Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin.

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

Pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the mechanism of IGFBP-4 proteolysis by PAPP-A.

Pregnancy-associated plasma protein-A (PAPP-A) has recently been identified as the proteinase responsible for cleavage of insulin-like growth factor binding protein (IGFBP)-4, an inhibitor of IGF action, in several biological fluids. Cleavage of IGFBP-4 by PAPP-A is believed to occur only in the presence of IGF. We here report that in addition to IGFBP-4, PAPP-A also cleaves IGFBP-5. Cleavage occurs at one site, between Ser-143 and Lys-144 of IGFBP-5. In the presence of IGF, IGFBP-4 and -5 are cleaved with similar rates by PAPP-A. Interestingly, cleavage of IGFBP-5 by PAPP-A does not require the presence of IGF, but is slightly inhibited by IGF. These findings have implications for the mechanism of proteolysis of IGFBP-4 by PAPP-A, suggesting that IGFBP-4 binds IGF, which then becomes a PAPP-A substrate. Using highly purified, recombinant proteins, we establish that (1) PAPP-A cleavage of IGFBP-4 can occur in the absence of IGF, although the rate of hydrolysis is very slow, and (2) IGF is unable to bind to PAPP-A. We thus conclude that IGF enhances proteolysis by binding to IGFBP-4, not by interaction with PAPP-A, which could not previously be ruled out.

Pregnancy-associated plasma protein-A2 (PAPP-A2), a novel insulin-like growth factor-binding protein-5 proteinase.

A novel metalloproteinase with similarity to pregnancy-associated plasma protein-A (PAPP-A), which we denoted PAPP-A2, has been identified. Through expression in mammalian cells we showed that recombinant PAPP-A2 polypeptide of 1558 residues resulted from processing of a 1791-residue prepro-protein. Unlike PAPP-A, PAPP-A2 migrated as a monomer (of 220 kDa) in non-reducing SDS-polyacrylamide gel electrophoresis. The prepro-parts of PAPP-A2 and PAPP-A are not homologous, but mature PAPP-A2 shares 45% of its residues with PAPP-A. Because PAPP-A specifically cleaves insulin-like growth factor-binding protein (IGFBP)-4, one of six known modulators of IGF-I and -II, we looked for a possible PAPP-A2 substrate among the members of this family. We showed that PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.

Expression of recombinant human pregnancy-associated plasma protein-A and identification of the proform of eosinophil major basic protein as its physiological inhibitor.

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

Selective blockade of leukotriene production by a single dose of the FPL 64170XX 0.5% enema in active ulcerative colitis.

5-Lipoxygenase products of arachidonic acid metabolism are thought to play a central role in the secondary amplification of the inflammatory response of several inflammatory diseases, including ulcerative colitis. FPL 64170XX is a selective inhibitor of the enzyme 5-lipoxygenase. Concentrations of leukotriene B4 and prostaglanding E2 in rectal dialysis fluid from 23 males with clinically and sigmoidoscopically active, distally located ulcerative colitis were measured by radioimmunoassays in a double-blind, placebo-controlled, parallel design study before and after rectal administration of an enema containing 0.5% of FPL 64170XX. Repeated measures analysis of leukotriene B4, after adjusting for baseline, showed a significant treatment effect (P = 0.0014). The concentration of leukotriene B4 from rectal dialysates in patients receiving the active drug dropped to 15% (95% confidence interval 5-40%) of the placebo level in the second dialysis following administration of FPL 64170XX 0.5%. By contrast, prostaglanding E2 concentrations doubled (P = 0.0068) in patients receiving FPL 64170XX 0.5% with no change in the placebo group. These findings demonstrate that a single dose of FPL 64170XX 0.5% enema selectively blocks the generation of the 5-lipoxygenase product, leukotriene B4, to a mean of 85% in the target tissue of inflammation. Topical administration of this new leukotriene synthesis inhibitor may prove to be a clinically useful approach to the treatment of active, distally located ulcerative colitis.

Omeprazole in the long-term treatment of gastro-oesophageal reflux disease. A double-blind randomized dose-finding study.

BACKGROUND: Omeprazole is effective in the treatment of reflux oesophagitis, and it is important to determine the lower dose limit with still appropriate clinical efficacy. METHODS: Patients with endoscopic oesophagitis grade 1-4 (N = 220) were randomized to double-blind treatment with 20 mg or 40 mg omeprazole daily for 4-8 weeks. Those healed after this initial treatment phase were re-randomized to double-blind treatment with 20 mg omeprazole daily (n = 67), 10 mg omeprazole daily (n = 68), or placebo (n = 33) for 6 months. Remission was defined as the absence of any endoscopic sign of oesophagitis. RESULTS: Healing rates were increased with 40 mg omeprazole, the therapeutic gain compared with the 20-mg dose being 15% after 4 and 8 weeks. The proportion of patients in remission after 6 months was 59% with 20 mg omeprazole, 35% with 10 mg omeprazole, and 0% with placebo. CONCLUSION: Maintenance treatment with 10 mg omeprazole can prevent recurrence of oesophagitis in about one-third of patients with all grades of oesophagitis, and 20 mg omeprazole in about twice as many.

Blockade of leukotriene production by a single oral dose of MK-0591 in active ulcerative colitis.

BACKGROUND: 5-Lipoxygenase products of arachidonic acid metabolism are thought to play a central role in the secondary amplification of the inflammatory response in a number of human inflammatory diseases, such as ulcerative colitis. MK-0591 (3-(1((4-chlorophenyl)methyl)-3((1,1-dimethyl-ethyl)thio)-5(quinolin+ ++-2ylmethyl-oxy)-1H-indol-2yl)-2,2-dimethyl-propanoate) exerts its effect by binding to the 5-lipoxygenase activating protein, thereby inhibiting the translocation and activation of 5-lipoxygenase. METHODS: Concentrations of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) in rectal dialysis fluid, ex vivo biosynthesis of LTB4 in whole blood, and urinary excretion of leukotriene E4 (LTE4) from 16 patients with mild to moderately active distally located ulcerative colitis were measured by use of radioimmunoassays in a double-blind, placebo-controlled parallel-design study before and after oral administration of a 250 mg dose of MK-0591 or placebo. RESULTS: The mean LTB4 concentration in rectal dialysis fluid was lowered after MK-0591 by > 90% (p < 0.05) from 4 to 8 hours, with a maximum inhibition of 97.5% +/- 3.4% (mean +/- SD) at 20 to 24 hours after dosing, whereas PGE2 was unchanged. In whole blood, MK-0591 decreased ex vivo biosynthesis of LTB4 (p < 0.01), with a maximum inhibition of 96.4% +/- 2.1% at 4 hours after dosing. Urinary excretion of LTE4 was reduced by more than 85% (p < 0.001) from 4 to 48 hours. No adverse events were observed. CONCLUSION: These findings show that a single oral 250 mg dose of MK-0591 results in nearly complete blockade of systemic leukotriene production and LTB4 formation in the target tissue of inflammation (the rectum). Controlled multiple-dose trials to assess the clinical efficacy of this novel 5-lipoxygenase-activating protein inhibitor seem to be worthwhile.

Efficacy of omeprazole in lower grades of gastro-oesophageal reflux disease.

Grade I oesophagitis is usually considered to be a less severe form of gastro-oesophageal reflux disease (GORD). However, with regard to symptom severity, patients without macroscopic mucosal lesions have been shown not to differ from those with more severe oesophagitis. A number of controlled trials on the efficacy of omeprazole in GORD have included patients with lower grades of the disease. The results show that the differences in efficacy between omeprazole and H2-receptor antagonists, which have been established for the treatment of erosive and ulcerative oesophagitis, also extend to patients with grade I oesophagitis (erythema and friability). In these studies, omeprazole provided more rapid symptom resolution and histological improvement than ranitidine. In one double-blind comparative trial, complete endoscopic normalization of the oesophageal mucosa was observed in 90% of patients with grade I oesophagitis within 4 weeks of treatment with omeprazole, 40 mg once daily, compared with 55% of those treated with ranitidine, 150 mg twice daily; at 8 weeks the mucosa in all patients in the omeprazole group had completely healed at endoscopy, while oesophagitis was still present in 21% of the patients receiving ranitidine. A separate 6-month, placebo-controlled maintenance study was performed in patients who had completed a short-term study and who had total relief from the major symptoms of GORD and complete healing of endoscopic oesophagitis. All patients given placebo had an endoscopic recurrence (i.e. endoscopic grade I or more) and this was associated with the return of symptoms in 75% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

[Chronic inflammatory bowel disease--current status]. / Kronisk inflammatorisk tarmsygdom--status.

Chronic inflammatory bowel disease (IBD) encompasses the disease entities, ulcerative colitis (UC) and Crohn's disease (CD). An aetiologic agent has not yet been defined and the diagnosis is based, therefore, on the sum of clinical, paraclinical, radiologic, endoscopic and histopathologic features. In recent years pathogenetic studies have focused on immune mechanisms, transmissible infectious agents, the potential role of the normal intestinal flora, dietary factors, enzymatic alterations and genetic features, in addition to vascular, neuromotor, allergic and psychologic factors. The prevalence of IBD is increased in first-degree relatives of patients and there is a high rate of disease concordance among monozygotic twins. Thus abnormal genes may encode for one or several immunoregulatory factors, while bacterial wall products seem to activate colonic inflammatory cells in a non-specific way, leading to increased production of cytokines, complement-derived peptides, eicosanoids, platelet activating factor, biogenic amines, kinins, chemotactic oligopeptides, and neuropeptides. The named soluble inflammatory mediators, in addition to free oxygen radicals, are considered responsible for the secondary amplification of the inflammatory process. The corner stones in medical therapy of IBD are still corticosteroids and sulphasalazine (SAZ). The new oral salicylates, which are analogues of SAZ or "slow release" preparations of 5-aminosalicylic acid (mesalazine), have provided a therapeutic progress, because they are tolerated better than SAZ. Immunosuppressive agents, such as azathioprine and 6-mercaptopurine, reduce the requirement for corticosteroids and are effective in refractory CD, but the effect is delayed up to several months. The therapeutic action of cyclosporine A is not sustained, but often associated with side effects. Metronidazole has a beneficial effect on perineal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

[Chronic inflammatory bowel disease]. / Kronisk inflammatorisk tarmsygdom.

Chronic inflammatory bowel disease (IBD) encompasses the disease entities, ulcerative colitis (UC) and Crohn's disease (CD). An aetiologic agent has not yet been defined and the diagnosis is based, therefore, on the sum of clinical, paraclinical, radiologic, endoscopic and histopathologic features. In recent years pathogenetic studies have focused on immune mechanisms, transmissible infectious agents, the potential role of the normal intestinal flora, dietary factors, enzymatic alterations and genetic features, in addition to vascular, neuromotor, allergic and psychologic factors. The corner stones in medical therapy of IBD are still corticosteroids and sulphasalazine (SAZ). The new oral salicylates, which are analogues of SAZ or "slow release" preparations of 5-aminosalicylic acid (mesalazine), have provided a therapeutic progress, because they are tolerated better than SAZ. Immunosuppressive agents, such as azathioprine and 6-mercaptopurine, reduce the requirement for corticosteroids and are effective in refractory CD, but the effect is delayed up to several months. The therapeutic action of cyclosporine A is not sustained, but often associated with side effects. Metronidazole has a beneficial effect on perineal disease. The efficacy of antimycobacterial drugs, sodium-cromoglycate, lidocaine, clonidine and sucralfate has been reported only in optimistic case stories and small open trials. A diet, rich in omega-3-fatty acids, modifies leukotriene (LT) production, but its clinical efficacy is insufficient. The first anti-leukotriene-drug, zileuton, has recently been evaluated and a significant, although insufficient, clinical response was obtained by a 70 per cent inhibition of rectal LTB4 synthesis. Dietary therapy may be useful as an adjunct to treatment of local complications in CD.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Lipoxygenase inhibitors for the treatment of inflammatory bowel disease.

The unique role of the enzyme 5-lipoxygenase (5-LO) in the production of leukotrienes (LTs) makes it a likely target for biochemical manipulation. The rationale for using 5-LO inhibitors for the treatment of inflammatory bowel disease (IBD) is based on the increased generation of LTs in the inflamed mucosa, LTB4 being the most potent chemotactic and chemokinetic metabolite of arachidonic acid. Furthermore, conventional drugs, such as corticosteroids, sulphasalazine, and 5-aminosalicylic acid, inhibit LT production and specific 5-LO inhibition accelerates healing in animal models of acute colitis. The compounds identified as 5-LO inhibitors can be divided into antioxidants, substrate-analogous, and a large miscellaneous group of inhibitors, where hydroxamic acids are potent and more selective inhibitors of 5-LO. The benzothiophene hydroxyurea, zileuton, is the first selective 5-LO inhibitor evaluated for the treatment of patients with IBD. An 800-mg oral dose of zileuton was shown to reduce LTB4, but not prostaglandin E2, concentrations by 75-85% in rectal dialysates from patients with active ulcerative colitis. The clinical efficacy of zileuton 800 mg b.i.d. has also been tested in a randomized, double-blind, placebo-controlled trial in similar patients. Zileuton significantly improved the symptom scores and the histology score, but not the sigmoidoscopy score, compared to pretreatment conditions and with response to placebo, the beneficial effects being most pronounced in patients not receiving concomitant sulphasalazine treatment. The mean inhibition of LTB4 in the target tissue of inflammation was 70%. The proof that any putative 5-LO inhibitor is blocking LT production is an important stage in assessing any such drug. The main disadvantage of existing new LT inhibitors relates to the high potency of LTs, and unless a higher level of inhibition can be achieved, endogenous LTs may still be present in sufficient amounts to produce their effects.

Omeprazole 20 mg three days a week and 10 mg daily in prevention of duodenal ulcer relapse. Double-blind comparative trial.

In a double-blind, parallel-group clinical trial of 195 patients with duodenal ulcers who after a short-term study had relief of pain and healed ulcers proved endoscopically, 65 were randomized to receive 20 mg omeprazole 3 days a week (once in the morning from Friday to Sunday), 64 to receive 10 mg omeprazole once daily in the morning, and 66 to receive placebo for up to 6 months. The patients underwent repeat endoscopy with biopsy of the gastric fundic mucosa (qualitative assessment of argyrophilic cell population), assessment of symptoms, and laboratory screening with measurement of basal serum gastrin concentrations at 3 and 6 months or more often if indicated by recurrence of symptoms. At 3 months, endoscopically proved ulcer relapse occurred in 16% receiving 20 mg omeprazole 3 days a week; 21% receiving 10 mg omeprazole daily; and 50% receiving placebo. At 6 months, corresponding rates were 23%, 27%, and 67% with 95% confidence intervals of difference between the placebo group and omeprazole groups of 28%-60% and 24%-56% (P less than 0.00001), respectively, and between omeprazole groups of -19%-11% (NS). No major clinical or laboratory side effects were noted. Thus both omeprazole regimens are effective and safe in preventing duodenal ulcer relapse.

Clinical pharmacokinetics of drugs used in the treatment of gastrointestinal diseases (Part II).

Part I of this article, which appeared in the previous issue of the Journal, covered the following agents: histamine H2-receptor antagonists (cimetidine, ranitidine, famotidine, nizatidine); muscarinic-M1-receptor antagonists (pirenzepine); proton pump inhibitors (omeprazole); site-protective agents (colloidal bismuth subcitrate, sucralfate); antacids and prostaglandin analogues; antiemetics and prokinetics (metoclopramide, domperidone, cisapride); and antispasmodics. In Part II, we consider the anti-inflammatory salicylates, nonspecific antidiarrhoeal agents, laxatives and cathartics.

Clinical pharmacokinetics of drugs used in the treatment of gastrointestinal diseases (Part I).

Drug treatment of gastrointestinal diseases, which was previously limited to the use of antacids, anticholinergics, antispasmodics, cathartics and laxatives, has changed markedly over the past decade. Histamine H2-receptor antagonists (e.g. cimetidine, ranitidine and more recently famotidine and nizatidine) have revolutionised the treatment of peptic acid disorders, but their role is currently challenged by muscarinic-M1-receptor antagonists (e.g. pirenzepine), proton pump inhibitors (e.g. omeprazole), prostaglandin analogues and site-protective drugs (e.g. colloidal bismuth subcitrate and sucralfate). Newer antiemetics with prokinetic properties (e.g. metoclopramide, domperidone and cisapride) have also been introduced in the management of gastrointestinal motility disturbances, and new anti-inflammatory salicylates (e.g. olsalazine and mesalazine) have been developed for the treatment of chronic inflammatory bowel diseases. Finally, diphenoxylate and loperamide have gained wide clinical application as nonspecific antidiarrhoeal agents. The basic pharmacokinetic properties of the above agents are briefly reviewed with the main emphasis on the newer and more important drugs in current use. Furthermore, the effects of age and disease on pharmacokinetics, in addition to drug interaction potentials and pharmacokinetic-pharmacodynamic relationships, are discussed. The anti-inflammatory salicylates, nonspecific antidiarrhoeal agents, laxatives and cathartics will be dealt with in Part II.

Selective 5-lipoxygenase inhibition in ulcerative colitis.

Leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) concentrations were measured in rectal dialysis fluid from ten patients with active ulcerative colitis before and after oral administration of 800 mg of the 5-lipoxygenase inhibitor A-64077. The median LTB4 level fell significantly, from 4.9 (range 0.6-20.4) ng/ml before treatment to 1.6 (0.3-5.7) ng/ml after 4 h and 0.7 (0.1-8.0) ng/ml after 8 h; it had returned to pretreatment levels by 28 h. The concentration of PGE2 did not change significantly. The increased generation of 5-lipoxygenase products, such as LTB4, in ulcerative colitis and the potent proinflammatory actions of these products suggest that they have an important role in the amplification of the inflammatory response. A controlled trial to assess the clinical efficacy of A-64077 seems worth while.

Cytoprotection of gastroduodenal mucous membrane with analogues of prostaglandins.

Analogues of E-type prostaglandins, such as arbaprostil, enprostil, misoprostol, rioprostil, and trimoprostil, suppress gastric acid secretion and (like native E-type prostaglandins) the chemically modified analogues enhance a number of gastroduodenal mucosal defence factors. These include regulation of the thickness and the composition of the mucous layer at the epithelial surface; modulation of active bicarbonate secretion; hydrophobicity of the surface epithelium; rapid cell proliferation and differentiation after mucosal damage; maintenance of interstitial bicarbonate; and the integrity of the mucosal microcirculation. In theory, this bimodal action makes prostaglandin analogues ideal drugs for treating and preventing lesions of the gastroduodenal mucous membrane. In practice, however, these expectations remain unfulfilled. First, in doses lower than those required to decrease acid secretion, prostaglandin analogues retain cytoprotective properties but are no better than placebo in healing peptic ulcers. Second, in fully antisecretory doses some prostaglandin analogues accelerate ulcer healing compared with placebo and provide healing rates about as high cimetidine, but less than achieved with ranitidine. Third, despite the fact that high doses of some analogues are superior to placebo in alleviating pain associated with ulcer disease, these agents proved inferior to cimetidine and ranitidine in relieving pain in most comparative trials. Fourth, enprostil and misoprostol in doses which combine antisecretory with cytoprotective properties perform significantly poorer than ranitidine in preventing relapse of peptic ulcer disease. Fifth, although several uncontrolled observations have suggested a therapeutic benefit of prostaglandin analogues in gastroduodenal haemorrhage, placebo-controlled trials show no effect of arbaprostil in stopping bleeding and in preventing rebleeding.(ABSTRACT TRUNCATED AT 250 WORDS)