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Since its introduction a decade ago, the Caco-2 in vitro model for testing intestinal permeability has found wide application, in particular for screening new molecules in the pharmaceutical industry, An important issue for the validation of the model is to verify integrity and proper functionality of the Caco-2 cells monolayer, to ensure reproducibility and consistency of results. Several of these methods are based on measuring the apparent permeability coefficients ([P.sub.app]) of well-characterized reference compounds, having known absorption characteristics, and comparing the observed values to those expected. Most of them use fluorescent or radioactive chemicals to test different parameters. The main limitation of such approaches is that each parameter to be tested is associated with a single specific method, so that multiple procedures are needed to adequately characterize the cell monolayer. This work describes the use of a unique LC-MS/MS method to simultaneously determine the [P.sub.app] of a set of reference substances having well-characterized absorption behavior, i.e., phenylalanine, atenolol, and propranolol. The method is routinely used in our laboratory to check on model reproducibility and proved useful in verifying the consistency of the results derived from the experiments.

We have compared the effect of bradykinin (BK) and the nonpeptide kinin B2 receptor agonist, FR 190997, in producing changes in membrane potential and tension in the circular muscle of guinea-pig colon by the sucrose gap technique. In the presence of atropine (1 µM), S-ketoprofen (3 µM) and apamin (0.1 µM), BK (1 µM for 20 s) induced a transient depolarization of the membrane with superimposed action potentials (spikes) and transient contraction. Nifedipine (1 µM) eliminated the spikes and markedly inhibited the BK-induced contractions. FR 190997 (3–10 µM for 20 s) induced a slowly developing sustained small depolarization associated with a slowly developing and sustained contraction but, contrary to BK, FR 190997 was unable to trigger spikes. Nifedipine had no effect on depolarization and contraction induced by FR 190997. In the presence of 1 µM nifedipine, the combined application of a blocker of receptor-operated cation channels, SKF 96365 (50 µM for 30 min), and of an inhibitor of sarcoplasmic reticulum calcium pump, cyclopiazonic acid (CPA 10 µM for 30 min), reduced the BK-induced depolarization and contraction by about 45%–60%. The same treatment induced about 40% reduction of the sustained contraction induced by FR 190997, whereas the concomitant depolarization was not significantly affected. The tyrosine kinase inhibitor genistein (40 µM for 20 min) had no effect on the BK- and FR 190997-induced depolarization and contraction in the presence of nifedipine. In radioligand binding experiments performed in membranes of colonic smooth muscle cells, both agonists displaced the [3H]BK specific binding with a pIC50 of 9.6 and 8.5 for BK and FR 190997, respectively. These findings indicate a substantial qualitative difference in mechanisms of excitation contraction coupling activated by BK and FR 190997 via B2 receptors in guinea-pig colon. [ABSTRACT FROM AUTHOR]

As part of a project aimed at the identification of a series of small, orally available antagonists for the hNK(2) receptor, starting from one of our capped dipeptide libraries, we succeeded in the chemical optimization of the first identified leads, finally producing a class of molecules with significant activity in our animal model after iv administration. We herein report the results of further chemical modifications made to reduce the overall peptide character of this series and the consequent improvement of their in vivo antagonist activity. The present work identified 6-methylbenzo[b]thiophene-2-carboxylic acid (1-[(S)-1-benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl]cyclopentyl)amide (10i), endowed with subnanomolar potency in all the in vitro tests and being highly potent and of long duration upon in vivo testing after both iv and id dosing.

Starting from 1 (MEN14268), a selective tachykinin NK2 receptor antagonist with an interesting in vitro pharmacological profile, a family of numerous antagonists was obtained through an optimization process focused on iterated structural modifications. The effects of the introduction of a wide variety of substituents on the lipophilic aromatic part of the molecule and the modulation of the structural constraint through the insertion of different achiral alpha,alpha-dialkylamino acids were investigated. In particular, aromatic and benzofused heteroaromatic moieties were introduced at the pseudo-N-terminal residue to replace the 2-benzothiophene moiety, and a systematic investigation of the best positioning of substituents onto the aromatic platform was reported for the benzothiophene core. Studies on the modulation of the length and the rigidity of the hydrophilic pseudo-C-terminal pendant are presented. Many heteroaliphatic groups are well tolerated by the receptor in this part of the ligand. The product 48f (MEN15596), bearing a methyl substituent on the benzothiophene and a tetrahydropyranylmethylpiperidine pendant, was finally selected for its good in vivo activity after intravenous, intraduodenal, and oral administration in guinea pigs.