title: Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT2R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies.
abstract: BACKGROUND
  The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging.
  
  
  RESULTS
  AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine.
  
  
  CONCLUSIONS
  The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. u.anand@imperial.ac.uk.|Nanomedicine Research Laboratory, Division of Medicine, Hammersmith Hospital, Imperial College London, BN5 Commonwealth Building, London, W12 0NN, UK. u.anand@imperial.ac.uk.
authors.forename: Uma
authors.initials: U
authors.lastname: Anand
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. y.yiangou@imperial.ac.uk.
authors.forename: Yiangos
authors.initials: Y
authors.lastname: Yiangou
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. marcosinisi@iol.it.|Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA7 4LP, UK. marcosinisi@iol.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Sinisi
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. drmikefox@aol.com.|Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA7 4LP, UK. drmikefox@aol.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Fox
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. antmacquillan@icloud.com.|Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA7 4LP, UK. antmacquillan@icloud.com.
authors.forename: Anthony
authors.initials: A
authors.lastname: MacQuillan
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. tom.quick@nhs.net.|Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore, Middlesex, HA7 4LP, UK. tom.quick@nhs.net.
authors.forename: Tom
authors.initials: T
authors.lastname: Quick
authors.affiliation: Nanomedicine Research Laboratory, Division of Medicine, Hammersmith Hospital, Imperial College London, BN5 Commonwealth Building, London, W12 0NN, UK. y.korchev@imperial.ac.uk.
authors.forename: Yuri E
authors.initials: YE
authors.lastname: Korchev
authors.affiliation: University of Oxford Structural Genomics Consortium, Old Road, Campus Research Building, Roosevelt Drive, Headington, Oxford, OX3 7DQ, UK. chas.bountra@sgc.ox.ac.uk.
authors.forename: Chas
authors.initials: C
authors.lastname: Bountra
authors.affiliation: Spinifex Pharmaceuticals Pty Ltd, Corporate One, Suite G5, 84 Hotham St, Preston, VIC, 3072, Australia. Tom.McCarthy@spinifexpharma.com.au.
authors.forename: Tom
authors.initials: T
authors.lastname: McCarthy
authors.affiliation: Peripheral Neuropathy Unit, Centre for Clinical Translation, Hammersmith Hospital, Imperial College London, Area A, Ground Floor, Du Cane Rd, London, W12 ONN, UK. p.anand@imperial.ac.uk.
authors.forename: Praveen
authors.initials: P
authors.lastname: Anand
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology; D057912:Angiotensin II Type 2 Receptor Blockers / Q000494:pharmacology / Q000627:therapeutic use*; D000818:Animals; D001559:Benzhydryl Compounds / Q000494:pharmacology / Q000627:therapeutic use*; D002118:Calcium / Q000378:metabolism; D005260:Female; D005727:Ganglia, Spinal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007546:Isoquinolines / Q000494:pharmacology / Q000627:therapeutic use*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D009417:Nerve Tissue / Q000378:metabolism; D009437:Neuralgia / Q000188:drug therapy* / Q000473:pathology; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D017208:Rats, Wistar; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D050916:TRPV Cation Channels / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D057912:Angiotensin II Type 2 Receptor Blockers; D001559:Benzhydryl Compounds; C000595242:EMA400; D007546:Isoquinolines; D044139:Receptor, Angiotensin, Type 2; D050916:TRPV Cation Channels; C494484:TRPV1 receptor; D000804:Angiotensin II; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium