Prostaglandin Receptor Activation Properties of Lubiprostone J. Cuppoletti*, D.H. Malinowska*, R. Ueno***Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH;**Sucampo Pharmaceuticals, Inc., Bethesda, MD Introduction Lubiprostone is used clinically to treat chronic idiopathic constipation and irritable bowel syndrome with constipation1. It is a prostone (derived from metabolites of prostaglandins) that activates ClC-2 Cl- of 20 nM2 and increaseschannels with an EC50 intestinal 3uid secretion. In the present study, the agonist and antagonist activity of lubiprostoneonclonedhumanEPandFPreceptorswasexaminedtomoredirectlydetermine for lubiprostone binding to these speci3c receptors. This reduces the need to usethe EC50 either EP receptor antagonists or complex biological processes such as contraction to infer binding of lubiprostone to prostaglandin receptors.Purpose The aim of the present study was to determine the activities of lubiprostone on recombinant prostaglandin receptors.Methods and FP receptors was assayed byLubiprostone binding to recombinant EP1, EP2, EP3, EP4 Millipore Corporation, Bioscience Division (St. Charles, MO), using Chemiscreen calcium optimized FLIPR cell lines containing high levels of the promiscuous G protein, Galpha15, to enhance coupling of the receptor to the calcium signaling pathway. These cells weretransfected with cDNA containing either full-length human EP1, EP2, splice variant 6 of or FP receptors. Triplicate assays of lubiprostone effects were carried out withEP3, EP4 as a positive control. In all cases, the readout was relative 3uorescencePGE2or PGF2_ units related to [Ca2+ through calcium release activated calcium ion channel (CRAC)]i measured by Fluo-4 relative 3uorescence. To measure antagonist effects of lubiprostone, to givecells with cloned human receptors were 3rst stimulated with either PGE2or PGF2_ about 80% of the maximum response and then lubiprostone was added.Results Positive controls:Measured agonist activity of on andPGE2EP1, EP2, EP3EP4 receptor-expressing cells generatedEC50values of 7.46, 49.82, 3.86 and 31.18 nM respectively. Agonist activity of on FP receptor-expressing cells gave anPGF2_EC50 of 3.40 nM. or FP receptors (Figure 1).Lubiprostone exhibited no agonist effects on cloned EP2, EP4 valuesThere was weak agonist activity of lubiprostone on EP1and EP3receptors with EC50 of 330 nM and 280 nM, respectively, which are 44 and 73 times higher than the agonist activity on the respective EP receptors.activity of PGE2 or FP receptorsLubiprostone did not demonstrate any detectable antagonist effects on EP2 receptors(Figure 2). However, there was weak antagonist effect of lubiprostone on EP4 =127 nM).(EC50
E001820 Figure 1. Agonist Effect of Lubiprostone on Cloned EP and FP ReceptorsFigure 1.and FPDose response curve for agonist activity of lubiprostone on cloned EP1, EP2, EP3, EP4 receptors expressed in cultured cells. Data represent the mean (± SE) of 3 determinations. Figure 2. Antagonist Effect of Lubiprostone on Cloned EP and FP ReceptorsFigure 2.and FPDose response curve for a ntagonist activity of lubiprostone on cloned human EP2, EP4 receptors expressed in cultured cells. Data represent the mean (± SE) of 3 determinations.Summary It has been shown in the present study that lubiprostone does not act as an agonist or FP receptors. The lack of agonist activity of lubiprostoneon cloned human EP2, EP4 on cloned human contradicts the 3nding that lubiprostone reduced electricallyEP4 stimulated neural contractions in rat and human colon circular muscle with anEC50 at near nanomolar levels that were inhibited by an (but not other EP) receptorEP4 antagonist3 receptor occupationand implies that these reported effects are not due to EP4 by lubiprostone. Agonist activity of lubiprostone on cloned was very low with anEP1EC50= 330 nM, a receptor. Moreover, this concentration isvalue 44 times higher than for PGE2on the EP1 for lubiprostone for activation of ClC-2 chlorideapproximately 15 times higher than the EC50 channels2. Lubiprostone remains mostly in the lumen of the gut and does not enter the circulation1. This means that lubiprostone will not reach sufficiently high concentrations to antagonists do not affectactivate EP1receptors in the stomach muscle layer. Since EP1 effects on the vagal nerve4, lubiprostone is unlikely to act on the stomach throughthe PGE2 (or other EP or FP) receptors causing vagal nerve stimulation. This study con3rmsEP1 the previous 3ndings of Uenoet al5 that demonstrated that lubiprostone does not have pharmacologically relevant activity on prostaglandin receptors.Conclusions At clinically relevant doses lubiprostone is unlikely to have signi3cant PG receptor activity. The results demonstrate that activation of ClC-2 chloride channels underlying the clinical receptor occupation.effects of lubiprostone is independent of EP or FP2_References1. Amitiza [package insert]. Bethesda, MD: Sucampo Pharmaceuticals, Inc., 2008.2. Cuppoletti J, Malinowska DH, Tewari KP, Li QJ, Sherry AM, Patchen ML, Ueno R. SPI-0211 activates T84 cell chloride transport and recombinant human ClC-2 chloride currents.Am J Physiol Cell Physiol. 2004 287(5):C1173-83.3. Bassil AK, Borman RA, Jarvie EM, McArthur-Wilson RJ, Thangiah R, Sung EZ, Lee K, Sanger GJ. Activation of prostaglandin EP receptors by lubiprostone in rat and human stomach and colon.Br J Pharmacol. 2008 154(1):126-35.4. Kan KK, Jones RL, Ngan MP, Rudd JA. Excitatory action of prostanoids on the ferret isolated vagus nerve preparation.Eur J Pharmacol. 2004 Apr 26;491(1):37-41.5. Ueno R, Engelke KJ, and Osama H. Effects of lubiprostone, a novel GI chloride channel activator, on isolated smooth muscle.Neurogastroenterol Motil. 2005 17(4): 625-626. Supported by Sucampo Pharmaceuticals, Inc. and Takeda Pharmaceutical Company, Ltd. Presented at The American College of Gastroenterology 2008 Scienti3c Meeting |
