Phase IIa study of the CD19 antibody MOR208 in patients with relapsed or refractory B-cell non-Hodgkin's lymphoma.

Background: This two-stage, phase IIa study investigated the antitumor activity and safety of MOR208, an Fc-engineered, humanized, CD19 antibody, in patients with relapsed or refractory (R-R) B-cell non-Hodgkin's lymphoma (NHL). CD19 is broadly expressed across the B-lymphocyte lineage, including in B-cell malignancies, but not by hematological stem cells. Patients and methods: Patients aged ≥18 years, with R-R NHL progressing after ≥1 prior rituximab-containing regimen were enrolled into subtype-specific cohorts: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), other indolent (i)NHL and mantle cell lymphoma (MCL). Treatment was MOR208, 12 mg/kg intravenously, weekly, for 8 weeks. Patients with at least stable disease could continue treatment for an additional 4 weeks. Those with a partial or complete response after 12 weeks could receive extended MOR208 treatment (12 mg/kg, either monthly or every second week) until progression. The primary end point was overall response rate. Results: Ninety-two patients were enrolled: DLBCL (n = 35), FL (n = 34), other iNHL (n = 11) and MCL (n = 12). Responses were observed in DLBCL, FL and other iNHL cohorts (26%, 29% and 27%, respectively). They lasted ≥12 months in 5/9 responding patients with DLBCL, 4/9 with FL and 2/3 with other iNHL. Responses in nine patients are ongoing (>26 months in five instances). Patients with rituximab refractory disease showed a similar response rate and progression-free survival time to patients with non-refractory disease. The most common adverse events (any grade) were infusion-related reactions (12%) and neutropenia (12%). One patient experienced a grade 4 infusion-related reaction and eight patients (9%) experienced grade 3/4 neutropenia. No treatment-related deaths were reported. Conclusions: MOR208 monotherapy demonstrated promising clinical activity in patients with R-R DLBCL and R-R FL, including in patients with rituximab refractory tumors. These efficacy data and the favorable safety profile support further investigation of MOR208 in phase II/III combination therapy trials in R-R DLBCL. ClinicalTrials.gov number: NCT01685008.

Increased susceptibility of gastric mucosa to ulcerogenic stimulation in diabetic rats--role of capsaicin-sensitive sensory neurons.

1. We examined the gastric mucosal blood flow (GMBF) and ulcerogenic responses following barrier disruption induced by sodium taurocholate (TC) in diabetic rats and investigated the role of capsaicin-sensitive sensory neurons in these responses. 2. Animals were injected streptozotocin (STZ: 70 mg kg(-1), i.p.) and used after 5, 10 and 15 weeks of diabetes with blood glucose levels of > 350 mg dl(-1). The stomach was mounted on an ex-vivo chamber under urethane anaesthesia and exposed to 20 mM TC plus 50 mM HCl for 30 min in the presence of omeprazole. Gastric transmucosal potential difference (PD), GMBF, and luminal acid loss (H+ back-diffusion) were measured before and after exposure to 20 mM TC, and the mucosa was examined for lesions 90 min after TC treatment. 3. Mucosal application of TC caused PD reduction in all groups; the degree of PD reduction was similar between normal and diabetic rats, although basal PD values were lower in diabetic rats. In normal rats, TC treatment caused luminal acid loss, followed by an increase of GMBF, resulting in minimal damage in the mucosa. 4. The increased GMBF responses associated with H+ back-diffusion were mitigated in STZ-treated rats, depending on the duration of diabetes, and severe haemorrhagic lesions occurred in the stomach after 10 weeks of diabetes. 5. Intragastric application of capsaicin increased GMBF in normal rats, but such responses were mitigated in STZ diabetic rats. The amount of CGRP released in the isolated stomach in response to capsaicin was significantly lower in diabetic rats when compared to controls. 6. The deleterious influences on GMBF and mucosal ulcerogenic responses in STZ-diabetic rats were partially but significantly antagonized by daily insulin (4 units rat(-1)) treatment. 7. These results suggest that the gastric mucosa of diabetic rats is more vulnerable to acid injury following barrier disruption, and this change is insulin-sensitive and may be partly accounted for by the impairment of GMBF response associated with acid back-diffusion and mediated by capsaicin-sensitive sensory neurons.

Role of nitric oxide in regulation of gastric acid secretion in rats: effects of NO donors and NO synthase inhibitor.

1. The role of nitric oxide (NO) in the regulation of acid secretion was examined in the anaesthetized rat. 2. A rat stomach was mounted in an ex vivo chamber, instilled with 2 ml of saline every 15 min, and the recovered sample was titrated at pH 7.0 against 0.1 N NaOH by use of an automatic titrator for acid secretion. Gastric mucosal blood flow (GMBF) was measured simultaneously by laser Doppler flowmeter. 3. Intragastric application of NO donors such as FK409 (3 and 6 mg ml[-1]) and sodium nitroprusside (SNP; 6 and 12 mg ml[-1]) as well as i.p. administration of cimetidine (60 mg kg[-1]), a histamine H2-receptor antagonist, significantly inhibited the increase in acid secretion in response to pentagastrin (60 microg kg(-1) h(-1), i.v.), in doses that increased gastric mucosal blood flow (GMBF). 4. Intragastric application of FK409 (6 mg ml[-1]) increased both basal and stimulated acid secretion induced by YM-14673 (0.3 mg kg(-1), i.v.), an analogue of thyrotropin-releasing hormone (TRH), but had no effect on the acid secretory response induced by histamine (4 mg kg(-1) h(-1), i.v.). 5. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg kg(-1), i.v.) did not affect basal acid secretion, but significantly potentiated the increase in acid secretion induced by YM-14673 and slightly augmented the acid secretory response to pentagastrin. 6. Both pentagastrin and YM-14673 increased the release of nitrite plus nitrate (NOx), stable NO metabolites, into the gastric lumen, and these changes were completely inhibited by prior administration of L-NAME (10 mg kg(-1), i.v.). 7. Pentagastrin caused an increase in luminal release of histamine and this response was significantly suppressed by intragastric application of FK409 (6 mg ml[-1]). 8. These results suggest that either exogenous or endogenous NO has an inhibitory action on gastric acid secretion through suppression of histamine release from enterochromaffin-like (ECL) cells.

Impaired duodenal bicarbonate secretion in diabetic rats. Salutary effect of nitric oxide synthase inhibitor.

We previously reported the impaired HCO3- secretion and the increased mucosal susceptibility to acid in the duodenum of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the salutary effect of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) on these changes and compared it with those of insulin. Animals were injected streptozotocin (STZ: 70 mg/kg, ip) and used after 1, 3-4, and 5-6 weeks of diabetes with blood glucose levels of > 300 mg/dL. Under urethane anesthesia the HCO3- secretion was measured in the proximal duodenal loop using a pH-stat method and by adding 10 mM HCl. L-NAME (20 mg/kg x 2) or insulin (4 units/rat) was administered sc for 4-5 weeks, starting 1 week after STZ treatment. The duodenal HCO3- secretory responses to various stimuli such as mucosal acidification (10 mM HCl for 10 min), 16,16-dimethyl prostaglandin E2 (dmPGE2: 10 micrograms/kg, i.v.), and vagal stimulation (0.5 mA, 2 ms, 3 Hz) were significantly decreased in STZ-treated rats, depending on the duration of diabetes. Repeated administration of L-NAME, starting from 1 week after STZ treatment, significantly reduced blood glucose levels toward normal values and restored the HCO3- responses to various stimuli in STZ rats, the effects being similar to those observed after supplementation of insulin. Diabetic rats developed duodenal lesions after perfusion of the duodenum with 150 mM HCl for 4 h, but this ulcerogenic response was significantly inhibited by the repeated treatment with L-NAME as well as insulin. We conclude that L-NAME is effective in ameliorating hyperglycemic conditions in STZ-diabetic rats, similar to insulin, and restores the impaired HCO3- secretion and the increased mucosal susceptibility to acid in diabetic rat duodenums.

Inhibition of gastric acid secretion by galanin in rats. Relation to endogenous histamine release.

Inhibitory effect of galanin on basal and secretagogs-stimulated gastric acid secretion was investigated in urethane-anesthetized rats. A rat stomach was mounted in an ex-vivo chamber, perfused with saline, and either gastric acid or alkaline secretion was determined by titrating the perfusate. Gastric mucosal blood flow (GMBF) was measured by a laser Doppler flowmeter. Intravenous infusion of galanin dose-dependently inhibited the increase of acid secretion induced by pentagastrin and carbachol but not by histamine, without any influence on the GMBF response. Galanin also reduced basal acid secretion while increasing GMBF, but did not evoke any change in basal gastric alkaline secretion. M15, which is a galanin receptor antagonist in the central nervous system but acts as a full agonist in the gastrointestinal smooth muscle, also suppressed pentagastrin-induced acid secretion, similar to galanin. In addition, pentagastrin increased the release of histamine into the gastric lumen, and this response was significantly inhibited by galanin. These results suggest that the inhibitory effect of galanin on acid secretion is mediated by suppression of endogenous histamine release from enterochromaffin-like cells and that the process may be related to the activation of the same subtype of galanin receptors as in the central nervous system and pancreatic beta-cells.

The role and interactions of nitric oxide (NO), carbon monoxide (CO), and prostanoids in the pathogenesis of postoperative ileus in rats.

The effects of heme oxygenase (HO) inhibitors, zinc-protoporphyrin-IX (ZnPP-IX), and tin protoporphyrin-IX (SnPP-IX) and their interactions with L-arginine/nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways were investigated in postoperative ileus in rats. Intestinal transit was measured as Evans blue migration after skin incision, laparotomy or laparotomy plus gut evisceration and handling. Laparotomy and small intestinal manipulations increased blood plasma nitrites/nitrates level 1.88-fold. N(omega)-nitro-L-arginine methyl ester, indomethacin, a selective COX-1 blocker (resveratrol) and COX-2 antagonists (nimesulide, DuP-697, NS-398) reversed the additional inhibitory effects of gut manipulation subsequent to laparotomy. In contrast, N-(3-(aminomethyl)benzyl)acetamidine or S-methylisothiourea, highly selective inducible NOS blockers, remained ineffective. ZnPP-IX and SnPP-IX overturned the effects of laparotomy on dye propulsion, but were only partially effective after laparotomy and gut handling attenuating the additional inhibitory influences of gut manipulation, the intestinal transit reaching 89.21%, 92.87%, 53.46%, and 48.56% of respective controls transit. Salutary effects of L-NAME, ZnPP-IX, and SnPP-IX were dose-dependent, L-arginine or hemin (HO substrate) sensitive. Administration of indomethacin and resveratrol subsequent to SnPP-IX reversed the inhibitory effects of laparotomy and manipulation, amounting to 93.91% and 87.43% of controls. On the other hand, L-NAME injected after SnPP-IX abolished the salutary effects of the latter, study dye migration reached 25.18% of control rat. Therefore we demonstrated that nitric oxide, carbon monoxide, and prostanoids play a role in the pathogenesis of postoperative ileus albeit in different mechanisms. Laparotomy stimulated HO activity, whereas gut manipulation led to an excessive constitutive NOS stimulation accompanied by augmented prostanoid synthesis by COX-1. Unaffected synthesis of either NO or CO enables a return of gastrointestinal transit during postoperative period, whereas a pharmacological blockade of two complementary metabolic pathways provides a most effective measure against postoperative ileus development.

Actions of galanin and some of its analogues on rat isolated gastric fundus.

The study was undertaken to characterize the effects of porcine galanin (pGal) and some of its analogues on rat gastric fundus muscle strips. pGal, galantide (M15) and pGal(1-14)-[Abu8]SCY-I evoked reproducible concentration-dependent contractions in concentrations of 1-300, 3-1,000 and 100-3,000 nM, respectively, with EC50 values of 13, 70 and 187 nM. Hill's coefficient for pGal is 1.03, indicating an interaction of one pGal molecule with one receptor, fulfilling criteria of classical receptor theory. For M15 and pGal(1-14)-[Abu8]SCY-I, Hill's coefficients are significantly different from 1, namely 0.73 and 1.56, so that one drug molecule may not interact with one receptor. The stimulatory effects of pGal were not modified by dibenamine 10 microM or glybenclamide 1 or 10 microM. Diltiazem 0.1, 1 and 10 microM, papaverine 0.1, 10 microM or dibutyryl cAMP (dib cAMP) 100 and 300 microM, blocked the contraction to pGal in a concentration-dependent manner, indicating an important role for the influx of extracellular calcium ions and regulation by cAMP the pGal-evoked contraction. Diltiazem, dibutyryl cAMP and papaverine were not competitive antagonists of pGal in the stomach smooth muscle.

Pharmacological characterization of the contractile effects of galanin (1-29)-NH2, galantide and galanin (1-14)-(alpha-aminobutyric acid8)scyliorhinin-I in the rat gastric fundus.

Porcine galanin (1-29)-NH2, galantide (M15) and galanin (1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I used in concentrations of 300, 1,000 and 3,000 nM respectively caused contractions of rat fundus strips. The contractile responses to galanin(1-29)-NH2 were not modified by atropine (10 microM), guanethidine (10 microM), naloxone (1 microM), a mixture of propranolol (10 microM) and phentolamine (10 microM), indomethacin (10 microM), a mixture of mepyramine (10 microM) and cimetidine (10 microM), saralasin (10 microM), and spantide (100 microM). The effects of M15 and galanin(1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I were significantly decreased by atropine for 36 and 18% and by spantide for 37 and 26% respectively. Indomethacin inhibited the muscle response to M15 without influence on the galanin (1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I-induced action. These results support findings that galanin (1-29)-NH2 contracts rat gastric fundus strips by stimulating specific receptors localized on the surface of smooth muscle cells. M15 and galanin(1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I seem to contract smooth muscles not only by acting at galanin receptors, but by interacting with muscarinic or tachykinin receptors or modulating the release of acetylcholine and substance P. Diltiazem (EC50 825 nM), dantrolene (EC50 30.2 microM) and the phospholipase C inhibitors U-73122 (EC50 549 microM) and U-73343 (EC50 751 microM) lowered the contraction to galanin(1-29)-NH2 in a concentration-dependent manner. These observations imply that though the extracellular Ca2+ influx plays a major role in the action of galanin(1-29)-NH2, the release of Ca2+ ions from the intracellular stores contributes to the response of smooth muscles of galanin(1-29) NH2. Norepinephrine (30, 60, 100 and 300 nM) concentration-dependently reduced the Emax to galanin (1-29)-NH2 and reduced the slopes of the concentration-contraction curves, without a notable change in EC50. Pertussis toxin pre-treatment (10 and 30 mg/kg intravenous [i.v.]), 120 h before the experiment, notably increased the maximal response of the rat gastric fundus to galanin(1-29)-NH2, without a significant change in the properties of the concentration-contraction curves (EC50, slopes). The observations may suggest that pertussis toxin-sensitive GTP-binding proteins are involved in the modulation of the excitatory effects of galanin(1-29)-NH2 in the rat gastric fundus.

Sources of activator Ca2+ for galanin-induced contractions of rat gastric fundus, jejunum and colon.

Galanin (Gal) evoked reproducible contractions of isolated rat gastric fundus, colon and jejunum longitudinal strips in concentrations ranging from 1 nM to 3 microM. EC50 of Gal equalled 12.63, 23.27 and 56.02 nM, respectively. Hill's coefficients were not different from unity in any of the tissues examined. Experiments have been performed in the presence of protease and peptidase inhibitors, a variety of specific antagonists and tetrodotoxin (TTX) to exclude the non-specific stimulatory or inhibitory action of Gal. Gal-evoked contractions were attenuated by diminished extracellular Ca2+ concentration and by diltiazem. Gal activity in gastric fundus and colon, but not in jejunum was inhibited by depleting intracellular Ca2+ stores, thapsigargin, dantrolene, ryanodine, TMB-8, neomycin and U-73122. Our data confirmed that Gal contracts rat fundus, jejunum and colon by stimulating specific receptors, which are coupled both to Ca2+ influx through the voltage-dependent calcium channels and intracellular Ca2+ release from ryanodine- and IP3-sensitive stores (stomach and colon) or the extracellular Ca2+ influx only (jejunum). Phosphatidylinositol-specific phospholipase C (PI-PLC) plays a crucial role in the former but not in the latter signal transduction cascade.

Actions of several substituted short analogues of porcine galanin on isolated rat fundus strips: a structure-activity relationship.

The activity of porcine galanin (Gal) fragments and analogues were tested in vitro using rat gastric fundus strips. The peptides contracted longitudinal smooth muscle in a concentration-dependent manner with the following order of potency: [Nle4]Gal(1-15), Gal(-15), [Cle4]Gal(1-15), [Hse6]Gal(1-15), [Va14]Gal(1-15), [Ile4]Gal(1-15), [endoTrip2a, Cle4]Gal(1-15), [desThr3, Cle4]Gal(1-15), [D-Leu4] Gal(1-15), [desLeu4]Gal(1-15). On the contrary [desTrp2, Val4]Gal (1-15) remained inactive up to 10 microM. The values of Hill's coefficients estimated from the appropriate concentration-contraction curves for all analogues except for [Val4]Gal(1-15), [Hse6]Gal(1-15), [endoTrp2a,Cle4]Gal(1-15), [desLeu4]Gal(1-15) and [D-Leu4] Gal(1-15) did not significantly differ from unity. Our results indicate that the integrity of the first four N-terminal amino acids of Gal molecule is essential for the full excitatory myogenic action of the peptide in rat gastric fundus. Similarly, substitution, addition or deletion of amino acid residues in positions two, three, four and six can considerably influence the ability of Gal analogues to interact with Gal receptors. The data acquired in the course of our structure-activity study suggest that both N- and C-terminals of Gal molecule contribute towards the affinity and activity of Gal in rat gastric smooth muscle cell receptors.

Endothelin receptor blockers protect against ischemia/reperfusion impairment of gastrointestinal motility in rats.

Intestinal ischemia/reperfusion (I/R) injury remains associated with high morbidity and mortality. The protective efficacy of the following endothelin (ET) receptor blockers: BQ-123 (ET(A) receptor), BQ-788 (ET(B)); tezosentan (dual ET blocker) was tested against the inhibition of gastrointestinal (GI) motility induced by intestinal I/R. Intestinal Evans blue transit was measured in untreated (UN) rats and animals subjected to skin incision (SI), I/R (1h superior mesenteric artery clamping followed by 2-24h reperfusion) or sham operation (SO). Surgical procedures were conducted under diethyl ether anesthesia. Anesthesia and SI did not affect the GI transit compared to UN rats. In contrast both SO and I/R significantly reduced GI motility, the latter evident at 2-24h of reperfusion. Tezosentan (1-10 mg/kg), BQ-123 and BQ-788 (0.1-1 mg/kg) protected against I/R-induced inhibition of intestinal motility in a time- and dose-dependent manner at the early and late stages of reperfusion. Furthermore tezosentan alleviated the I/R-induced decrease in the contractile response of the longitudinal jejunal smooth muscle strips to carbachol in vitro. The serum ET(1-21) level was increased at 2h but not 24h of reperfusion compared to SO animals and ET(1-21) was higher in tezosentan pretreated rats.

Salutary effects of tachykinin receptor antagonists in a rat model of postoperative ileus.

BACKGROUND: Postoperative ileus (PI) is a common surgical complication treated mainly with supportive measures. Tachykinins control gastrointestinal motility and modulate somatic and visceral pain sensation; therefore, the effect of tachykinin receptor antagonists in a rat model of PI using NK(1-3) antagonists, SR140333, SR48968, and SR142801, was investigated. MATERIALS AND METHODS: Intestinal transit was measured as Evans blue migration after varied nociceptive stimuli: skin incision (SI), laparotomy (LAP), or laparotomy plus gut manipulation (L + M) in anesthetized rats. RESULTS: Diethyl ether anesthesia and SI did not influence the intestinal transit of the dye in comparison to untreated animals--UN: 61.17 +/- 5.47, 62.10 +/- 8.30, and 56.70 +/- 4.10 cm, respectively. In contrast LAP and L + M have significantly reduced intestinal motility to 26.40 +/- 2.07 and 9.70 +/- 1.15 cm, respectively. SR140333 (3-30 microg/kg), SR48968 (1-30 microg/kg), and SR142801 (3-10 microg/kg) reversed the additional inhibitory effects of gut manipulation subsequent to LAP dose-dependently, the dye transit returning with the use of the most effective antagonist doses up to 25.28 +/- 1.08, 21.70 +/- 0.19, and 25.0 +/- 1.34 cm. The combinations of submaximal doses of NK(1) and NK(3), NK(2) and NK(3) and NK(1), and NK(2) and NK(3) antagonists were not more effective than a single-agent regimen. On the other hand SR140333 and SR48968 (NK(1) + NK(2) antagonists) acted additively, the intestinal transit reaching 26.60 +/- 0.85 cm. SR140333, SR48968, and SR142801 have not affected the intestinal passage in UN rats or those undergoing SI or LAP. CONCLUSIONS: SR140333, SR48968, and SR142801 exert a salutary action on suppressed gut motility following surgical manipulation of the gut, the combination of NK(1) and NK(2) antagonists being most beneficial.

The contractile effects of several substituted short analogues of porcine galanin in isolated rat jejunal and colonic smooth muscle strips.

The activity of short porcine galanin (Gal) analogues was tested in vitro using rat jejunal and colonic smooth muscle strips. Peptides evoked concentration-dependent tissue contractions yielding typical response curves in concentration range from 0.3 nM to 300 microM, with a characteristic fall-down effect at the supramaximal concentrations. Gal(1-15) was less potent than Gal(1-29). Furthermore, [D-Trp(2)]Gal(1-15), [endo-Trp(2),Cle(4)]Gal(1-15), [D-Leu(4)]Gal(1-15), [des-Leu(4)]Gal(1-15), [Hse(6)]Gal(1-15), [Dab(14)]Gal(1-15), [Dpr(14)]Gal(1-15) or [Arg(14)]Gal(1-15) showed a considerable decrease in potency compared to Gal(1-15) in jejunal and/or colonic smooth muscle cells. Functional evidence confirmed that the integrity of both N- and C-terminals must be preserved in order to preserve a full excitatory myogenic potential of the peptide in rat jejunum and colon. Besides, amino acids located in positions 2, 4, 6 and 14 play a crucial role in recognition and activation of molecular domains responsible for Gal action in the intestinal smooth muscle.

Intracerebroventricular pertussis toxin enhances sensitivity to chemical convulsants and decreases the protective efficacy of carbamazepine in mice.

The effects of pretreatment with pertussis toxin on pentylenetetrazole-, bicuculline-, aminophylline- and pilocarpine-induced seizures were investigated in mice. In animals treated intracerebroventricularly with pertussis toxin (0.5 microgram animal-1 120 h prior to testing), the CD50 (convulsive dose in 50%) values were considerably decreased in comparison with the CD50 in sham-treated animals. CD50 values of pentylenetetrazole, bicuculline, pilocarpine and aminophylline were calculated to be 39.9, 2.0, 262 and 141 mg kg-1, whereas they were calculated to be 57.7, 2.7, 324 and 230 mg kg-1 in sham-treated animals. The observations suggest that the enhanced sensitivity to a number of chemical convulsants irrespective of their mode of action possibly results from a functional suppression of inhibitory transmission at receptors coupled to pertussis toxin sensitive G proteins, rather than a direct action on G protein linked excitatory neurotransmission. Pertussis toxin significantly decreased the protective action of carbamazepine, increasing its ED50 (effective dose in 50%) from 14.8 to 20.1 mg kg-1 in a maximal electroshock convulsive test. It influenced the ED50 of neither diphenylhydantoin nor diazepam. The diminution of carbamazepine's efficacy might result from a summation effect of adenosine receptor antagonist properties of the drug and a suppression of transmission at adenosine receptors coupled to G proteins sensitive to pertussis toxin. Pertussis toxin pretreatment remained without any significant influence on the total plasma levels of carbamazepine, diphenylhydantoin and diazepam. This may lead to the conclusion that the interaction between pertussis toxin and carbamazepine does not seem to be of a pharmacokinetic nature and occurs probably at neuronal level.

Influence of intracerebroventricular administration of tetanus toxin on experimental seizures and protection afforded by some antiepileptic drugs in mice.

The dose to the intracerebroventricularly administered (i.c.v.) tetanus toxin (Tetx) evoking the death of 50% of experimental mice (LD50) was estimated to be 18.0 (11.5-28.2) times the minimal lethal dose (MLD). MLD is defined as the lowest does of Tetx necessary to kill a 20-g albino mouse within 96 h after intraperitoneal treatment. Tetx (0.25 and 0.5 LD50) increased the convulsive threshold of electric current from 24 to 96 and 120 h, respectively, following i.c.v. administration. Both doses of Tetx diminished convulsant potencies of pentylenetetrazole, bicuculline, aminophylline and pilocarpine 24 h after application. At the same time Tetx (0.5 LD50) increased the protection afforded by carbamazepine, valproate, phenobarbital and diazepam in maximal electroshock (MES) by approximately 36, 11, 21 and 26%, respectively, without affecting total blood plasma levels of antiepileptic drugs. No marked changes in gamma-aminobutyric acid (GABA) concentration and total activity of L-glutamic acid decarboxylase (GAD) assessed in the whole-brain homogenates resulted from Tetx treatment. Our results seem to indicate that low doses (< LD50) of i.c.v. administered Tetx may lead to a relative prevalence of inhibitory over excitatory processes in the central nervous system suggesting a complex action of Tetx at the neuronal level.

Role of nitric oxide in pathogenesis of serotonine-induced gastric lesions in rats.

We investigated the role of nitric oxide (NO) in the development of gastric mucosal lesions induced by serotonine (5-HT) in rats. Repeated subcutaneous administration of 5-HT (20 mg kg-1) produced damage in the stomach with severe edema in the submucosa. Gastric lesions induced by 5-HT were prevented by simultaneous administration of aminoguanidine, a selective inducible NO synthase (iNOS) inhibitor, as well as by methysergide, a 5-HT antagonist. In addition, the lesions were inhibited by pretreatment with the antioxidative drugs, such as allopurinol (a xanthine oxidase inhibitor) and hydroxyurea (a neutrophil reducing agent). Following 5-HT treatment, the Ca(2+)-independent NOS activity in the gastric mucosa was significantly increased within 6 h and remained elevated for 2 days thereafter. The serum NOx levels increased 12 h after the administration of 5-HT, reaching a peak 24 h later. Gastric mucosal thiobarbituric acid (TBA) reactants and myeloperoxidase (MPO) activity were also significantly increased after 2 days treatment with 5-HT. Our results suggest that: (1) the repeated administration of 5-HT induced inflammatory gastric lesions in the rat stomach; (2) iNOS is upreguated during 5-HT treatment, and NO produced by iNOS contributes to development of gastric lesions in response to 5-HT, in addition to the oxyradical formation, and (3) the deleterious role of NO in this model may be accounted for by a cytotoxic action of peroxynitrite that is formed in the presence of NO and superoxide radicals.

Role of eicosanoids, nitric oxide, and afferent neurons in antacid induced protection in the rat stomach.

The mechanism underlying the mucosal protective effect of antacids is still unclear. This study shows that in rats the aluminum containing antacid, hydrotalcit, induces dose dependent protection against gastric mucosal damage caused by ethanol or indomethacin which is considerably enhanced by acidification. Hydrotalcit did not increase gastric mucosal formation or the intraluminal release of prostaglandins, and did not prevent the increase in mucosal leukotriene C4 formation in response to ethanol. Pretreatment with indomethacin did not attenuate the protective effect of unmodified or acidified hydrotalcit. Furthermore, hydrotalcit significantly reduced the gastric damage caused by indomethacin even when it was administered up to 2 hours after the ulcerogen. In indomethacin treated rats, simultaneous administration of hydrotalcit did not affect the concentrations of indomethacin in serum or inflammatory exudates nor did it attenuate the inhibition of prostaglandin release into the exudates. In hydrotalcit treated rats there was no attenuation of the increase in sulphidopeptide leukotriene release or decrease in leukocyte influx into inflammatory exudates elicited by indomethacin administration. Functional ablation of afferent neurons and inhibition of endogenous nitric oxide partially antagonised the protective effect of unmodified, but not of acidified, hydrotalcit. It is concluded that (i) the protective effect of unmodified and acidified hydrotalcit is independent of the eicosanoid system; (ii) protection against indomethacin induced gastric lesions does not require treatment before dosing of the ulcerogen and does not interfere with absorption and anti-inflammatory actions of indomethacin; (iii) endogenous nitric oxide and afferent neurons contribute partly to the effect of unmodified, but not of acidified, hydrotalcit suggesting that different mechanisms mediate their mucosal protective activity.

Polaprezinc exerts a salutary effect on impaired healing of acute gastric lesions in diabetic rats.

We examined the influence of diabetes mellitus (DM) on the healing of HCl-induced gastric lesions and the healing promoting effect of polaprezinc [N-(3-aminopropionyl)-L-histidinato zinc] on these lesions. Studies were performed on rats injected intraperitoneally with streptozotocin (STZ, 70 mg/kg) five weeks prior to experiments. Diabetic rats had blood glucose levels (BGLs) higher than 350 mg/100 ml. Randomly chosen animals were treated subcutaneously with insulin (4 IU/day/rat) starting 1 week after STZ. Animals were given 1 ml of 0.6 N HCl by oral gavage (per os) following 18 hr of fasting; they were fed normally from 1 hr later and killed at various time points after HCl administration. Polaprezinc (3-30 mg/kg) or its components ZnSO4/7H2O and L-carnosine were given orally, twice daily for four days following HCl treatment. Gastric lesions induced by HCl healed macroscopically to quiescence within 10 days. DM and insulin did not affect the development of HCl-invoked gastric lesions, but the healing of such lesions was markedly impaired in animals with DM. Daily administration of insulin returned high BGLs to significantly lower ranges (190-208 mg/100 ml) and markedly antagonized the healing impairment. Polaprezinc (>10 mg/kg) significantly reversed the delay observed in diabetic rats without any notable effects on BGLs or acid secretion. Similar trends were observed with ZnSO4/7H2O or a mixture of ZnSO4/7H2O and L-carnosine, but not by L-carnosine alone. The mucosal expression of insulin-like growth factor-1 (IGF-I) mRNA was significantly lower in diabetic rats, a dysregulation partially corrected by insulin and polaprezinc. In addition, the delayed healing in diabetic rats was also significantly promoted by the repeated subcutaneous administration of rhIGF-I (>10 microg/kg, twice daily) without any notable effect on BGLs or acid secretion. These results suggest that DM exerted a deleterious influence on the healing of acute gastric lesions in both insulin- and zinc-sensitive manner. The salutary effects of polaprezinc on the impaired healing of gastric lesion in STZ-diabetic animals may at least be partly explained by enhancement of mucosal IGF-I mRNA expression in the stomach.

Exogenous insulin-like growth factor (IGF)-1 improves the impaired healing of gastric mucosal lesions in diabetic rats.

BACKGROUND: We examined the influence of diabetes mellitus (DM) on the healing of HCl-induced gastric lesions and the healing promoting effect of IGF-1 on these lesions. METHODS: Experiments were performed on rats injected with streptozotocin (STZ, 70 mg kg(-1), i.p.) 5 weeks prior to the experimental session. Diabetic animals had blood glucose levels (BGLs) higher than 350 mg dl(-1). Randomly chosen rats were treated with insulin (4 IU day(-1) rat(-1)) starting 1 week after STZ. Animals were given 1 ml of 0.6 m HCl by gavage following 18 h of fasting. Normal feeding was started 1 h later. Animals were killed at various time points after HCl. Recombinant human IGF-1 (rhIGF-1) at doses 10-400 microg kg(-1) was injected subcutaneously twice daily for 4 days following HCl treatment. RESULTS: Gastric lesions induced by HCl healed macroscopically within 10 days. DM and insulin regimen did not affect the development of HCl-invoked gastric lesions. However, DM significantly delayed the healing of such lesions. Daily administration of insulin returned the high BGLs to significantly lower ranges (190-210 mg dl(-1)) and markedly antagonized the delayed healing. Likewise, the repeated administration of rhIGF-1 (>/=10 microg kg(-1)) significantly enhanced the healing of gastric lesions in diabetic rats, without any notable effect on BGLs or gastric acid secretion. The mucosal expression of IGF-1 mRNA was lower in diabetic rat stomachs as compared to normal rats, although the expression was apparently restored after insulin treatment. CONCLUSION: These results suggest that DM has a deleterious influence on the healing of acute gastric lesions in both insulin- and IGF-1-sensitive manner. The systemic administration of rhIGF enhanced the rate of healing of gastric lesions observed in the healing-impaired STZ-diabetic animals.

Nitric oxide synthase activity, viability and cyclic GMP levels in rat colonic epithelial cells: effect of endotoxin challenge.

The present study has examined whether epithelial cells could be a source of the inducible form of nitric oxide synthase (iNOS) in the colon of rats challenged with E. coli lipopolysaccharide and whether such excessive endogenous nitric oxide (NO) or exogenous NO released from NO donors could affect their viability. Epithelial cells were isolated from rat colon, and cell viability was determined by trypan blue exclusion. The appearance of a calcium-independent iNOS determined by the conversion of radiolabeled L-arginine to citrulline was observed in cells harvested from lipoplysaccharide (3 mg/kg for 4 hr)-treated rats, with a 10-fold increase in total NOS activity compared with control. This was accompanied by a 3-fold decrease in epithelial cell viability. Levels of iNOS and of cellular injury were not significantly affected in rats made neutropenic by the administration of antineutrophil serum. Induction of NOS was also associated with an increase in epithelial cyclic guanylate monophosphate levels. Both iNOS activity and cell injury were inhibited by in vivo pretreatment with dexamethasone (1 mg/kg i.v., for 4 hr) or the NOS inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg s.c.) in a dose that itself reduced viability. The incubation of epithelial cells with the NO donors, nitroprusside, S-nitroso-N-acetyl penicillamine or S-nitroso-N-glutathione (0.1-1 mM) produced concentration-dependent cytotoxicity. These findings indicate that the induction of NOS in colonic epithelial cells is not dependent upon infiltration of neutrophils and is accompanied by a reduction in cellular viability, an effect mimicked in vitro by NO donors.(ABSTRACT TRUNCATED AT 250 WORDS)