Inhibition of human mast cell activation with the novel selective adenosine A 2B receptor antagonist 3-isobutyl-8-pyrrolidinoxanthine (IPDX) Igor Feoktistov a,b , Emily M. Garland b , Anna E. Goldstein a , Dewan Zeng c , Luiz Belardinelli c , Jack N. Wells b,1 , Italo Biaggioni a,b, * a Department of Medicine, Vanderbilt University, Nashville, TN 37232, USA b Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA c CV Therapeutics, 3172 Porter Drive, Palo Alto, CA 94303, USA Received 21 November 2000; accepted 15 March 2001 Abstract The antiasthmatic drug enprofylline was the first known selective, though not potent, A 2B antagonist. On the basis of structure–activity relationships (SARs) of xanthine derivatives, we designed a novel selective adenosine A 2B receptor antagonist, 3-isobutyl-8-pyrrolidinox- anthine (IPDX), with potency greater than that of enprofylline. IPDX displaced [ 3 H]ZM241385 ([ 3 H]4-(2-[7-amino-2-(2- furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol) from human A 2B adenosine receptors with a K i value of 470  2 nM and inhibited A 2B -dependent cyclic AMP (cAMP) accumulation in human erythroleukemia (HEL) cells with a K B value of 625  71 nM. We found that IPDX was more selective than enprofylline toward human A 2B receptors. It was 38-, 55-, and 82-fold more selective for human A 2B than for human A 1 (K i value of 24  8 M), human A 2A (K B value of 36  8 M), and human A 3 (K i value of 53  10 M) adenosine receptors, respectively. IPDX inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced interleukin-8 secretion in human mast cells (HMC-1) with a potency close to that determined for A 2B -mediated cAMP accumulation in HEL cells, thus confirming the role of A 2B adenosine receptors in mediating human mast cell activation. Since adenosine triggers bronchoconstriction in asthmatic patients through human mast cell activation, IPDX may become a basis for the development of new antiasthmatic drugs with improved properties compared with those of enprofylline. Our data demonstrate that IPDX can be used as a tool to differentiate between A 2B and other adenosine receptor-mediated responses. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Receptors purinergic; P1; Mast cells; Asthma; Adenosine; Xanthines; Interleukin-8 1. Introduction The endogenous nucleoside adenosine can be released to, or formed in, the extracellular space under hypoxic and inflammatory conditions. Once generated, adenosine acts as an autocoid by interacting with adenosine receptors belong- ing to the seven transmembrane G-protein-coupled group of cell-surface receptors. Four subtypes of adenosine receptors have been cloned: A 1, A 2A, A 2B, and A 3. Significant ad- vancement has been made in the understanding of the mo- lecular pharmacology and the physiological relevance of adenosine receptors, but our knowledge of A 2B receptors lags behind that of other receptor subtypes. Lack of selec- tive pharmacological probes has hindered research in this area. NECA, a non-selective adenosine analog remains the most potent A 2B agonist. Characterization of A 2B receptors * Corresponding author. Mailing address: Italo Biaggioni, M.D., Clin- ical Trails Center, Vanderbilt University, 3500 The Village at Vanderbilt, 1500 21st Avenue South, Nashville, TN 37212-8210. Tel.: +1– 615-343– 1787; fax: +1– 615-343–7929. E-mail address: italo.biaggioni@mcmail.vanderbilt.edu (I. Biaggioni). 1 Send reprint requests to: Jack N. Wells, Ph.D., 446 RRB, Department of Pharmacology, Vanderbilt University, Nashville, TN 37232. Tel.: +1– 615-343–3542. E-mail address: jack.wells@mcmail.vanderbilt.edu Abbreviations: cAMP, cyclic, AMP; DPCPX, 1,3-dipropyl-8-cyclo- pentylxanthine; DPSPX, 1,3-dipropyl-8-p-sulfophenylxanthine; FBS, fetal bovine serum; IB-MECA, N 6 -(3-iodobenzyl)-N-methyl-5'-carbamoylad- enosine; IL-8, interleukin-8; IPDX, 3-isobutyl-8-pyrrolidinoxanthine; NECA, 5'-N-ethylcarboxamidoadenosine; PDE, 3',5'-cyclic nucleotide phosphodiesterase; SAR, structure–activity relationship. Biochemical Pharmacology 62 (2001) 1163–1173 0006-2952/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0006-2952(01)00765-1