TSPO) Ligands and Benzodiazepines

Original Paper Phpsy/2014-08-0369/3.2.2015/MPS Enhancing Neurosteroid Synthesis – Relationship to the Pharmacology of Translocator Protein (18 kDa) (TSPO) Ligands and Benzodiazepines Authors L. Wolf1, A. Bauer1, D. Melchner1, H. Hallof-Buestrich1, P. Stoertebecker1, E. Haen1, 2, M. Kreutz3, N. Sarubin1, V. M. Milenkovic1, C. H. Wetzel1, R. Rupprecht1, C. Nothdurfter1 Affiliations 1 Department of Psychiatry and Psychotherapy, University Regensburg, Regensburg, Germany 2 Institute of Pharmacy, University of Regensburg, Regensburg, Germany 3 Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany Key words Abstract Results: In BV-2 microglia and C6 glioma cells ●▶ TSPO ligands ▼ all compounds significantly enhanced TSPO ●▶ benzodiazepines Introduction: The treatment of anxiety dis- protein expression. Radioligand binding assays ●▶ pregnenolone orders is still a challenge; novel pharmacologi- revealed the highest binding affinity to TSPO ●▶ neurosteroids cal approaches that combine rapid anxiolytic for XBD173, followed by diazepam and etifox- ●▶ anxiolytic efficacy with fewer side effects are needed. A ine. Pregnenolone synthesis was most potently promising target for such compounds is the mito- enhanced by etifoxine. chondrial translocator protein (18 kDa) (TSPO). Discussion: Etifoxine turned out to be the TSPO plays an important role for the synthesis of most potent enhancer of neurosteroidogenesis, neurosteroids, known to modulate GABAA recep- although its binding affinity to TSPO was lowest. tors, thereby exerting anxiolytic effects. These results indicate that the efficacy of TSPO Methods: We investigated the pharmacological ligands to stimulate neurosteroid synthesis, profile of 2 well established TSPO ligands (XBD173 thereby leading to anxiolytic effects cannot be and etifoxine) compared to the benzodiazepine concluded from their binding affinity to TSPO. diazepam with regard to TSPO binding affinity, TSPO expression and neurosteroidogenesis. Introduction Currently, one of the most promising novel drug ▼ targets for the treatment of anxiety disorders is Mental disorders are widespread and highly the translocator protein 18 kDa (TSPO) [3]. TSPO disabling with a considerable socioeconomic is a 5 helical transmembrane protein located in received 15.07.2014 revised 25.11.2014 impact. Among these, anxiety disorders belong to the outer mitochondrial membrane. It promotes accepted 27.11.2014 the most frequent. A 2004 WHO commissioned the transport of cholesterol into the mitochon- study of EU citizens found that 13.6 % of the pop- drial matrix, which is the rate-limiting step in Bibliography ulation suffer from these disorders [1]. However, neurosteroid synthesis. Cholesterol is then DOI http://dx.doi.org/ the pharmacological treatment of anxiety disor- metabolized to pregnenolone by CYP 11A1 (cho- Downloaded by: IP-Proxy University of California Irvine, California. Copyrighted material. 10.1055/s-0034-1398507 ders is still a challenge [2]. First line treatment lesterol side-chain cleavage enzyme, P450scc). Published online: 2015 options in acute states are benzodiazepines (e. g., Pregnenolone is then further metabolized to Pharmacopsychiatry diazepam). These drugs have a rapid onset of other neurosteroids, such as allopregnanolone © Georg Thieme Verlag KG Stuttgart · New York action, but they are sedative and quickly induce and 3α,5α-THDOC (3alpha,5alpha-tetrahydrode- ISSN 0176-3679 tolerance and abuse liability. For long-term oxycorticosterone) which are potent modulators treatment, certain antidepressants, such as of GABAA receptor function, thereby exerting Correspondence selective-serotonin-reuptake-inhibitors (SSRIs) anxiolytic effects [3]. TSPO is expressed in many C. Nothdurfter, MD are preferred because they are not sedative and organs, the highest expression levels are found in Department of Psychiatry and do not cause tolerance. However, these drugs steroid-synthesizing cells, e. g., adrenal, gonad Psychotherapy have a delayed onset of action of several weeks. It and brain cells [4]. In the central nervous system University Regensburg is obvious that novel compounds for the treat- (CNS), TSPO is mainly expressed in microglia [5] Universitätsstrasse 84 D-93053 Regensburg ment of anxiety disorders are needed that com- and in reactive astrocytes [6]. Germany bine rapid anxiolytic efficacy with a preferable Most TSPO ligands are primarily used in neuro- Caroline.Nothdurfter@klinik. side effect profile. imaging as diagnostic tools for brain inflamma- uni-regensburg.de tion [7]. However, some TSPO ligands already Wolf L et al. Enhancing Neurosteroid Synthesis … Pharmacopsychiatry Phpsy/2014-08-0369/3.2.2015/MPS Original Paper play a role in the pharmacological treatment of anxiety disor- added directly to the culture medium. Solvent (ethanol) concen- ders. The benzoxazine etifoxine was the first TSPO ligand that tration was 0.5 % in each well. After 24 h cells were harvested by showed clinical anxiolytic effects comparable to those of the scrapping cells in 1 mL HEPES buffer [20 mM HEPES, 5 mM EDTA, benzodiazepine lorazepam in patients suffering from adjust- 1 M NaCl supplemented with a protease inhibitor cocktail ment disorders with anxiety [8]. Etifoxine (trade name Stresam) (Sigma-Aldrich)] and subsequent sonication. Lysates were stored is used as an anxiolytic and anticonvulsant. However, this drug is at − 20 °C until further use. also a weak direct GABAA receptor modulator [9]. The later developed compound XBD173 (AC-5 216/emapunil) is a phenyl- Western blotting purine that is rather selective for TSPO; it has also been shown to Protein concentrations of cell lysates were quantified by means exert potent anxiolytic efficacy in animals as well as in humans of the Bradford method [13] with the Bio-Rad Protein Assay Dye without causing sedation [10]. Because the proof of concept Reagent Concentrate (Bio-Rad Laboratories, Munich, Germany). study with XBD173 using the cholecystokinin tetrapeptide 10 µg of protein lysates were separated by sodium dodecyl sul- (CCK-4) challenge paradigm in healthy volunteers was positive, fate gel electrophoresis (SDS-PAGE) with 15 % polyacrylamide a phase II trial in patients with generalized anxiety disorder was gels and were then transferred onto a nitrocellulose membrane. conducted which, however, did not show superiority over pla- Membranes were blocked with 5 % non-fat dry milk in TBST cebo (unpublished data). Diazepam is a frequently prescribed (10 mM TRIS, 150 mM NaCl, 0.1 % Tween 20; pH 7.4). TSPO was anxiolytic belonging to the group of benzodiazepines, which are detected with a goat-anti-TSPO antibody (My Biosource, San direct GABAA receptor modulators. They bind to the benzodiaz- Diego, CA, USA) and ß-actin with a rabbit-anti-ß-actin (Sigma- epine binding site (located at the interface of α and γ subunits) Aldrich) overnight at 4 °C. Blots were washed with TBST and thereby increasing the frequency of the chloride ion channel incubated with secondary horseradish peroxidase (HRP)-conju- opening in the presence of GABA [11]. However, some benzodi- gated antibodies. Bands were detected with the Chemilumines- azepines have also been reported to bind to TSPO [3]. cence Substrate Super Signal West Pico (Fisher Scientific, In summary, there is a considerable overlap between different Schwerte, Germany) and visualized with a digital imaging sys- classes of anxiolytic compounds with regard to their pharmaco- tem (Image Quant LAS 4000, GE Healthcare Europe, Freiburg, logical mechanisms of action. However, their clinical effects and Germany). Densitometry analysis was performed with ImageJ also side effects differ notably. To get a better insight into the Software (Wayne Rasband, National Institute of Health, USA). pharmacology of these drugs we characterized the mechanism TSPO values were normalized to ß-actin values of the same sam- of action of the TSPO ligands XBD173 and etifoxine in compari- ple and to control (cells treated with solvent). son to the benzodiazepine diazepam with regard to TSPO binding affinity, TSPO expression and neurosteroid production. Pregnenolone ELISA BV-2 and C6 cells were seeded onto 24-well plates. 80 % conflu- ent cells were treated for 21 h with etifoxine, XBD173 or diaze- Materials and Methods pam directly added to the medium at concentrations of 0.1 µM, ▼ 1 µM, 3 µM and 10 µM, respectively. These concentrations were Chemicals chosen according to concentrations reached in the cerebrospinal Etifoxine and diazepam were purchased from Sigma-Aldrich fluid of humans treated with diazepam [12]. After this first (Seelze, Germany). XBD173 was obtained by custom synthesis period of incubation plates were washed once with phosphate (APAC Pharmaceuticals, Columbia, USA). All compounds were buffered saline (PBS, Sigma-Aldrich) and then filled with 1mL dissolved in ethanol as stock solutions of 10 mM. pregnenolone assay buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgSO4, 10 mM glucose and 10 mM HEPES) [14] sup- Cell culture plemented with 0.1 % BSA (bovine serum albumin, Sigma- BV-2 mouse microglia cells were a generous gift from Dr. Marcus Aldrich) and 25 µM trilostane (Sigma-Aldrich). Cells were treated Karlstetter (Center of Ophtalmology, Experimental Immunology again with the above mentioned compounds for further 3 h. of the Eye, University of Cologne, Germany). C6 rat glioma cells Afterwards, supernatants were used in an enzyme-linked were a generous gift from Dr. Barbara Di Benedetto (Max- immunosorbent assay for pregnenolone

Load more