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Norfenfluramine is a full agonist at human 5-HT2B receptors; evidence from a pre-clinical bioassay that predicts 5-HT2B agonism in man Cardiac valvulopathy associated with norfenfluramine (the active metabolite of fenfluramine) led to the withdrawal of fenfluramine from clinical use (Fitzgerald et al., 2000). This is proposed to be mediated via agonism of cardiac 5-HT2B receptors. The human colon, a tissue known to express 5-HT2B receptors, has previously been characterised as a suitable functional in vitro preparation to detect compounds that bind to 5-HT2B receptors (Borman et al., 2002). Therefore, the aim of this study was to confirm these findings and to investigate the functional effects of norfenfluramine and YM348 (Kimura et al., 2003) in the human colon longitudinal smooth muscle preparation. With informed consent and local Ethics Committee approval, macroscopically normal human colon was obtained from 24 donors (9 female, 15 male, 27-83 years) undergoing abdominal surgery. Upon arrival at the laboratory, longitudinal smooth muscle strips (2 x 10 mm) were isolated from inter-taenial sections. Strips were mounted in 15 ml tissue baths containing oxygenated (95%O2/5%CO2) Krebs solution at 37°C and connected to isometric force transducers with a resting tension of 1.5 g. Electrical field stimulation (EFS) was applied with 10 s stimulation every 60 seconds, at 10 V and with a 1 ms pulse width, at a range of frequencies from 0.1 to 40 Hz. Sub-maximal frequency was determined by performing a frequency response curve in each tissue. Following a 60 min rest period a single cumulative concentration-response curve (CCRC) to agonist was performed. For antagonist experiments, the compounds were pre-incubated for 30 min prior to the addition of the agonist. Data were calculated as a percentage of the response to 100 nM alpha-methyl-5-HT added to the bath at the end of the CCRC. Data are mean pEC50/Emax/Hill slope/pKb ± s.e.mean, n-values refer to the number of donors. Data were compared using Student’s t-test with statistical significance set at P<0.05. The EFS responses were inhibited by 1 μM tetrodotoxin and 1 μM atropine, suggesting the involvement of cholinergic nerves. Increasing the stimulation frequency resulted in increased phasic contractions, with a half-maximal frequency response of 5 Hz. Cumulative application of 5-HT, alpha-methyl-5-HT, YM348 or norfenfluramine (0.1 nM–10 μM) resulted in concentration-dependent potentiation of the EFS responses in some donors with rank order of potency of alpha-methyl-5-HT ³ 5-HT = YM348 >> norfenfluramine (see Table 1). In addition, these data suggest no gender difference in response to the agonists tested. SB206553 (5-HT2B antagonist, Hoyer et al., 2002) 100 nM, produced parallel rightward shifts of the alpha-methyl-5-HT CCRC with a mean pKb of 7.99 ± 0.17 (n=3). Table 1. Mean pEC50, Emax and Hill slope ± s.e.mean values for test compounds.
x = responding donors to test agent, n= total number of donors tested. Non-responders are defined by the lack of CCRC to 5-HT / alpha-methyl-5-HT, or to 100 nM alpha-methyl-5-HT at the end of the study in the case of norfenfluramine or YM348. In conclusion, this study has shown that both norfenfluramine and YM348 are potent full agonists at native human 5-HT2B receptors. Additionally, the potency of norfenfluramine is in good agreement with the reported clinical exposure that results in valvulopathy (100 nM, Fitzgerald et al., 2000) and therefore may suggest that this assay has the potential to predict compounds that could be associated with cardiac valvulopathy in man.
Borman, R.A. et al., (2002) Br.J.Pharmacol., 135 , 1144-51.
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