Antonio Triolo

Differential effect of ethanol and hydrogen peroxide on barrier function and prostaglandin E2 release in differentiated Caco‐2 cells: Selective prevention by growth factors

The present study investigates the effects of ethanol and hydrogen peroxide (H(2)O(2)) on the barrier function and prostaglandin E(2) (PGE(2)) 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 PGE(2) release by enzyme immunoassay. Ethanol and H(2)O(2) decreased TEER and increased the transport of lucifer yellow without affecting that of propranolol and phenylalanine. Only the effects of ethanol were accompanied by PGE(2) production and were reversible without causing long-term cytotoxicity. The cyclooxygenase-2 inhibitor, NS-398, prevented the effect of ethanol on both PGE(2) 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 PGE(2) release. Their combination prevented the effect of H(2)O(2). In conclusion, ethanol and H(2)O(2) increased paracellular permeability in differentiated Caco-2 cells without affecting transcellular and active transport. Cyclooxygenase-2 stimulated PGE(2) 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.

Increased Paracellular Absorption by Bile Salts and P-Glycoprotein Stimulated Efflux of Otilonium Bromide in Caco-2 Cells Monolayers as a Model of Intestinal Barrier

The present study investigates the intestinal permeability of otilonium bromide, a spasmolytic drug used to treat irritable bowel syndrome, across Caco-2 cell monolayers. The amount of otilonium bromide transported was determined by high-performance liquid chromatography-mass spectrometry. Epithelial barrier integrity was estimated by measuring transepithelial electrical resistance and the transport of reference compounds, P-glycoprotein activity by measuring rhodamine 123 efflux. Results showed that the apparent permeability of otilonium bromide was comparable to that of our zero permeability marker, inulin, in the apical-to-basal direction and similar to that of rhodamine 123 in the basal-to-apical direction. The P-glycoprotein substrate, verapamil, prevented otilonium bromide efflux and, conversely, otilonium bromide inhibited P-glycoprotein activity. Bile salts induced a transient opening of tight junctions, as measured by selective increase of paracellular transport, and significantly enhanced the absorption of otilonium bromide. In turn otilonium bromide potentiates the effect of bile salts on tight junctions without modifying their critical micellar concentration or altering cell viability. In conclusion, otilonium bromide is a paracellularly transported drug whose absorption, in amounts sufficient to exert a spasmolytic effect, is favoured by bile salts. P-glycoprotein, by stimulating efflux, contributes to remove excess compound, restraining its distribution and site of action to the intestinal wall.

SOLID-PHASE AMINO-ZINC-ENOLATE CYCLIZATION FOR THE SYNTHESIS OF 3-SUBSTITUTED PROLINES

Abstract Preparation of 3-substituted prolines was achieved by extension to Solid Phase Organic Synthesis (SPOS) of the amino-zinc-enolate cyclization we have recently described. 1 Functionalizations of proline at C-3 were considered by nucleophilic substitutions of an iodo derivative obtained on the resin.

Successful bridging from a peptide to a non peptide antagonist at the human tachykinin NK-2 receptor.

Non peptide products have been found to show nanomolar binding and functional affinities at the human tachykinin NK-2 receptor. The new antagonists do not possess stereogenic centers and their thermal behaviour in solution is featured by a peculiar set of conformational stereoisomers. A macroscopic viewpoint is preferentially adopted to rationalize the obtained results.

Side reactions in peptide synthesis: Dehydration of C-terminal aspartylamide peptides during side chain to side chain cyclization

Abstract Side chain to side chain cyclization in solution of C-terminal aspartylamide peptides with PyBOP or HBTU produced concomitant extensive dehydration of the C-terminal amide to the corresponding nitrile. This side reaction is effectively prevented by HOBT.

Modulation on C‐ and N‐Terminal Moieties of a Series of Potent and Selective Linear Tachykinin NK2 Receptor Antagonists

Herein we describe the synthesis of a series of new potent tachykinin NK2 receptor antagonists by the modulation of the C‐ and N‐terminal moieties of ibodutant (MEN 15596, 1). The N‐terminal benzo[b]thiophene ring was replaced by different substituted naphthalenes and benzofurans, while further modifications were evaluated at the C‐terminal tetrahydropyran moiety. Most compounds demonstrated a high affinity for the human NK2 receptor and high in vitro antagonist potency, indicating that a wide range of substituents at both termini can be incorporated in the molecule without detrimental effects on the interactions with the NK2 receptor. Selected compounds were tested in vivo confirming their activity as NK2 antagonists. In particular, after both iv and id administration to guinea pig, compound 61 b was able to antagonize NK2‐induced colonic contractions with a potency and duration‐of‐action fully comparable to the reference compound 1 (MEN 15596, ibodutant).

Solid-phase synthesis of MEN 11270, a new cyclic peptide kinin B2 receptor antagonist.

An efficient synthesis of the cyclic decapeptide MEN 11270 [H‐𝒹Arg1‐Arg2‐Pro3‐Hyp4‐Gly5‐Thi6‐Dab7‐𝒹Tic8‐Oic9‐Arg10 c(7γ–10α)] was developed. Two three‐dimensional orthogonal strategies were applied and compared: Fmoc/Tos/Boc (procedure A) and Fmoc/Pmc/Dde (procedure B). Both resulted in a 23‐step strategy comprising the stepwise solid‐phase chain assembly of the linear protected peptide, partial deprotection, solution‐phase cyclization and final full deprotection. The stepwise assembly of the linear peptide was optimized by double coupling and acylation time prolongation for critical residues (Tic, Dab, Thi, Pro). O‐(7‐azabenzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium (HATU) was preferred as coupling reagent for Dab. In the cyclization step, the partial racemization of Arg10 (31% using 1‐ethyl‐3‐(3′‐dimethylaminopropyl)‐carbodiimide/1‐hydroxybenzotriazole (EDC/HOBt) as activation system) was reduced to 3% with HATU. The final deprotection was performed in the presence of dimethylsulfide (procedure A) and thiocresol (procedure B) as scavengers, to avoid the sulfation of Hyp side chain. The final compound and the main by‐products were characterized by mass spectroscopy (MS), nuclear magnetic resonance (NMR) and racemization test. Procedure B produced operationally simpler and more efficient results than A (28% overall yield versus 4%). Copyright

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

It has recently been shown that the biological activity of the second generation kinin B1 receptor selective antagonist, desArg10 HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg10 HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide DTic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg10 HOE 140 to that of an agonist.

Solid support-dependent alkylation of tryptophan residues in SPPS using a 2-methoxybenzyl alcohol-based linker

Several side reactions can be encountered in the synthesis of Trp-containing peptides, due to molecular species originating from side chain-protecting groups or from the linker during acidolytic cleavage of the peptide from the resin. The linker can be the source of both alkylation in solution of the indole moiety of the tryptophan side chain and permanent readdition of the cleaved peptide to the resin. We report that both these reactions occur at a high level during the synthesis of Trp-containing peptides on a PEG-PS resin containing a 2-methoxybenzyl alcohol-based linker, in spite of the presence of suitable scavengers in the TFA-based cleavage mixture. Both side reactions are efficiently prevented by the use of a protected analogue of tryptophan, namely Nim-Boc-Trp, previously reported for the synthesis of peptides containing tryptophan and arginine residues.

Structural characterization of the regioisomers of di(hydroxybutyl) ether by GC-EI MS and theoretical calculations†

Di(hydroxybutyl) ether (DHBE), a liver protecting drug, is composed of a mixture of three regioisomers: 4-(3-hydroxybutoxy)-2-butanol (1), 3-(4-hydroxy-2-butoxy)-1-butanol (2), and 3-(3-hydroxybutoxy)-1-butanol (3). Unequivocal differentiation of each regioisomer of DHBE was rapidly obtained without isolation of the single components, using GC-MS with electron ionization (EI).The mass spectrum of 1 showed a rearrangement ion at m/z 118, characteristic of the 3-hydroxybutyl chain, deriving from loss of acetaldehyde from the molecular ion, whereas 2 and 3 were characterized by the ion at m/z 117, expected from alpha-cleavage of the 4-hydroxy-2-butyl chain. The species at m/z 118, in turn, loses a water molecule via a mechanism involving both alcohol hydrogens, as shown by deuterium exchange experiments. Both this finding and theoretical calculations support a mechanism in which the loss of acetaldehyde in 1 occurs via a cyclic intermediate, stabilized by a strong hydrogen bond between the alcohol oxygen bearing the charge and the other alcohol oxygen, and involves initial hydrogen transfer from the former to the latter.The EI spectrum of 2, having two 4-hydroxy-2-butyl chains, showed the fragmentations expected from classical fragmentation rules of aliphatic ethers and alcohols, whereas the EI spectrum of 3, bearing one 4-hydroxy-2-butyl and one 3-hydroxybutyl chain, showed essentially the characteristic fragments of both chains.