WO2020061636A1 - Treatment of neuropathic pain - Google Patents

Abstract

The present invention relates to the treatment of neuropathic pain using NFE2L2 activator compounds of Formula (I), where R₁ is selected from C₁-C₃ alkyl and R₂ is selected from optionally substituted C₁-C₃ alkyl, or a pharmaceutically acceptable salt, solvate or isomer thereof. Preferred compounds of Formula (I) include dimethyl fumarate, tepilamide fumarate and diroximel fumarate.

Description

TREATMENT OF NEUROPATHIC PAIN

FIELD

The present invention relates to the treatment of neuropathic pain.

BACKGROUND

Neuropathic pain, caused by nervous system lesion or disease, has an estimated prevalence of 7-10% in the general population and is a tremendous burden to the economy and the patient’s quality of life. Pharmacological treatment of such pain relies primarily upon monoamine reuptake inhibitors, anticonvulsant agents, and opioids. First line treatments include amitriptyline, duloxetine, gabapentin and pregabalin. Serotonin and norepinephrine reuptake inhibitors (SNRIs) are a drug class often recommended of which duloxetine is a member. These drags have only modest efficacy and are also plagued by adverse effects and risk for misuse and abuse. Several strategies have been proposed to realize new and non-addictive treatments for chronic pain, including development of drugs that target endogenous pain-resolution mechanisms and that simultaneously modify multiple pathophysiological mechanisms that underlie pain.

Because oxidative stress appears to be a key mechanistic node for neuropathic pain, the present inventors focussed on the master regulator of the antioxidant response-nuclear factor erythroid 2-related factor 2 (NFE2L2; Nrf2)-as an alternative target for neuropathic pain. The inventors aimed to test whether certain compounds would activate NFE2L2 and promote antioxidant activity to reverse mitochondrial dysfunction and neuroinflammation.

There have been many attempts to pharmacologically control oxidative stress in chronic pain states; to date, none are in clinical use. Supplementation of individual antioxidants has not only failed due to unfavourable pharmacokinetics, but also because numerous antioxidants are required to restore homeostasis by coliaborativeiy catabolizing reactive oxygen species.

Investigation of the disease-modifying potential of NFE2L2 activation for neuropathic pain is limited. NFE2L.2 activators sulforaphane and cobalt protoporphyrin are anti -nociceptive in several rodent models of neuropathic pain. To date, these agents have not been clinically translated, and there has been little Investigation of the therapeutic effects on known pain mechanisms

The present invention seeks to address some of the shortcomings of the prior art therapeutics and is directed to a specific class of compounds which activate NFE2L2 and are now shown, for the first time, to be useful in the alleviation of neuropathic pain.

SUMMARY OF THE INVENTION

In one aspect the invention provides a method of treating neuropathic pain including the step of administering to a subject in need thereof a compound of formula (I):

wherein

Ri is selected from C1-C3 alkyl;

R₂ is selected from optionally substituted C1-C3 alkyl,

or a pharmaceutically acceptable salt, solvate, or isomer thereof.

In an embodiment the method relates to the treatment of peripheral neuropathic pain. DESCRIPTION OF THE FIGURES

Figure 1. Effects of dimethyl fumarate treatment on reflexive and operant nociceptive measures following SNI. (A) Beginning 14 days after SNI or sham surgery, dimethyl fumarate (DMF) or vehicle was orally administered q.d. for 7 days (Day 1 and 2: 30 mg kg-l; Day 3 and 4: 100 mg kg-l, Day 5-7: 300 mg kg-l). Mechanical allodynia was assessed using the von Frey test. SNI-induced allodynia was attenuated by dimethyl fumarate in a dose-dependent fashion. n=4 rats/group. (B, C) Beginning 14 days after SNI or sham surgery, dimethyl fumarate or vehicle was orally administered for 5 days (300 mg kg ¹ q.d.). (B) Von Frey thresholds for mechanical allodynia. n=6 rats/group. (C) Latency to cross noxious probes and enter the dark compartment during the final 300 s mechanical conflict- avoidance test after 4 days of treatment. n=5 in the naive-dimethyl fumarate group, n=6 rats in all other groups. Data are mean ± SD or individual data points; *P<0.05, **P<0.0l, ***P<0.00l. Figure 2. NFE2L2 activation in lumbar DRG. (A-D) Ipsilateral L4/5 DRG were collected after 5 days of oral dimethyl fumarate treatment (300 mg kg ¹ q.d.) or vehicle, which began 14 days after SNI/sham surgery. (A) NFE2L2 and nuclei were

immunofluorescently labelled in DRG sections. The number of D API-positive nuclei colocalized with NFE2L2 are expressed as a percentage of the total number of nuclei in each section. (B) Representative fluorescent photomicrographs are presented (40x). Scale bar = 50 pm. (C) NFE2L2 protein levels were assayed in nuclear extracts using Western blotting. Blot density (normalized to histone H3 loading control) and relative to the sham- vehicle condition is presented. (D) Representative blots are presented. Data are mean ±

SD; *P<0.05, **R<0.01, ***P<0.00l; DRG from n=6 rat/group.

Figure 3. Effects of dimethyl fumarate treatment on antioxidants and oxidized DNA/RNA after SNI. Ipsilateral L4/5 DRGs were collected after 5 days of oral dimethyl fumarate (DMF) treatment (300 mg kg ¹ q.d.) or vehicle (Veh), which began 14 days after SNI/sham surgery. mRNA expression was quantified for enzymes involved in glutathione synthesis and activity (A) Gclm, (B) Gclc, (C) Gsr, (D) as well as total glutathione protein levels. mRNA for superoxide dismutase (SOD) isoforms (E) Sodl, (F) Sod2 and (G) total SOD activity were quantified. (H) Levels of 8-oxo-dG/8-oxo-G, a marker of DNA/RNA oxidative damage. Data are mean ± SD; *P<0.05, **R<0.01, ***P<0.00l; DRG from n=6 rats/group.

Figure 4. Effects of NFE2L2 on anti-nociceptive effects of dimethyl fumarate. (A)

Beginning 14 days after SNI or sham surgery, the NFE2L2 inhibitor trigonelline (300 mg kg ¹ b.i.d.) or vehicle was administered together with dimethyl fumarate (300 mg kg ¹ q.d.) or vehicle for 5 days. Von Frey thresholds for mechanical allodynia are presented n = 5 rats in the sham-vehicle-trigonelline and SNI-vehicle-trigonelline groups; n=6 rats in all other groups (B, C) Ipsilateral L4/5 DRGs were collected after 5 days of oral

coadministration of the NFE2L2 inhibitor trigonelline (300 mg kg ¹ b.i.d.) or vehicle with dimethyl fumarate (300 mg kg ¹ q.d.) or vehicle (Veh), which began 14 days after SNI surgery. NFE2L2 and nuclei were immunofluorescently labelled in DRG sections. (B) The number of DAPI-positive nuclei colocalized with NFE2L2 are expressed as a percentage of the total number of nuclei in each section. DRG from n=6 rats/group. (C) Representative fluorescent photomicrographs are presented (40x). Scale bar = 50 pm. (D) Beginning 7 days after SNI surgery, dimethyl fumarate (300 mg kg ¹ q.d.) or vehicle was administered for 5 days to wild-type or Nfe2l2-I- male and female mice (n=4 per sex). Von Frey thresholds for mechanical allodynia are presented, with data pooled from both sexes. Data are mean ± SD; *P<0.05, **R<0.01, ***P<0.00l.

Figure 5. ATF3 levels in lumbar DRG. Ipsilateral L4/5 DRGs were collected after 5 days of oral dimethyl fumarate treatment (300 mg kg ¹ q.d.) or vehicle, which began 14 days after SNI/sham surgery. ATF3 and DAPI were immunofluorescently labelled in DRG sections. (A) The number of nuclei colocalized with ATF3 are expressed as a percentage of the total number of nuclei in each section. (B) Representative fluorescent

photomicrographs are presented (40x). Data are mean ± SD; *P<0.05, ***P<0.00l; DRG from n=6 rats/group.

Figure 6. Effects of dimethyl fumarate treatment on mitochondrial bioenergetics after

SNI. Ipsilateral L4/5 DRGs were collected and dissociated after 5 days of oral dimethyl fumarate treatment (300 mg kg ¹ q.d.) or vehicle, which began 14 days after SNI/sham surgery. (A) A summary of mitochondrial bioenergetics is presented, and (B) basal respiration, (C) ATP-linked respiration (response to oligomycin), (D) maximal

mitochondrial respiration (response to FCCP), and (E) spare respiratory capacity

(difference between maximal and basal OCR) were quantified. Data are mean ± SD;

*P<0.05, **R<0.01, ***P<0.00l; DRG neurons from n=5 rats in the sham-dimethyl fumarate and SNI-dimethyl fumarate groups, n=6 rats in the sham- vehicle group, and n=7 rats in the SNI- vehicle group.

Figure 7. Effects of dimethyl fumarate treatment on expression of proinflammatory cytokines. Ipsilateral L4/5 DRGs were collected after 5 days of oral dimethyl fumarate treatment (300 mg kg ¹ q.d.) or vehicle, which began 14 days after SNI/sham surgery. mRNA expression was quantified for (A) Illb, (C) Ccl2, and (E) Tnf; protein levels for (B) IL-lp and (D) CCL2 were assayed. Data are mean ± SD; *P<0.05, **R<0.01, ***P<0.00l; DRG from n=6 rats/group.

Figure 8. Effects of tepilamide fumarate and diroximel fumarate relative to

gabapentin treatment on reflexive nociceptive measures following SNI. Beginning 14 days after SNI, tepilamide fumarate (100 mg kg ¹ q.d.), diroximel fumurate (100 mg kg ¹ q.d.), gabapentin (30 mg kg ¹ q.d.) or vehicle was orally administered for 5 days. Von Frey thresholds for mechanical allodynia. n=6 rats/group.

DETAILED DESCRIPTION OF THE INVENTION

In a further aspect of the invention there is provided a pharmaceutical composition for treating neuropathic pain, the composition comprising an effective amount of a compound of formula (I) and related formulae as herein defined or a pharmaceutically acceptable salt thereof and optionally a carrier or diluent.

In an embodiment Ri is methyl.

In an embodiment R₂ is methyl or optionally substituted ethyl.

In certain embodiments the compounds of formula (I) are represented by formula (la):

where R₂ is selected from:

a) CEL; b) CH2CH3;

wherein:

Y represents a N-containing optionally substituted heterocyclyl;

R³ and R⁴ are independently C₁-C₃ alkyl; and n is 1 or 0.

In certain embodiments n is 1.

In certain embodiments R² and R³ are both CH2CH3.

In certain embodiments Y is a 5 or 6 membered optionally substituted heterocyclyl.

In certain embodiments Y is selected from the following:

Representative compounds of formula (I)/(Ia) include:

O (dimethyl fumurate (DMF))

(Tecfidera) The salts of the compounds of the invention are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts.

The pharmaceutically acceptable salts include acid addition salts, base addition salts, and the salts of quaternary amines and pyridiniums. The acid addition salts are formed from a compound of the invention and a pharmaceutically acceptable inorganic or organic acid including but not limited to hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, toluenesulphonic, benzenesulphonic, acetic, propionic, ascorbic, citric, malonic, fumaric, maleic, lactic, salicylic, sulfamic, or tartaric acids. The counter ion of quaternary amines and pyridiniums include chloride, bromide, iodide, sulfate, phosphate, methansulfonate, citrate, acetate, malonate, fumarate, sulfamate, and tartrate. The base addition salts include but are not limited to salts such as sodium, potassium, calcium, lithium, magnesium, ammonium and alkylammonium. Also, basic nitrogen-containing groups may be quaternised with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others. The salts may be made in a known manner, for example by treating the compound with an appropriate acid or base in the presence of a suitable solvent.

The compounds of the invention may be in crystalline form and/or as solvates (e.g. hydrates) and it is intended that both forms be within the scope of the present invention. The term "solvate" is a complex of variable stoichiometry formed by a solute (in this invention, a compound of the invention) and a solvent. Such solvents should not interfere with the biological activity of the solute. Solvents may be, by way of example, water, ethanol or acetic acid. Methods of solvation are generally known within the art.

It will be appreciated that the compounds of the invention may have at least one asymmetric centre, and therefore are capable of existing in more than one stereoisomeric form. The invention extends to each of these forms individually and to mixtures thereof, including racemates. The isomers may be separated conventionally by chromatographic methods or using a resolving agent. Alternatively, the individual isomers may be prepared by asymmetric synthesis using chiral intermediates.

The invention also includes where possible a salt or pharmaceutically acceptable derivative such as a pharmaceutically acceptable ester, solvate and/or prodrug of the above mentioned embodiments of the invention.

In another aspect of the invention, there is provided a pharmaceutical composition that comprises a therapeutically effective amount of one or more of the aforementioned compounds or pharmaceutically acceptable salts thereof, including pharmaceutically acceptable derivatives thereof, and optionally a pharmaceutically acceptable carrier or diluent.

The term "composition" is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.

The pharmaceutical compositions or formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal, intrathecal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

The compounds of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.

Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.

The compounds of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt of a compound of the invention.

For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispensable granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilisers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid that is in a mixture with the finely divided active component.

In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration. For preparing suppositories, a low melting wax, such as an admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

Sterile liquid form compositions include sterile solutions, suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent or a mixture of both.

The compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, eg. sterile, pyrogen-free water, before use.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.

Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.

Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump. To improve nasal delivery and retention the compounds according to the invention may be encapsulated with cyclodextrins, or formulated with other agents expected to enhance delivery and retention in the nasal mucosa.

Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.

Alternatively, the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 5 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation.

When desired, formulations adapted to give sustained release of the active ingredient may be employed.

The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The invention also includes the compounds in the absence of carrier where the compounds are in unit dosage form. The amount of the compound of the invention to be administered may be in the range from about 10 mg to 2000 mg per day, depending on the activity of the compound and the disease to be treated.

Liquids or powders for intranasal administration, tablets or capsules for oral administration and liquids for intravenous administration are the preferred compositions.

The pharmaceutical preparations of the compounds according to the present invention may be co-administered with one or more other active agents in combination therapy. For example, the pharmaceutical preparation of the active compound may be co-administered (for example, separately, concurrently or sequentially), with one or more other agents used to treat pain.

The compounds of the invention have been shown to be beneficial in treating neuropathic pain. The inventors have focused on the transcription factor nuclear factor erythroid 2- related factor 2 (NFE2L2; Nrf2). Under physiological conditions, NFE2L2 is sequestered in the cytosol by Kelch-like ECH associated-protein 1 (Keapl) and ubiquitinated for degradation. However, oxidants and electrophiles trigger release of NFE2L2 from Keapl, translocation to the nucleus, and binding to the antioxidant response element that initiates transcription of >200 antioxidant-related genes. Thus, NFE2L2 was considered to be an attractive therapeutic target to stimulate endogenous production of the multiple antioxidants required to simultaneously detoxify a range of reactive oxygen species. Here, the inventors evaluated the therapeutic actions of dimethyl fumarate (Tecfidera) and related compounds in the rat spared nerve injury (SNI) model of neuropathic pain. Dimethyl fumarate is EMA- and FDA-approved for relap sing-remitting multiple sclerosis. It was observed that these compounds had the ability to reverse neuropathic pain behaviours, activate NFE2L2, and resolve mechanistic pathways that maintain neuropathic pain. Accordingly, for the first time, dimethyl fumarate and related fumurates of formulae (I) or (la) have been shown to be useful non-opioid alternatives to the treatment of neuropathic pain.

Neuropathic (nerve) pain is caused by damage, injury or dysfunction of nerves due to trauma, surgery, disease or chemotherapy. It is often described as burning, painful, cold or akin to electric shocks and may manifest with tingling, pins and needles, numbness or itching. Neuropathic pain can be the primary symptom of a particular condition or disease state, such as cancer, complex regional pain syndrome or post herpetic neuralgia. It can also be associated with other medical conditions or other forms of pain, including pelvic pain, fibromyalgia and orofacial pain. Phantom pain following a limb amputation is also a type of neuropathic pain.

It is also contemplated that the term neuropthatic pain also encompasses "peripheral neuropathic pain" which is generally defined as pain arising as a direct or indirect consequence of a lesion or disease affecting the peripheral somatosensory system. Peripheral neuropathic pain includes all types of peripheral neuropathic pain, caused by for instance peripheral diabetic neuropathy type 1 or 2, induced by various noxious substances such as alcohol, caused by various deficiencies such as vitamin Bl, B6 and/or B12 deficiency, various intoxications, such as hypervitaminosis B6, caused by hypothyroidism, chemotherapy induced polyneuropathy (CIPN) (due to chemotherapeutic agents such as : alkylating agents, such as cis-platinum(II)-diaminedichloride (platinol or cisplatin); oxaliplatin (Eloxatin or Oxaliplatin Medac); and carboplatin (Paraplatin); antitumour antibiotics, including those selected from the group comprising anthracyclines, such as doxorubicin (Adriamycin, Rubex); antimetabolites, including folic acid analogues such as pyrimidine analogues such as 5-fluorouracil (Fluoruracil, 5-FU), gemcitabine (Gemzar), or histone deacetylase inhibitors (HDI) for instance, Vorinostat (rINN); natural alkaloids, including paclitaxel (Taxol); inhibitors of protein tyrosine kinases and/or serine/threonine kinases including Sorafenib (Nexavar), Erlotinib (Tarceva), Dasatanib (BMS-354825 or Sprycel)), drug-induced neuropathy, some compounds for the treatment of infectious diseases (e.g. streptomycin, didanosine or zalcitabine), or other other physiologically toxic compounds. Other peripheral neuropathies that can cause peripheral neuropathic pain include: small fiber neuropathy (SFN), hereditary motor and sensory neuropathies (HMSN), chronic inflammatory demyelinating polyneuropathy (CIDP), trigeminal neuralgia, post-herpetic neuralgia, intercostal neuralgia, entrapment neuropathies (e.g. carpal tunnel syndrome, tarsal tunnel syndrome, abdominal cutaneous nerve entrapment syndrome), sciatic pain, chronic idiopathic axonal polyneuropathy (CIAP), vulvodynia, proctodynia, neuropathy due to infectious disease conditions, such as post-polio syndrome, AIDS or HIV-associated, lyme associated, Sjogren-associated, lymphomatous neuropathy, myelomatous neuropathy, carcinomatous neuropathy, vasculitic/ischaemic neuropathy and other mono- and polyneuropathies.

In an embodiment the invention contemplates the treatment of neuropathic pain associated with chemotherapy - often a side-effect when treating solid tumors. Examples of solid tumors include adrenocortical carcinoma, anal tumor/cancer, bladder tumor/cancer, bone tumor/cancer (such as osteosarcoma), brain tumor, breast tumor/cancer, carcinoid tumor, carcinoma, cervical tumor/cancer, colon tumor/cancer, endometrial tumor/cancer, esophageal tumor/cancer, extrahepatic bile duct tumor/cancer, Ewing family of tumors, extracranial germ cell tumor, eye tumor/cancer, gallbladder tumor/cancer, gastric tumor/cancer, germ cell tumor, gestational trophoblastic tumor, head and neck tumor/cancer, hypopharyngeal tumor/cancer, islet cell carcinoma, kidney tumor/cancer, laryngeal tumor/cancer, leiomyosarcoma, leukemia, lip and oral cavity tumor/cancer, liver tumor/cancer (such as hepatocellular carcinoma), lung tumor/cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal tumor/cancer, neuroblastoma, oral tumor/cancer, oropharyngeal tumor/cancer, osteosarcoma, ovarian epithelial tumor/cancer, ovarian germ cell tumor, pancreatic tumor/cancer, paranasal sinus and nasal cavity tumor/cancer, parathyroid tumor/cancer, penile tumor/cancer, pituitary tumor/cancer, plasma cell neoplasm, prostate tumor/cancer, rhabdomyosarcoma, rectal tumor/cancer, renal cell tumor/cancer, transitional cell tumor/cancer of the renal pelvis and ureter, salivary gland tumor/cancer, Sezary syndrome, skin tumors (such as cutaneous t-cell lymphoma, Kaposi's sarcoma, mast cell tumor, and melanoma), small intestine tumor/cancer, soft tissue sarcoma, stomach tumor/cancer, testicular tumor/cancer, thymoma, thyroid tumor/cancer, urethral tumor/cancer, uterine tumor/cancer, vaginal tumor/cancer, vulvar tumor/cancer, and Wilms' tumor. In one embodiment, the pain is associated with treating the following cancers: bladder cancer, breast cancer, colon cancer, gastroenterological cancer, kidney cancer, lung cancer, including non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, proximal or distal bile duct cancer, or melanoma. In another embodiment the present invention contemplates the treatment of neuropathic pain associated with pain complications arising from an infection such as a bacterial, fungal or viral infection. Examples include shingles, HIV/AIDS, etc.

In still a further embodiment the present invention contemplates the treatment of neuropathic pain associated with back pain, rheumatoid arthritis, trigeminal neuralgia, or diabetic neuropathy.

In another further embodiment the present invention contemplates the treatment of neuropathic pain caused by nerve compression (trapped nerve). Examples include carpal tunnel syndrome or sciatica.

In another further embodiment the present invention contemplates the treatment of neuropathic pain associated with stroke.

For certain of the abovementioned conditions it is clear that the compounds may be used prophylactically as well as for the alleviation of symptoms. Thus references herein to "treatment" or the like are to be understood to include such prophylactic treatment, as well as therapeutic treatments.

DEFINITIONS

The term "alkyl" as used alone or in combination herein refers to a straight or branched chain saturated hydrocarbon group. The term "Ci-io alkyl" refers to such a group containing from one to ten carbon atoms. Examples include methyl ("Me"), ethyl ("Et"), n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like.

The term "heterocyclyl" refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur, oxygen, selenium or phosphorous within the ring.

Examples of 5-membered monocyclic heterocyclyl groups include tetrahydrofuran, pyrrolinyl, pyrrolidinyl, pyrazolyl, and imidazolinyl. Examples of 6-membered monocyclic heterocyclyl groups include piperidinyl, 1,4- dioxanyl, morpholinyl, l,4-dithianyl, thiomorpholinyl, and piperazinyl.

The term "optionally substituted" means that a group may include one or more substituents. One or more hydrogen atoms on the group may be replaced by substituent groups independently selected from halogens (for example halo alkyl such as -CF₃), Ci-₆ alkyl, C_2-6 alkenyl, C2-6 alkynyl, -(CH₂)_pC_3-7 cycloalkyl, -(CH₂)pC₄-7 cycloalkenyl, -(CH₂)_P aryl, -(CH₂)_P heterocyclyl, -(CH₂)_P heteroaryl, -C₆H₄S(0)_qCi-₆ alkyl, -C(Ph)₃, -CN, -OR^c, - 0-(CH₂)I-₆-R^c, -0-(CH₂)I-₆-0R^c, -0C(0)R^c, -C(0)R^c, -C(0)0R^c, -0C(0)NR^dR^e, -NR^dR^e, -NR^cC(0)R^d, -NR^cC(0)NR^dR^e, -NR^cC(S)NR^dR^e, -NR^cS(0)₂R^d, -NR^cC(0)0R^d, -C(NR^c)NR^dR^e, -C(=NOR^d)R^c, -C (=N OH)NR^dR^e, -C(0)NR^dR^e, -C(=NCN)-NR^dR^e, -C(=NR^c)NR^dR^e, -C(=NR^d)SR^e, -NR^dC(=NCN)SR^e, -C0NR^cS0₂R^d, -C(S)NR^dR^e, - S(0)_qR^c, -S0₂NR^dR^e, -S0₂NR^cC(0)R^d, -0S(0)₂R^c, -PO(OR^c)₂ and -N0₂;

where p is 0-6, q is 0-2 and each R^c, R^d and R^e is independently selected from H, C1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C₃-7 cycloalkyl, C₄-7 cycloalkenyl, aryl, heterocyclyl, heteroaryl, C1-6 alkylaryl, C1-6 alkylheteroaryl, and C1-6 alkylheterocyclyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, C1-6 alkylaryl, C1-6 alkylheteroaryl, or C1-6 alkylheterocyclyl, may be optionally substituted with one to six of same or different groups selected from halogen, hydroxy, lower alkyl, lower alkoxy, -C0₂H, CF₃, CN, phenyl, NH₂ and -N0₂; or when R^d and R^e are attached to the same nitrogen atom, they may, together with the atom to which they are attached, form a 5 to 7 membered nitrogen containing heterocyclic ring.

A list of preferred optional substituents includes: fluoro, C1-6 alkyl, C1-6 alkoxy, C_2-6 alkenyl, C_2-6 alkynyl, Ci-₆ haloalkyl (in particular -CF₃), Ci-₆ haloalkoxy (such as -OCF₃), - OH, phenyl, benzyl, phenoxy, benzyloxy, benzoyl, -NH₂, -NHCI-₄ alkyl, -N(CI-₄ alkyl)₂, -CN, -N0₂, mercapto, -P=0(OH)(NH₂), -S(0)₂NH₂, -NHS(0)₂NH₂, -S(0)₂NHCi_₄ alkyl, - S(0)₂N(Ci-₄ alkyl)₂, Ci-6 alkylcarbonyl, Ci-6 alkoxycarbonyl, C0₂H, -S(0)R"' (where R'" is lower alkyl or cycloalkyl) and -S(0)₂R"' (where R'" is lower alkyl, cycloalkyl or OH).

Compounds of the invention may be prepared according to the following general scheme A below: Scheme A:

R¹OH, acid

Eq. 1

or R’X

Esterification

Eq. 2

R²OH or R²X

1. R¹OH

2. AICI₃ O Esterification O

Eq. 3

Equation 1 outlines the synthesis of (I) through acid catalysed reaction of fumaric acid (1) with alcohol R'OH, in the case where R¹ = R². Acids such as sulfuric acid and hydrochloric acid are known to affect this transformation. Esterification could also be carried out through reaction of fumaric acid (1) with R^xX (where X represents halogen atoms, chlorine, bromine or iodine and R¹ = R²) in the presence of a base such as potassium carbonate.

Equation 2 outlines the synthesis of (I) through the conversion of monomethyl fumarate (2) to an acid chloride via reaction with thionyl chloride or oxalyl chloride, which can then be reacted with alcohol R²OH to give ester (I). Numerous alternative esterification procedures could be used such as direct coupling of carboxylic acid (2) with alcohol R²OH in the presence of dicyclohexyldiimide (DCC) or other diimides using Steglich conditions or utilising other coupling agents such as 0-(7-azobenzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexa fluorophosphate (HATU) or propylphosphonic anhydride (T3P) or via the Mitsunobu reaction. Ester (I) could also be generated from carboxylic acid (2) through the generation of a mixed anhydride and coupling with alcohol R²OH. Esterification could also be carried out through reaction of carboxylic acid (2) with R²X (where X represents halogen atoms, chlorine, bromine or iodine) in the presence of a base such as potassium carbonate. Equation 3 outlines the reaction of maleic anhydride (3) with alcohol R'OH, followed by reaction with aluminium trichloride to give carboxylic acid (4). Esterification of carboxylic acid (4) can be achieved using the same methodology used to esterify carboxylic acid (2).

Another variation is to add, remove or modify the substitutents of the product to form new derivatives. This could be achieved again by using standard techniques for functional group interconversion, well known in the industry such as those described in Comprehensive Organic Transformations: A Guide to Functional Group Preparations by Larock R C, New York, VCH Publishers, Inc. 1989.

The invention will now be further described based on the following non-limiting examples.

EXAMPLES

Materials and Methods

Animals

Pathogen-free adult male Sprague Dawley rats (10 weeks old on arrival, Envigo, USA) were used. Rats were housed 2-3 per cage in a light- and temperature-controlled room (l2:l2-h light-dark cycle, lights on at 7:00 am) with food and water available ad libitum. Pre-treatment weights ranged from 309-342 g. Male and female (10-14 weeks old) wild- type (WT) and Nfe2l2 ' mice on a C57BL/6J genetic background (Jackson Laboratory, Bar Harbor, ME) were housed (5 per cage) and bred at The University of Texas MD Anderson Cancer Center. The animal facility is pathogen-free and AAALAC-accredited. Pre treatment weights ranged from 19-26 g. To detect a reversal of von Frey threshold from 0.4 g to 4 g with a standard deviation of 0.41 g, a two-tailed power calculation indicated that n=5 rats per group would be sufficient with Type I error set at 0.05 and the power at 90%. The expected variance for biochemical outcomes is smaller, and therefore a group size of 5 rats would be sufficient for these measures as well. The rats were randomly assigned to groups using a random number generator (Graphpad, San Diego, CA, USA). No animals were excluded from this study for any reason. All procedures were approved by the MD Anderson Cancer Center Institutional Animal Care and Use Committee and conformed to the National Academy of Science Guide for the Care and Use of Laboratory Animals. Spared nerve injury (SNI) surgery

SNI surgery was performed as described for rats and mice, leaving the sural nerve intact, under inhaled isoflurane anesthesia (2-4 % vol in 1 liters min^-1 oxygen). The surgical plane of anesthesia was verified by areflexia. Ophthalmic ointment was applied to the eyes before surgery commenced. Identical procedures were used for sham surgery, but the nerves were not ligated or transected. Naive rats were left undisturbed in their home cages. Animals were monitored post-operatively until fully ambulatory prior to return to their home cage. All surgeries took place between 9:00 am and 2:00 pm. Postoperative analgesia was not provided so as not to confound the endpoints under study.

Drug administrations

Dimethyl fumarate (Sigma-Aldrich, St. Louis, MO, USA) was suspended with

methylcellulose (viscosity 15 cP, 2% w v ¹ in water; ACROS, Geel, Belgium) and administered by oral gavage. In our preliminary experiment, rats received escalating daily doses of dimethyl fumarate (Days 1 and 2: 30 mg 5 ml ¹ kg ¹; Days 3 and 4: 100 mg 5 ml ¹ kg ¹; Days 5-7: 300 mg 5 ml ¹ kg ¹) beginning 14 days after SNI/sham surgery. In all subsequent experiments, rats and mice were administered dimethyl fumarate for 5 days (300 mg 5 ml ¹ kg ¹, q.d.), beginning 14 days after SNI/sham surgery. Dosing took place from 8:00-10:00 am. Trigonelline (Cayman Chemical, Ann Arbor, MI, USA) was suspended with methylcellulose and administered to rats by oral gavage for 5 days (300 mg 5 ml ¹ kg ¹, b.i.d.), beginning 14 days after SNI/sham surgery. Dosing took place from 8:00-10:00 am and 4:00-6:00 pm. Equivolume methylcellulose (2% w v ¹) was used as vehicle control for both drugs. An independent investigator dosed the rats in order to maintain blinding to treatment groups for the other investigators who performed the behavioral testing and assays.

Tepilamide fumurate and Diroximel fumurate were prepared based on the synthetic protocols outlined in Published International Patent applications W02010022177 and WO2014152494 respectively. The administration protocol for these two other fumurates is as outline above for DMF except the dosage amounts where lOOmg/kg (cf DMF

300mg/kg). Mechanical allodynia

Testing was conducted blind with respect to group assignment. Rats received at least three 60-minute habituations to the test environment prior to behavioral testing. Rodents were placed in a small plexiglass enclosure on a mesh stand. The von Frey test was performed as described previously for rats, and mice, between 2 and 4 h of dosing and finishing before 12:00 pm. The behavioral responses were used to calculate absolute threshold (the 50% probability of response) by fitting a Gaussian integral psychometric function using a maximum-likelihood fitting method.

Mechanical conflict-avoidance

Voluntary aversion to a noxious stimulus was assessed using a commercial 3-chambered apparatus, the Mechanical Conflict-avoidance System (Noldus, Leesburg, Virginia, USA). The apparatus presents rats with a choice in responding to two aversive stimuli— either to remain exposed to an aversive bright light in one chamber or to escape the light by crossing a middle chamber having a floor covered by a dense array of sharp probes, in order to reach a dark, safe chamber. Longer latencies to escape the light chamber indicate increased motivation to avoid the probes, and this escape latency is currently the most common measure of pain -related behavior in this test. We performed the operant mechanical conflict- avoidance test on rats with modifications as recently described in detail. Naive rats were used in the control group, as we have previously shown that this assay is highly sensitive to postoperative pain after sham surgery. The test was performed over two days, with each day consisting of three 300 s trials. Testing was performed between 8:00 am and 2:00 pm. In each trial 1) a rat was placed inside the light chamber with the lid closed, the light off, and the exit door closed; 2) after 20 seconds the light was turned on; 3) after 15 seconds the exit door was opened when (or if) the rat faced the exit; 4) the rat freely explored all 3 chambers in the apparatus for 300 s, 5) the rat was returned to its home cage, and 6) the device was thoroughly cleaned with 70% ethanol and distilled water in preparation for the next trial. On day 1 (third day of drug treatment), the probe height was set to 0 mm for all three trials. On day 2 (fourth day of drug treatment), the probe height was set to 0 mm for the first trial, and then raised to 4 mm for the second and third trials. Data are presented as the latency to enter the dark compartment during the third trial on day 2. Tissue collection

Within 4 h of the final dose on day 5, rats were deeply anaesthetized with Beuthanasia-D (approximately 100 mg kg ¹ pentobarbital, 13 mg kg ¹ phenytoin; i.p.; Merck, USA) and then perfused with ice-cold saline. The ipsilateral lumbar DRG (L4/5) were then isolated. For immunohistochemistry, the saline perfusion was followed by ice-cold 4%

paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).

Immunohistochemistry

L4/5 DRG were post-fixed in 4% paraformaldehyde overnight at 4°C. Tissues were cryoprotected stepwise in 15%, 22% and 30% sucrose in 0.1 M phosphate buffer, supplemented with 0.01% sodium azide (Sigma- Aldrich). After freeze-mounting in O.C.T compound (Sakura Finetek, Torrance, CA, USA), tissues were serially sectioned at 10 pm across 10 slides. Each slide contained 5 sections per DRG so that 10 sections, per animal, were analyzed. Sections were incubated with blocking buffer (2% bovine serum albumin (Sigma- Aldrich), 5% normal goat serum (Abeam, USA), and 0.1% saponin (Sigma- Aldrich) in phosphate buffered saline) overnight at 4°C. Primary antibodies were added after washing and incubated at 4°C for 21 h. Subsequently, sections were washed, then incubated with secondary antibodies for 2 h at room temperature. Primary antibodies used were anti-NFE2L2 antibody (1:500; rabbit polyclonal IgG; Abeam) and anti-ATF3 antibody (1:500; mouse monoclonal IgGl; Santa Cruz Biotechnology, USA). Secondary antibodies used were goat anti-rabbit antibody and donkey anti-mouse antibody (1:500; ThermoFisher Scientific, Waltham, MA, USA). Fluorescent photomicrographs were captured using an SPE Leica Confocal Microscope (Leica Microsystems, Buffalo Grove, IL, USA) and an Olympus BX53 microscope with a mercury lamp (U-HGLGPS; Olympus Corporation, Tokyo, Japan). Sections were analysed using LAS X software (Leica) and cellSens imaging software (Olympus).

Western blotting

Nuclear fractions from ipsilateral L4/5 DRG of each rat were isolated with a NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific), according to manufacturer instructions. Western blotting was performed as previously described. In brief, extracted nuclear proteins were subjected to NuPAGE Bis-Tris (4-12%) gel electrophoresis under reducing conditions (ThermoFisher Scientific) and then electrophoretically transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA,

USA). Nonspecific binding sites on the membrane were blocked with Superblock buffer in TBS containing 0.1% Tween-20, 0.05% Tris-Chloride, and 0.03% 5 M NaCl (TBS-T) for 1 h at 22- 24 °C. Membranes were subsequently incubated overnight at 4 °C with primary anti-NFE2L2 antibody (1:1,000; rabbit polyclonal IgG; Abeam) and anti-histone H3 antibody (1:2,000; rabbit polyclonal IgG; Abeam) (loading control) in blocking buffer containing 0.1% Tween-20. The membranes were then washed with PBS containing 0.1% Tween-20, and probed with HRP secondary antibody (1:5,000; goat polyclonal IgG;

Jackson ImmunoResearch, West Grove, PA, USA) in blocking buffer containing 0.1% Tween-20 for 1 h at 22-24 °C. After washing with PBS containing 0.1% Tween-20, membranes were developed with enhanced chemiluminescent substrate (ThermoFisher Scientific), and scanned on an ImageQuant LAS 4000 mini (GE, Boston, MA, USA). Densitometry analysis was performed using ImageQuant TL software (GE). Data were normalized to loading control (histone H3).

Real-time polymerase chain reaction (PCR)

Total RNAs were extracted using TRIzol (ThermoFisher Scientific) from the DRG tissues. One pg RNA was used for reverse transcription with iScript Reverse Transcription Supermix (Bio-Rad). Real-time PCR was carried out in a final volume of 20 pL with iTaq Universal SYBR Green Supermix (Bio-Rad) containing 2 pL of 5 times diluted cDNA and monitored by CFX Connect Real-Time PCR Detection System (Bio-Rad). The following cycling parameters were used: 95 °C for 3 min; 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Primer sequences are reported in Table Sl. The level of the target mRNA was quantified relative to the housekeeping gene ( Gapdh ) using the AACT method. Gapdh was not significantly different between treatments.

Assay kits

DRG were dissociated with a mortar and pestle in tissue extraction reagent supplied with protease and phosphatase inhibitors as previously described. Assay kits for glutathione (703002, Cayman Chemical, Ann Arbor, MI, USA) (detection range: 0.25-16 pM) superoxide dismutase (706002, Cayman Chemical) (detection range: 0.005-0.050 U/ml), DNA/RNA oxidative damage (589320, Cayman Chemical) (detection range: 10.3-3,000 pg/ml), IL- 1 b (RLB00, R&D Systems, Minneapolis, MN) (detection range: 5-2,000 pg/ml), TNF (RTA00, R&D Systems) (detection range: 5-800 pg/ml), and CCL2

(abl00778, Abeam) (detection range: 24.67-18,000 mM), were used according to manufacturer instructions. Results were normalized to total protein levels (Bradford protein assay).

Mitochondrial bioenergetics

Analysis of mitochondrial bioenergetics was performed in dissociated DRG neurons using the Seahorse assay as previously described. Oxygen consumption rate (OCR), including basal respiration, ATP-linked respiration, proton leak, and maximal respiratory capacity, were determined as described previously by subtracting nonmitochondrial respiration.

Total protein levels quantified using standard Bradford protein assay in each well were used to normalize each OCR value.

Statistics

The Shapiro-Wilk test was performed to increase confidence that the data were normally distributed, while the Grubb’s test indicated that there were no outliers in our dataset. Mechanical allodynia was analyzed as the interpolated 50% thresholds (absolute threshold). Where appropriate, one-way ANQVAs or unpaired t tests were used to confirm that there were no baseline differences in absolute thresholds between treatment groups. Differences between treatment groups were determined using repeated measures two-way or three-way ANOVA, followed by Tukey’s post hoc tests, where appropriate. For the mechanical conflict avoidance assay, the rats were divided into two groups: those that entered the dark compartment at any time during the 300 s trial and those that did not. Data were analyzed using Chi-square tests. For biochemical assays, data were analyzed either by unpaired t tests or by two-way ANOVA followed by Tukey’s post hoc tests, as appropriate. Analyses were performed using Prism 8 (Graphpad). Complete statistical methods, comparisons, and results (F-statistics, P-values and 95% confidence intervals) are presented in Table S2. There were no missing data. Results are expressed as mean + standard deviation (SD). P<0.05 was considered statistically significant.

Results

Effects of dimethyl fumarate on reflex and operant nociceptive measures We assessed the anti-nociceptive effect of oral dimethyl fumarate treatment on established pain-related behaviors induced by SNI. In a preliminary experiment, cumulative dose escalation of dimethyl fumarate was evaluated to identify an effective dose. Dimethyl fumarate attenuated SNI-induced allodynia (time x treatment: F₆, 36 = 2.7859, P = 0.025; time: F₃, 36 = 2.451, P = 0.079; treatment: F₂, 36 = 166.9, P < 0.001). Post hoc analyses revealed that this drug was effective at 300 mg kg ¹, ( P = 0.007), but not at lower doses (Fig. 1A). To confirm the therapeutic efficacy of this dose in an independent experiment, dimethyl fumarate was administered daily for 5 days, beginning 14 days after SNI or sham surgery. Within three days, dimethyl fumarate treatment completely reversed mechanical allodynia, compared to vehicle treatment, an effect that was maintained until dosing conclusion (Fig. 1B; time x injury x treatment: F₄, so = 17.4, P < 0.001; injury x treatment: Fi, 20 = 96.6, P < 0.001; time x treatment: F₄, so = 28.0, P < 0.001; time x injury: F₄, so = 21.6, P < 0.001; treatment: Fi, 20 = 138, P < 0.001; injury: Fi, 20 = 467, P < 0.001; time: F₄, so = 23.5, P < 0.001) . In this experiment, we also noted a reduction in body weight in the dimethyl fumarate treated group (274 ± 13 g), compared to the vehicle treated group (325 ± 9 g) (P< 0.001).

The operant mechanical conflict- avoidance test was used to further evaluate the anti nociceptive properties of dimethyl fumarate in a task where mechanical stimulation is voluntary. Only 2/6 rats from the SNI-vehicle group crossed the 4 mm probes and entered the dark compartment. In contrast, 6/6 rats from the SNI-dimethyl fumarate group entered the dark compartment; this increased proportion of rats that crossed the noxious probes was statistically significant compared to the SNI-vehicle group ( P = 0.013) (Fig. 1C). At 0 mm (i.e., probes absent), all rats entered the dark compartment within 15 s (data not shown).

NFE2L2 activation by dimethyl fumarate in DRG

To determine whether dimethyl fumarate could activate NFE2L2 in the DRG as predicted (that is, induce nuclear translocation), we next examined NFE2L2 colocalization with the nuclear stain DAPI using immunohistochemistry (injury x treatment: Fi, 22 = 7.01, P = 0.015; injury: Fi, 22 = 0.428, P = 0.520; treatment: Fi, 22 = 72.07, P < 0.001). Dimethyl fumarate treatment increased the proportion of NFE2F2⁺ nuclei in both the sham (51 NFE2F2⁺ nuclei/81 total nuclei ± 22/29) (P<0.00l) and SNI groups (60/125 ± 17/52) (P = 0.002), compared to the sham-vehicle group (18/114 ± 10/34) (Fig. 2A). We found that the proportion of NFE2L2⁺ nuclei increased in the SNI- vehicle group (22/102 ± 8/34), compared to sham-vehicle was not statistically significant (P=0.538). Representative photomicrographs are presented in Fig. 2B.

We also measured NFE2L2 protein levels in the nuclear extracts using Western blotting as an orthogonal approach (Fig. 2C, D; injury x treatment: Fi, _l6 = 0.35, P = 0.561; injury: Fi, 16 = 0.01, P = 0.966; treatment: Fi, _l6 = 33.0, P < 0.001). Post hoc tests showed that dimethyl fumarate treatment increased levels of NFE2L2 in the sham group ( P = 0.002) and SNI group ( P = 0.011). NFE2L2 levels were not increased in the SNI-vehicle group, compared to sham-vehicle ( P = 0.969). Representative blots are presented in Fig. 2D.

Effects of dimethyl fumarate on antioxidant expression and activity

Because NFE2L2 activation drives expression of antioxidants, we evaluated the effect of dimethyl fumarate treatment on both the expression and activity of two representative antioxidants in DRG after SNI: glutathione and superoxide dismutase (SOD). These antioxidants are responsible for catabolism of superoxide, hydrogen peroxide and hydroxyl radicals. The expression of several genes involved in glutathione synthesis and activity were quantified. Dimethyl fumarate treatment increased expression of Gclm (Fig. 3A; injury x treatment: Fi, 20 = 4.78, P = 0.041; injury: Fi, 20 = 3.83, P = 0.065; treatment: Fi,

20 = 11.13, P = 0.003) and Gclc (Fig. 3B; injury x treatment: Fi, 20 = 0.26, P = 0.616;

injury: Fi, 20 = 1.65, P = 0.214; treatment: Fi, 20 = 14.0, P = 0.001), which encode subunits of glutamate-cysteine ligase (the first rate limiting enzyme in glutathione synthesis). The gene encoding glutathione reductase, Gsr, which catalyzes the reduction of glutathione disulfide to glutathione sulfhydryl, was not altered by dimethyl fumarate treatment (Fig. 3C; injury x treatment: F_I, 20 = 0.95, P = 0.341; injury: Fi, 20 = 0.17, P = 0.687; treatment: Fi , 20 = 1.94, P = 0.179). Gclm, but not Gclc or Gsr, was decreased in the SNI-vehicle group compared to sham-vehicle (P = 0.038) (Figs. 3A-C). Dimethyl fumarate treatment increased total glutathione protein levels in the DRG (Fig. 3D; injury x treatment: Fi, 20 = 7.11, P = 0.015; injury: Fi, 20 = 1.91, P = 0.183; treatment: Fi, 20 = 15.44, P < 0.001). Furthermore, total glutathione protein levels were decreased by SNI compared to sham- vehicle ( P = 0.044) (Fig. 3D). There was no effect of dimethyl fumarate in the sham groups. Dimethyl fumarate treatment rescued expression of Sod / (Figs. 3E; injury x treatment: Fi, 20 = 2.80, P = 0.110; injury: Fi, 20 = 78.800, P = 0.010; treatment: Fi, 20 = 13.82, P <

0.001) and Sod2 (Figs. 3F; injury x treatment: Fl,₂₀ = 10.5, P = 0.004; injury: Fi, 20 = 3.23, P = 0.087; treatment: Fi, 20 = 27.3, P < 0.001), which respectively encode the superoxide dismutase isoforms found in the cytosol and mitochondria (Figs. 3E, F). Compared to shamvehicle, Sodl ( P = 0.022) and Sod2 ( P = 0.010) levels were both downregulated by SNI (Figs. 3E, F). Activity levels of total SOD were also restored by dimethyl fumarate treatment, though enzyme activity was not attenuated by SNI compared to sham-vehicle (Fig. 3G; injury x treatment: Fi, 20 = 15.19, P < 0.001; injury: Fi, 20 = 0.33, P = 0.573; treatment: Fi, 20 = 24.72, P < 0.001). Once again, there was no effect of dimethyl fumarate in the sham groups.

Finally, to test whether dimethyl fumarate treatment could attenuate oxidative stress, we examined oxidized DNA/RNA. 8-oxoguanine (8-oxo-G) and 8-oxo-2-deoxyguanonsine (8- oxo-dG) are common nucleic acid lesions induced by reactive oxygen species. Dimethyl fumarate treatment reduced DNA/RNA oxidation (Fig. 3H; injury x treatment: Fi, 20 =

5.04, P = 0.036; injury: Fi, 20 = 2.91, P = 0.104; treatment: Fi, 20 = 13.44, P = 0.002). SNI non-statistically significantly increased DNA/RNA oxidation compared to sham-vehicle (P=0.05l). There was no effect of dimethyl fumarate in the sham group.

Role of NFE2L2 in the anti-nociceptive effects of dimethyl fumarate

The next experiments aimed to determine whether activation of the NFE2L2 antioxidant pathway mediated the anti-nociceptive effects of dimethyl fumarate. First, we administered the NFE2L2 inhibitor trigonelline together with dimethyl fumarate. Trigonelline prevented the reversal of allodynia induced by dimethyl fumarate (Fig. 4A; time x treatment: F₄, 40 = 18.33, P < 0.001; time: F2.541, 25.41 = 20.53, P < 0.001; treatment: Fi, 10 = 17.19, P =0.002). Trigonelline did not alter von Frey thresholds in the absence of dimethyl fumarate in the sham (F1.397, 5.587 = 0.95, P = 0.405) or the SNI conditions (Fig. 4A; F2.583, 10.33 = 0.278, P = 0.813). Compared to dimethyl fumarate treatment alone, trigonelline co-treatment also reduced the percentage of NFE2L2⁺ nuclei in the DRG from rats with SNI (Fig. 4B).

Representative photomicrographs are presented in Fig. 4C. Next, we administered dimethyl fumarate to wild-type and Nfe2l2 ' male and female mice, beginning 7 days after SNI when allodynia is fully established in this model. Dimethyl fumarate treatment failed to reverse allodynia in mice lacking Nfe2l2, whereas allodynia was alleviated by the treatment in wild-type mice (Fig. 4D; time x treatment: F₄, 56 =

38.26, P < 0.001; time: F2_.607, 36.50 = 37.85, P < 0.001; genotype: Fi, 14 = 92.70, P < 0.001). In this experiment, we also found that allodynia returned several days after the conclusion of dimethyl fumarate treatment (Fig. 4D). There were no baseline differences in von Frey threshold between genotypes ( P = 0.486). As there were no differences between the sexes, data were pooled for analysis.

Effects on injured DRG neurons

Activating transcription factor 3 (ATF3) is a commonly used surrogate marker for neuronal damage after peripheral nerve injury. Strikingly, treatment with dimethyl fumarate reduced the proportion of ATF3⁺ nuclei in the SNI group (10 ATF3⁺ nuclei/l02 total nuclei ± 1/30) compared to the SNI-vehicle group (39/107 ± 13/15) (Fig. 5A; injury x treatment: Fi, 24 = 50.50, P < 0.001; injury: Fi, 24 = 141.3, P < 0.001; treatment: Fi, 24 = 53.88, P< 0.001). In agreement with a previous study, we found that the percentage of ATF3⁺ nuclei in the DRG was increased by ~38 fold after SNI alone, compared to sham- vehicle (1/81 ± 1/30) and sham-dimethyl fumarate (1/118 ± 1/24) (P<0.00l) (Fig. 5A). Representative photomicrographs are presented in Fig. 5B.

Impact to mitochondrial bioenergetics in DRG neurons

Redox imbalance disrupts mitochondrial bioenergetics, which is believed to contribute to neuropathic pain. Therefore, we investigated whether dimethyl fumarate treatment could restore mitochondrial bioenergetics after SNI in DRG neurons. Oxygen consumption rates (OCR) were measured in cultured rat DRG neurons under different conditions (Fig. 6A). Dimethyl fumarate treatment completely restored SNI-induced deficits in basal respiration (Fig. 6B; injury x treatment: Fi, is = 5.95, P = 0.025; injury: Fi, is = 11.19, P = 0.004; treatment: Fi, is = 3.78, P = 0.068), ATP-linked respiration (Fig. 6C; injury x treatment: Fi, is = 3.71, P = 0.070; injury: Fi, is =14.18, P = 0.001; treatment: Fi, is = 11.58, P = 0.003), maximal mitochondrial respiration (Fig. 6D; injury x treatment: Fi, is = 7.17, P = 0.015; injury: Fi, is = 4.41, P = 0.050; treatment: Fi, is = 4.41, P = 0.017) and spare respiratory capacity (Fig. 6E; injury x treatment: Fi, is = 5.28, P = 0.034; injury: Fi, is = 1.90, P = 0.185; treatment: Fi, is = 5.68, P = 0.028). Dimethyl fumarate treatment had no effect in the sham group.

Effects on cytokine mRNA expression and protein levels

Proinflammatory cytokines and chemokines are induced downstream of reactive oxygen species, and promote dysfunctional synaptic plasticity that drives neuropathic pain. We therefore quantified gene expression and protein levels of the cytokines I L- 1 b and TNF, and the chemokine CCL2, which have a well-characterized role in neuropathic pain.

Compared to SNI- vehicle, treatment with dimethyl fumarate reduced expression of Illb mRNA (Fig. 7A; injury x treatment: Fi, 20 = 0.35, P = 0.559; injury: Fi, 20 = 15.42, P < 0.001; treatment: Fi, 20 =17.00, P < 0.001) and levels of IL- 1 b protein (Fig. 7B; injury x treatment: Fi, i₆ = 6.42, P = 0.022; injury: Fi, i₆ =20.6, P < 0.001; treatment: Fi, i₆= 27.8, P < 0.001). Illb mRNA and IL- I b protein were increased after SNI, compared to sham (Fig. 7A-C). SNI-induced increases in Ccl2 mRNA (Fig. 7C; injury x treatment: Fi, 20 = 6.75, P = 0.017; injury: Fi, 20 = 32.34, P < 0.001; treatment: Fi, 20 = 16.03, P < 0.001) and CCL2 protein levels (Fig. 7D; injury x treatment: Fi, i₆ = 43.41, P < 0.001; injury: Fi, i₆ = 140.5, P < 0.001; treatment: Fi, ½ = 54.67, P < 0.001) were also alleviated by dimethyl fumarate treatment. TnfmRNA expression was reduced by dimethyl fumarate (Fig. 7E; injury x treatment: Fi, 20 = 1.25, P = 0.28; injury: Fi, 20 = 5.70, P = 0.027; treatment: Fi, 20 = 20.53, P < 0.001), but TNF protein was not detected in any sample.

Effects of diroximel fumarate and tepilamide fumarate on mechanical allodynia

We assessed the anti-nociceptive effect of oral diroximel fumarate and tepilamide fumarate treatment on established pain-related behaviors induced by SNI. Diroximel fumarate and tepilamide fumarate attenuated SNI-induced allodynia (Fig. 8). As a comparison, the gold- standard treatment for neuropathic pain, gabapentin, was also assessed at the highest non sedating dose (30 mg/kg). Both diroximel fumarate and tepilamide fumarate produced superior antinociception relative to gabapentin (Fig. 8).

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of treating neuropathic pain including the step of administering to a subject in need thereof a compound of formula (I):

wherein

Ri is selected from C1-C3 alkyl;

R2 is selected from optionally substituted C₁-C₃ alkyl,

or a pharmaceutically acceptable salt, solvate, or isomer thereof.

2. A method of claim 1 wherein Ri is methyl.

3. A method of claim 1 or claim 2 wherein R₂ is methyl or optionally substituted ethyl

4. A method of claim 1 wherein formula (I) is represented by formula (la):

where R₂ is selected from:

a) CH₃; b) CH2CH3;

wherein:

Y represents a N-containing optionally substituted heterocyclyl;

R³ and R⁴ are independently C₁-C₃ alkyl; and

n is 1 or 0.

5. A method of claim 4 wherein n is 1.

6. A method of claim 4 or claim 5 wherein R² and R³ are both CH₂CH₃.

7. A method of claim 4 wherein Y is a 5 or 6 membered optionally substituted heterocyclyl.

8. A method of claim 7 wherein Y is selected from the following:

9. A method of claim 1 wherein formula (I) is represented by one of the following compounds:

or a pharmaceutically acceptable salt, solvate, or isomer thereof.

10. A method according to any one of claims 1 to 9 wherein the neuropathic pain is peripheral neuropathic pain.

11. A method according to any one of claims 1 to 9 wherein the neuropathic pain is pain associated with chemotherapy.

12. A method according to any one of claims 1 to 9 wherein the neuropathic pain is associated with back pain, theumatoid arthritis, trigeminal neuralgia, or diabetic neuropathy.

13. A method according to any one of claims 1 to 9 wherein the neuropathic pain is caused by nerve compression (trapped nerve).

14. A method according to any one of claims 1 to 9 wherein the neuropathic pain is associated with pain complications arising from an infection such as a bacterial, fungal or viral infection.

15. Use of a compound of formula (I):

wherein

Ri is selected from C₁-C₃ alkyl;

R₂ is selected from optionally substituted C1-C3 alkyl,

or a pharmaceutically acceptable salt, solvate, or isomer thereof,

for treating neuropathic pain.

16. Use of a compound of formula (I):

wherein

Ri is selected from C₁-C₃ alkyl;

R₂ is selected from optionally substituted C1-C3 alkyl,

or a pharmaceutically acceptable salt, solvate, or isomer thereof,

in the manufacture of a medicament for treating neuropathic pain.

17. Use according to claim 15 or 16 wherein the compound of formula (I) is as defined in any one of claims 2 to 9.