A simple in chemico method for testing skin sensitizing potential of chemicals using small endogenous molecules.

Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical in chemico. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.

Cysteamine-colon and cysteamine-duodenum lesions in rats. Attenuation by gastric pentadecapeptide BPC 157, cimetidine, ranitidine, atropine, omeprazole, sulphasalazine and methylprednisolone.

Recently, we showed cysteamine-duodenal lesions without gastric acid, since they were induced also in gastrectomized rats, as in naive rats, and they were inhibited by the novel stomach pentadecapeptide BPC 157 as well as standard antiulcer drugs (i.e. cimetidine, ranitidine, omeprazole, bromocriptine, atropine). Therefore, as an advantage of considering cysteamine as a directly acting cytotoxic agent and mentioned agents as direct cytoprotective agents, the present focus was on the ulcerogenic effect of cysteamine and protective effect of gastroduodenal antiulcer agents outside upper gastrointestinal tract (i.e. in colon). Intrarectal administration of the cysteamine (200 or 400 mg/kg b.w) produced severe colon lesions (i.e. transmural inflammation with serosal involvement) in rats (30 min-72 h-experimental period), apparently distinctive from smaller lesions after non-specific irritant enema [diluted HCl solution, pH 3.8 (adjusted to pH of cysteamine solution (pH 3.8)]. All of the tested antiulcer agents were applied simultaneously with cysteamine enema (8 cm from the anus, in a volume of the 1.0 ml/rat) intraperitoneally (i.p.), intragastrically (i.g.) or intrarectally (i.r.). Pentadecapeptide BPC 157 (10 microg or 10 ng/kg b.w.), given in either regimen, previously shown to have, besides others, a particular beneficial activity just in the intestinal mucosa, inhibited these cysteamine colon lesions (assessed after 30 min, 60 min, 180 min, 24 h, 48 h, 72 h following cysteamine in a dose of either 200 or 400 mg/kg i.r.). Cysteamine-colon lesions were also attenuated by standard antiulcer agents (mg/kg b.w.), given i.p., i.g., or i.r., such as ranitidine (10), cimetidine (50), omeprazole (10), atropine (10), together with methylprednisolone (1), and sulphasalazine (50, i.r.), assessed 30 min following application of 200 mg of cysteamine. Finally, standard cysteamine duodenal lesions (assessed 24 h after a subcutaneous application of 400 mg/kg of cysteamine) were also attenuated by these agents application (given in the same doses, i.p., 1 h before cysteamine), with only exception to sulphasalazine. Thus, the extended cysteamine specific ulcerogenic effect, cysteamine colon/duodenum lesion-link and an extenuation of agents protection from upper to lower part of gastrointestinal tract (i.e. stomach pentadecapeptide BPC 157, standard antiulcer agents, cimetidine, ranitidine, atropine, omeprazole) and vice versa (remedies for inflammatory bowel disease) evidenced in the present study may be potentially important for both further experimental and clinical research.

Effects of Cistus laurifolius L. flowers on gastric and duodenal lesions.

The flowers and flower buds of Cistus laurifolius L. are used for the treatment of peptic ulcers. Through the bioassay-guided fractionation of the material, using water immersion and immobilisation-induced stress ulcer model, the EtOH-precipitated part from the aqueous extract (E-H2O decreases) was determined to be the active fraction. For the evaluation of the mode of action, the activity of E-H2O decreases was tested using various ulcer models in rats and mice and this fraction was found active against pylorus ligation-, abs. ethanol-, indomethacin-, indomethacin plus HCl/EtOH-induced gastric and cysteamine-induced duodenal lesions while ineffective against serotonin-induced gastric lesions. The active fraction showed its activity not only on per os administration but also after subcutanous injection. According to the results of biochemical studies, the active fraction showed a potent antiacid activity. In addition, histopathological, and toxicological studies were conducted with the active fraction.

Differences in action of topical and systemic cysteamine on gastric blood flow, gastric acid secretion and gastric ulceration in the rat.

The effect of cysteamine on gastric blood flow and on the indomethacin-induced gastric mucosal damage was studied. In anesthetized rats, cysteamine (280 mg/kg) given subcutaneously (s.c.) decreased gastric blood flow measured by the laser Doppler flowmetry technique. In contrast, cysteamine (1-60 mg/ml) applied topically to the serosal surface of the stomach evoked a concentration-dependent and long-lasting increase in gastric blood flow. At 60 mg/ml, cysteamine increased blood flow by 166.8 +/- 26.1% of predrug control value. Pretreatment with indomethacin (20 mg/kg, s.c.), intravenous (i.v.) atropine (1 mg/kg), propranolol (1 mg/kg, i.v.), combined H1 and H2-blockade or bilateral cervical vagotomy alone or combined with i.v. guanethidine (8 mg/kg), or pretreatment with the capsaicin analogue resiniferatoxin did not reduce the vasodilator response to cysteamine. The vasodilator response to topical capsaicin, was not reduced after s.c. cysteamine (280 mg/kg) pretreatment. In conscious pylonus-ligated rats, s.c. cysteamine (100 or 280 mg/kg) given simultaneously with indomethacin inhibited gastric acid output but had variable effects on the indomethacin-induced gastric mucosal damage. Cysteamine (100 or 280 mg/kg) administered s.c. 4 h prior to indomethacin enhanced gastric injury by s.c. indomethacin, but did not prevent the gastroprotective action of capsaicin. In contrast, orally administered cysteamine (60 mg/ml) reduced gastric injury induced by s.c. indomethacin plus intragastric HCl. These data provide the first evidence for the effect of cysteamine on gastric microcirculation in the rat and suggest a direct vasodilator effect for topical cysteamine. The microvascular effects of cysteamine are largely responsible for the different effects of this agent on experimental gastric injury.

The gastrin/cholecystokinin-B receptor antagonist L-365,260 reduces basal acid secretion and prevents gastrointestinal damage induced by aspirin, ethanol and cysteamine in the rat.

L-365,260, a nonpeptide antagonist of gastrin/CCK-B receptors, was evaluated in receptor binding, antisecretory and gastrointestinal damage assays. L-365,260 binds potently and stereo-selectively to gastrin and CCK-B sites in guinea pig tissue. In contrast, L-365,260 binds to the isolated canine parietal cell gastrin receptor weakly, and without stereoselectivity. In the pylorus-ligated rat, low doses of L-365,260, given i.v., attenuated pentagastrin-stimulated acid secretion, whereas higher doses were required to inhibit both histamine-stimulated and basal acid secretion. In an aspirin-induced gastric damage model, L-365,260 was 2.4-fold less potent than the standard histamine H2 antagonist cimetidine in preventing gastric damage when given i.v., and was 8.3-fold less potent than cimetidine when given p.o. Moreover, the ED50 value for L-365,260, given i.v., in prevention of aspirin-induced gastric damage (11.5 mg/kg) agreed well with its ED50 value for inhibition of basal acid secretion (12.6 mg/kg). At doses as great as 100 mg/kg p.o., neither L-365,260 nor cimetidine had an effect on ethanol-induced gastric damage. L-365,260, although p.o. less bioavailable relative to cimetidine in the aspirin gastric damage model, was as potent as cimetidine in the prevention of cysteamine-induced duodenal ulcers in the rat. We conclude that the gastrin/CCK-B receptor antagonist L-365,260, at doses supramaximal for the inhibition of pentagastrin-stimulated secretory responses in vivo, inhibits gastrointestinal damage in models of peptic ulcer disease by an antisecretory mechanism of action.

Inhibition of recA induction by the radioprotector 2-mercaptoethylamine.

Our earlier finding that the radioprotective action of 2-mercaptoethylamine (MEA) is counteracted by ascorbate suggests a biochemical mechanism of action, which is supported by observations that MEA is not radioprotective in Rec- E. coli strains. In this study we show that MEA inhibits the induction of the recA gene by UV- or gamma-irradiation or by nalidixic acid in Escherichia coli strain GE94, which contains a recA-lacZ fusion. This effect, which may be counteracted by cysteine, indicates that in general MEA inhibits the induction of SOS functions.

Prevention of cysteamine-induced myoclonus blocks the long-term inhibition of kindled seizures.

In kindled rats, the administration of cysteamine (CSH, 200 mg/kg, i.p.) 4 h prior to a kindled seizure leads to long-term (up to 10 days) inhibition of kindled seizures. CSH (200 mg/kg, i.p.) also induces myoclonic seizures in kindled rats. We suggest that the long-term inhibition of kindled seizures might be the result of the myoclonus, not the somatostatin depletion as previously suggested. Prior administration of the short-acting benzodiazepine midazolam (5 mg/kg, i.p.) eliminated the CSH-induced myoclonus and prevented the long-term inhibition of kindled seizures. These results suggest that the CSH-induced long-term inhibition of kindled seizures is the result of an interaction between the myoclonic seizure and a subsequent kindled seizure.

The effect of the new H2-receptor antagonist mifentidine on gastric secretion, gastric emptying and experimental gastric and duodenal ulcers in the rat: comparison with cimetidine and ranitidine.

The new H2-receptor antagonist mifentidine (DA 4577) was tested for its antisecretory and gastric motor effects in comparison with cimetidine and ranitidine. The Shay rat preparation (5 h) was used for studying gastric secretion; the gastric emptying of a liquid meal was chosen for studying gastric motility. All the three compounds inhibited acid secretion in a dose-dependent fashion. Calculated ED50s were 2.3, 12.2 and 92.8 mg X kg-1 for mifentidine, ranitidine and cimetidine, respectively. Therefore, in this animal model, mifentidine was about 40 times more potent than cimetidine and 5 times more potent than ranitidine. As far as gastric emptying is concerned, the effect of equiactive antisecretory doses (i.e. the respective ED50s calculated from the previously established dose-response curves) of all the three antagonists was completely different. Cimetidine delayed emptying rate, whereas ranitidine accelerated it and mifentidine was completely ineffective. However, at higher doses, also this compound affected emptying rate by reducing it dose-dependently. Gastric and duodenal ulcers were induced in the rat by dimaprit (100 mg X kg-1 intravenously) and cysteamine (250 mg X kg-1 subcutaneously), respectively. As far as gastric ulcer is concerned, the ED50s (the effective dose which protected 50% of the animals from lesions) were 0.23, 4.40 and 9.70 mg X kg-1 for mifentidine, ranitidine and cimetidine, respectively. As regards duodenal ulcer, the ED50 was 4.48 for mifentidine and 150.00 mg X kg-1 for ranitidine. In this animal model, the efficacy of cimetidine was very low. Therefore an ED50 could not be determined. In conclusion, results of the present investigation demonstrated that mifentidine is a potent antisecretory compound and an effective anti-ulcer agent in the rat.

Protection against duodenal ulceration by somatostatins.

The purpose of our investigation was to study the effect of two somatostatin analogs (SMS 201-995 and 008) on duodenal ulcer disease induced by cysteamine in rats. Male Wistar rats were given cysteamine (28 mg/100 g body wt.) by rubber stomach tube three times in a single day. All animals of the test group were injected subcutaneously either with SMS 201-995 in different doses or with 008. On the 3rd day stomach, duodenum, and adrenal glands were macroscopically examined for lesions (duodenal ulceration, gastrointestinal bleeding, adrenal hemorrhage). Our results showed a dose-dependent effect of SMS 201-995 on the mortality, incidence, and intensity of cysteamine-induced duodenal lesions in rats. The incidence and the intensity of gastrointestinal bleeding and adrenal hemorrhage were also dose-dependently reduced by SMS 201-995. There was no definite effect of 008 on the mortality, incidence, or intensity of cysteamine-induced duodenal ulceration.

Gastric mucosal protection by somatostatins.

Somatostatin and somatostatin derivatives were tested for their ability to prevent gastric hemorrhagic erosions induced by ethanol. The somatostatin analogues were cyclohexapeptides with the rearranged amino acids 7-14 of somatostatin (S-14): Phe-Thr-Lys(Z)-Trp-Phe-D-Pro(I), Phe-Thr-Lys-Trp-Phe-D-Pro(II), Phe-Thr-Lys-D-Trp-Phe-Tyr(III) and Tyr-Phe-D-Trp-Lys-Thr-Phe(IV). In Spraque-Dawley rats receiving ethanol alone, the lesions involved 18.1 +/- 3.2% of the glandular stomach while after S- 14 (10(-7) mol/rat) the lesioned area was reduced to 6.3 +/- 1.1% (p less than 0.05). Peptide I and peptide II (doses 10(-7) -10(-9) mol/rat) decreased the area of erosions to less than 5%. Peptide III was less active and peptide IV was inactive. In rats with chronic gastric fistula S- 14 and peptide II decreased the cysteamine-stimulated acid secretion without affecting the pepsin output. We also continuously measured the intraluminal pH in the stomach of Wistar rats which develop gastric erosions after subcutaneous injection of cysteamine. The erosions were reduced by S- 14 or SMS while the intraluminal pH did not change under the influence of cysteamine or the combination of cysteamine plus S- 14 or SMS. Thus some of the peptides derived from S- 14 exert prominent gastric mucosal protection without influencing gastric secretion.

[Interrelation between the analgesic and ulcerogenic action of cysteamine]. / Vzaimosviaz' mezhdu anal'geticheskim i ul'tserogennym deistviem tsisteamina.

Interrelationship was studied between the influence of cysteamine on pain threshold and ulcerogenic effect on the duodenum. Cysteamine (350 mg/kg) induced analgesia in mice which was prevented by naloxone (1.5 mg/kg). In rats, cysteamine produced duodenal ulcers with concomitant analgesia. The intensity of ulceration was higher in animals with lower basal pain threshold. The correlation between central and peripheral effects of endogenous opioids in the development of experimental duodenal ulcers is discussed.

Gastric cytoprotective properties of SCH 32651, a novel antiulcer agent.

The antisecretory activity of SCH 32651 (3-amino-2-methyl-8-phenylmethoxyimidazo[1,2-a] pyrazine HCl . 1/3 H2O) has been described in a preceding paper. This report describes its gastric cytoprotective properties. SCH 32651 inhibited the gastric lesions due to ethanol in a dose-dependent manner (ID50 = 5 mg/kg p.o.) and exerted its maximal effect when given 30 min before ethanol and had a duration of at least 90 min at 30 mg/kg p.o. The activity of SCH 32651 against ethanol was unaffected by indomethacin pretreatment. Furthermore, SCH 32651 produced significant increases in gastric mucus release (10-100 mg/kg p.o.) and rapidly reversed ethanol-induced (30-100 mg/kg p.o.) fall in gastric potential difference (PD). SCH 32651 displayed significant antiulcer activity in indomethacin-(30-100 mg/kg p.o.), aspirin - (30-100 mg/kg p.o.), "aspirin + acid" - (10-100 mg/kg p.o.), reserpine - (10-100 mg/kg p.o.), stress - (30-100 mg/kg p.o.), and cysteamine (10-100 mg/kg p.o.) ulcers. The present data in conjunction with those reported in a preceding paper indicate that SCH 32651 is an orally effective novel antiulcer drug with both gastric antisecretory and cytoprotective properties.

Effects of a gastric antisecretory-cytoprotectant 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (Sch 28 080) on cysteamine, reserpine and stress ulcers in rats.

Prostaglandin E2 and carbenoxolone, putative cytoprotective agents, were tested in cysteamine, reserpine and stress ulcers in rats. In cysteamine-induced duodenal ulcer, PGE2 was inactive at 0.1 and 0.5 mg/kg p.o.; carbenoxolone at 100 mg/kg p.o. decreased the incidence but not the severity of the ulcer. PGE2 at 5.0 mg/kg p.o. and carbenoxolone at 300 mg/kg p.o. showed moderate effects, but the dosage also inhibited cysteamine-stimulated acid secretion. PGE2 (0.1 and 0.3 mg/kg p.o.) was inactive and carbenoxolone (100 and 300 mg/kg p.o.) further aggravated the gastric ulceration caused by reserpine or cold-restraint stress. In contrast, atropine (3 and 10 mg/kg p.o.) and cimetidine (30, 100 and 300 mg/kg p.o.) were active in all three ulcer models. But the results with cimetidine in stress ulcer were somewhat variable. 2-methyl-8-(phenylmethoxy) imidazo [1,2-a] pyridine-3-acetonitrile (Sch 28 080), a novel structure with both cytoprotective and antisecretory activity, was highly efficacious in cysteamine, reserpine and stress ulcers (1-30 mg/kg p.o.), which was presumably adequately accounted for by its potent antisecretory activity. It is concluded that cysteamine, reserpine and stress ulcers may not be appropriate models for testing the potential antiulcer effect of primarily cytoprotective compounds.

The effect of intracisternally administered bombesin on cysteamine-induced duodenal ulcers in rats.

Cysteamine (420 mg/kg s.c.) increased gastric acid secretion in rats 3-6 h post-injection; duodenal pH fell from 6.0 +/- 0.2 (S.E.M.) at 0 time to 2.2 +/- 0.3 at 12 h. A high (60-80%) incidence of duodenal ulcerations was observed 18 h after cysteamine injection. Intracisternal (i.c.) administration of bombesin (1 microgram) significantly inhibited both cysteamine-induced increases in gastric acid secretion and the development of duodenal ulcers. This effect of bombesin was dose-dependent and appears to be relatively specific since i.c. neurotensin (1 or 30 micrograms) was not cytoprotective. Peripheral cholinergic (muscarinic) blockade with atropine methylbromide (10 mg/kg i.p.) was as effective as i.c. bombesin (1 microgram) in inhibiting cysteamine-induced gastric hypersecretion and duodenal ulcers.

The mechanism of gastrin release in cysteamine-induced duodenal ulcer.

Duodenal ulcer can be induced in rats by a single dose of cysteamine. The ulcer formation is accompanied by acid hypersecretion and elevated serum gastrin levels. This study was performed to elucidate the mechanisms of gastrin release after an ulcerogenic dose of cysteamine. Cysteamine induced a rise in serum gastrin from 29 +/- 5 pg/ml to a maximum of 203 +/- 62 pg/ml after 3 h in unoperated rats, whereas no rise was seen in vagotomized or antrectomized rats. The beta-adrenergic blocking agent propranolol strongly inhibited cysteamine-induced gastrin release, whereas atropine dependent on an intact vagus and may be mediated by beta-adrenergic receptors.

Dopamine disorder in duodenal ulceration.

Cysteamine-induced duodenal ulcers in rats were prevented by the dopamine agonists bromocriptine, lergotrile, and apomorphine, whereas both the severity of duodenal ulcers and the mortality among cysteamine-treated rats were raised by the dopamine receptor antagonist, haloperiodol. Bromocriptine and lergotrile greatly reduced gastric-acid output in cysteamine-treated rats. A review of the literature shows a high incidence of duodenal ulcers in patients with Parkinson's disease (associated with dopamine deficiency) and a low occurrence in schizophrenics (associated with dopamine excess and/or hyperactivity). Thus, changes in peripheral and/or central dopamine concentrations and/or receptor activity may have a role in the pathogenesis of duodenal ulceration.

Suppression of induced beta-galactosidase synthesis by cysteamine and its reversion by gamma-irradiation in the presence of ascorbate.

The induced synthesis of beta-galactosidase in E. coli was found to be inhibited by cysteamine. This inhibitory effect of the SH compound was antagonized by the addition of ascorbate followed by gamma-irradiation with relatively low doses. The cAMP level which, it has been suggested, plays a role in the radioprotective action of cysteamine, is stabilized by ascorbate against changes induced by irradiation.