Influence of a Brazilian sewage outfall on the toxicity and contamination of adjacent sediments.

The submarine sewage outfall of Santos (SSOS) is situated in the Santos Bay (São Paulo, Brazil) and is potentially a significant source of contaminants to the adjacent marine ecosystem. The present study aimed to assess the influence of SSOS on the sediment toxicity and contamination at Santos Bay. At the disposal site, sediments tended to be finer, organically richer and exhibited higher levels of surfactants and metals, sometimes exceeding the "Threshold Effect Level" values. The SSOS influence was more evident toward the East, where the sediments exhibited higher levels of TOC, total S and metals during the summer 2000 sampling campaign. Sediment toxicity to amphipods was consistently detected in four of the five stations studied. Amphipod survival tended to correlate negatively to Hg, total N and % mud. This work provides evidence that the SSOS discharge affects the quality of sediments from Santos Bay, and that control procedures are warranted.

Demonstration of specific E-type prostaglandin receptors using enriched preparations of canine parietal cells and [3H]misoprostol free acid.

High-affinity, E-type prostaglandin binding sites in enriched canine parietal cell preparations were identified with [3H] misoprostol free acid, a prostaglandin E1 analogue. Saturable, reversible, and highly stereospecific binding was identified, with approximately 8,000 binding sites per cell. Prostaglandin I and F bound weakly, and cimetidine and histamine did not bind. The results indicate that [3H] misoprostol free acid binds to E-type prostaglandin receptors, which suggests that the ulcer-healing inhibition of gastric acid secretion by misoprostol results from its interaction with a specific E-type prostaglandin receptor.

2-Oxo-1,8-naphthyridine-3-carboxylic acid derivatives with potent gastric antisecretory properties.

The syntheses of 2-oxo-1,8-naphthyridine-3-carboxylic acid derivatives having potent gastric antisecretory properties in the pyloric-ligated (Shay) rat model are described. Two of the more potent compounds tested that were selected for more detailed dose-response evaluation were 4-amino-1-ethyl-1,2-dihydro-2-oxonaphthyridine-3-carboxylic acid ethyl ester (35) and 1-ethyl-1,2-dihydro-7-methyl-4-(4-methyl-1-piperazinyl)-2- oxo-1,8-naphthyridine-3-carboxylic acid ethyl ester (77). These compounds lowered total acid output in the rat in a dose-related fashion. Both compounds were more potent than cimetidine when tested in the rat. Both 35 and 77 showed inhibitory activity in food-stimulated acid secretion in the Pavlov-pouch, conscious dog. The mechanism of action for this series is not known. Details of structure-activity relationships are described.

Synthesis and gastrointestinal pharmacology of a 3E,5Z diene analogue of misoprostol.

A stereospecific synthesis and the gastric antisecretory and diarrheal activity of a 3E,5Z diene analogue of misoprostol are described. The key intermediate in the synthesis was an alpha chain truncated acetylene that was obtained by a cuprate/enolate capture procedure on the corresponding cyclopentenone. Palladium-catalyzed coupling of the acetylene with methyl 4-iodo-3(E)-butenoate provided the conjugated enyne. Although selective hydrogenation of the enyne with Lindlar catalyst failed, the desired 3E,5Z diene was obtained with P-2 nickel as catalyst. The diene was about 3 times more potent than misoprostol in inhibiting gastric acid secretion in dogs and also in producing diarrhea in rats.

18-cycloalkyl analogues of enisoprost.

By use of standard cuprate methodology, a series of 18-cycloalkyl analogues of enisoprost was prepared in an effort to impede omega chain metabolism and prolong duration of gastric antisecretory activity. An initial product of omega chain oxidation, the C-20 hydroxy analogue, was also synthesized for pharmacological comparison. The cyclopropyl, cyclobutyl, and cyclopentyl analogues were approximately one-fourth as potent as enisoprost in inhibiting gastric acid secretion, while the cyclohexyl and cycloheptyl analogues showed very weak activity, and the 20-hydroxy compound was inactive at a dose 100 times the ED50 of enisoprost. The cyclobutyl compound had a longer duration of antisecretory action than enisoprost and the other cycloalkyl analogues. The cycloalkyl analogues unexpectedly possessed low diarrheogenic activity in rats.

Synthesis and gastric antisecretory properties of alpha chain diene derivatives of misoprostol.

The synthesis and gastric antisecretory activity in dogs of seven alpha chain diene derivatives of misoprostol are described. The key intermediates in the preparation of these compounds were C-9 tert-butyldimethylsilyl enol ethers that were obtained by in situ silylation of cuprate enolates derived from alpha chain unsaturated cyclopentenones. Selenylation chemistry on these intermediates provided the C2-C3 trans dienes that, where possible, were also deconjugated to produce the corresponding C3-C4 dienes. The most interesting structure in this series is the C5-C6 cis, C3-C4 cis/trans (1:1) diene that could not be readily separated chromatographically into its individual geometric isomers. The gastric antisecretory activity of the mixture of isomers was approximately 3 times greater than that of misoprostol by intragastric administration. The separation of undesired diarrheogenic effects from antisecretory activity was significantly improved relative to misoprostol.

Identification and regulation of alpha 2-adrenergic receptors in rabbit ileal mucosa.

The alpha 2-adrenergic receptors in rabbit ileal mucosal membranes can be identified by using [3H]clonidine. [3H]Clonidine bound to a homogeneous population of sites (30-120 fmoles/mg protein) with a KD of 2.2 nM at 25 degrees. Alpha-adrenergic agonists and antagonists competed with [3H] clonidine for the binding sites with an order of potency typical for alpha 2-receptors. Mg2+, Ca2+, or Mn2+ (2.4 mM) markedly increased the binding of [3H]clonidine. At the maximally effective concentration, Mg2+ increased both the binding affinity of [3H]clonidine and the number of receptor sites. Both NaCl and GppNHp, the guanyl nucleotide, inhibited [3H]clonidine binding. NaCl decreased the binding affinity of [3H]clonidine, with no appreciable effect on the number of receptor sites. These findings indicate that ileal mucosal alpha 2-receptors can exist in multiple affinity states, which can be regulated by divalent cations, NaCl, and guanyl nucleotides. It appears that NaCl and GppNHp regulate alpha 2-receptors in ileal mucosa by different mechanisms.

12(R)-hydroxyeicosatetraenoic acid is a neutrophil chemoattractant in the cavine, lapine, murine and canine dermis.

Psoriasis is a disease state characterized by epidermal proliferation, neutrophil infiltration, along with release of the proinflammatory mediators leukotriene-B4(LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE]. LTB4 and 12(R)-HETE are chemoattractant to the neutrophil, the latter approximately 1000x less potent. LTB4 and 12(R)-HETE are present in psoriatic scale, the latter in quantities so much greater than LTB4 that it is proposed as a primary mediator of neutrophil infiltration in psoriasis. 12(R)-HETE, synthesized in optically pure form by a new, shorter route, was injected into the dermis of the cavine, lapine, canine, mouse and rat. At doses up to 50 mu gm per intradermal site, 12(R)-HETE was chemoattractant to the neutrophil (as assessed by dermal myeloperoxidase levels) with response in the cavine greater than canine greater than lapine greater than mouse greater than rat.

Misoprostol preclinical pharmacology.

Misoprostol, a synthetic derivative of prostaglandin E1, was tested and shown to be an effective gastric antisecretory agent against histamine-, pentagastrin-, and meal-stimulated acid secretion in dogs. Misoprostol reduced the volume of acid secretion as well as the hydrogen ion concentration. Misoprostol did not reduce gastric mucosal blood flow, nor did it alter meal-stimulated serum gastrin levels. Misoprostol inhibited acid secretion in histamine-stimulated isolated gastric glands indicating a direct antisecretory effect on parietal cells. The potency of misoprostol was greatest when administered in direct contact with the gastric mucosa indicating local absorption and action. Misoprostol strengthened the gastric mucosal barrier as shown by the attenuation of aspirin-induced lowering of transmucosal electrical potential differences. Misoprostol protected the gastric mucosa of rats subjected to ethanol-, taurocholate-, pyloric ligation-, stress- and indomethacin-induced damage. Misoprostol also protected against indomethacin-induced intestinal lesions in rats and reduced duodenal ulcer formation in guinea-pigs and cats. The doses of misoprostol required to protect against gastric damage were about one-tenth of those required to inhibit acid secretion. The results of these and other studies indicate that misoprostol is a safe agent with unique properties that should provide a new approach for treatment of ulcer diseases of the gastrointestinal tract.

Gastric secretion with feeding and restraint in conscious rhesus monkeys.

Four chair-housed rhesus monkeys (Macaca mulatta) were surgically prepared with vagally-innervated fundic gastric pouches. As in man, basal acid secretion was evident in all subjects. In response to a test meal, acid output rose twofold over basal values and remained elevated up to 6 h. Compared to control values, food-stimulated acid output was reduced significantly during periods of restraint.

Propagation of mouse mammary tumor cell lines and production of mouse mammary tumor virus in a serum-free medium.

Five different mouse mammary tumor cell lines were propagated in a serum free medium. Evaluation of growth characteristics, including logarithmic growth, cell population increase, protein production and days to confluency, showed serum-free medium comparable to serum-containing medium. Mouse mammary tumor virus expression and production, in C3H and GR tumor cell lines, as determined by virus particle counting and RNA dependent DNA polymerase assays, subsequent to dexamethasone stimulation revealed equivalent to higher levels of virus in serum-free medium as compared to serum-containing medium.

Expression of gastric antisecretory and prostaglandin E receptor binding activity of misoprostol by misoprostol free acid.

In enriched canine parietal cell preparations, misoprostol, an analog of prostaglandin E1 methyl ester, was rapidly deesterified to misoprostol free acid. Under this circumstance, misoprostol and misoprostol free acid exhibited equal antisecretory potency against histamine-stimulated acid secretion and bound equally well to prostaglandin E receptors. When the deesterification of misoprostol was inhibited by paraoxon, an esterase inhibitor, the antisecretory and receptor binding activity of misoprostol was markedly reduced, with potency much less than misoprostol free acid. These results indicate that misoprostol free acid is the active biological form of misoprostol that binds to prostaglandin E receptors and mediates the antisecretory action of misoprostol.

Antisecretory activity of misoprostol, its racemates, stereoisomers and metabolites in isolated parietal cells.

Inhibition of histamine-stimulated acid secretion by misoprostol was compared to its racemates, stereoisomers and metabolites in isolated canine parietal cell preparations. The concentration of misoprostol required to inhibit 50% of maximal histamine-stimulated acid secretion (IC50) was 3.8 +/- 0.3 nM. One racemate of misoprostol was at least 1000 times more potent than the other. Of the four misoprostol stereoisomers, the 11R, 16S isomer exhibited the most potent activity against histamine with an IC50 value of 1.4 +/- 0.1 nM. The acid metabolite of misoprostol was equally potent as misoprostol. In contrast to the acid metabolite, the beta-oxidation metabolites of misoprostol lacked significant activity at 1 microM. The results indicate that: 1) the acid metabolite of misoprostol may play a significant role in the antisecretory activity of misoprostol, and 2) the high degree of stereo-specificity associated with the antisecretory effects indicates that the activity of misoprostol may be receptor mediated.

E-type prostaglandin binding sites in isolated canine parietal cells: elucidation with (3H) misoprostol free acid.

(3H) Misoprostol free acid, a prostaglandin E1 analog, was used to identify high affinity binding sites for E-type prostaglandins in enriched canine parietal cell preparations. Saturable, reversible, and highly stereospecific binding, with an estimated number of 8000 binding sites per cell, was demonstrated. The binding sites have high affinity for misoprostol, misoprostol free acid, and other E-type prostaglandins. Prostaglandins of the I- and F-type bind weakly, and chemically unrelated compounds, such as histamine and cimetidine, do not bind. The results indicate that (3H) misoprostol free acid binds to E-type prostaglandin receptors, and that the ulcer-healing drug, misoprostol, inhibits gastric acid secretion through interaction with a specific E-type prostaglandin receptor.

Effect of misoprostol on histamine-stimulated acid secretion and cyclic AMP formation in isolated canine parietal cells.

Isolated canine parietal cells were used to study the ability of misoprostol to inhibit acid secretion in the presence of a number of acid secretagogues. Misoprostol inhibited histamine-stimulated acid secretion in a dose-dependent and noncompetitive manner. A concentration of 2-3 X 10(-9) M misoprostol inhibited maximal histamine-stimulated acid secretion by one half. Misoprostol had little to no effect on acid secretion stimulated by carbachol and dibutyryl cAMP, had no effect on the acid secretion directly stimulated by pentagastrin, and only modestly inhibited acid secretion stimulated by forskolin. Misoprostol noncompetitively inhibited cAMP formation in response to histamine, with an IC50 value similar to that for the inhibition of histamine-stimulated acid secretion. These results indicate that: (1) misoprostol specifically inhibits histamine-stimulated acid secretion in parietal cells, and (2) the antisecretory action of misoprostol is closely related to the reduction of histamine-stimulated cAMP formation with the site of major action most likely in the coupling process between histamine H2 receptor sites and histamine-sensitive adenylate cyclase.

Comparative mucosal protective properties of misoprostol, cimetidine, and sucralfate.

Misoprostol, a PGE1 derivative that inhibits gastric acid secretion in rats, was compared with cimetidine and sucralfate in several rat experimental ulcer models. Gastric lesions were produced by aspirin, indomethacin, stress, sodium taurocholate, and ethanol. In all tests, misoprostol (50, 100, and 200 micrograms/kg) and cimetidine and sucralfate (50, 100, and 200 mg/kg) were administered intragastrically. Misoprostol protected against gastric lesions in all five experimental ulcer models at lower than gastric antisecretory doses. Cimetidine protected in the indomethacin, aspirin, and stress models, but only at gastric antisecretory doses, and did not protect against lesion formation in the ethanol and taurocholate models. Sucralfate, over the dose range tested, was not consistently protective in any of the five experimental ulcer models. It is concluded that misoprostol provides gastric mucosal protection against a wide variety of noxious agents by means of a unique mechanism and that reduction of gastric acid secretion is not required, as it is with cimetidine, for the protective effect.

Misoprostol attenuates aspirin-induced changes in potential difference and associated damage in canine gastric mucosa.

Aspirin induces ulceration, cellular exfoliation, and blood loss associated with decreases in gastric mucosal potential difference (PD). Certain prostaglandins prevent the development of experimental gastric and duodenal ulcers and modify indices related to ulceration. Misoprostol, a synthetic PGE1 derivative with gastric antisecretory and mucosal protective activities, was examined at gastric antisecretory doses in dogs with Heidenhain pouches, to determine its effect on aspirin-associated changes in PD, K+ efflux, blood loss, and cell shedding, as measured by DNA release. These parameters were examined before, during, and up to 4 hours after exposure of the pouches to aspirin. Disruption of the gastric mucosal barrier (GMB) by aspirin was associated with a fall in PD and losses of K+, DNA, and blood into the pouches. Misoprostol inhibited the fall in PD and prevented blood loss over the entire period examined. Cell loss was inhibited only during the recovery period immediately following aspirin. The effect of misoprostol on GMB is consistent with studies in which prostaglandins preserve the GMB and prevent necrotic ulcerations while allowing superficial cell damage.

The effect of leukotriene-B4 receptor antagonist, SC-41930, on acetic acid-induced colonic inflammation.

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8-p ropyl- 2H-1-benzopyran-2-carboxylic acid, is a potent in vitro leukotriene-B4 (LTB4) receptor antagonist. LTB4 levels are elevated in colonic tissue of inflammatory bowel disease (IBD) patients which may account for the high degree of neutrophil (PMN) infiltration. The guinea pig acetic acid-induced colonic inflammation model has characteristics of IBD including PMN infiltration, edema, ulceration and necrosis. The model was used to evaluate the effect of SC-41930. SC-41930 was given orally, 30 min before and after intrarectal administration of 3% acetic acid. The PMN marker enzyme, myeloperoxidase, was measured along with histological evaluation to assess inflammation. Both parameters showed significantly less inflammation in SC-41930 treated animals with an oral ED50 of 20 mg/kg. These study results with an LTB4 receptor antagonist indicate a role for LTB4 in colonic inflammation and that an LTB4 receptor antagonist may be beneficial for treatment of IBD.

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions.

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions.

A leukotriene B4 (LTB4) analog, 20-trifluoromethyl LTB4 (20CF3-LTB4), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF3-LTB4 was equipotent to LTB4 as a chemoattractant (EC50, 3 nM), produced 50% of maximal activity of LTB4, and competed with [H] LTB4 for binding to intact human neutrophil LTB4 receptors. In contrast to chemotactic activity, 20CF3-LTB4 in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 microM on LTB4-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB4, 20CF3-LTB4 induced neutrophil desensitization to degranulation by LTB4. The results indicate that hydrogen atoms at C-20 of LTB4 are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF3-LTB4 is a partial agonist for chemotaxis and an antagonist for degranulation suggests that different LTB4 receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB4 can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.