WO2018190713A1 - Inhibiteurs de lysine méthyltransférase destinés au traitement de la douleur - Google Patents

Inhibiteurs de lysine méthyltransférase destinés au traitement de la douleur Download PDF

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WO2018190713A1
WO2018190713A1 PCT/NL2018/050226 NL2018050226W WO2018190713A1 WO 2018190713 A1 WO2018190713 A1 WO 2018190713A1 NL 2018050226 W NL2018050226 W NL 2018050226W WO 2018190713 A1 WO2018190713 A1 WO 2018190713A1
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fam173b
inhibitor
pain
mitochondrial
specific inhibitor
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PCT/NL2018/050226
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Hanneke Lucia Dorothea Maria WILLEMEN
Niels EIJKELKAMP
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Umc Utrecht Holding B.V.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/549—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect

Definitions

  • the invention is in the field of medical treatment methods and therapeutic compositions for use in such methods.
  • the invention relates to therapeutic compounds for treatment of pain, especially persistent inflammatory and/or neuropathic pain.
  • spinal cord microglia have an activated phenotype and produce inflammatory mediators that trigger or maintain the long-lasting changes in nociceptive circuitry, thereby contributing to persistent pain (Clark and Malcangio, 2014. Front Cell Neurosci 8, 121; Graeber and Christie, 2012. Exp. Neurol. 234, 255-261; Milhgan and Watkins, 2009. Nat Rev Neurosci 10, 23-36; Old et al., 2015. Handb Exp Pharmacol 227, 145-170; Ren and Dubner, 2010. Nat Med 16, 1267-1276).
  • ROS reactive oxygen species
  • ROS levels in the dorsal root ganglia (DRG) and/or spinal cord contribute to chronic pain development in different rodent models (Fidanboylu et al., 2011. PLoS One 6, e25212; Flatters, 2015. Prog Mol Biol Transl Sci 131, 119- 146; Kim et al, 2008. Neurosci Lett 447, 87-91; Schwartz et al., 2009. J. Neurosci 29, 159- 168) and altered ROS levels are associated with chronic pain development in humans (Meeus et al., 2013. Expert Opin Ther Targets 17, 1081-1089; Sanchez- Dominguez et al., 2015. Mitochondrion 21, 69-75; Tan et al., 2011. Eur J Pain 15, 708-715).
  • SNPs single nucleotide polymorphisms
  • CWP chronic widespread pain
  • GWAS GWAS
  • Two top intronic SNPs on chromosome 5pl5.2 were shown to be associated with a 30% higher risk of developing CWP.
  • This genomic region encodes Chaperonin Containing TCP1 Subunit 5 (CCT5) and the hitherto functionally uncharacterized FAM173B protein, indicating potential novel pain genes.
  • CCT5 Chaperonin Containing TCP1 Subunit 5
  • FAM173B hitherto functionally uncharacterized FAM173B protein
  • FAM173B was identified as a lysine-specific protein methyltransferase that controls the development of chronic pain. Protein FAM173B resides in
  • Chronic pain was found to be mediated by increased expression of neuronal FAM173B methyltransferase activity, and involves reactive oxygen species-dependent pathway and/or activation of glial cells.
  • the invention therefore provides a specific inhibitor of FAM173B
  • mitochondrial lysine methyltransferase for use as a medicament, especially for use in a method of treatment of chronic pain, preferably of persistent inflammatory and/or neuropathic pain.
  • the invention further provides a use of a specific inhibitor of FAM173B mitochondrial lysine methyltransferase in the manufacture of a medicament, especially a medicament for the treatment of chronic pain, preferably of persistent inflammatory and/or neuropathic pain.
  • Said inhibitor preferably is a specific inhibitor of the family of seven-6-strand lysine methyltransferase, more preferably a specific inhibitor of FAM173B lysine methyltransferase.
  • Said inhibitor preferably is a peptide or peptide analogue.
  • Said peptide or peptide analogue preferably comprises a cell-penetrating domain.
  • Said inhibitor preferably is a specific inhibitor of mitochondrial FAM173B lysine methyltransferase such as mitochondria-targeted chaetocin or a functional analogue thereof.
  • Said inhibitor may be provided to primary sensory neurons of an individual suffering from chronic pain, preferably by systemic or local administration, including percutaneous injection and/or infusion, such as intrathecal,
  • a specific inhibitor of FAM173B mitochondrial lysine methyltransferase preferably is combined with a ROS inhibitor compound and/or a mitochondrial inhibitor.
  • Said ROS inhibitor compound preferably is selected from ascorbic acid, L-galactonic acid-g-galactone, pyruvate, mannitol, Trolox, a- tocopherol, Ebselen, uric acid and/or imidazole.
  • Said mitochondrial inhibitor preferably is metformin.
  • a specific inhibitor of FAM173B mitochondrial lysine methyltransferase may further be combined with the provision of microglia cells of the individual with a glial cell activation modulator compound.
  • the invention further provides a pharmaceutical composition comprising a specific inhibitor of FAM173B mitochondrial lysine methyl transferase and a pharmaceutically acceptable excipient.
  • Said pharmaceutical composition may further comprise an inhibitor of the generation of reactive oxygen species (ROS), a mitochondrial inhibitor and/or an inhibitor of glial cell activation.
  • ROS reactive oxygen species
  • Said pharmaceutical composition preferably is for use in a method of treatment of chronic pain, preferably of persistent inflammatory and neuropathic pain.
  • the invention further provides a method of typing an individual suffering from pain, the method comprising providing a sample of said individual;
  • the invention further provides a method of treating an individual suffering rom pain, the method comprising typing an individual according to the method of the invention; and treating an individual with a specific inhibitor of FAM173B, optionally combined with a ROS inhibitor and/or glial cell activation modulator compound, if the level of expression of FAM173B is enhanced in said individual when compared to a reference.
  • FIG. 1 Sensory neuron FAM173B promotes chronic pain
  • CFA complete Freund's adjuvant
  • mice received intrathecal injections of mFaml73b-AS or mismatch (MM) ODN (MM- ODN) at days 5, 6, 7, 9 and 10 during inflammatory pain or day 1-9 after SNI.
  • Mouse Faml73b is expressed in sensory neurons and intraplantar injections with HSV-amplicons (day 1 and 3) encoding either for hFAM173B or nothing (EV) induced hFAM173B expression selectively in sensory neurons at day 4.
  • Scale bar is 100 ⁇ .
  • f-g Intraplantar HSV-hFAM173B injection at day 5 and 7 rescued mFaml73b-AS-mediated attenuation of (1) thermal and (g) mechanical
  • n 8 in the CFA-model of persistent inflammatory pain. Mice received intrathecal asODN at day 5, 6, 7, 9 and 10 after CFA.
  • j-k Intraplantar HSV- liFAM173B injections at 3 and 1 day prior to an unilateral intraplantar
  • a Topology diagram of archetypical sevens-strand methyltransferase with alpha- helices (boxes) and beta-strands (arrows), b, Protein sequence alignment of FAM173A/B from Homo sapiens (h), Mus musculus (m) and the aKMT homolog of FAM173B (FAM173hom) from Sulfolobus islandicus (Si). Predicted secondary structure of mFaml73b above alignment, coded as in a. Bars with white stripe indicate predicted N- and C-terminus of mFaml73b. Motif I and Post I (boxed) are involved in binding of SAM.
  • Asp94 (*) was mutated to generate an enzymatically inactive protein.
  • the first residue (Thr56) in recombinant truncated hFAM173B (FAM173BA55) is also indicated (vertical arrow), c, Fluorography of HEK293- extracts incubated with [3H]-SAM and recombinant hFAM173BA55.
  • Statistical analysis was performed by Holm-Sidak multiple comparison test (d) or Student's t test (e).
  • hFAM173B-GFP co-localized with the mitochondrial dye MitoTracker, but not with endoplasmic reticulum (PDI) or Golgi (PGM130).
  • PDI endoplasmic reticulum
  • Golgi Golgi
  • Scale bar 10 ⁇ g Electron microscopy GFP-tagged hFAM173B- expressing HEK293.
  • N Nucleus
  • M mitochondrion
  • dotted line boundary between non-transfected (left) and transfected cell (right).
  • Cultured primary sensory neurons were stained for mFaml73b and the mitochondrial marker COX IV.
  • Intrathecal anti-TNFa 100 ⁇ g/mouse
  • Mice received intraplantar CFA to induce persistent hyperalgesia and received intrathecal mFaml73b-AS at day 5, 6, 7, 9, and 10.
  • Wild-type hFAM173B but not hFAM173B-D94A expression in sensory neurons prolonged carrageenan-induced (a) thermal and (b) mechanical hypersensitivity (n 6).
  • FAM173R' refers to a gene on human chromosome 5 in the 5pl5.2 region. Said gene encodes a protein with Uniprot accession number Q6P4H8. The protein sequence was found to have homology to the family of 7 beta strand S-adenosyl-L-methionine-dependent, lysine-specific methyltransferases. This family of methyltransferases structurally differs from the family of SET domain comprising methyltransferases. Seven-6-strand methyltransferases methylate a wide range of substrates, including small metabolites, lipids, nucleic acids and proteins. Until recently, the histone-specific Dotl/DOTIL was the only identified eukaryotic seven-6-strand methyltransferase (Falnes et al., 2016.
  • the encoded protein FAM173B is a mitochondrial lysine methyltransferase.
  • Frnl 73b refers to the homologous gene of mouse.
  • chronic pain refers to a pain in humans without apparent biologic value that has persisted beyond the normal tissue healing time (usually taken to be three months) that lasts more than 12 weeks.
  • Chronic pain also termed persistent pain, also refers to pain in rodent observed in model of persistent pain, for example persistent neuropathic or inflammatory pain models, that persists beyond normal tissue healing time or that persists during ongoing damage (at least several days).
  • Chronic pain may arise from an initial injury, damage or inflammation, such as a back sprain, or from an ongoing cause, such as illness. However, often there is no clear cause. Chronic pain often results in disability and despair.
  • peptide refers to a molecule with an amino acid chain of between 5 and 100 amino acid residues, preferably between 10 and 50 amino acid residues.
  • peptide includes a peptide in which one or more of the amino acid monomers have been modified, for example by acetylation, amidation and/or glycosylation.
  • peptide analogue refers to peptidomimetics which are or which comprise small peptide-like chains such as peptoids and 6-peptides designed to mimic a peptide.
  • the altered chemical structure is preferably designed to adjust one or more properties such as, for example, stability, of a peptide, cell-penetrating domain.
  • chaetocin refers to a natural product from
  • Chaetocin is known as a broad spectrum, non-specific lysine methyltransferase inhibitor, especially of SET domain comprising methyltransferases.
  • lysine methyltransferase inhibitors including lysine methyltransferase inhibitor of the epidithiodiketopiperazine class, BIX01294 (N-(l-benzylpiperidin-4-yl)-6,7- dimethoxy-2-(4-methyl-l,4-diazepan- l-yl)quinazolin-4-amine;trihydrochloride), UNC0224 (7-[3-(dimethylamino)propoxy]-6-methoxy-2-(4-methyl- l,4-diazepan-l- Yl)-N-(l-methylpiperidin-4-Yl)quinazohn-4-amine), UNC0321 (7- [2- [2- (dimethylamino)ethoxy]ethoxy]-6-methoxy-2-(4-methyl-l,4-diaze
  • Said functional analogue preferably is a 7 beta strand lysine
  • methyltranferase inhibitor preferably a specific inhibitor of a 7 beta strand lysine methyltranferase, of which class FAM173B is a member.
  • Said functional analogue preferably is specific inhibitor of FAM173B.
  • specific inhibitor of FAM173B refers to an methyltransferase-inhibiting compound that has a binding affinity Kd for a seven- 6-strand methyltransferase such as FAM173B that is at least 10 fold lower than the Kd for a SET domain containing methyltransferase.
  • a specific inhibitor of FAM173B preferably has a Kd of less than 10 7 M, preferably less than 1( 8 M.
  • Said specific inhibitor preferably is selected from the group consisting of adenosine-2',3'- dialdehyde (Adox) and, more preferably, specific inhibitors of DOT1L such as 7-[5- deoxy-5- [[3- [[[[4-(l, l-dimethylethyl)phenyl] amino]carbonyl] amino]propyl] time thLylethyl)amino]-6-I)-ribofuranosyl] ⁇
  • Adox adenosine-2',3'- dialdehyde
  • a more general methyltransferase inhibitor such as, for example, chaetocin may be rendered into a specific inhibitor of FAM173B by targeting the inhibitor to mitochondria.
  • mitochondria have a strong negative internal potential of about -180mV. Cationic molecules are attracted to negatively charged mitochondria. Therefore, a molecule such as a general methyltransferase inhibitor can be targeted to mitochondria by conjugating this molecule to one or more cell-penetrating, lipophilic peptides such as
  • rhodamine-based and cyanine-based fluorophores Zielonka et al., 2017. Chemical Reviews 117: 10043-10120
  • oligoguanidinium or triphenylphosphonium moieties.
  • 2,2,6, 6-tetramethyl-4-[[5-(triphenylpliospliomo)pentyl]oxy]- l- piperidinyloxy, monobromide is a mitochondria-targeting superoxide dismutase mimetic that combines an antioxidant moiety (TEMPO L, also known as 4-hydroxy- TEMPO) with a lipophilic cation triphenylphosphonium, which allows it to pass through lipid bilayers and accumulate in mitochondria.
  • TEMPO L antioxidant moiety
  • 4-hydroxy- TEMPO lipophilic cation triphenylphosphonium
  • chaetocin or a functional analogue of chaetocin, may rendered into a specific inhibitor of FAM173B by targeting the inhibitor to mitochondria, for example by conjugating this molecule to one or more cell-penetrating, lipophilic peptides such as rhodamine-based and cyanine-based fluorophores,
  • a further preferred specific inhibitor of FAM173B is based on the nucleic acid sequence of the FAM173B-encoding gene, or based on the amino acid sequence of FAM173B.
  • Said sequence -based specific inhibitor of FAM173B preferably is provided to a person in need thereof by intraplantar and/or intrathecal
  • administration preferably by lumbar intrathecal injection.
  • Said sequence-based specific inhibitors of FAM173B include antisense oligodeoxynucleotides (asODN) and small interfering RNA (siRNA) molecules mediating RNA interference, which are 18-to 23-nucleotide dsRNA molecules with 2 nucleotide-long 3' overhangs.
  • AsODN and/or siRNA molecules preferably target human FAM173B.
  • Said asODN and/or siRNA molecules may be provided to relevant cells as in vitro synthesized oligonucleotide.
  • Said oligonucleotide may be modified, for example by phosphorothioate (PS) bonds, to prevent degradation of the oligonucleotide.
  • PS phosphorothioate
  • said oligonucleotide is targeted to mitochondria by conjugating the oligonucleotide to one or more cell- penetrating, lipophilic peptides such as rhodamine-based and cyanine-based fluorophores (Zielonka et al., 2017. Chemical Reviews 117: 10043-10120), oligoguanidinium, or triphenylphosphonium moieties.
  • lipophilic peptides such as rhodamine-based and cyanine-based fluorophores (Zielonka et al., 2017. Chemical Reviews 117: 10043-10120), oligoguanidinium, or triphenylphosphonium moieties.
  • Said expression cassette preferably comprises a polymerase III enhancer/promoter.
  • a preferred polymerase III enhancer/promoter is selected from the U6 and HI promoter.
  • Said expression cassette preferably is provided in a vector, preferably a viral vector that is able to transduce neural cells, preferably sensory neurons.
  • Said viral vector preferably is a recombinant adeno-associated viral vector, a herpes simplex virus-based vector, or a lentivirus-based vector such as a human immunodeficiency virus-based vector.
  • Said viral vector most preferably is a herpes simplex virus-based vector.
  • FAM173B is an antibody or antibody mimetic that specifically binds and inhibits FAM173B.
  • the term antibody includes a single heavy chain variable domain antibody such as a camelid VHH, a shark immunoglobulin-derived variable new antigen receptor, a scFv, a tandem scFv, a scFab, and an improved scFab (Koerber et al., 2015. J Mol Biol 427: 576-86).
  • antibody mimetic refers to a molecule that can specifically bind FAM173B, but that is not structurally related to an antibody.
  • Antibody mime tics include a designed ankyrin repeat protein, a binding protein that is based on a Z domain of protein A, a binding protein that is based on a fibronectin type III domain, engineered lipocalin, and a binding protein that is based on a human Fyn SH3 domain (Skerra, 2007. Current Opinion Biotechnol 18: 295-304; Skrlec et al., 2015. Trends Biotechnol 33: 408-418).
  • cell-penetrating domain refers to peptides that facilitate cellular intake/uptake of various molecules.
  • suitable cell- penetrating domains are penetratin or Antenapedia (N-terminus
  • RQIKWFQNRRMKWKK TAT (N-termmus YGRKKRRQRRR), SynBl (N- terminus RGGRLSYSRRRFSTSTGR), SynB3 (N-termmus RRLSYSRRRF), PTD-4 (N-terminus PIRRRKKLRRLK), PTD-5 (N-terminus RRQRRTSKLMKR), FHV Coat-(35-49) (N-terminus RRRRNRTRRNRRR VR) , BMV Gag-(7-25) (N-terminus KMTRAQRRAAARRNRWTAR) , HTLV-II Rex-(4-16) (N-terminus
  • TRRQRTRRARRNR D-Tat (N-termmus GRKKRRQRRRPPQ), R9-Tat (N- terminus GRRRRRRRRRPPQ), transportan (N-termmus
  • GALFLGWLGAAGSTMGAWSQPKKKRKV GALFLGWLGAAGSTMGAWSQPKKKRKV
  • mitochondrial targeting peptides are MLSLRQSIRFFKPATRTLCSSRYLL, amino acid residues 289- 18 of the C terminus of APE 1
  • ROS inhibitors refers to a molecule that may quench free radicals and/or inhibits production of reactive oxygen species (ROS).
  • ROS inhibitors include Trolox (6- hydroxy-2,5,7,8-tetramethylchiOman-2-carboxylic acid), a combination of Trolox and ascorbate, dihydroethidium (DHE), TEMPOL (2,2,6, 6-tetramethyl-4- piperidinol-N-oxyl), Tiron (disodium;4,5-dihydroxybenzene- 1,3-disulfonate), phenyl-N-t-butyl nitrone (PBN) and ebselen (1.2-phenyl- l,2-benzoisoselenazol- 3(2H)-one).
  • Trolox 6- hydroxy-2,5,7,8-tetramethylchiOman-2-carboxylic acid
  • DHE dihydroethidium
  • TEMPOL 2,2,6, 6-tetramethyl-4- piperid
  • Said term includes specific mitochondrial ROS inhibitors such as MitoQ (l()-(4,5-dimethoxy-2-methyl-3,6-dioxo-l,4-cyclohexadien- l- yl)decyl](triphenyl)phosphonium methanesulfonate), a mixture of [10-(4,5- dimethoxy-2-methyl-3,6-dioxocyclohexa-l,4-dienyl)decyl] triphenylphosphonium bromide and [l()-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)decyl]triphenyl phosphonium bromide ⁇ , MitoVitE (vitamin E attached to a triphenylphosphonium cation), mitoTEMPO (2-(2,2,6,6-tetramethylpiperidin- l-oxyl-4-ylamino)-2- oxoethyl)triphenyl
  • IC50 refers to a concentration of a compound that inhibits an enzymatic activity to 50% of the maximal activity.
  • An IC50 value typically is expressed as a molar concentration.
  • the present invention is based on the finding that FAM173B is a
  • Chronic pain may occur after an injury or after a disease, or it may occur without any known physical cause. In some cases, it is accompanied by a tissue pathology such as chronic inflammation in some types of arthritis. Chronic pain is a very general concept and there are several varieties of chronic pain related to the musculoskeletal system, visceral organs, skin, and nervous system.
  • Examples of known diseases that may cause chronic pain include:
  • fibromyalgia irritable bowel syndrome, chronic arthropathy, post herpetic neuralgia, trigeminal neuralgia, neuropathy including mononeuropathy and polyneuropathy, small fiber neuropathy, metabolic neuropathy, diabetes-induced neuropathy, post-surgical pain syndrome, spinal nerve compression syndrome, migraine, chemotherapy-induced neuropathy, cancer-induced neuropathy, kidney and/or thyroid-induced neuropathy and rheumatoid and osteoarthritis.
  • chronic pain especially chronic widespread pain, chronic inflammatory and/or chronic neuropathic pain.
  • oligodeoxynucleotides resulted in a reduction of Faml73b mRNA expression and, consequently, also in a reduction of the steady state level of the encoded protein in lumbar dorsal root ganglia, without affecting spinal cord Fam l73b mRNA expression.
  • This intrathecal administration abrogated thermal and mechanical hyperalgesia.
  • Intrathecal injections of different Faml 73b asODN, but not of control ODNs consistently abrogated thermal and mechanical hyperalgesia after inflammation and/or nerve ligation, indicating that
  • Faml73b attenuates persistent neuropathic and inflammatory pain.
  • a mitochondrial lysine methyltransferase is required for development and/or sustainment of chronic pain provides an inhibitor, preferably a specific inhibitor, of said a mitochondrial lysine methyltransferase for use in a method of treatment of chronic pain, preferably of persistent inflammatory and/or neuropathic pain.
  • Said inhibitor of mitochondrial lysine methyltransferase preferably specific inhibitor of FAM173B, may be an organic or inorganic compound, a peptide, a polynucleotide, a lipid, or a hormone or hormone analog.
  • Said organic or inorganic compound preferably is characterized by a relatively low molecular weight.
  • a low molecular weight compound i.e. with a molecular weight of 500 Dalton or less, is likely to have good absorption and permeation in biological systems and is consequently more likely to be a successful drug candidate than a compound with a molecular weight above 500 Dalton (Lipinski et al., 1997. Advanced Drug Delivery Reviews 23: 3-25).
  • Synthetic compound libraries e.g. LOP ACTM, Sigma Aldrich
  • natural compound libraries Specs, TimTec
  • the invention further relates to a method for identifying a compound that inhibits FAM173B mitochondrial lysine methyltransferase, comprising (a) contacting a compound with a FAM173B mitochondrial lysine methyltransferase polypeptide, (b) determining a binding affinity of the compound to the polypeptide,
  • said inhibitor is a peptide or peptide analogue, preferably a peptide or peptide analogue that inhibits the lysine methyltransferase of
  • FAM173B Said inhibition of the lysine methyltransferase of FAM17B preferably is selective over inhibition of other lysine methyl transferase, whereby the term selective indicates that the IC50 for inhibition of FAM173B is more than 2x lower than the IC50 for inhibition of another lysine methyltransferase, preferably said IC50 for inhibition of FAM173B is more than 4x lower than the IC50 for inhibition of another lysine methyltransferase, more preferably more than lOx lower.
  • Inhibition might be achieved by competition with a peptide substrate and/or by competition with a co-factor, S-adenosyl methionine. Said inhibition preferably is achieved by competition with a peptide substrate or a peptide analogue of a substrate.
  • said peptide substrate or peptide analogue of a substrate preferably comprises a cell- penetrating domain. Said cell-penetrating domain preferably is present at the N- terminus of the peptide or peptide analogue. Said cell-penetrating domain preferably is selected from penetratin, TAT, SynBl and SynB3.
  • said peptide substrate or peptide analogue of a substrate may be expressed by a viral vector, preferably a viral vector that is able to transduce neural cells, preferably sensory neurons.
  • Said viral vector preferably is a recombinant adeno-associated viral vector, a herpes simplex virus -based vector, or a lentivirus-based vector such as a human immunodeficiency virus-based vector.
  • Said viral vector most preferably is a herpes simplex virus-based vector.
  • Said peptide substrate or a peptide analogue of a substrate preferably additionally comprises a mitochondrial targeting moiety, preferably a
  • a preferred mitochondrial targeting moiety is a cell-penetrating, lipophilic peptide such as rhodamine-based and cyanine-based fluorophore, an oligoguanidinium, or a triphenylphosphonium moiety.
  • a preferred mitochondrial targeting peptide comprises amino acid residues 289-318 of the C terminus of APEl (HSLLPALCDSKIRSKALGSDHC PITLYLAL). Said mitochondrial targeting peptide preferably is present at the C-terminus of the peptide substrate or a peptide analogue of a substrate.
  • Said inhibitor may also be a known inhibitor of a lysine methyltransferase, preferably a broad spectrum inhibitor of lysine methyltransferases.
  • the term broad spectrum refers to an inhibitor that is able to inhibit multiple different lysine methyltransferases with a similar IC50 concentration.
  • Said broad spectrum inhibitor of lysine methyltransferases, such as chaetocin is preferably targeted to mitochondria, for example by conjugating the inhibitor to one or more cell-penetrating, lipophilic peptides such as rhodamine-based and cyanine-based fluorophores, oligoguanidinium, or triphenylphosphonium moieties.
  • a preferred specific inhibitor of FAM173B lysine methyltransferase is a mitochondria targeted chaetocin or functional analogue thereof.
  • An inhibitor of lysine methyltransferase preferably a specific inhibitor of FAM173P
  • Said primary sensory neurons preferably include thermoreceptor neurons, somatosensory neurons visceral neurons and/or mechanoreceptor neurons.
  • the cell bodies of these neurons are located in ganglia throughout the spine.
  • Methods of providing a compound such as a peptide or peptide analogue, or a small molecule, to primary sensory neurons, preferably to cell bodies of primary sensory neurons are known in the art.
  • Said methods include systemic administration, and local administration by percutaneous injection such as intrathecal, paravertebral, intraforaminal and transforaminal administration, as will be specified herein below.
  • Inhibition might further be achieved by alteration of the gene encoding
  • FAM173B in vivo preferably in primary sensory neurons.
  • Said alteration includes silencing of the gene, for example by expression of ZFP transcription factors attached to a "gene repression" domain in order to down-regulate (repress) the expression of FAM173B in vivo, preferably in primary sensory neurons; introducing inactivating alterations in the gene encoding FAM173B in vivo, preferably by alteration, including deletion of part or all, of the active domain of the lysine methyl transferase, especially of amino acid aspartic acid (D) at position 94 of the human FAM173B amino acid sequence within Motif 1, as depicted in Figure 2B.
  • D amino acid aspartic acid
  • Said alteration may be accomplished, for example, by CRISPR-CAS (Sander and Joung, 2014. Nature Biotech 32, 347-355) and exon skipping (McNally et al., 2016. J Clin Invest 126: 1236-1238).
  • An inhibitor of lysine methyltransferase preferably a specific inhibitor of FAM173B, preferably is combined with a ROS inhibitor compound.
  • a ROS inhibitor compound preferably is combined with a ROS inhibitor compound.
  • activation of FAM173B by overexpression of FAM173B in sensory neurons results in increased production of ROS.
  • Inhibition of the production of ROS, or at least a decrease of the production of ROS was shown to attenuate chronic pain such as persistent neuropathic pain.
  • a combination of an inhibitor of lysine methyltransferase, preferably a specific inhibitor of FAM173B, with a ROS inhibitor compound is likely to result in both immediate and sustained treatment of said chronic pain.
  • a ROS inhibitor compound likely provides immediate, transient attenuation of chronic pain, while an inhibitor of lysine
  • methyltransferase preferably a specific inhibitor of FAM173B
  • a preferred ROS inhibitor compound is selected from ascorbic acid, DHE, L- galactonic acid-g-galactone, pyruvate, mannitol, Trolox, TEMPOL, mitoTEMPO, PBN, a-tocopherol, Ebselen, uric acid and/or imidazole.
  • methyltransferase preferably a specific inhibitor of FAM173B, optionally in combination with a ROS inhibitor, may additionally be combined with the provision of a mitochondrial inhibitor.
  • Said inhibitor preferably is a biguanide, such as metformin (3-(diaminomethylidene)- l, l-dimethylguanidine) and
  • phenformin (l-(diaminomethylidene)-2-(2-phenylethyl)guanidine).
  • methyltransferase preferably a specific inhibitor of FAM173B, optionally in combination with a ROS inhibitor and/or a mitochondrial inhibitor, may
  • glial cell activation modulator preferably is selected from minocycline
  • interleukin- 10 delivery product Studies have shown that one aspect of chronic pain is that activated glia in the spinal cord release proinflammatory products that drive pain and weaken the effects of opioid analgesia. Administration of
  • minocycline, ibudilast, ATL313, synerkine and/or XT101 will reverse glial cell activation and may further provide attenuation of chronic pain.
  • Said inhibitor may further find use, alone or in combination with a ROS inhibitor compound, a mitochondrial inhibitor, and/or a glial cell activation modulator compound, in the treatment of other neuroimmune-related diseases such as Alzheimer, Parkinson and multiple sclerose, in which microglia and activation of reactive oxygen species also play a role.
  • Said inhibitor of lysine methyltransferase preferably a specific inhibitor of
  • FAM173B alone or in combination with a ROS inhibitor compound, a
  • mitochondrial inhibitor and/or a glial cell activation modulator compound, can be administered by a number of routes.
  • Said inhibitor of lysine methyltransferase preferably a specific inhibitor of
  • FAM173B alone or in combination with a ROS inhibitor compound, a
  • mitochondrial inhibitor and/or a glial cell activation modulator compound preferably is provided to a patient in need thereof by systemic, or local
  • administration including intrathecal, paravertebral, transforaminal and intraforaminal administration.
  • systemic administration refers to oral, intravenous, intramuscular, intra-articular, intra- arterial, intramedullary, intrathecal, epidural, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, inhalational, intraocular, intra-aural or rectal injection or infusion, preferably intravenous or intramuscular infection or infusion.
  • intradiscal administration also termed intradiscal
  • administration refers to administration in or through the foramen intervertebrale, preferably such that the inhibitor, optionally in
  • injection or infusion refers to injection or infusion of said inhibitor, optionally in combination with one or more compounds, into the spinal canal, or into the subarachnoid space so that it reaches the cerebrospinal fluid.
  • Said injection or infusion may be accomplished by use of external pump or of a fully implantable device.
  • Said external pump is preferably equipped with a percutaneous catheter, tunneled or not tunneled, or equipped with a subcutaneous injection port and an implanted catheter.
  • An implantable drug delivery system with a constant flow may be beneficial for long-term delivery of said inhibitor of lysine methyltransferase, preferably a specific inhibitor of FAM173B, alone or in combination with a ROS inhibitor compound and/or a glial cell activation modulator compound.
  • paravertebral administration refers to injection or infusion into a space immediately lateral to where the spinal nerves emerge from the intervertebral foramina.
  • administration refers to injection or infusion into the opening at the side of the spine where a nerve root exits. This opening is known as a foramen. There is a small sleeve of the epidural space that extends out over the nerve root for a short distance.
  • the invention further provides a pharmaceutical composition comprising a specific inhibitor of FAM173B mitochondrial lysine methyltransferase as defined herein above and a pharmaceutically acceptable excipient.
  • Said pharmaceutically acceptable excipient preferably is selected from diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, lactose such as a-lactose monohydrate, anhydrous a-lactose, anhydrous ⁇ -lactose, spray-dried lactose, and/or agglomerated lactose, sugars such as dextrose, maltose, dextrate and/or inulin, glidants (flow aids) and lubricants, and combinations thereof.
  • a carbohydrate such as starch
  • a starch derivative such as starch acetate and/or maltodextrin
  • a polyol such as xylitol, sorbitol and/or mann
  • Said pharmaceutical composition for intrathecal administration preferably is a sterile isotonic solution.
  • Said buffer preferably is citrate-based buffer, preferably lithium-, sodium-, potassium-, or calcium- citrate monohydrate, citrate trihydrate, citrate tetrahydrate, citrate pentahydrate, or citrate heptahydrate; lithium, sodium, potassium, or calcium lactate; lithium, sodium, potassium, or calcium phosphate; lithium, sodium, potassium, or calcium male ate; lithium, sodium, potassium, or calcium tartarate; lithium, sodium, potassium, or calcium succinate; or lithium, sodium, potassium, or calcium acetate, or a combination of two or more of the above.
  • the pH of said buffer may be adjusted, preferably to a pH of 7.27 - 7.37 by hydrochloric acid, sodium hydroxide, citric acid, phosphoric acid, lactic acid, tartaric acid, succinic acid, or a combination of two or more of the above.
  • the volume of may range from 0.5 ml to 5 ml.
  • Said excipient preferably is selected from, but not limited to, urea, L-histidine, L-threonine, L-asparagine, L-serine, L- glutamine, polysorbate, polyethylene glycol, propylene glycol, polypropylene glycol, or a combination of two or more of the above.
  • a pharmaceutical composition as defined herein above may further comprise an inhibitor of the generation of reactive oxygen species (ROS), a mitochondrial inhibitor, and/or an inhibitor of glial cell activation.
  • ROS reactive oxygen species
  • a preferred ROS inhibitor compound is selected from ascorbic acid, DHE, TEMPOL, mitoTEMPO, PBN, L- galactonic acid-g-galactone, pyruvate, mannitol, Trolox, a- tocopherol, Ebselen, uric acid and/or imidazole.
  • Said mitochondrial inhibitor preferably is metformin.
  • Said glial cell activation modulator compound preferably is selected from minocycline, ibudilast, ATL313, synerkme and/or XT 101.
  • Said pharmaceutical composition comprising a specific inhibitor of FAM173B mitochondrial lysine methyltransferase and, optionally, an inhibitor of reactive oxygen species (ROS) generation, a mitochondrial inhibitor, and/or an inhibitor of glial cell activation may be provided as a kit of parts, comprising two or more receptacles comprising said inhibitor of FAM173B mitochondrial lysine
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • a pharmaceutical composition as defined herein above preferably is for use in a method of treatment of chronic pain, preferably of persistent inflammatory and neuropathic pain.
  • Said composition preferably is administered to a person in need thereof by systemic or local administration to the spinal canal, or to the subarachnoid space by injection or by infusion.
  • Said injection or infusion may be accomplished by use of external pump or of a fully implantable device.
  • Said external pump is preferably equipped with a percutaneous catheter, tunneled or not tunneled, or equipped with a subcutaneous injection port and an implanted catheter.
  • An implantable drug delivery system with a constant flow may be beneficial for long-term delivery of said composition.
  • the invention further provides a method of typing an individual suffering from pain, the method comprising providing a sample of said individual;
  • Said sample preferably is a liquid biopsy.
  • liquid biopsy refers to a liquid sample that is obtained from a subject.
  • Said liquid biopsy is preferably selected from blood, urine, milk, cerebrospinal fluid, interstitial fluid, lymph, amniotic fluid, bile, cerumen, feces, female ejaculate, gastric juice, mucus pericardial fluid, pleural fluid, pus, saliva, semen, smegma, sputum, synovial fluid, sweat, tears, vaginal secretion, and vomit.
  • a preferred liquid biopsy is a cerebrospinal fluid.
  • a proteinaceous sample may be prepared from the liquid sample, as is known to a person skilled in the art.
  • Said proteinaceous sample may be fractionated used standard techniques such as chromatography methods including ion exchange chromatography and/or size-exclusion chromatography, as is known to the skilled person.
  • FAM173B may be concentrated by affinity chromatography, for example by employing affinity partners such as antibodies or functional parts thereof that bind specifically to FAM173B.
  • Said concentration step preferably removes proteins and/or peptides that interfere with the subsequent detection of FAM173B.
  • a preferred method for determining a level of FAM173B comprises high performance liquid chromatography (HPLC), preferably coupled to tandem mass spectrometry (LC-MS MS).
  • HPLC high performance liquid chromatography
  • LC-MS MS tandem mass spectrometry
  • the LC-MS/MS analysis may be performed, for example by using a HPLC chromatographic system coupled to a triple-quadrupole mass- spectrometer.
  • the invention further provides a method of treating an individual suffering from pain, the method comprising typing an individual according to a method of the invention, and treating an individual with a specific inhibitor of FAM173B, whether or not combined with a ROS inhibitor, a mitochondrial inhibitor, and/or a glial cell activation modulator compound, if the level of expression and/or activity of FAM173B is enhanced in said individual when compared to a reference.
  • Said reference is a liquid biopsy, preferably a cerebrospinal fluid, from one or more individuals, preferably at least 5, more preferably at least 10 individuals, not suffering from chronic pain.
  • a similarity score may be provided, which similarity score may vary between +1, indicating a prefect similarity, and - 1, indicating a reverse similarity, with a reference.
  • an arbitrary threshold is used to type a sample as likely from an individual not suffering from chronic pain.
  • a similarity score is preferably displayed or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.
  • said reference may also be a liquid biopsy, preferably a cerebrospinal fluid, from one or more individuals, preferably at least 5, more preferably at least 10 individuals, that are suffering from chronic pain.
  • an individual will be treated with a specific inhibitor of FAM173B, whether or not combined with a ROS inhibitor, a
  • mitochondrial inhibitor and/or a glial cell activation modulator compound, if the level of expression of FAM173B is similar in said individual when compared to the reference.
  • a specific inhibitor of FAM173B whether or not combined with a ROS inhibitor, a mitochondrial inhibitor, and/or a glial cell activation modulator compound will be determined by the individual to which the dose is administered, in light of factors related to the individual's requiring treatment. Said dosage preferably is between 1 microgram and 10 milligram per kg per hour. Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired therapeutic effect.
  • Factors that can be taken into account include the severity of the pain and other factors, including the general health of the subject, age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy, as is known to a person skilled in the art.
  • the FAM173B protein preferably is expressed in a host cell.
  • Commonly used expression systems for heterologous protein production include E. coli, baculovirus, yeast, Chinese
  • Hamster Ovary cells CHO
  • human embryonic kidney (HEK) cells and derivatives thereof including HEK293 cells including HEK293T, HEK293E, HEK-293F and HEK-293FT (Creative Biolabs, NY, USA), PER.C6® cells (Thermo Fisher
  • FAM173B protein may be produced using prokaryotic cells or eukaryotic cells, preferably E. coli, or fungi, most preferably filamentous fungi or yeasts such as
  • FAM173B protein in filamentous fungi is preferably performed as described by Joosten et al., 2005. J Biotechnol 120:347-359, which is included herein by reference.
  • a preferred method for producing FAM173B protein in S. cerevisiae is according to the method as described by v. d. Laar et al., 2007. Biotech
  • FAM173B protein production is by expression in Pichia pastoris as described by Rahbarizadeh et al., 2006. J Mol Immunol 43:426-
  • Said FAM173B protein is preferably produced by the provision of a nucleic acid encoding said the FAM173B protein to a cell of interest.
  • Said nucleic acid preferably DNA
  • Said nucleic acid is preferably produced by recombinant technologies, including the use of polymerases, restriction enzymes, and ligases, as is known to a skilled person.
  • said nucleic acid is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant technologies, as is known to the skilled person.
  • Said nucleic acid is preferably codon-optimised to enhance expression of FAM173B protein in the selected cell or cell line.
  • optimization preferably includes removal of cryptic splice sites, removal of cryptic polyA tails and/or removal of sequences that lead to unfavourable folding of the mRNA.
  • the presence of an intron flanked by splice sites may encourage export from the nucleus.
  • the nucleic acid preferably encodes a protein export signal for secretion of the FAM173B protein out of the cell into the periplasm of prokaryotes or into the growth medium, allowing efficient purification of the FAM173B protein.
  • the single transmembrane domain which is located between ammo acids 38 - 58 of the human protein (UniProt Q6P4H8 (F173B_HUMAN)), may be amended or deleted, for example to prevent incorporation of FAM173B protein in inclusion bodies.
  • a preferred FAM173B protein comprises a protein export signal and lacks an intact transmembrane domain, for example by deletion of amino acid residues 1-55.
  • FAM173B protein Methods for purification of FAM173B protein are known in the art and are generally based on chromatography, such as protein A affinity for antibody purification, and ion exchange, to remove contaminants.
  • chromatography such as protein A affinity for antibody purification, and ion exchange, to remove contaminants.
  • recombinant FAM173B protein may be tagged with a specific tag by genetic engineering to allow the protein attach to a column specific to the tag and therefore be isolated from impurities.
  • the purified protein is then exchanged from the affinity column with a decoupling reagent. The method has been increasingly applied for purifying recombinant protein.
  • tags for proteins such as histidine tag
  • an affinity column that specifically captures the tag (eg., a Ni-IDA column for Histidine tag) to isolate the protein from other impurities.
  • the protein is then exchanged from the column using a decoupling reagent according to the specific tag (eg., immidazole for histidine tag). This method is more specific, when compared with traditional purification methods.
  • Suitable further tags include c-myc domain (EQKLISEEDL), hemagglutinin tag (YPYDVPDYA), and maltose-binding protein, glutathione-S- transferase, maltose-binding protein, FLAG tag peptide, biotin acceptor peptide, streptavidin-binding peptide and calmodulin-binding peptide, as presented in Chatterjee, 2006. Cur Opin Biotech 17, 353-358). Methods for employing these tags are known in the art and may be used for purifying FAM173B protein.
  • Methods for determining lysine methyl transferase activity of FAM173B are known in the art and include methods for detecting products of methyl transferase activity, methods for detecting residual substrate peptide, and methods for detecting a decrease in concentration of the methyl donor, S-adenosyl-L-methionine (SAM).
  • SAM S-adenosyl-L-methionine
  • the methods available for detection and assay of methyl transferase activity vary in their simplicity, rapidity, range of detection and sensitivity. Said methods comprise qualitative assays such as protein agar plate assay, radial diffusion and thin layer enzyme assay and, preferred, quantitative assays which provide a measure of the methyl transferase activity of the enzyme.
  • the commonly used methods employ natural or synthetic substrates using techniques such as enzyme-linked immunosorbent assay-based assays (ELISA), spectrophotometry, fluorimetry, and radiometry. Said methods may be used for identification of an individual suffering from chronic pain who may benefit from treatment aimed at reducing expression and/or activity of FAM173B in relevant cells of the individual, and for screening and identifying inhibitors, preferably specific inhibitors, of FAM173B.
  • ELISA enzyme-linked immunosorbent assay-based assays
  • a peptide or analog preferably is labeled, for example with a chromogenic group, a (chemo)luminescent group, a radiolabel and/or, most preferred, a fluorescent group.
  • a labeled peptide is preferably used in spectrophotometry, fluorimetry, and radiometry. Said label is preferably present at a terminus of the peptide. Said terminus is either the amino-terminus (N-terminus) or the carboxy- terminus (C-terminus).
  • the term "terminus" indicates that the label preferably is present on one or more of the first five amino acid monomers from the N-terminus, and/or on one or more of the last five amino acid monomers at the C-terminus.
  • Said label is preferably present at the N-terminal amino acid monomer, and/or at the C- terminal acid monomer.
  • the skilled person will understand that said label can be indirectly coupled to the N- or C-terminus, for example through a linker that is attached to N-terminus and/or C-terminus.
  • Said linker preferably is an amino acid residue, for example a glutamic acid residue at the N-terminus, and/or an aspartic acid or a cysteine residue at the C-terminus.
  • a preferred chromogenic group is 2'-azino-bis-(3-ethylbenzothiazoline-6- sulfonic acid (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), p-nitroanilide, paranitrophenol and/or 5-bromo-4-chloro-3-hydroxyindole.
  • ABTS o-ethylbenzothiazoline-6- sulfonic acid
  • OPD o-phenylenediamine
  • TMB 3,3',5,5'-tetramethylbenzidine
  • p-nitroanilide paranitrophenol and/or 5-bromo-4-chloro-3-hydroxyindole.
  • a preferred (chemo)luminescent group is a dioxetane derivative such as 1,2- dioxetanedione (C204), 3,3,4,4-tetramethyl- l,2-dioxetane and 3,3,4-trimethyl-l,2- dioxetane, 3-(4-methoxyspiro[l,2-dioxetane-3,2'-tricyclo[3,3, l, 13,7]decan]-4-yl- )- l- aniline and luminol.
  • a preferred radiolabel is 35S and/or 14C.
  • the labeled methylated products are preferably separated from the unmethylated substrate, for example by employing magnetic beads.
  • a peptide or analog preferably is labeled with one or more fluorescent group.
  • Fluorescent groups are known and have been described, for example, in U.S. Pat. No. 7,256,012 and U.S. Pat. No. 7,410,769.
  • Some non-limiting examples of fluorescent groups include coumarin derivatives such as 7-amino-4- methylcoumarin (AMC), 7-acetoxy-4-methylcoumarin (7-AC-4-MC) and 7- hydroxycoumarin, fluorescein, tetramethylrhodamine, rhodamine B, lissamine, rhodamine X, Texas Red, cyanine dyes, Dabcyl, BODIPY dyes, alexa dyes, QSY 7 and QSY 9 dyes, and other fluorescent dyes commonly available from, for example, Invitrogen Corp (Carlsbad, Calif.). Other dyes known to those skilled in the art may also be used.
  • a preferred fluorescence-based protease assay is simple, inexpensive and sensitive.
  • Said protease assay preferably comprises a soluble fluorescein
  • FITC isothiocyanate
  • mice C57BL/6 mice (Harlan Laboratories), since we did not overt sex differences during pain behaviour measurements.
  • Mice received an intraplantar injection unilateral or in both hind paws of 5 ⁇ ⁇ -carrageenan (1% w/v, Sigma-Aldrich) to induce transient inflammatory pain (Wang et al., 2013a. J Clin Invest 123, 5023-5034) or 20 ⁇ Complete Freund's Adjuvant (CFA, Sigma-Aldrich) to induce persistent inflammatory pain (Ren and Dubner, 1999. Nat Med 16, 1267-1276).
  • Spared nerve injury (SNI) was performed as described previously (Decosterd and Woolf, 2000. . Pain 87, 149-158; Willemen et al., 2012.
  • conditioned place preference was calculated by subtracting the mean time spent in the white room during pre-conditioning (days 1 and 2) with the time spent in the white room (day 5) after 2 days of conditioning (day 3-4) with intraperitoneal injections of gabapentin (100 mg/kg, Sigma-Aldrich).
  • mFaml73b (NM_ 026546.1) and hFAM 173B (NM_199133.3) were cloned into several vectors including pAcGFP-Nl, pIRES2-AcGFPl, bacterial expression vector pET28a and pCMV6 containing a myc-tag at the C-terminal of human or mouse FAM173B (Origene).
  • pIRES2-Ac,GFP vectors were used for functional experiments and GFP expression was used to verify successful transfection.
  • the pCMV6 and pAcGFP-Nl vectors were used for identification of cellular and subcellular localization of FAM173B and pET28a was used for the production of recombinant FAM173B in Escherichia coli.
  • HSV herpes simplex virus
  • mice were inoculated twice (day -3 and day -1 prior to carrageenan or at day 5 and 7 after CFA) with 2.5 ⁇ of 1.4 x 107 pfu ml (intraplantar) or 5 ⁇ 5 x 106 pfu/ml (intrathecal).
  • Drug administration 2.5 ⁇ of 1.4 x 107 pfu ml (intraplantar) or 5 ⁇ 5 x 106 pfu/ml (intrathecal).
  • mice received an intraperitoneal injection (day 5 after carrageenan) with 100 ⁇ phenyl-N-t-butyl nitrone (PBN, 100 mg/kg, Sigma- Aldrich).
  • PBN phenyl-N-t-butyl nitrone
  • mice received two PBN injections (2 hours apart) at one month after carrageenan. Spinal cords were collected 2 hours after the last PBN administration.
  • Intrathecal injections (5 ⁇ 1) with minocycline (6 g l, Sigma- Aldrich), neutralising TNF antibody (20 mg/ml, Enbrel®) and (Cy3-labeled, setl) asODN (3 ⁇ g l day 5,6,7,9 and 10, Sigma Aldrich), were performed under light isoflurane anaesthesia as described (Eijkelkamp et al., 2010. J Neurosci 30, 2138- 2149; Hylden and Wilcox, 1980. Eur J Pharmacol 67, 313-316.). The following phosphorothioated asODN sequences were used that specifically target niFam ,173b and not hFAM173B:
  • HEK293 and mouse neuroblastoma Neuro2a (N2a) cells were kept in Dulbecco's Modified Eagle medium (DMEM) with Glutamax-1 containing 4,5g/L D-Glucose, pyruvate and 10% fetal calf serum.
  • DMEM Dulbecco's Modified Eagle medium
  • FAAI173B expression was downregulated (100 ⁇ asODN) or overexpressed with plasmids as described above using Lipofectamin 2000 (Life Technology) according to manufactures instructions.
  • the cells were incubated for 20-30 minutes with 50 nM Mitotracker Red FM (Life technology) or 50 nM TMRM (Sigma-Aldrich) 2 days after transfection and following manufacturer's instructions.
  • DRGs were collected and primary sensory neurons were cultured as described (Eijkelkamp et al., 2013. Nat Commun 4, 1682). Twenty-four hours after plating, sensory neuron cultures were inoculated with HSV (10,000 pfu) for 3 days. The anti-mitotic fluoro-deoxyuridine (FDU 13.3 microg/ml, Sigma-Aldrich) was added to inhibit satellite glia cell growth in the neuronal cultures. Sensory neurons were stimulated with 100 ng recombinant TNFa (Peprotech) with or without phenyl-N-t- butyl nitrone (PBN 2 niM, Sigma-Aldrich). Six hours after neuronal TNFa stimulation (+/- PBN) the cultures were washed for three times with media
  • DMEM fetal calf serum
  • new media was added and after 15 hours supernatants were collected.
  • the collected supernatants were diluted 1: 1 with DMEM and added to spinal microglia cultures for 24 hours.
  • Spinal microglia were cultured as described previously (Yip et al., 2009).
  • IL6 and TNFa contents were determined by ELISA according to manufacturer's protocol (R&D systems).
  • the detection limit of IL-6 was 15 pg/ml and of TNFa 31 pg/ml.
  • HEK293 and N2A cells were grown in 6-well plates and transfected with pCMV6- FAM173Bmyc as described above. The cells were treated as described previously (Slot and Geuze, 2007. Nat Protoc 2, 2480-2491). Briefly, cells were chemically fixed using 2% formaldehyde (FA), 0.2% glutaraldehyde in 0.1M phosphate buffer pH 7.4 (Pi) for 2 hours and stored overnight in 1% FA in Pi. After rinsing with PBS (3x) and PBS-0.15 M glycine 1 ml, a 1 % gelatin solution was put on the cells and using a cell scraper the cells were removed from the Petri dish, transferred to an Eppendorf vial and spun down.
  • FA formaldehyde
  • Pi glutaraldehyde
  • PBS-0.15 M glycine 1 ml a 1 % gelatin solution was put on the cells and using a cell scraper the cells were removed from the Petri dish,
  • DHE dihydroethidium
  • MitoTrackerRedCM-H2XROS fluorescence was analyzed in small diameter neurons ⁇ 25 ⁇ and large diameter neurons > 25 ⁇ .
  • mice were deeply anesthetized with an overdose of sodium pentobarbital and transcardially perfused with PBS followed by 4% paraformaldehyde and spinal cords and DRGs were collected. Tissues were postfixed, cryoprotected in sucrose, embedded in OCT compound (Sakura), and frozen at -80°C. Cryosections (10 pm) of lumbar DRG and of lumbar spinal cord segments L3-L5 were stained with anti- Ibal (1:500, Wako). DRGs were stained with rabbit anti-NF200 (1:200, Millipore), biotinylated anti-IB4 (1:25, Vector laboratories ) and rabbit anti-GFAP (1:2000, Dako).
  • N2A cells were stained with anti-PDl (1: 100, Enzo life sciences) and anti- pGM130 (1: 100, BD Transduction laboratories).
  • DRG's sciatic nerves and hind paws staining's for FAM173B (1:500, Biorbyt), biotinylated anti-IB4 (1:25, Vector laboratories ), GFP (1:3000, Abeam) and periplierin (1: 100, Sigma Aldrich), tissues were fresh frozen, cut and post-fixed in PFA prior for staining. Staining's were visualized by using Alexafluor 488-(streptavidin) or 594-conjugated secondary antibody's. Nuclei were stained with DAPI.
  • Photographs were captured with a confocal laser scanning microscope LSM700 (co-localization experiments) or with a Zeiss Axio Observer microscope (Zeiss) using identical exposure times for all slides within one experiment. Fluorescence intensity was analyzed with ImageJ software.
  • FAM173A NP_076422.1
  • FAM173B NP_954584.2
  • Mus musculus Faml73a NP_663385.2
  • Faml73b NP_()80822.1
  • FAM173hom homolog of FAM173 proteins from the Archaea Sulfolobus islandicus (Si) (gi I 227827841) were used for the alignment.
  • the alignment was generated using the MUSCLE algorithm embedded in Jalview (Edgar, 2004. Nucleic Acids Res 32, 1792- 1797; Waterliouse et al., 2009. Bioinformatics 25, 1189-1191) and prediction of protein secondary structure was performed with Jpred 3 (Cole et al., 2008.
  • FAM173BA55-D94A (enzymatically inactive), were cloned into pET28a and expressed as N-terminally hexahistidine tagged proteins in Escherichia coli BL21- CodonPlus(DE3)-RIPL cells (Agilent) and purified using nickel-nitrilotriacetic acid- agarose (Qiagen), according to manufacturer's instructions and as described (Malecki et al, 2015. J Biol Chem 290, 423-434). Eluted protems were buffer- exchanged (Malecki et al., 2015. J Biol Chem 290, 423-434) and protein purity was asses by SDS-PAGE and Coomassie staining. Protein concentrations were measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific).
  • Methyltransferase reactions contained 10 ⁇ g of homopolymers or equivalent amounts of cell extracts from adenosine dialdehyde (AdOx)- treated HEK293 cells (Davydova et al., 2014. J Biol Chem 289, 30499-30510), [3H]-SAM (2 ⁇ (3 ⁇ 4 and recombinant hFAM173B (100 pmol) in 50 ⁇ reactions and were incubated for 1 hour at 37°C, as described (Jakobsson et al., 2015. PLoS One 10, e0131426;
  • Protein concentrations of the total cell lysates or mitochondrial/cytosol fractions were determined using a Bradford assay (Bio-Rad). Protein samples (20 ⁇ g) were separated by 12% SDS-PAGE and transferred to a PVDF membrane (Immobilon-P, Millipore). Membrane was stained with 1: 1000 goat anti-FAM173B, 1: 1000 mouse anti-COXIV (Invitrogen) or 1: 1000 goat ant- ⁇ -actin, followed by incubation with 1:5000 donkey anti goat-HRP (others all Santa Cruz Biotechnology). Specific bands were visualised by chemiluminescence (ECL, Advansta) and imaging system Proxima (Isogen life sciences) Real-time RT-PCR
  • RNA from freshly isolated DRGs and spinal cords was isolated using TRizol and RNeasy mini kit (Qiagen).
  • cDNA was synthesized using Reverse Transcriptase (Biorad)(Peters et al., 2013).
  • Quantitative real-time PCR reaction was performed with an I-cycler iQ5 (Bio-Rad) as described. The following primers were used: mFaml73b forward: TggTgTgCCCCAgATgAT
  • TNFa forward gCggTgCCTATgTCTCAg
  • GFAP forward ACAgACTTTCTCCAACCTCCAg
  • GAPDH forward TgAAgCAggCATCTgAggg
  • cDNA was synthesized from 1 ⁇ g total RNA (Clontech) and PGR was performed using Phusion polymerase (ThermoFisher Scientific) following manufacturing instructions. Human and mouse FAM173B was detected in a tissue panel
  • hFAMl 73B forward gTAgCCACgCCgTTTgTAAC
  • Faml 73b expression in vivo by lumbar intrathecal injections of a nuclease resistant antisense oligodeoxynucleotide (asODN), a method that has been shown to reduce mRNA expression and protein translation (Dias and Stein, 2002. Mol Cancer Ther 1, 347-355).
  • asODN nuclease resistant antisense oligodeoxynucleotide
  • Intrathecal injection of a fluorescent labeled Faml 73b asODN targeted almost all sensory neurons in the DRG and some other cells including Ibal and GFAP positive cells in the DRG (data not shown).
  • Intrathecal administration of Fain 173b asODN at day 5 till 10 in the CFA model of persistent inflammatory pain abrogated thermal and mechanical hyperalgesia (Fig. Vole).
  • Intraplantar injection of 5 ⁇ 1% carrageenan induced transient hyperalgesia (Aley et al, 2000. J Neurosci 20, 4680-4685; Wang et al., 2013. J Clin Invest 123, 5023- 5034) that resolved within 4-6 days (Fig. lh/i).
  • Fig. lh/i/j Intraplantar or intrathecal (data not shown) administration of HSY-hFAMl 73B prior to the induction of transient inflammatory pain markedly prolonged carrageenan-induced evoked thermal and mechanical hyperalgesia after intraplantar carrageenan as
  • FAM173B harbors characteristic motifs involved in binding of the methyl donor S adenosyl-L- methionine (SAM). Moreover it shows similarities for a subclass of SAM173B
  • hFAM173B displayed significant methyltransferase activity on poly-L-lysine but not on poly-L-arginine (Fig. 2d).
  • hFAM173B-D94A a putative ly enzymatically inactive mutant of hFAM173B (hFAM173B-D94A), generated by mutating a key conserved residue (Asp94) in the SAM-binding Motif I of
  • FAM173B and the methyltransferase death mutant FAM173B-D94A in mitochondria was further confirmed by western blot analysis of mitochondrial and cytosol fractions of N2A cells (data not shown). Electron microscopy of immuno-gold labeling of GFP-tagged hFAM173B showed that hFAM173B was predominantly present in the cristae of mitochondria when expressed in HEK293 (Fig. 2g) or N2A cells (data not shown).
  • HSV-mediated expression of hFAM173B in sensory neurons increased MitoTrackerRedCM-H2XROS fluorescence in small ( ⁇ 25 ⁇ ) diameter 3 and 6 days after intraplantar carrageenan (Fig. 31), indicating liFAM173B expression in sensory neurons promotes ongoing mitochondrial superoxide production in vivo.
  • ROS scavenger phenyl-N-t-butylnitrone (PBN) during hFAM173B-induced persistent inflammatory hyperalgesia.
  • microglia/macrophages contributes to the FAM173B-mediated prolongation of inflammatory pain.
  • Intrathecal injection of the glial cell inhibitor minocycline at day 7 completely inhibited hFAM173B-induced persistent inflammatory hyperalgesia (Fig. 4c).
  • FAM173B-mediated prolongation of inflammatory pain we asked whether in vivo overexpression of hFAM173B would engage glial cells after induction of inflammatory pain.
  • Expression of hFAM173B specifically in sensory neurons using HSV amplicons significantly increased the Ibal+ immunofluorescence in DRG and spinal cord at 5 and 10 days after carrageenan-treatment compared to mice treated with empty HSV amplicons (Fig. 4d-f).
  • This neuronal FAM173B-mediated spinal microglia activation in vivo was attenuated after inhibition of ROS with the ROS scavenger PBN (data not shown).
  • hFAM173B-D94A did not affect mitochondrial membrane potential, in contrast to wt hFAM173B which increased ⁇ (Fig. 5e).
  • expression of wt hFAM173B but not hFAM173B-D94A increased the fluorescence of the ROS-sensitive dye DHE in small ( ⁇ 25 ⁇ ) diameter neurons at day 5 during carrageenan-induced inflammatory hyperalgesia, indicating FAM173B-mediated increase in ⁇ and ROS production is also methyltransferase dependent (Fig. 5f).
  • culturing spinal microglia with supernatants of TNFa- stimulated sensory neurons expressing wt FAM173B increased IL6 (Fig.

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  • Rheumatology (AREA)
  • Pain & Pain Management (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un inhibiteur spécifique de la lysine méthyltransférase mitochondrienne FAM173B pour l'utilisation comme médicament, plus spécifiquement pour l'utilisation dans un procédé de traitement de la douleur chronique. L'invention concerne en outre une composition pharmaceutique comprenant un inhibiteur spécifique de la lysine méthyltransférase mitochondrienne (FAM173B), l'utilisation de cette composition dans un procédé de traitement de la douleur chronique, des procédés de typage d'une personne souffrant de douleur, et des procédés de traitement d'une personne souffrant de douleur.
PCT/NL2018/050226 2017-04-12 2018-04-12 Inhibiteurs de lysine méthyltransférase destinés au traitement de la douleur WO2018190713A1 (fr)

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