Ion channel binding

Abstract: Efforts to define precisely the role of 5-HT2B receptors in normal and disease processes have been hindered by the absence of selective antagonists. To address this deficiency, we developed a series of naphthylpyrimidines as potentially useful 5-HT2B receptor antagonists. RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine) was found to have nanomolar affinity for the 5-HT2B receptor (pKi=9.5±0.1) and 1,000 fold selectivity for this receptor as compared to numerous other receptor and ion channel binding sites. In cells expressing human recombinant 5-HT2B receptors, RS-127445 potently antagonized 5-HT-evoked formation of inositol phosphates (pKB=9.5±0.1) and 5-HT-evoked increases in intracellular calcium (pIC50=10.4±0.1). RS-127445 also blocked 5-HT-evoked contraction of rat isolated stomach fundus (pA2=9.5±1.1) and (±)α-methyl-5-HT-mediated relaxation of the rat jugular vein (pA2=9.9±0.3). RS-127445 had no detectable intrinsic activity in these assays. In rats, the fraction of RS-127445 that was bioavailable via the oral or intraperitoneal routes was 14 and 60% respectively. Intraperitoneal administration of RS-127445 (5 mg kg−1) produced plasma concentrations predicted to fully saturate accessible 5-HT2B receptors for at least 4 h. In conclusion, RS-127445 is a selective, high affinity 5-HT2B receptor antagonist suitable for use in vivo. The therapeutic potential of this molecule is being further evaluated. British Journal of Pharmacology (1999) 127, 1075–1082; doi:10.1038/sj.bjp.0702632

Abstract: Background QT prolongation is an important biomarker of the arrhythmia torsades de pointes and appears to be related mainly to blockade of delayed inward cardiac rectifier potassium currents. The aim of this study was to quantify the relationship between in vitro human ether-a-go-go-related gene (hERG) potassium channel blockade and the magnitude of QT prolongation in humans for the class III antiarrhythmic dofetilide. Methods The in vitro affinity and activity of dofetilide were determined in recombinant cell cultures expressing the hERG channel, and the QT-prolonging effect of dofetilide was assessed in 5 clinical studies (80 healthy volunteers and 17 patients with ischemic heart disease). A population pharmacokinetic-pharmacodynamic analysis of the in vitro and in vivo data was performed in NONMEM by use of the operational model of pharmacologic agonism to estimate the efficiency of transduction from ion channel binding to Fridericia-corrected QT response. Results A 3-compartment pharmacokinetic model with first-order absorption characterized the time course of dofetilide concentrations. On the basis of an in vitro potency of 5.13 ng/mL for potassium current inhibition and predicted unbound dofetilide concentrations, the estimated transducer ratio (τ) of 6.2 suggests that the QT response plateaus before currents are fully blocked. In our study population, 10% hERG blockade corresponds to a QT prolongation of 20 ms (95% confidence interval, 12–32 ms). With long-term dofetilide administration, tolerance develops with a half-life of 4.7 days. Conclusions The current mechanism-based pharmacokinetic-pharmacodynamic model quantified the relationship between in vitro hERG channel blockade and clinical QT prolongation for dofetilide. This model may prove valuable for assessing the risk of QT prolongation in humans for other drugs that selectively block the hERG channel on the basis of in vitro assays and pharmacokinetic properties. Clinical Pharmacology & Therapeutics (2005) 77, 572–582; doi: 10.1016/j.clpt.2005.02.004

Abstract: The actions of pyridostigmine (Pyr), an anticholinesterase agent, were studied on the acetylcholine (ACh) receptor-ion channel complex and on the electrically excitable membrane of the frog cutaneous pectoris and sartorius muscles and the chronically denervated soleus muscle of the rat. Pyr at concentrations of 0.2-0.4 mM potentiated the indirect evoked muscle twitch and at concentrations greater than or equal to 0.8 mM depressed the indirect twitch with an IC50 of about 2 mM. Twitch depression produced by Pyr was reversed slowly, and after a 60-min wash only 59% of the control muscle twitch had returned. Pyr did not affect either the membrane potential or the muscle action potential. Pyr had several effects at the neuromuscular junction of the frog and rat. It decreased the peak amplitude of the end-plate current (EPC) in a voltage- and concentration-dependent manner. In contrast to diisopropylfluorophosphate, which depresses the EPC amplitude and induces a double exponential decay of the EPC and miniature end-plate current (MEPC), Pyr produced a marked prolongation of the time constants of EPC and MEPC decay while maintaining a single exponential decay. The decrease caused by Pyr of indirect twitch tension, EPC amplitude, and ACh sensitivity indicates mechanisms which limit the number and/or properties of conducting channels. The drug decreased channel conductance and prolonged channel lifetime as revealed by Fourier analysis of ACh-induced end-plate current fluctuations. An altered form of the conducting species induced by Pyr appears to be responsible for either the apparent agonist-induced depolarization or its ability to increase the affinity of ACh for its recognition site. Pyr was also found to inhibit the binding of ACh and alpha-bungarotoxin to receptor-rich membrane from the electric organ of Torpedo nobiliana, and to have a higher affinity for the receptor than for the ion channel binding sites. These actions are distinct from acetylcholinesterase inhibition caused by the agent. Strong evidence suggests that the direct influences of the agent on neuromuscular transmission involve at least three distinct, although possibly interacting, mechanisms: (a) a weak agonist action, (b) the formation of desensitized receptor-complex intermediates, and (c) the alteration of the conductance properties of active channels.