-21.

[00175] 24. Sloka S, Metz LM, Hader W, Starreveld Y, Yong VW. Reduction of microglial activity in a model of multiple sclerosis by dipyridamole. Journal of

neuroinflammation. 2013; 10:89.

[00176] 25. Montalban XH, B.; Rammohan, K.; Giovannoni, G.; de Seze, J.; Bar- Or, A.; Arnold, D.L.; Sauter, A.; Kakarieka, A.; Masterman, D.; Chin, P.; Garren, H.; Wolinsky, J.; on behalf of the ORATORIO Clinical Investigators. Efficacy and safety of ocrelizumab in primary progressive multiple sclerosis - results of the placebo-controlled, double-blind, Phase III ORATORIO study. ECTRIMS Online Library. 2015.

[00177] 26. Kappos L, Bar-Or, A.; Cree, B.; Fox, R.; Giovannoni, G.; Gold, R.; Vermersch, P.; Arnould, S.; Sidorenko, T.; Wolf, C; Wallstroem, E.; Dahlke F. Efficacy and safety of siponimod in secondary progressive multiple sclerosis - Results of the placebo controlled, double-blind, Phase III EXPAND study. ECTRIMS Online Library. 2016. [00178] 27. Kutzelnigg A, Lucchinetti CF, Stadelmann C, et al. Cortical demyelination and diffuse white matter injury in multiple sclerosis. Brain. 2005 Nov; 128(Pt 11):2705-12.

[00179] 28. Baker D, O'Neill JK, Gschmeissner SE, Wilcox CE, Butter C, Turk JL. Induction of chronic relapsing experimental allergic encephalomyelitis in Biozzi mice. Journal of neuroimmunology. 1990;28(3):261-70.

[00180] 29. Metz LM, Zhang Y, Yeung M, et al. Minocycline reduces gadolinium- enhancing magnetic resonance imaging lesions in multiple sclerosis. Ann Neurol. 2004 May;55(5):756.

[00181] 30. Metz LM, Li D, Traboulsee A, et al. Glatiramer acetate in combination with minocycline in patients with relapsing-remitting multiple sclerosis: results of a Canadian, multicenter, double-blind, placebo-controlled trial. Multiple sclerosis (Houndmills,

Basingstoke, England). 2009 Oct; 15(10): 1 183-94.

[00182] 31. Sorensen PS, Sellebjerg F, Lycke J, et al. Minocycline added to subcutaneous interferon beta-1a in multiple sclerosis: randomized RECYCLINE study.

European journal of neurology. 2016 May;23(5):861-70.

[00183] 32. van den Borne BE, Dijkmans BA, de Rooij HH, le Cessie S, Verweij CL. Chloroquine and hydroxychloroquine equally affect tumor necrosis factor-alpha, interleukin 6, and interferon-gamma production by peripheral blood mononuclear cells. The Journal of rheumatology. 1997 Jan;24(1):55-60.

[00184] 33. Sperber K, Quraishi H, Kalb TH, Panja A, Stecher V, Mayer L.

Selective regulation of cytokine secretion by hydroxychloroquine: inhibition of interleukin 1 alpha (IL-1-alpha) and IL-6 in human monocytes and T cells. The Journal of rheumatology. 1993 May;20(5):803-8.

[00185] 34. Lu JQ, Ringrose J, Gross D, Emery D, Blevins G, Power C. Multifocal inflammatory demyelination in a patient with rheumatoid arthritis and treatment

complications. Journal of the neurological sciences. 2016 Aug 15;367:305-7.

[00186] 35. Bertolaccini ML, Contento G, Lennen R, et al. Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome. Journal of autoimmunity. 2016 May 6. [00187] 36. Watkins LM, Neal JW, Loveless S, et al. Complement is activated in progressive multiple sclerosis cortical grey matter lesions. Journal of neuroinflammation. 2016; 13(1):161.

[00188] 37. Romme Christensen J, Bornsen L, Ratzer R, et al. Systemic inflammation in progressive multiple sclerosis involves follicular T-helper, Th17- and activated B-cells and correlates with progression. PloS one. 2013;8(3):e57820.

[00189] 38. Fitzner B, Hecker M, Zettl UK. Molecular biomarkers in cerebrospinal fluid of multiple sclerosis patients. Autoimmunity reviews. 2015 Oct; 14(10):903-13.

[00190] 39. Komori M, Lin YC, Cortese I, et al. Insufficient disease inhibition by intrathecal rituximab in progressive multiple sclerosis. Annals of clinical and translational neurology. 2016 Mar;3(3):166-79.

[00191] 40. Wells JE, Hurlbert RJ, Fehlings MG, Yong VW. Neuroprotection by minocycline facilitates significant recovery from spinal cord injury in mice. Brain. 2003 Jul;126(Pt 7):1628-37.

[00192] 41. Goncalves DaSilva A, Yong VW. Matrix metalloproteinase-12 deficiency worsens relapsing-remitting experimental autoimmune encephalomyelitis in association with cytokine and chemokine dysregulation. Am J Pathol. 2009 Mar;174(3):898- 909.

[00193] Although the disclosure has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.

[00194] All publications, patents and patent applications mentioned in this

Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.