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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.

Abstract: Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca2+]i mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B1 receptor agonist, Lys-[des-Arg9]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B2 receptor antagonist, fasitibant chloride, but not by the B1 selective antagonist, Lys-[Leu8][des-Arg9]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B2 receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B2 receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride. [Copyright &y& Elsevier]

The present study investigates the effects of ethanol and hydrogen peroxide (H2O2) on the barrier function and prostaglandin E2 (PGE2) release in differentiated Caco-2 cells. Epithelial barrier integrity was estimated by measuring transepithelial electrical resistance (TEER), the transport of reference compounds and lactate dehydrogenase leakage, the PGE2 release by enzyme immunoassay. Ethanol and H2O2 decreased TEER and increased the transport of lucifer yellow without affecting that of propranolol and phenylalanine. Only the effects of ethanol were accompanied by PGE2 production and were reversible without causing long-term cytotoxicity. The cyclooxygenase-2 inhibitor, NS-398, prevented the effect of ethanol on both PGE2 release and TEER, while inhibition of both cyclooxygenase-2 and tyrosine kinase drastically compromised cell viability and TEER recovery. Hepatocyte growth factor, keratinocyte growth factor or insulin prevented the effect of ethanol on cell permeability, but not on PGE2 release. Their combination prevented the effect of H2O2. In conclusion, ethanol and H2O2 increased paracellular permeability in differentiated Caco-2 cells without affecting transcellular and active transport. Cyclooxygenase-2 stimulated PGE2 release mediated the reversible effect of ethanol on tight junctions and, meanwhile, contributed to cell survival. Growth factors, normally present in the intestine, exerted a selective protective effect toward paracellular permeability increase induced by irritants. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:713–727, 2009 [ABSTRACT FROM AUTHOR]

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]

In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK2 receptor splice variant named β isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the α isoform. Moreover, neurokinin A was unable to inhibit the [3H]SR48968 binding to the β isoform up to μM concentrations. In cells expressing the human tachykinin NK2 receptor β isoform, contrary to those expressing the α isoform, natural or selective tachykinin receptor agonists (1 μM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca2+]i. The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca2+]i increase in cells expressing the α or β isoform of the human tachykinin NK2 receptor. The present data indicate that the human tachykinin NK2 receptor β isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK2 receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands. [Copyright &y& Elsevier]

Abstract: The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pK i 10.1) and potently antagonized (pK B 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pK i 6.1) and NK3 (pK i 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pK B 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pK B 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2 ≤6). In anaesthetized guinea-pigs, MEN15596 inhibited in a dose-related and persistent manner colon contractions induced by the selective tachykinin NK2 receptor agonist, [βAla8]neurokinin A(4–10) (3 nmol/kg i.v.), either after intravenous (ED50 0.18 μmol/kg), intraduodenal (ED50 3.16 μmol/kg) or oral administration (10–30 μmol/kg) without affecting, at 3 μmol/kg, i.v., the colonic contractions produced by the NK1 receptor selective agonist [Sar9]substance P sulfone (3 nmol/kg i.v.). In addition MEN15596 was effective in inhibiting bronchoconstriction produced by i.v. administration of [βAla8]neurokinin A(4–10). Overall the results indicate that MEN15596 is a potent and selective tachykinin NK2 receptor antagonist possessing high affinity and potency for guinea-pig, pig and human receptor, long duration of action in in vivo experiments and good oral bioavailability. [Copyright &y& Elsevier]

Abstract: NK2 antagonists have been reported to be potentially useful for the treatment of a number of chronic diseases, such as asthma, irritable bowel syndrome, cystitis, and depression. Starting from an in-house prepared library of capped dipeptides, we have identified a series of molecules with subnanomolar binding affinity for the hNK2 receptor. These molecules are composed by three well-defined regions: a planar aromatic acyl system as N-terminal capping group, a rigid and quite lipophilic core, and a flexible and relatively hydrophilic C-terminal capping group. Here we report how we were able to manipulate the N-terminal capping group to obtain significant in vivo activity after iv and id administration. [Copyright &y& Elsevier]

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.