Enhanced Ca(2+) response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines.

Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1ß enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.

Comparison between oral and intra-articular antinociceptive effect of dexketoprofen and tramadol combination in monosodium iodoacetate-induced osteoarthritis in rats.

Dexketoprofen and tramadol, alone or in combination, were evaluated after oral or intra-articular administration on knee osteoarthritis nociception induced by intra-articular (i.ar.) monosodium iodoacetate (MIA, 1 mg/25 µl) in the rat right knee while the left knee received saline (25 µl). Seven days after MIA treatment, dexketoprofen, tramadol, their combination or the vehicle were administered. Nociception was evaluated as alteration in hind limb weight distribution with Incapacitance tester at different time-points after drug administration. Oral dexketoprofen (0.1-1 mg/kg) or tramadol (0.5-5 mg/kg) induced maximal antinociception at 1 and 5 mg/kg, respectively. Their combination dose-dependently increased the intensity and duration of antinociception, that was additive and lasted up to 3 days. Also the intra-articular administration of dexketoprofen or tramadol (10-100 µg/25 µl) inhibited MIA-induced nociception, and the combination of the lower doses (10 µg/25 µl) produced a long lasting more than additive antinociceptive effect indicating a synergistic interaction between the two drugs. This effect was significantly reduced by naloxone (10 µg/25 µl, i.ar.) co-administered with both compounds. The intra-articular administration of both drugs at 10 µg/25 µl in the contralateral control knee joint provoked a marked synergistic antinociceptive effect indicating significant systemic diffusion through synovial membrane. The oral or intra-articular combination of dexketoprofen and tramadol produced additive or synergistic antinociceptive effects, respectively, in the model of MIA-induced osteoarthritis in rats, that might allow to obtain therapeutic advantages with lower side effects.

Characterization of a novel proinflammatory effect mediated by BK and the kinin B2 receptor in human preadipocytes.

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 [Ca²âº](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.

Characterization of ibodutant at NK(2) receptor in human colon.

We have characterized the pharmacological profile of the nonpeptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) through radioligand binding and contractility assays in the human colon smooth muscle. The antagonist affinity of ibodutant was evaluated through concentration-dependent inhibition curves at the [(125)I]NKA specific binding by using membranes prepared from human colon smooth muscle. In this assay the affinity of ibodutant (pKi 9.9) was compared to that of other two selective NK2 receptor antagonists, nepadutant (pKi 8.4) and saredutant (pKi 9.2). The antagonist potency of ibodutant was evaluated towards the [ßAla(8)]NKA(4-10)-mediated contractions of human colon smooth muscle strips. In this assay ibodutant (3, 10, 30 and 100 nM) induced a concentration-dependent rightward shift of the [ßAla(8)]NKA(4-10) concentration-response curves without depressing the maximal contractile effect. The analysis of the curves yielded a Schild-plot linear regression with a slope not different from unity (1.02), thus indicating a surmountable antagonist behavior. The calculated apparent antagonist potency as pKB value was 9.1. No sex related differences were observed in NK2 receptor pharmacology for [ßAla(8)]NKA(4-10) or ibodutant in colonic strips obtained from male or female patients. Reversibility experiments of tachykinin NK2 receptor blockade indicated that the inhibition of the agonist-induced contractions in preparations pre-exposed to ibodutant, and afterwards subjected to repeated washing cycles remained almost constant showing no sign of recovery during the 3h observation period. Overall, the present study indicates ibodutant as a potent tachykinin NK2 receptor antagonist in the human colon tissue, also endowed with a persistent duration of action.

Antagonist profile of ibodutant at the tachykinin NK(2) receptor in guinea pig isolated bronchi.

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK(2) receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK(2) receptor agonist [ßAla8]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300 nM) induced a concentration-dependent rightward shift of the [ßAla8]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65-1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pK(B) value was 8.31 ± 0.05. Ibodutant (0.3-100 nM) produced a concentration-dependent inhibition of the nonadrenergic-noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100 nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95 ± 4% inhibition), the calculated IC(50) value was 2.98 nM (95% c.l. 1.73-5.16 nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100 nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK(2) receptor antagonist in guinea pig airways.

Design and synthesis of novel sulfonamide-containing bradykinin hB(2) receptor antagonists. Synthesis and structure-relationships of α,α-tetrahydropyranylglycine.

A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R'). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.

Fasitibant chloride, a kinin B2 receptor antagonist, and dexamethasone interact to inhibit carrageenan-induced inflammatory arthritis in rats.

BACKGROUND AND PURPOSE: Bradykinin, through the kinin B2 receptor, is involved in inflammatory processes related to arthropathies. B2 receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated. EXPERIMENTAL APPROACH: Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1ß, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h. KEY RESULTS: The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg9-[Leu8]-bradykinin (B2 receptor, leukotriene, catecholamine and B1 receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS: Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B2 receptor, or through the indirect effects mediated by release of eicosanoids and cytokines.

hNK2 receptor antagonists. The use of intramolecular hydrogen bonding to increase solubility and membrane permeability.

Starting from in-house capped tripeptide libraries, we have developed two series of compounds as potent antagonists of the hNK(2) receptor with a reduced peptide character. These two series maintained a crucial amide bond, which could not be methylated or substituted with classical isostere without a dramatic loss in binding affinity, very likely due conformational changes. We report here the planning, synthesis and evaluation of molecules belonging to the selected chemical series, which contain a strategically placed hydrogen bond acceptor. The aim of the work was to improve membrane permeability via the formation of an intramolecular hydrogen bonding, and at the same time to maintain the structural characteristics geometry and polarity of the amide linkage so as to retain a relevant binding affinity for the biological target.

Differences between zofenopril and ramipril, two ACE inhibitors, on cough induced by citric acid in guinea pigs: role of bradykinin and PGE2.

Dry and persistent cough is one of the commonest side effects experienced by patients treated with angiotensin-converting enzyme (ACE) inhibitors for the therapy of hypertension and congestive heart failure. The present study investigated the effect of zofenopril and ramipril on cough induced by citric acid in guinea pig and the involvement of bradykinin (BK) and prostaglandin E2 (PGE2) in mediating the responses of these drugs. Zofenopril (10 mg/kg) or ramipril (3-10 mg/kg), which is threefold more potent than zofenopril, on a mg basis, in lowering blood pressure, was orally administered daily in drinking water for 2 weeks. At the end of this period, aerosol of citric acid solution (0.1 M) was performed and the number of cough counted for 10 min. The role of the kinin B(2) receptor was also investigated. BK and PGE2 levels in the bronchoalveolar lavage (BAL) fluid were measured after repeated oral treatment with zofenopril or ramipril (10 mg/kg). Ramipril (3-10 mg/kg) increased citric acid-induced cough by 40% and 60%, respectively, as compared to the vehicle control group (15.0 ± 1.8), while zofenopril (10 mg/kg) was without effect. The enhancement of citric acid-induced cough caused by ramipril (10 mg/kg) was reduced by the kinin B(2) receptor antagonist MEN16132 (0.25 mg/kg ip). BK and PGE2 levels in the BAL fluid were increased, in comparison to the control group, after ramipril treatment, while they were unchanged after zofenopril administration. Zofenopril, contrary to ramipril, did not affect either citric acid-induced cough in the guinea pigs or BK and PGE2 production in the airways.

Structure-activity relationships of 6-methyl-benzo[b]thiophene-2-carboxylic acid (1-[(S)-1-benzyl-4-[4-(tetrahydropyran-4-ylmethyl)piperazin-1-yl]butylcarbamoyl]cyclopentyl)amide, potent antagonist of the neurokinin-2 receptor.

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.

Radioligand binding characterization of the bradykinin B(2) receptor in the rabbit and pig ileal smooth muscle.

Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and

Modulation on C- and N-terminal moieties of a series of potent and selective linear tachykinin NK(2) receptor antagonists.

Herein we describe the synthesis of a series of new potent tachykinin NK(2) 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 NK(2) 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 NK(2) receptor. Selected compounds were tested in vivo confirming their activity as NK(2) antagonists. In particular, after both iv and id administration to guinea pig, compound 61 b was able to antagonize NK(2)-induced colonic contractions with a potency and duration-of-action fully comparable to the reference compound 1 (MEN 15596, ibodutant).

Effect of Intra-articular 4-(S)-amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl} piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132), a kinin B2 receptor antagonist, on nociceptive response in monosodium iodoacetate-induced experimental osteoarthritis in rats.

The present study was designed to investigate the role of bradykinin (BK) in the knee joint osteoarthritis induced by intra-articular (i.ar.) administration of monosodium iodoacetate (MIA) in the rat, and to determine the efficacy of the kinin B(2) receptor antagonists, 4-(S)-amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl} piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132) and icatibant, in reducing pain. Rats received MIA (1 mg/25 microl i.ar.) in the right knee. MEN16132, icatibant (1, 3, and 10 microg/25 microl i.ar.), or saline were administered 7 days after MIA treatment, and their antinociceptive effect was observed for 2 weeks. MEN16132 induced a marked and sustained reduction of incapacitation produced by MIA, approximately 56% inhibition of pain at 3 microg/knee. MEN16132 analgesia was more potent and longer lasting, up to 10 days, than icatibant. MEN16132 (3 microg/knee), at different time points from MIA treatment in separate groups of animals, produced comparable maximal antinociceptive effects, whereas the pain response induced by MIA was unaffected if MEN16132 (10 microg/knee) was administered in the contralateral knee. Indomethacin at high doses (100-625 microg/knee) inhibited by approximately 40% but with a short duration the MIA-induced pain. MIA treatment produced a significant increase of BK and prostaglandin E(2) (PGE(2)) metabolite levels in synovial fluid up to 21 days, and PGE(2) metabolite levels were reduced almost to basal values by MEN16132. In conclusion the potent and long-lasting analgesic effect of MEN16132 in MIA-induced osteoarthritis indicates an important role for BK in osteoarthritic pain, and suggests that MEN16132 can be a candidate for the treatment of this chronic disease.

Chemically distinct HDAC inhibitors prevent adipose conversion of subcutaneous human white preadipocytes at an early stage of the differentiation program.

The present study investigated the effect of HDAC inhibitors on the differentiation of human subcutaneous white adipocytes. Results showed that trichostatin A, suberoylanilide hydroxamic acid, valproic acid and MS-275 inhibited triglyceride accumulation, GPDH activity and FABP4 protein expression in adipocytes, as well as leptin and VEGF release, while cells retained a fibroblast-like morphology. HDAC inhibitors exerted their antiadipogenic effect without inducing apoptosis or affecting cell viability and number, while 1-2 log unit higher concentrations were mostly required to exert an antiproliferative effect or to reduce LDH activity. A brief exposure to HDAC inhibitors at the beginning of the differentiation program was sufficient to observe the antiadipogenic effect while differentiation restarted after compound withdrawal and further exposure to inducers of differentiation demonstrating reversibility of the events. HDAC inhibitors hyperacetylated histone H4, but only hydroxamate-based compounds produced a massive acetylation of alpha-tubulin, indicating that this latter event is not required to prevent adipose conversion. HDAC inhibitors induced a significant reduction of the expression of the transcription factor C/EBPalpha, an early marker of differentiation, and a diminution of fibronectin immunoreactivity was also observed. In conclusion, HDAC inhibitors from different chemical classes potently inhibited human adipose conversion at an early stage of the differentiation program.

Pharmacological characterization of the bradykinin B2 receptor antagonist MEN16132 in rat in vitro bioassays.

The pharmacological profile of the bradykinin B(2) receptor antagonist MEN16132 at the rat B(2) receptor has been investigated and compared with that of icatibant (formerly Hoe 140). Antagonist affinity has been measured through radioligand binding experiments with membranes prepared from uterine and airway tissue. MEN16132 inhibited [(3)H]bradykinin binding with subnanomolar affinity (pK(i) values 10.4 and 10.1 in the uterus and airways, respectively), and was about 3-fold less potent than icatibant (pK(i) values 10.9 and 10.5). Antagonist potency has been estimated towards bradykinin-induced contractility of uterine and urinary bladder smooth muscle preparations. In these assays MEN16132 (pK(B): 9.7 both in uterus and bladder) was about 10-fold more potent than icatibant [pK(B): 8.8 in uterus, and pK(B) 8.0 in urinary bladder, as from Meini, S., Patacchini, R., Giuliani, S., Lazzeri, M., Turini, D., Maggi, C.A., Lecci, A., 2000a. Characterization of bradykinin B(2) receptor antagonists in human and rat urinary bladder. Eur. J. Pharmacol. 388, 177-182]. Washout experiments conducted in the uterine preparation indicated for MEN16132 (100 nM) a slower reversibility than icatibant (300 nM).Altogether present results indicate that MEN16132 displays high affinity and potency also for the rat bradykinin B(2) receptor, and thus is suitable for further investigations in pathophysiological models in this species.

Multifaceted approach to determine the antagonist molecular mechanism and interaction of ibodutant ([1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]-amide) at the human tachykinin NK2 receptor.

Ibodutant (MEN15596, [1-(2-phenyl-1R-[[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl]-ethylcarbamoyl)-cyclopentyl]-amide) is a tachykinin NK(2) receptor (NK(2)R) antagonist currently under phase II clinical trials for irritable bowel syndrome. This study focuses on the ibodutant pharmacodynamic profile at the human NK(2)R and compares it with two other antagonists, nepadutant (MEN11420, (cyclo-[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)]) and saredutant [SR48968, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]. In functional experiments (phosphatidylinositol accumulation) in Chinese hamster ovary cells expressing the human NK(2)R, ibodutant potency measured toward concentration-response curves to neurokinin A as pK(B) was 10.6, and its antagonism mechanism was surmountable and competitive. In the same assay, antagonism equilibration and reversibility experiments of receptor blockade indicated that ibodutant quickly attains equilibrium and that reverts from receptor compartment in a slower manner. Kinetic properties of ibodutant were assessed through competitive binding kinetics experiments performed at [(3)H]nepadutant and [(3)H]saredutant binding sites. Determined K(on) and K(off) values indicated a fast association and slow dissociation rate of ibodutant at the different antagonist binding sites. Last, by radioligand binding experiments at some mutated human tachykinin NK(2)Rs, the amino acidic determinants crucial for the high affinity of ibodutant were identified at the transmembrane (TM) level: Cys167 in TM4; Ile202 and Tyr206 in TM5; Phe270, Tyr266, and Trp263 in TM6; and Tyr289 in TM7. These results indicated an extended antagonist binding pocket in the TM portion of the receptor, which is conceived crucial for TM3 and 6 arrangement and leads to G protein-coupled receptor activation. By combining this information and molecular modeling, the docking mode of ibodutant-human NK(2)R complex is proposed.

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.

Excitatory and inhibitory urinary bladder reflexes induced by stimulation of cervicovaginal capsaicin-sensitive sensory fibers in rats.

We have investigated the effect of intravaginal application of capsaicin on micturition reflex in female rats. Urinary bladder contractility was measured by transurethral pressure recording at isovolumetric and subthreshold conditions in anaesthetized rats. The intravaginal application of capsaicin (15 microg/50 microl rat) induced reproducible bladder phasic contractions, without desensitization upon repeated applications, that were blocked by intravenous atropine (1 mg/kg) or hexamethonium (5 mg/kg) and prevented by removal of paracervical ganglia or systemic capsaicin pretreatment (125 mg/kg, s.c.). The inhibition of sympathetic transmission by guanethidine (30 mg/kg, s.c.) produced significant increase of the bladder reflex contractions activated by intravaginal capsaicin. Intravenous administration of the TRPV1 antagonist, capsazepine (3 mg/kg), significantly reduced the excitatory reflex response to capsaicin. Intravaginal administration of capsaicin (15 microg/50 microl), during distension-induced reflex bladder contractions, produced a transient block of reflexes, unaffected by guanethidine pretreatment. In conclusion, the stimulation of capsaicin-sensitive sensory nerve endings in the rat cervix-vagina induced a dual excitatory or inhibitory bladder response in anaesthetized female rats depending on the degree of bladder distension.

Cinnamic acids and mono-substituted benzoic acids as useful capping groups for the preparation of hNK2 receptor antagonists.

NK(2) 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 hNK(2) 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 i.v. and i.d. administration.

alpha,alpha-Cyclopentaneglycine dipeptides capped with biaryls as tachykinin NK(2) receptor antagonists.

The NK(2) receptor belongs to the family of tachykinin neurotransmitters. It has been reported to be involved in several pathological conditions, and selective antagonists are potentially useful drugs for the treatment of asthma, irritable bowel syndrome, cystitis, and depression. Starting from in-house capped dipeptide libraries, we were able to identify a number of molecules with sub-nanomolar binding affinity for the hNK(2) receptor. All were characterized by a rigid core structure with a strong constraint induced by an alpha,alpha-cyclopentaneglycine fragment. Herein we report the further elaboration of three initial basic structures. The planar benzothiophene group was substituted with a series of biphenyl and heterobiphenyl moieties that are well tolerated in terms of receptor affinity. The new compounds also maintained good antagonist potency in an in vitro functional assay, and a number of them showed significant in vivo activity after intravenous administration in our guinea pig model.