Clinical trial: alvimopan for the management of post-operative ileus after abdominal surgery: results of an international randomized, double-blind, multicentre, placebo-controlled clinical study.

BACKGROUND: Post-operative ileus (POI) affects most patients undergoing abdominal surgery. AIM: To evaluate the effect of alvimopan, a peripherally acting mu-opioid receptor antagonist, on POI by negating the impact of opioids on gastrointestinal (GI) motility without affecting analgesia in patients outside North America. METHODS: Adult subjects undergoing open abdominal surgery (n = 911) randomly received oral alvimopan 6 or 12 mg, or placebo, 2 h before, and twice daily following surgery. Opioids were administered as intravenous patient-controlled analgesia (PCA) or bolus injection. Time to recovery of GI function was assessed principally using composite endpoints in subjects undergoing bowel resection (n = 738). RESULTS: A nonsignificant reduction in mean time to tolerate solid food and either first flatus or bowel movement (primary endpoint) was observed for both alvimopan 6 and 12 mg; 8.5 h (95% CI: 0.9, 16.0) and 4.8 h (95% CI: -3.2, 12.8), respectively. However, an exploratory post hoc analysis showed that alvimopan was more effective in the PCA (n = 317) group than in the non-PCA (n = 318) group. Alvimopan was well tolerated and did not reverse analgesia. CONCLUSION: Although the significant clinical effect of alvimopan on reducing POI observed in previous trials was not reproduced, this trial suggests potential benefit in bowel resection patients who received PCA.

A randomized trial measuring fecal blood loss after treatment with rofecoxib, ibuprofen, or placebo in healthy subjects.

PURPOSE: Gastrointestinal microbleeding, as assessed by the measurement of (51)chromium-labeled red blood cells, is a marker of the mucosal injury associated with the use of nonsteroidal anti-inflammatory drugs. This study tested the hypotheses that cyclooxygenase-2 specific inhibition with rofecoxib would cause less fecal blood loss than a therapeutic dose of ibuprofen and would be equivalent to placebo. SUBJECTS AND METHODS: In this randomized, double-blind group study, gastrointestinal blood loss was assessed by measurement of fecal (51)chromium radioactivity during a 1-week placebo baseline period and during 4 weeks of treatment with rofecoxib (25 mg or 50 mg once daily), ibuprofen (800 mg three times daily), or placebo in 67 healthy subjects. Gastrointestinal blood loss during treatment weeks 2 to 4 (versus the baseline period) was expressed as the geometric mean ratio of fecal radioactivity in weeks 2 to 4 compared with baseline. RESULTS: Ibuprofen caused significantly (P <0.001) greater gastrointestinal blood loss (geometric mean ratio of 5.2, 95% confidence interval [CI]: 4.2 to 6.3) than the 25-mg dose of rofecoxib (2.6, 95% CI: 2.2 to 3.1), the 50-mg dose of rofecoxib (2.6, 95% CI: 2.2 to 3.0), or placebo (2.1, 95% CI: 1.8 to 2.5). In contrast, gastrointestinal blood loss with both doses of rofecoxib were equivalent to placebo by a predetermined clinical similarity bound. CONCLUSIONS: In healthy subjects, treatment with rofecoxib, at 2 to 4 times the doses that are currently recommended for the treatment of patients with osteoarthritis, produced significantly less fecal blood loss than a therapeutic dose of ibuprofen and was equivalent to placebo.

Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis.

Sp1 nuclear levels have been shown to directly correlate with the proliferative state of the cell. We therefore studied changes in the abundance of Sp1 in a rat pituitary cell line GH4 whose growth rate is regulated by epidermal growth factor (EGF). Nuclear extracts from GH4 cells treated with 10 nM EGF for at least 16 h showed a 50% decrease in Sp1 binding to a GC-rich element present in the gastrin promoter. The decrease in binding correlated with a decrease in cell proliferation, a loss of nuclear Sp1 protein and a 50-60% decrease in Sp1-mediated transactivation through an Sp1 enhancer element in transfection assays. Okadaic acid, a phosphatase inhibitor, was synergistic with the effect of EGF on Sp1 protein levels suggesting that the loss of Sp1 was mediated by phosphorylation events. This result was confirmed by showing a 2-fold increase in orthophosphate-labeled Sp1 with EGF and okadaic acid. Cycloheximide prevented the expected loss of Sp1 mediated by EGF and okadaic acid suggesting that the synthesis of a protease may mediate these events. This hypothesis was tested directly by showing that the cysteine protease inhibitor leupeptin prevented Sp1 degradation. Using the PEST-FIND computer program, the computed PEST score for human and rat Sp1 is 10.4 and 13.7, respectively, indicating that Sp1 has a domain with a high concentration of proline, glutamic acid, serine, and threonine residues as reported for a number of proteins with inducible rates of degradation. Collectively, these results indicate that sustained stimulation of GH4 cells by EGF initiates a cascade of phosphorylation events that promotes Sp1 proteolysis, decreased Sp1 nuclear levels and decreased cellular proliferation.

Overexpression of ZBP-89, a zinc finger DNA binding protein, in gastric cancer.

ZBP-89 is a Krüppel-type zinc finger protein that binds to the gastrin EGF response element (gERE). Sp1 binds to the same DNA element and transactivates gastrin promoter activity, whereas ZBP-89 competes for Sp1 binding and prevents EGF induction. Both transcription factors mediate growth factor signals originating from the EGF receptor and thus were studied in normal and neoplastic tissues or cell lines. When compared to normal tissue from the same patient, ZBP-89 protein expression was increased in neoplastic tissue from the stomach antrum and in malignant cell lines. RT-PCR analysis of ZBP-89 mRNA correlated with protein overexpression. Immunocytochemical studies confirmed that ZBP-89 expression is elevated in neoplastic tissue and chronic gastritis, whereas Sp1 expression was nearly unchanged. These results suggest that the transcription factor ZBP-89, like Sp1, may be a marker for neoplastic transformation in some gastric cancers.

ZBP-89, a Krüppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter.

We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene.

Epidermal growth factor stimulation of the human gastrin promoter requires Sp1.

Growth factors coordinately regulate a variety of different genes to stimulate cellular proliferation. In the stomach, gastrin, epidermal growth factor (EGF), and transforming growth factor-alpha all mediate gastric mucosal homeostasis by promoting cell renewal. We have previously shown that EGF and phorbol esters stimulate the human gastrin promoter through a novel GC-rich DNA element 5'-(68)GGGGCGGGGTGGGGGG-53 called gERE (gastrin EGF response element). In this report, we show that three factors bind to this element, the transcription factor Sp1 and two fast migrating complexes designated gastrin EGF response proteins (gERP 1 and 2). To understand how these factors bind and confer EGF responsiveness, mutations of gERE were tested in vitro for protein binding and in vivo for promoter activation. Both gel shift assays and UV cross-linking studies revealed that the factors bind to overlapping domains, Sp1 to the 5' half-site and gERP 1 and 2 to the 3' half-site. Placing either the 5' or 3' mutations upstream of a minimal gastrin promoter abolished EGF induction. Therefore both the 5' and 3' domains were required to confer EGF induction. Collectively, these results demonstrate that complex interactions between Sp1 and other factors binding to overlapping gERE half-sites confer EGF responsiveness to the gastrin promoter.

Guanosine nucleotides regulate hormone binding of insulin receptors.

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5'-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.

The alpha beta monomer of the insulin receptor has hormone-responsive tyrosine kinase activity.

Insulin receptors from turkey erythrocyte membranes exist as monomers and dimers when membranes are solubilized with detergent. We examined the ability of monomers and dimers to act as protein kinases to effect both autophosphorylation of the receptor and phosphorylation of an exogenous substrate. After separation by sucrose-density-gradient centrifugation, only receptor dimers show significant basal and insulin-stimulated kinase activity, whereas material at the position of receptor monomers is not active. Partial reduction of the membrane-bound receptors with dithiothreitol, however, produces a receptor monomer containing an alpha and a beta chain which has protein kinase activity similar to that of the original dimers. With rat adipocyte plasma membranes, which in the absence of reducing agents only contain receptor dimers, reduction with dithiothreitol also produces monomers with receptor kinase activity. Receptor monomer hormone-dependent kinase activity is insensitive to receptor concentration and shows stimulation after immobilization on an affinity support.