Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1 Thomas Christoph a, * , Clemens Gillen a , Joanna Mika b,c , Arnold Gru ¨nweller d,e , Martin K.-H. Scha ¨fer d , Klaus Schiene a , Robert Frank a , Ruth Jostock a , Gregor Bahrenberg a , Eberhard Weihe d , Volker A. Erdmann e , Jens Kurreck e a Research & Development, Gru ¨nenthal GmbH, Zieglerstr. 6, 52078 Aachen, Germany b Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps University Marburg, D-35033 Marburg, Germany c Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krako ´w, Poland d Institute for Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35037 Marburg, Germany e Institute for Chemistry and Biochemistry, Free University Berlin, Thielallee 63, 14195 Berlin, Germany Received 21 July 2006; received in revised form 28 August 2006; accepted 31 August 2006 Available online 12 October 2006 Abstract To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED 50 value of 10.6 mg/ kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain. # 2006 Elsevier Ltd. All rights reserved. Keywords: Antisense oligonucleotides; Capsaicin receptor; Thioxo-BCTC; Neuropathic pain; Vanilloid receptor 1. Introductory statement The capsaicin or vanilloid receptor VR1 is a member of the transient receptor potential (TRP) family and is thus also referred to as TRPV1 (Montell et al., 2002). It is predominantly expressed by nociceptive sensory neurons and functions as a cation channel (Caterina et al., 1997; Tominaga et al., 1998). TRPV1 is activated by a wide array of pain-producing stimuli including capsaicin, noxious heat and low pH (for reviews, see Cortright and Szallasi, 2004; Tominaga and Tominaga, 2005) and is furthermore sensitized by pain-associated intracellular signaling pathways (e.g. protein kinase A, protein kinase C, ATP, phosphatidylinostol diphosphate (PIP 2 )) in a way that makes it a potentially pivotal regulator of nociceptor activity (Benham et al., 2002; Di Marzo et al., 2002). TRPV1 is therefore regarded as a promising target for the development of new analgesic strategies. The receptor agonist capsaicin is already being used for treatment of neuropathic pain, but its initial pungency hinders its broad application (The Capsaicin Study Group, 1991). The crucial involvement of TRPV1 to drive inflammatory pain is well established by analysis of TRPV1 knock-out mice (Caterina et al., 2000; Davis et al., 2000) as well as by investigations employing TRPV1 antagonists (Garcia-Martinez et al., 2002; Walker et al., 2003). Chemical ablation of TRV1 www.elsevier.com/locate/neuint Neurochemistry International 50 (2007) 281–290 * Corresponding author. Tel.: +49 241 569 2421; fax: +49 241 569 2852. E-mail addresses: thomas.christoph@grunenthal.de, thomas.christoph@grunenthal.com (T. Christoph). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2006.08.017