Considerations on nonclinical approaches to modeling risk factors of suicidal ideation and behavior.

Given the serious nature of suicidal ideation and behavior (SIB) and the possibility of treatment-emergent SIB, pharmaceutical companies are now applying more proactive approaches in clinical trials and are considering the value of nonclinical models to predict SIB. The current review summarizes nonclinical approaches to modeling three common risk factors associated with SIB: aggression, impulsivity, and anhedonia. For each risk factor, a general description, advantages and disadvantages, species considerations, nonclinical to clinical translation, and pharmacological validation with respect to treatments associated with SIB are summarized. From this review, several gaps were identified that need to be addressed before use of these nonclinical models can be considered a viable option to predict the relative risk for SIB. Other future directions that may compliment these nonclinical approaches, including the use of selectively-bred or genetically-modified rodent models, transgenic models, gene expression profiling, and biomarker analysis, are discussed. This article was developed with the support of the DruSafe Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ, www.iqconsortium.org).

Altered contractility of circular and longitudinal muscle in TNBS-inflamed guinea pig ileum.

Intestinal motility disorders are often associated with gut inflammation. We evaluated, in vitro under isometric conditions, changes in contractility of longitudinal and circular muscle layers from guinea pig ileum after 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis. TNBS treatment did not modify length-active tension relationships for both muscle layers, whereas a significant increase in passive tension was observed in the circular muscle response to stretching. Moreover, in both control and inflamed strips at optimal stretch, concentration-response curves to KCl were similar for both layers. In contrast, contractile responses to receptor agonists were differentially altered in both layers in comparison with controls. Thus, in longitudinal strips from TNBS-treated ileum, there was a twofold increase in maximal response (Emax) induced by carbachol and histamine without modification of 50% effective concentration (EC50) values; responses to 5-hydroxytryptamine (5-HT) were not modified; both alpha 1- and alpha 2-adrenoceptor-mediated responses to epinephrine were abolished. In circular strips, inflammation did not affect the Emax induced by carbachol and histamine but led to increased EC50 values; Emax to 5-HT was reduced without change in EC50 values. Moreover, in the dose range used (0.1 nM to 0.1 mM), a maximal response to carbachol was not obtained in inflamed circular strips. The results indicate that in the guinea pig model of TNBS-induced ileitis, the in vitro contractile responses of circular and longitudinal smooth muscle to the stimulation of various receptors are differentially altered, whereas non-receptor-mediated contraction to KCl depolarization is not modified.

Functional evidence for NO-synthase activation by substance P through a mechanism not involving classical tachykinin receptors in guinea-pig ileum in vitro.

1. This study tested the hypothesis that a nitric oxide synthase (NOS) was activated in guinea-pig ileum in vitro in response to substance P (SP), and attempted to characterize the tachykinin receptor involved in this activation by the use of selective receptor agonists and antagonists. 2. Strips of guinea-pig ileum (8 x 2 mm) were superfused (Krebs, 37 degrees C, 2 ml min-1) with: (i) tachykinin receptor agonists: SP, GR 73,632 (NK1), GR 64,349 (NK2), senktide (NK3), and neuropeptide (NP) gamma; (ii) tachykinin receptor antagonists: CP 99,994 (NK1), SR 48,968 (NK2), SR 142,801 (NK3); (iii) nerve-related agents: carbachol (CCh), atropine, tetrodotoxin (TTX), hexamethonium; (iv) NOS inhibitors: N omega-nitro-L-arginine-methyl-ester (L-NAME), N omega-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG); (v) NO-related agents, L-arginine (L-Arg), D-arginine (D-Arg), sodium nitroprusside (NaNP) and methaemoglobin. Muscle contractility was recorded isometrically and quantified as integrated area of activity. 3. SP, tachykinin receptor agonists and NP gamma (10 pM to 10 microM), produced concentration-dependent contractions of ileal strips, with EC50s in the nanomolar range, and maximal responses (Emax) attained at 0.1 microM for SP and 1 microM for the other agonists. The Emax response to SP equalled that to KCl (60 mM) taken as a 100% control (99.3% [93.0-105.7]; mean and 95% CI; n = 12); a comparable Emax contraction was obtained with the other tachykinin receptor agonists (1 microM) as well as with CCh (1 microM). 4. Under baseline conditions, L-NAME (1 microM), L-NMMA (1 microM) and AG (1 microM), failed to contract the muscle strip. In contrast, when superfused for 3 min, 10 min after SP (0.1 microM), they induced a transient contraction of the strip (e.g. for 1 microM L-NAME: 50 to 70 s duration; amplitude 73 +/- 12%, n = 24). 5. The NOS inhibitor-induced contractile response was not obtained after KCl (60 mM), GR 73,632, GR 64,349, senktide or CCh (all up to 1 microM). In contrast, this contractile response was obtained after NP gamma (1 microM). 6. Blockade of tachykinin NK1, NK2 and NK3 receptors by continuous superfusion of CP 99,994, SR 48,968 and SR 142,801 (1 microM) respectively, starting 5 min before SP, did not modify the response to L-NAME, superfused 10 min after SP (0.1 microM). The contractile response to L-NAME (1 microM) was blocked by atropine (1 microM), superfused either before or after SP. In contrast, it persisted after TTX or hexamethonium (1 microM) superfused in the same conditions. 7. The amplitude of NOS inhibitor-induced contraction (1 microM) was dependent on the concentration of priming SP (1 pM to 1 microM). In contrast, the contractile response to NOS inhibitors (1 nM to 10 microM) of the ileum strip primed with SP (0.1 microM) was not concentration-related. 8. L-NAME-induced contraction was prevented by continuous superfusion of L-Arg (1 microM), but not D-Arg (1 microM). In addition, the NO donor, sodium nitroprusside (1 microM) and the NO scavenger, methaemoglobin (10 micrograms ml-1), both prevented the contractile response to L-NAME. 9. In summary, SP and to a lesser extent NP gamma, exert a permissive action allowing contractile stimulating effects of L-NAME, L-NMMA and AG, in guinea-pig ileum in vitro, by a mechanism which apparently does not involve tachykinin NK1, NK2 and NK3 receptors. This action is likely to result from the activation of a NO-synthase by SP in the vicinity of intestinal myocytes. Thus, L-NAME, L-NMMA or AG, by blocking this SP-induced NO production, unveiled a smooth muscle contraction which involves a cholinoceptor (atropine-sensitive) mechanism.

Intraluminal excretion of PAF, lysoPAF, and acetylhydrolase in patients with ulcerative colitis.

PAF-acether (PAF) is a phospholipid synthesized by numerous inflammatory cells. PAF can produce several pathological changes in various organs, especially in the colon. In animals PAF causes colonic ulceration and inflammation, which are similar to the anatomic lesions seen in human ulcerative colitis. The aim of this study was to measure in vivo colonic production of PAF in active ulcerative colitis using a modified colonic perfusion method. Ten patients with active ulcerative colitis and six control patients were investigated. A colonic segment was continuously perfused with a buffer and the liquid was recovered 20 cm distally, after a 45-min period of equilibration, at 20-min intervals. PAF, lysoPAF, and acetylhydrolase were measured in the colonic samples. PAF and lysoPAF outputs were significantly higher in patients with active ulcerative colitis compared to controls patients. There was a significant correlation between colonic PAF output and, respectively, macroscopic mucosal lesions and myeloperoxidase colonic output. We thus conclude: (1) the colonic perfusion method allows in vivo study of the metabolism of PAF during ulcerative colitis and could also be used to study the efficiency of PAF antagonists in UC; and (2) colonic production of PAF is increased during ulcerative colitis and correlated to local injury and inflammation. Whether or not PAF plays a role in the pathogenesis of ulcerative colitis remains open for further investigations.

Evidence for mast cell, leukotriene and nitric oxide involvement in the regulation of the adrenoceptor number of inflamed small intestine in guinea pigs.

Changes in the populations of neurotransmitter receptors involved in the control of intestinal smooth muscle function have been associated with the altered motility of the inflamed gut. Thus, trinitrobenzenesulphonic acid (TNBS)-induced gut inflammation is accompanied by an increase in alpha- and a decrease in beta-adrenoceptor numbers in guinea pig small intestine. In the present study, we investigated the effects of anti-inflammatory compounds (cyclooxygenase inhibitor indomethacin, lipooxygenase inhibitor MK-886, nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (L-NAME), mast cell stabilizer doxantrazole) on TNBS-induced adrenoceptor changes. Smooth muscle adrenoceptor populations, labelled by subtype-specific radioligands 6 days after TNBS, were significantly different from those of sham-treated controls: alpha 1- and alpha 2-adrenoceptor numbers increased by more than 50%, while beta-adrenoceptor numbers decreased by more than 50%. These changes, associated with severe inflammation as assessed histologically and by myeloperoxidase assay, were prevented by doxantrazole or L-NAME, and only partly by MK-886. In contrast, indomethacin did not prevent these changes. It appears then that: (a) mast cell mediators, nitric oxide and leukotrienes are likely to contribute to TNBS-induced changes in adrenoceptor populations in the guinea pig inflamed intestine; (b) there is no evidence for prostanoid involvement in this process. It was suggested that changes in smooth muscle adrenoceptor populations may be an important mechanism by which gut inflammation alters intestinal motility.

Inverse regulation of alpha- and beta-adrenoceptors during trinitrobenzenesulfonic acid (TNB)-induced inflammation in guinea-pig small intestine.

The hypothesis has been raised that intestinal motor disturbances induced by inflammation of the digestive tract may reflect alterations in intestinal cell-membrane receptors. This question has been addressed herein for adrenoceptors by performing [3H]prazosin, [3H]rauwolscine and [3H]DHA binding studies on guinea-pig jejunal smooth-muscle membrane preparations from both healthy controls and 3, 6, and 10 days after TNB-induced intestinal inflammation. Each of the adrenoceptor subtype-selective radioligands used bound selectively to a single saturable class of sites, with no significant (p < 0.05) variation of dissociation constant (KD) values along the inflammatory process. In contrast maximal binding capacities (Bmax) for the different radioligands varied moderately but significantly (p < 0.05 and p < 0.01) according to the time after TNB injection. The alpha-adrenoceptors were significantly upregulated, respectively from (mean +/- SE in fmoles/mg of proteins) 27 +/- 3.8 (controls) to 91 +/- 3.1 (day 10) for the alpha 1-subtype, and from 26 +/- 2.7 (controls) to 102 +/- 5.9 (day 10) for the alpha 2-subtype. In contrast beta-adrenoceptors were down-regulated from 384 +/- 34.6 (controls) to 158 +/- 17.2 (day 10). These findings highlighted an apparently inverse pathological regulation of intestinal alpha- and beta- adrenoceptor densities. They suggest that changes in intestinal adrenoceptors, probably resulting from functional denervation of the inflamed bowel tissue, may contribute to the altered intestinal motility observed in inflammatory bowel diseases.