ORIGINAL ARTICLE In vitro pharmacological profiling of R406 identifies molecular targets underlying the clinical effects of fostamatinib Michael G. Rolf, Jon O. Curwen, Margaret Veldman-Jones, Cath Eberlein, Jianyan Wang, Alex Harmer, Caroline J. Hellawell & Martin Braddock AstraZeneca R&D Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom Keywords Abstract Blood pressure elevation, fostamatinib, in vitro pharmacological profiling, R406, SYK Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug Correspondence discovery; there are fewer reports addressing the relevance of broad profiles to Michael G. Rolf, AstraZeneca R&D Molndal,€ clinical adverse effects. Here, we have characterized the pharmacological profile Pepparedsleden 1, 431 83 Mo¨ lndal, Sweden. of the active metabolite of fostamatinib, R406, linking an understanding of drug Tel: +46 31 776 60 40; Fax: +46 31 776 37 selectivity to the increase in blood pressure observed in clinical studies. R406 60; E-mail: [email protected] was profiled in a broad range of in vitro assays to generate a comprehensive Funding Information pharmacological profile and key targets were further investigated using func- No funding information provided. tional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links Received: 9 April 2015; Accepted: 14 July between the profile of R406 and clinical side effects. R406 was selective outside 2015 the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase Pharma Res Per, 3(5), 2015, e00175, doi: 10.1002/prp2.175 domain, having activity at many protein kinases at therapeutically relevant con- centrations when tested in multiple in vitro systems. Systematic literature analy- doi: 10.1002/prp2.175 ses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile estab- lishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development. Abbreviations 2-Cl-IB-MECA, (2S,3S,4R,5R)-5-[2-chloro-6[(3-iodophenyl)methylamino]purin-9- yl]-3,4-dihydroxy-N-methyloxolane-2-carboxamide; 2D, two-dimensional; 3D, three-dimensional; CHO, chinese hamster ovary; DAPK1, death-associated protein kinase 1; DMSO, dimethylsulphoxide; FLT3, Fms-like tyrosine kinase 3; GTPcS, guanosine 50-O-[gamma-thio]triphosphate; HPLC, high-performance liquid chro- matography; HUVEC, human umbilical vein endothelial cell; ITP, immune throm- bocytopenic purpura; KIT, mast/stem cell growth factor receptor; RA, rheumatoid arthritis; SRC, proto-oncogene tyrosine-protein kinase Src; STK16, serine-threonine protein kinase 16; SYK, spleen tyrosine kinase; TYRO3, tyrosine-protein kinase receptor TYRO3. ª 2015 Astrazeneca Ltd. Pharmacology Research & Perspectives publishedbyJohnWiley&SonsLtd, 2015 | Vol. 3 | Iss. 5 | e00175 British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. Page 1 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Pharmacological Profile of Fostamatinib M. G. Rolf et al. Introduction cascade and is thought to mediate a diverse set of cellular responses in RA and in the periphery. Spleen tyrosine A new drug is typically developed to target a specific bio- kinase signaling is an integral component of auto-antibody logical molecule in order to treat the symptoms or modu- activation of immune cells (Wong et al. 2004) and inhibi- late the underlying causes of a disease. However, very few tion of SYK may, therefore, reduce the autoimmune if any small molecule drugs are truly specific for the response (Braselmann et al. 2006). Fostamatinib modulates intended primary target and unintended off-target inter- immune signaling in multiple cell types involved in inflam- actions may drive adverse drug reactions or, in some mation and tissue damage in rheumatoid arthritis and so cases, contribute to the efficacy of the drug. In vitro phar- may inhibit key steps in the progression of this disease macological profiling has been demonstrated to add value (Wong et al. 2004). In phase II clinical studies for RA in the drug discovery process by influencing chemical (Weinblatt et al. 2008, 2010), fostamatinib has been associ- design with the objective of selecting a compound with low ated with an increase in systolic blood pressure of approxi- promiscuity, maximizing the chances of success and mini- mately 3 mmHg between baseline at month 1, as compared mizing the risk of intolerable side effects (Bowes et al. with a decrease of 2 mmHg with placebo. Preclinical stud- 2012). Off-target pharmacology may not be restricted to ies suggest that alterations in nitric oxide production in the targets closely related to the intended therapeutic target endothelium may underlie these increases (Skinner et al. (Paolini et al. 2006), and consideration should be given to 2014). Fostamatinib itself is a prodrug, rapidly metabolized the most appropriate technologies and test systems to use in vivo to the pharmacologically active moiety R406 (Bowes et al. 2012). Prospectively, the data can be used to (Baluom et al. 2013) and therefore in vitro studies investi- differentiate between different chemical series and to gating the mechanisms underlying the in vivo actions of predict possible adverse drug reactions (Whitebread et al. fostamatinib utilize R406. As a member of the tyrosine 2005; Peters et al. 2012). Retrospectively, the profile can kinase class inhibitor class of compounds, R406 like other provide mechanistic insight into the effects seen in preclini- members of the class exhibits activity at other kinase targets cal and clinical studies. Linking an adverse effect to a (Metz et al. 2011; Davis et al. 2011), although this is molecular target can enable informed decisions to be made reduced when compared with SYK inhibition in assays of about study exclusion criteria to ensure patients’ safety, or cellular function (Braselmann et al. 2006). In a limited mitigate the need for broad-based warnings in labeling. panel of nonkinase targets, R406 had few off-target activi- Off-target pharmacology can also support additional indi- ties outside of the kinase space (Braselmann et al. 2006), cations for marketed drugs. For example, the inhibitory and this study did not identify a molecular target that activity of quetiapine fumarate and particularly its metabo- could link to the increases in blood pressure seen in pre- lite N-desalkylquetiapine on the noradrenaline transporter clinical and clinical studies. However, this assessment that was initially detected via broad in vitro pharmacologi- focused on receptors, ion channels, and transporters, cal profiling provides part of the basis for the efficacy of thereby omitting a number of target classes known to be this compound in depressive disorders (Goldstein et al. commonly hit by kinase inhibitors, for example, phospho- 2007; Jensen et al. 2008). diesterases (Luke et al. 2008). A broader profiling exercise, Fostamatinib (previously known as R788) is the first utilizing multiple technologies, is therefore required. In the small molecule oral spleen tyrosine kinase (SYK) inhibitor current study, we present the most comprehensive in vitro (Braselmann et al. 2006; Riccaboni et al. 2010; Singh pharmacological profile published to date for a compound et al. 2012), which has completed phase III clinical trials in late-stage clinical trials and describe the links between in patients with rheumatoid arthritis (RA; Weinblatt et al. individual targets and the observed effects of fostamatinib 2013) and is currently in phase 3 trials for immune in patients. thrombocytopenia purpura (ITP) (http://www.rigel.com/ rigel/pipeline). Materials and Methods Spleen tyrosine kinase is expressed in cells of the hematopoietic lineage such as mast cells, basophils, B-cells, Determination of the solubility of R406 T-cells, neutrophils, dendritic cells, macrophages, mono- cytes, erythrocytes, and platelets in addition to non- R406 was synthesized by AstraZeneca. The solubility of hematopoietic cells such as osteoclasts, vascular endothelial R406 in 100% DMSO and in eight assay buffers represen- cells, fibroblasts, hepatocytes, and neuronal cells (reviewed tative of the compositions used in the in vitro assays was in De Castro 2011; Singh et al. 2012). Spleen tyrosine determined. kinase is an intracellular protein kinase and is a key media- An R406 DMSO solution was diluted with the buffer tor of Fc and B-receptor signaling found on the surface of used in kinase assay until the solubility limit was inflammatory cells where it acts at the head of a signaling achieved. The resulting mixture was vortexed well. An ali- 2015 | Vol. 3 | Iss. 5 | e00175 ª 2015 Astrazeneca Ltd. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, Page 2 British Pharmacological Society