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OBJECTIVE: The aim was to investigate the possible interactions of the two peripheral hormones, leptin and ghrelin, that regulate the energy balance in opposite directions. METHODS: Leptin-receptor mutated Zucker diabetic fatty (ZDF) and lean control rats were treated with the ghrelin-receptor ligand, tabimorelin (50 mg/kg p.o.) for 18 days, and the effects on body weight, food intake and body composition were investigated. The level of expression of anabolic and catabolic neuropeptides and their receptors in the hypothalamic area were analysed by in situ hybridization. RESULTS: Tabimorelin treatment induced hyperphagia and adiposity (increased total fat mass and gain in body weight) in lean control rats, while these parameters were not increased in ZDF rats. Treatment with tabimorelin of lean control rats increased hypothalamic mRNA expression of the anabolic neuropeptide Y (NPY) mRNA and decreased hypothalamic expression of the catabolic peptide pro-opiomelanocortin (POMC) mRNA. In ZDF rats, the expression of POMC mRNA was not affected by treatment with tabimorelin, whereas NPY mRNA expression was increased in the hypothalamic arcuate nucleus. CONCLUSION: This shows that tabimorelin-induced adiposity and hyperphagia in lean control rats are correlated with increased hypothalamic NPY mRNA and decreased POMC mRNA expression. The elimination of tabimorelin-induced adiposity and hyperphagia in ZDF rats may be due to lack of POMC mRNA downregulation. In conclusion, we suggest that ghrelin-receptor ligands exert their adipogenic and orexigenic effects via hypothalamic mechanisms that are dependent on intact leptin-receptor signalling.

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BACKGROUND: In inflammatory bowel disease (IBD) the disease activity correlates with colonic concentrations of leukotrienes (LTs). The enzyme 5-lipoxygenase (5-LO) is responsible for the enzymatic production of LTs. It has previously been demonstrated in experimental models of inflammation, that 5-LO is activated through intracellular translocation of the pre-formed enzyme, and increased constitutive activation of 5-LO has been demonstrated in idiopathic pulmonary fibrosis. The objective of the present study was to investigate whether de novo synthesis of 5-LO is increased in patients with quiescent IBD, or is induced during acute exacerbations of IBD. METHODS: Sixty-one individuals were included in the study. Twenty-eight had ulcerative colitis (UC), 21 had Crohn's disease (CD), and 12 were healthy controls. A standard rigid rectoscopy was performed in all individuals. The degree of inflammation was assessed using a semi-quantitative scale. A mucosal biopsy was taken from the most inflamed area as judged macroscopically. mRNA for 5-LO was detected using a RT-PCR technique, and the assay applied was evaluated by control experiments. RESULTS: The expression of mRNA for 5-LO in colonic biopsies was similar in IBD patients with quiescent disease and healthy controls. When grouped according to endoscopically assessed disease activity the fraction of patients demonstrating 5-LO mRNA in colonic biopsies showed no significant change (p > 0,6; chi2 -test for trend). CONCLUSIONS: This study demonstrates no significant relationship between endoscopically assessed disease activity and relative presence of mRNA for 5-LO in colonic biopsies. Thus, there is no evidence of increased expression of 5-LO mRNA in either quiescent or active stages of IBD.

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NN703 is an orally active and selective growth hormone secretagogue (GHS) that was derived from growth hormone-releasing peptide-1(GHRP-1) via ipamorelin by a peptidomimetic approach and has now entered into phase II clinical trials. When the disposition in rats of NN703 and GHRP-6 was studied using whole-body autoradiography following administration of an iv dose of radiolabeled material, we found that a substantial amount of these secretagogues accumulate in the glandular part of the stomach. Because this is the site of synthesis and secretion of ghrelin, the endogenous GHS, we investigated the effect of resection of the gastrointestinal (GI) tract on growth hormone (GH) release induced by GHRP-6. This procedure significantly attenuated the GH secretion response by 60-70%. By contrast, the effect of GH-releasing hormone on GH release was not inhibited. The binding of GHRPs to the glandular part of the stomach and the blunted GH response to GHRP-6 following resection of the GI tract suggest a role for ghrelin as a mediator of part of the GH-releasing effect of GHRPs.

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The growth hormone (GH)/insulin-like growth factor-1 axis is not only of importance for linear body growth during childhood, but it is also one of the major determinants of adult bone mass. Studies show that GH treatment increases bone mass in rodents as well as in adult GH-deficient humans, but the effect of GH treatment on bone mass in healthy humans has so far not been impressive. Recently, a new class of GH secretagogues (GHSs) has been developed. In humans, GHS treatment affects biochemical markers of bone turnover and increases growth velocity in selected short children with or without GH deficiency. In rodents, GHS treatment increase bone mineral content, but it has not yet been shown that GHS treatment can affect bone mass in adult humans.

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Growth hormone (GH) is of importance for normal bone remodelling. A recent clinical study demonstrated that MK-677, a member of a class of GH secretagogues (GHSs), increases serum concentrations of biochemical markers of bone formation and bone resorption. The aim of the present study was to investigate whether the GHSs, ipamorelin (IPA) and GH-releasing peptide-6 (GHRP-6), increase bone mineral content (BMC) in young adult female rats. Thirteen-week-old female Sprague-Dawley rats were given IPA (0.5 mg/kg per day; n=7), GHRP-6 (0.5 mg/kg per day; n=8), GH (3.5 mg/kg per day; n=7), or vehicle administered continuously s.c. via osmotic minipumps for 12 weeks. The animals were followed in vivo by dual X-ray absorptiometry (DXA) measurements every 4th week. After the animals were killed, femurs were analysed in vitro by mid-diaphyseal peripheral quantitative computed tomography (pQCT) scans. After this, excised femurs and vertebrae L6 were analysed by the use of Archimedes' principle and by determinations of ash weights. All treatments increased body weight and total tibial and vertebral BMC measured by DXA in vivo compared with vehicle-treated controls. However, total BMC corrected for the increase in body weight (total BMC:body weight ratio) was unaffected. Tibial area bone mineral density (BMD, BMC/area) was increased, but total and vertebral area BMDs were unchanged. The pQCT measurements in vitro revealed that the increase in the cortical BMC was due to an increased cross-sectional bone area, whereas the cortical volumetric BMD was unchanged. Femur and vertebra L6 volumes were increased but no effect was seen on the volumetric BMDs as measured by Archimedes' principle. Ash weight was increased by all treatments, but the mineral concentration was unchanged. We conclude that treatment of adult female rats with the GHSs ipamorelin and GHRP-6 increases BMC as measured by DXA in vivo. The results of in vitro measurements using pQCT and Archimedes' principle, in addition to ash weight determinations, show that the increases in cortical and total BMC were due to an increased growth of the bones with increased bone dimensions, whereas the volumetric BMD was unchanged.

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The discovery of a new class of compounds that stimulate the release of growth hormone (GH) in a manner distinctly different from growth hormone-releasing hormone (GHRH) is advancing the understanding of the mechanisms that control GH secretion. These compounds, the GH secretagogues, act at both pituitary and hypothalamic levels, and might even elicit effects in the CNS and peripheral systems. A receptor with high affinity for the GH secretagogues has been identified and several observations suggest the presence of additional receptors. The existence of these specific endogenous receptors could indicate that the mechanism of GH release is not yet fully understood. Several potential indications have been explored clinically and, as some of these compounds are orally active, they could offer attractive alternatives to recombinant human growth hormone (hGH) in treating GH disorders such as growth hormone deficiency (GHD), age-related conditions, obesity and catabolic conditions.

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Growth hormone releasing peptides (GHRP) are synthetic hexapeptides that physiologically stimulate GH release through two different pathways: 1) central and 2) direct action on somatotropic cells. Animal experiments and first clinical trials show that synthetic GHRP and synthetic analogues could be useful substitutes to recombinant GH in the treatment of GH deficiency, and in pathological conditions which may benefit from amplification of the GH-IGF I axis activity.

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The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in vitro in both NkO and CEO irrespective of the inhibitory substance used. In contrast, the oxysterols and cholesterol had no significant biological activity on this oocyte function, and consequently we found no correlation between LXRalpha activation and meiosis stimulation.

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The synthesis of leukotriene B4 by A23187-stimulated rat peritoneal leukocytes was studied in the presence of 0.1% normal human serum, serum from patients treated with NSAIDs for either an inflammatory (rheumatoid arthritis, RA) or a non-inflammatory condition (lumbar disc protrusion, LDP), and serum from RA patients drawn one week after withdrawal from NSAID treatment. The capacity for LTB4 synthesis was significantly lower in the presence of serum from NSAID treated patients: thirty per cent less than observed in presence of normal serum in the RA group, and fifty per cent in the LDP group. When NSAIDs were withdrawn from RA patients, the LTB4 production in presence of serum increased, but was not completely normalized after one week. These results indicate that NSAID treatment may down-regulate the capacity for leukotriene synthesis by an indirect effect.

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Cytokines, particularly the proinflammatory cytokines, whose role and natural regulation in inflammatory bowel disease are reviewed here, are produced by many cell types, including immune cells. Cytokines function as important hormones of the immune system, and many act both regionally and systemically via specific receptors. The demonstration of increased circulating and mucosal levels of proinflammatory (and other) cytokines (and receptors) in active inflammatory bowel disease does not by itself constitute any proof as to the primary involvement of these mediators. However, they may contribute significantly to disease manifestations, and specific therapeutic intervention at the cytokine or cytokine receptor level may show up to be clinically most relevant. This is underscored by the increasing evidence that proven therapies of inflammatory bowel disease to a great extent seem to function through cytokine modulation.

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Activated polymorphonuclear leucocytes, which are accumulated in inflammatory lesions of inflammatory bowel disease, produce tissue destructive, oxygen derived free radicals and other inflammatory mediators. The PMN superoxide production elicited by formyl-methionyl-leucyl-phenylalanine or the complement split product 5a were compared in IBD and healthy volunteers. Significantly reduced superoxide production was found in PMNs from patients with Crohn's disease as compared to normal controls, when fMLP or CSa were used as stimulants (p < 0.001 and p < 0.01, respectively), whereas no differences were found when ulcerative colitis patients were compared to normal controls (p > 0.05). The enhanced oxygen derived free radical production previously reported in active IBD, and especially in CD intestinal lesions, may either be due to an accumulation of productive phagocytes or to a change of the inflammatory profile of these cells when migrating into intestinal lesions, possibly due to interaction with other mediators (e.g. adhesion molecules and interleukins).

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ETH615 (4-(2-quinolylmethoxy)-N-(3-fluorobenzyl-phenyl-amino-methyl -4- benzoic-acid), a synthetic inhibitor of leukotriene B4 production and activities, was tested for its effect on the production of and biological responses towards human interleukin-8. We found that ETH615 inhibits lipopolysaccharide-induced (LPS-induced) expression of interleukin-8 messenger-RNA (mRNA) and interleukin-8 production in human peripheral blood mononuclear cells. We also observed that ETH615 completely inhibited interleukin-8 as well as leukotriene B4 directed chemotaxis of human neutrophils in a dose-dependent manner. A moderate effect on fMLP-directed neutrophil chemotaxis was observed. Further, no significant effect on either interleukin-8, leukotriene B4 or fMLP-directed T-cell migration was observed. These results further support the concept of a cytokine-leukotriene regulatory circuit and encourage the establishment of clinical trials testing the effect of ETH615 on inflammatory skin diseases, which are characterized by high levels of interleukin-8 and leukotriene B4 in lesional skin.

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Activated polymorphonuclear leukocytes (PMNs), which are found in the inflammatory lesions of chronic inflammatory bowel disease, produce tissue-destructive oxygen-derived free radicals. The influence of 5-aminosalicylic acid (5-ASA), its acetylated metabolite (Ac-5-ASA), sulfasalazine (SAZ), and olsalazine (OLZ) (5-ASA dimer linked by an azo group) in pharmacologically relevant concentrations (0.1-10 mM) were tested on PMN superoxide production with either the receptor-specific agent formyl-methionyl-leucyl-phenylalanine (fMLP) or the protein kinase C activator phorbol myristate acetate (PMA). Inhibition of receptor-specific superoxide production occurred at 0.07, 0.32, and 0.63 mM (IC50 values) for 5-ASA, SAZ, and OLZ, respectively. No inhibitory effects of SAZ and OLZ were observed when PMA was applied as stimulus for PMN superoxide production. The results indicate that the signal to which PMNs respond by generating superoxide is primarily due to calcium release from intracellular stores. They further suggest that SAZ and OLZ may affect the oxygen-derived free radical production in human PMNs by unspecific cytotoxicity or by interference with the nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidase system, whereas 5-ASA itself is a free radical scavenger.

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ETH615 (4-[2-quinolylmethoxy]-N-[3-fluorobenzyl]-phenylaminometh yl-4-benzoic acid) is a potent inhibitor of leukotriene biosynthesis in A23187-stimulated leukocytes, and of IL-8 gene expression in LPS-stimulated PBMC. It shows anti-inflammatory activity in a canine model of dermal inflammation. A topical formulation is present in phase II clinical trials. In the present study the effect of ETH615 on oxazolone-induced acute inflammation and phorbol ester-induced chronic inflammation in the mouse ear was investigated. Betamethasone (0.04 mg/ear) and ETH615 (1-1.5 mg/ear) significantly inhibited both the oedema formation and the PMN infiltration. The cream and ointment formulations of ETH615 developed for clinical studies were equally active. ETH615 is thus an anti-inflammatory agent in these murine models of dermatosis.

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Ulcerative colitis and Crohn's disease are chronic inflammatory bowel diseases. The most widely prescribed drug for treatment of these diseases, sulfasalazine, has been shown to inhibit the activity of free radicals, as the active moiety of sulfasalazine, 5-aminosalicylic acid, is a radical scavenger. This effect of 5-aminosalicylic acid may be of clinical relevance, as a recent study has shown that 5-aminosalicylic acid reacts with oxygen-derived free radicals formed in the intestine in this disease. Reaction with free radicals does not, however, occur in patients with rheumatoid arthritis treated with the same agent. Furthermore, a significant correlation exists between the activity in the intestine of free radicals, as measured by the rate of lipid peroxidation, and the disease activity.

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This investigation was performed in order to examine the role of sulfidopeptide-leukotrienes in a chronic inflammatory bowel disease, ulcerative colitis, by use of the recently developed LTD4/LTE4 antagonist, SR 2640 (2-[3-(2-quinolylmethoxy)phenylamino]benzoic acid). Eight ulcerative colitis patients with a mild to moderate disease activity were included in this open and uncontrolled study and SR 2640, 250 mg t.i.d., was administered for 6 weeks. Treatment of the patients with SR 2640 reduced the inhibitory effect of LTD4 on LTB4-directed chemotaxis of neutrophils purified from their blood. This indicates that the dose administered was sufficiently high to obtain systemic LTD4 receptor antagonism. Three of the 8 patients were in clinical remission at the end of the study, and the lack of clinical symptoms persisted for at least 2 months after discontinuing the drug. The condition of 3 patients was unchanged, and that of 2 patients deteriorated after 5 weeks, requiring treatment with sulphasalazine and steroids. SR 2640 was well tolerated by all patients. In a previously published study dealing with 4 weeks sulphasalazine treatment in the same category of patients, remission rates of 5% and 25% were found in the placebo and sulphasalazine groups, respectively, and the remission rate of SR 2640 thus seems to be of the same magnitude as that of sulphasalazine. The serum and faecal concentrations of SR 2640, and its metabolite, the beta-glucuronide, were found to be lower in ulcerative colitis patients as compared to healthy volunteers, and it is therefore possible that altered pharmacokinetics of SR 2640 is present in patients with chronic inflammatory bowel disease.(ABSTRACT TRUNCATED AT 250 WORDS)

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