On the central mechanism underlying ghrelin's chronic pro-obesity effects in rats: new insights from studies exploiting a potent ghrelin receptor antagonist.

In the present study, we explore the central nervous system mechanism underlying the chronic central effects of ghrelin with respect to increasing body weight and body fat. Specifically, using a recently developed ghrelin receptor antagonist, GHS-R1A (JMV2959), we investigate the role of GHS-R1A in mediating the effects of ghrelin on energy balance and on hypothalamic gene expression. As expected, in adult male rats, chronic central treatment with ghrelin for 14 days, when compared to vehicle-treated control rats, resulted in an increased body weight, lean mass and fat mass (assessed by dual X-ray absorptiometry), dissected white fat pad weight, cumulative food intake, food efficiency, respiratory exchange ratio and a decrease of energy expenditure. Co-administration of the ghrelin receptor antagonist JMV2959 suppressed/blocked the majority of these effects, with the notable exception of ghrelin-induced food intake and food efficiency. The hypothesis emerging from these data, namely that GHS-R1A mediates the chronic effects of ghrelin on fat accumulation, at least partly independent of food intake, is discussed in light of the accompanying data regarding the hypothalamic genes coding for peptides and receptors involved in energy balance regulation, which were found to have altered expression in these studies.

Ghrelin control of GH secretion and feeding behaviour: the role of the GHS-R1a receptor studied in vivo and in vitro using novel non-peptide ligands.

Energy homeostasis is controlled by a complex regulatory system of molecules that affect food intake and that are critical for maintaining a stable body weight during life. Ghrelin is a peptide of 28 amino acid synthesized predominantly by the stomach and the gut, which activate the type 1a growth hormone (GH) secretagogue receptor (GHS-R1a), a G-protein coupled receptor. The acylated form of ghrelin potently stimulates GH secretion both in vitro and in vivo in several animal species, including humans. Beside the endocrine effect, ghrelin shows also extraendocrine activities, including stimulation of feeding behaviour. Several classes of small synthetic peptide and non-peptide ligands of the GHS-R1a have been described and are able to release GH and stimulate food intake. However, in time, it appeared that the stimulating effects on GH secretion could be divorced from those on food intake, suggesting that more than a single receptor might be involved. Several experimental data have even questioned the physiological role of ghrelin in the control of GH secretion and energy metabolism. By using novel agonists, partial agonists, and antagonists for the GHS-R1a receptor, we have studied whether the stimulation of this receptor could account for the purported physiological role of ghrelin. Our results demonstrate that the ability to bind in vitro the GHS-R1a is not predictive of the in vivo biological activity of the compounds and that the endocrine and extraendocrine effects could be mediated also by receptors different from the GHS-R1a.

Synthesis and pharmacological evaluation of new (indol-3-yl)alkylamides and alkylamines acting as potential serotonin uptake inhibitors.

A series of new indolylalkylamides 3-18 and alkylamines 19-26 has been prepared in the search of novel 5-hydroxytryptamine (5-HT) uptake inhibitors. Synthesis of N-2,3 or 4-pyridinyl-(indol-3-yl) acetamides and propionamides 3-10 was achieved starting from the corresponding Ph3P/BrCCl3 or DCC-activated acids. Reduction of the pyridine nucleus led to the N-piperidinylalkylamides 15-18 via the tetrahydropyridinyl derivatives 11-14, and LiAlH4 reduction afforded the desired amines 19-26. The affinity of these compounds for 5-HT and also dopamine (DA) and noradrenaline (NA) uptake sites was measured. Among the 16 studied amides only N-(methylpiperidin-3-yl)-(indol-3-yl) propionamide 16 exhibited a moderate 5-HT uptake inhibitory effect: 38% at 10 mu mol/l. In contrast the N-pyridinyl-(indol-3-yl)alkylamines 19-26 exerted high inhibition at this concentration and two of them, 23 and 24, remained very efficient at 0.1 mu mol/l. Optimal activity was observed in the 4-pyridinyl subseries and was compatible with variation (n = 1, 2) of the length of the interspacing alkylamino chain. Although 23 and 24 were about 17-fold less active than indalpine as 5-HT uptake inhibitors, they demonstrated, like indalpine, excellent selectivity for the 5-HT uptake site versus the DA uptake site. Both amines inhibited tetrabenazine-induced hypothermia and potentiated 5-HTP-induced behavioural effects in mice. The absence of 3,4-dioxyphenylalanine (dopa)-induced behavioural effects with compound 24 suggests possible antidepressant activity through selective inhibition of central neuronal serotonin uptake and/or increased monoamine release.

Morphine 6-glucuronide and morphine 3-glucuronide as molecular chameleons with unexpected lipophilicity.

Morphine 6-glucuronide, but not morphine 3-glucuronide, is a highly potent opiate receptor agonist. In fact, there is converging evidence that much of the analgesic effect occurring after morphine treatment in humans is due to this metabolite rather than to the parent drug. Yet glucuronides as a rule are considered as highly polar metabolites unable to cross the blood-brain barrier and rapidly excreted by the urinary and/or biliary routes. Here, we report that morphine 6-glucuronide, and to a lesser extent morphine 3-glucuronide, are far more lipophilic than predicted, and in fact not much less lipophilic than morphine itself. Force-field and quantum mechanical calculations indicate that the two glucuronides can exist in conformational equilibrium between extended and folded forms. The extended conformers, because they efficiently expose their polar groups, must be highly hydrophilic forms predominating in polar media such as water; in contrast, the folded conformers mask part of their polar groups, thus being more lipophilic and likely to predominate in media of low polarity such as biological membranes.

COR3224--a new antidepressant: pharmacokinetics and metabolism in rat.

COR3224 is a new 2-amino-2 oxazoline derivative with antidepressant properties. The distribution, excretion and metabolism of radiochemically labelled 14C-COR3224 has been investigated in the rat after intravenous injection. The compound was widely distributed and rapidly excreted. Hydroxylation and sulphate conjugation are involved in the COR3224 elimination.

Flavonoids as inhibitors of rat liver monooxygenase activities.

Flavanone and six hydroxylated derivatives, and cianidanol and eight ethers and esters thereof, were investigated as inhibitors of cytochrome P-450 mediated reactions in rat liver microsomes. The IC50 values towards aminopyrine N-demethylation varied over a 20-fold range and were shown to depend on the pattern of hydroxylation (flavanone derivatives) and on lipophilicity (cianidanol derivatives). In the latter case, a bilinear relationship exists, the optimal log P being 2.92. Using selected compounds, IC50, Km and Vmax values were determined for aminopyrine N-demethylation, biphenyl 4-hydroxylation, and biphenyl 2-hydroxylation. Depending on the inhibitor and on the activity examined, non-competitive, competitive, or mixed inhibition was seen. Interaction with cytochrome P-450 was also studied spectrally and was always found to result in a modified type II difference spectrum (ligand binding). A dual binding mode is postulated, involving electrostatic and lipophilic interactions.

Protective effect of malotilate against galactosamine intoxication in the rat.

The influence of malotilate was tested in a model of galactosamine (GalN) intoxication in the rat; two different experiments were carried out: (1) an acute intoxication (GalN at a single dose of 500 mg/kg s.c.): after pretreatment with malotilate (100 and 200 mg/kg p.o.) a marked reduction of hepatocellular injury, as shown by decrease of ASAT, ALAT and GlDH, was observed; (2) a subacute intoxication, GalN at 3 x 300 mg/kg s.c. for 3 consecutive days, which induced a more moderate hepatic necrosis: malotilate, 200 mg/kg/day p.o. for 4 days beginning the day before the first GalN injection, decreased significantly the disturbances of the biochemical measured parameters (plasma total proteins, prothrombin time, fibrinogen level, BSP clearance and hepatic total lipids) and accelerates the return to normal. Pathology studies revealed that pretreatment with malotilate increased markedly the number of mitoses observed in the hepatic cells after intoxication. In addition, the time of appearance of these mitoses was earlier in malotilate treated animals. From these studies, it can be concluded that malotilate protects against hepatocellular injury induced by GalN in the rat and that it stimulates the regenerative capacity of the liver after intoxication.

Inhibiting or potentiating effects of flavonoids on carbon tetrachloride-induced toxicity in isolated rat hepatocytes.

Carbon tetrachloride (CCl4) added to isolated rat hepatocytes produces toxic effects which were assessed by monitoring the release of aspartate aminotransferase (ASAT). CBrCl3 was equitoxic with CCl4, while CHCl3 was inactive, suggesting solvent properties not to be involved. The CCl4-mediated toxicity was markedly decreased by carbon monoxide, indicating possible activation by cytochrome P 450. 55 flavonoid compounds were tested for their ability to interfere with CCl4-induced release of ASAT. The compounds are cianidanol, 3-ethers and 3-esters thereof, flavanones, flavanolols, flavones and flavanols. The more hydrophilic compounds inhibit the CCl4-induced toxicity, while the lipophilic derivatives are potentiators. No other structure-activity relationships are apparent. The results are discussed in terms of the mechanisms of action of the compounds and of the validity of the technique as a screening test for hepatotropic agents.

Effect of malotilate on chronic liver injury induced by carbon tetrachloride in the rat.

The effect of malotilate, a new drug proposed for the treatment of chronic liver diseases, was studied in carbon tetrachloride (CCl4)-induced chronic liver injury in the rat. Treatment with CCl4 (0.5 ml/kg twice per week, intraperitoneally for 6 or 9 weeks) led to marked necrosis, steatosis and fibrosis, as shown by both biochemical and histological examinations, and a significant decrease of the bromosulfophtaleine (BSP) clearance test. Malotilate (50 mg/kg p.o., 5 days per week given simultaneously with CCl4 for 6 weeks), suppressed the increase of plasma aminotransferase activity and decreased significantly the accumulation of lipid and collagen in the liver; histology confirmed this protective effect of malotilate. The BSP clearance test returned to normal values and the rise in hepatic collagen synthesis activity in the malotilate-treated and intoxicated rats was reduced as compared with intoxicated control rats. The same effect was found when malotilate (100 mg/kg, p.o., 5 days per week), was given for 3 weeks to rats already intoxicated during the 6 previous weeks. Malotilate was able to prevent the increase of hepatic alterations that appeared during the last 3 weeks of CCl4 intoxication. These results show clearly that malotilate can markedly reduce the hepatic disorders induced by a chronic CCl4 intoxication in the rat.

The effect of 3-palmitoyl-(+)-catechin on Kupffer cells in guinea pig liver.

We report a morphometric study on the effect of 3-palmitoyl-(+)-catechin on phagocytic activity of latex particles by Kupffer cells in guinea pig liver. We found an increase in the density of latex particles in Kupffer cells of animals treated with 3-palmitoyl-(+)-catechin. The volume and density of Kupffer cells of treated animals increased (p less than 0.01); there was no significant difference in surface density of the plasma membrane. None of the parameters measured was altered in endothelial cells. These results show that 3-palmitoyl-(+)-catechin produces morphological and functional changes in Kupffer cells.

Antinecrotic effect of 3-palmitoyl-(+)-catechin against liver damage induced by galactosamine or ethanol in the rat.

The antinecrotic potential of a new drug, 3-palmitoyl-(+)-catechin (PC), which is a derivative of (+)-cyanidanol-3, was studied in two different experimental models of necrosis of the liver in the rat: acute hepatitis induced by galactosamine and liver damage induced by a combination of chronic ethanol feeding and hypoxia. The extent of liver damage was assessed by histological examination and by measuring blood hepatic enzyme and bilirubin levels. Intraperitoneal and oral treatments with PC were carried out at different dosages and times, before acute galactosamine intoxication. PC treatment decreased the extent of diffuse necrosis and inflammation in liver tissue and reduced galactosamine-induced biochemical deterioration. The lowest active doses of PC were 25 mg/kg, i.p. and 500 mg/kg by mouth. In experiments with ethanol, we confirmed that a 6-h period of hypoxia produced necrosis of the liver in rats undergoing chronic treatment with ethanol. Simultaneous treatment with PC (80 mg/kg/day) for 21 days during ethanol feeding gave significant protection against histological and biochemical deterioration induced by ethanol and hypoxia. The anti-necrotic effect of PC in two models, which are recognized as producing part of the biochemical and/or histological deterioration induced by viruses and ethanol in man, indicates that it is a potentially useful agent for the treatment of necrosis of the human liver.

Influence of palmitoyl-3-catechin and heptyl-3-catechin on the leucocyte migration inhibition test carried out in the presence of PPD and hepatitis B surface antigen (HBsAg).

It has been shown that (+)-cyanidanol-3 [+)-catechin) is able to stimulate in vitro the cell-mediated immune response specific for hepatitis B surface antigen (HBsAg), and might contribute to the elimination of hepatitis B virus (HBV) during HBV infection. In the present study, the possible action of two of its derivatives, palmitoyl-3-catechin and heptyl-3-catechin, on this type of immunity was investigated by adding the substances to leucocyte migration inhibition tests performed in presence of PPD and HBsAg, with leucocytes from individuals sensitized to these antigens. In normal individuals sensitized to PPD, the addition of palmitoyl-3-catechin and heptyl-3-catechin amplified the inhibition of migration by resp. 7.2% (p less than 0.05) and 13.5% (p less than 0.001). In patients previously infected by HBV and sensitized to HBsAg, the maximum amplification was resp. 12.7% (p less than 0.001) and 7.6% (p less than 0.05). This effect was dose-dependent. These substances did not modify the leucocyte migration measured in the absence of antigen. Palmitoyl-3-catechin and heptyl-3-catechin therefore seem capable of amplifying the cell-mediated immune response. The effect of the two derivatives, which were selected because of their liposolubility, was more pronounced than the effect of (+)-cyanidanol-3. It is thus possible that the two new catechin-derivatives, not yet in therapeutical use, will also stimulate cell-mediated immunity to HBsAg, and that a more marked clinical effect might be expected.

The effect of cianidanol on rat hepatic monooxygenase activities.

The antinecrotic hepatoprotective agent cianidanol ((+)-catechin, (+)-cyanidan-3-ol, Catergen) has been tested for its ability to interfere with rat liver monooxygenase activities. In vitro, the drug was found to inhibit biphenyl 4-hydroxylation in microsomes from uninduced rats, and aminopyrine N-demethylase in microsomes from uninduced and PB-treated rats, with IC50 just below 1 x 10(-3) mol/l. The compound is less active in inhibiting biphenyl 2- and 4-hydroxylation and ethoxyresorufin 0-deethylation in microsomes from 3-methylcholanthrene-treated rats. Kinetically, cianidanol behaves as a non-competitive inhibitor in all reactions investigated. It binds to oxidized cytochrome P-450 as a ligand (modified type II binding spectrum). The drug does not decrease levels of cytochrome P-450, but rather protects the enzyme from lipoperoxidation-mediated destruction. In vivo, a single dose of cianidanol does not inhibit [14C]-aminopyrine metabolism. Only after 5 days administration a weak but statistically significant inhibitory effect is detected. The mechanism of action of cianidanol is discussed.

The effect of carbon tetrachloride on isolated rat hepatocytes.

Isolated rat hepatocytes were incubated with carbon tetrachloride (CCl4) at a concentration of 0.2 mol CCl4/ml of incubation medium. The ultrastructural alterations and release of lactate dehydrogenase (LDH) and glutamate-oxaloacetate transaminase (GOT), were recorded after different periods of incubation. After 5 min incubation with CCl4, morphological changes observed by electron microscopy, involved the plasma membrane. The endoplasmic reticulum and mitochondria were altered later. These morphological alterations were accompanied by an early release of LDH and GOT into the incubation medium. It is concluded that, in contrast with its in vivo effects, in vitro CCl4 can induced an early morphological alteration of the hepatocyte plasma membrane before damaging the endoplasmic reticulum.

Protective effect of (+)cyanidanol-3 in acute liver injury induced by galactosamine or carbon tetrachloride in the rat.

Pretreatment of rats with (+)cyanidanol--3 decreases the alterations in liver function tests (transaminase and bilirubin) as well as the accumulation of hepatic triglycerides induced by acute doses of galactosamine or carbon tetrachloride. This action was related to the dose of (+)cyanidanol-3 administered. The lowest effective doses were 3 intraperitoneal injections of 125 mg/kg administered before galactosamine or carbon tetrachloride. In the case of carbon tetrachloride intoxication, in vivo administration of (+)cyanidanol-3 reduced to a slight extent the absorption of conjugated dienes produced in liver microsomal lipid. This observation confirms that (+)cyanidanol-3 is able to prevent lipid peroxidation in vivo. As the protective effect of (+)cyanidanol-3 becomes apparent in two types of intoxication which are very different in their primary mechanisms of action, it is suggested that, the flavonoid also acts on a later stage of the process leading to necrosis and steatosis of the liver.

Inhibition of galactosamine induced edemas in the rat by drugs preventing macromolecular leakage from the hamster cheek pouch microvasculature.

We have shown that terbutaline and dimethpyrindene, 2 drugs which prevent macromolecular leakage in the in vivo--bio-assay of Hamster Cheek Pouch, either by direct action onendothelial cells or by blocking histamine receptors at the microvascular leakage sites, can also prevent macromolecular leakage in vivo in 2 different models of generalized edema.