Development and validation of an LC-MS/MS-ESI method for the determination of lorglumide, a CCK-1 antagonist in mouse plasma: application to a pharmacokinetic study.

A highly sensitive, rapid assay method was developed and validated for the estimation of lorglumide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in positive-ion mode. The assay procedure involves extraction of lorglumide and phenacetin (internal standard, IS) from mouse plasma with simple protein precipitation. Chromatographic separation was achieved using an isocratic mobile (0.2% formic acid solution-acetonitrile, 20:80, v/v) at a flow-rate of 0.5 mL/min on an Atlantis dC18 column maintained at 40 °C with a total run time of 4.0 min. The MS/MS ion transitions monitored were 459.2 → 158.4 for lorglumide and 180.1 → 110.1 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.42 ng/mL and the linearity range extended from 0.42 to 500 ng/mL. The intra- and inter-day precisions were in the ranges of 1.47-10.9 and 3.56-7.53, respectively.

Effects of loxiglumide on pancreatic exocrine secretion stimulated by meal in conscious dogs.

The effects of loxiglumide (CAS 107097-80-3, CR 1505), a novel cholecystokinin-A(CCK-A) receptor antagonist, on pancreatic exocrine secretion stimulated by meal were examined in conscious dogs with chronic pancreatic fistula. Pancreatic exocrine secretion was stimulated by intraduodenal infusion of a liquid test meal and postprandial plasma CCK levels were apparently elevated. Loxiglumide inhibited the meal-stimulated outputs of pancreatic protein, amylase and bicarbonate at an intravenous dose of 10 mg/kg/h (p < 0.05). However, loxiglumide did not show apparent inhibition of pancreatic juice volume and trypsin output. These results show that the selective CCK-A antagonist loxiglumide may inhibit the increase of pancreatic exocrine secretion based on selective blockade of receptor binding of CCK endogenously induced by meal in dogs.

Effect of chronic oral administration of the CCK receptor antagonist loxiglumide on exocrine and endocrine pancreas in normal rats.

CONCLUSION: In normal adult rats, administration of a low dose of loxiglumide for 7 d had no significant effect on exocrine and endocrine pancreatic function, whereas a high dose of loxiglumide decreased pancreatic enzyme output without inducing insulin resistance and diabetes mellitus. BACKGROUND: There is a possibility that chronic administration of cholecystokinin receptor antagonists not only inhibits the growth of the pancreas but also alters exocrine and endocrine pancreatic function. METHODS: Loxiglumide at a dose of 50, 100, or 200 mg/kg body weight, or the same volume of saline, was given by an orogastric tube twice daily for 7 d (13 successive times). Biochemical and functional changes were determined on d 8 at 24 h after the last administration of loxiglumide and 18 h fasting. Pancreatic exocrine and endocrine function was simultaneously determined following an intravenous injection of a mixed solution of 0.5 g/kg body weight glucose plus 100 ng/kg body weight cerulein. RESULTS: Pancreatic weight and protein content were dose-dependently decreased by loxiglumide, whereas DNA content was decreased only by the highest dose of loxiglumide. Loxiglumide caused dose-dependent decreases in pancreatic fluid and protein output. Total pancreatic insulin content in rats treated with loxiglumide was not significantly different from that in the control rats. However, insulin concentration relative to DNA content was significantly increased in rats treated with 200 mg/kg body weight loxiglumide compared with that in other groups of rats. Glucose-stimulated insulin release was significantly low in rats treated with the highest dose of loxiglumide compared with that in other groups of rats, although there was no difference of serum glucose concentrations among these four groups of rats.

Effects of cholecystokinin receptor antagonist loxiglumide on rat exocrine pancreas.

Effects of long-term administration of the cholecystokinin receptor antagonist loxiglumide on exocrine pancreas were studied in adult rats. Plasma concentrations of loxiglumide at 8 h after a single subcutaneous injection of 50 mg/kg body weight of loxiglumide were 3.2 +/- 0.8 microgram/ml, which were comparable to those at 12 h after oral administration of the same dose (3.7 +/- 0.9 microgram/ml). Eight hours' prior subcutaneous injection of loxiglumide (50 mg/kg body weight) significantly suppressed pancreatic exocrine secretion stimulated by an intravenous bolus injection of 50 ng/kg body weight caerulein compared with the control rats. Based on these results, in the first experiment, loxiglumide at a dose of 50 mg/kg body weight was given subcutaneously three times a day (low dose) for 6 days to adult rats fed a standard laboratory diet. Low dose of loxiglumide significantly decreased pancreatic wet weight (-14%) and pancreatic contents of protein (-26%), trypsin (-38%), and lipase (-68%), while having no significant effect on pancreatic contents of DNA and amylase. In the second experiment, three times higher dose of loxiglumide (150 mg/kg body weight) was given by an orogastric tube twice daily for 6 days. High dose of loxiglumide significantly decreased pancreatic weight (-11%) and contents of protein (-20%) and DNA (-22%), whereas it significantly increased amylase (+92%) and trypsin content (+20%).(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of plasma cholecystokinin (CCK) concentrations by bioassay and radioimmunoassay in man. A critical evaluation.

The present investigation was designed to perform a direct comparison of a rat pancreatic acini bioassay system and a specific CCK radioimmunoassay (antiserum G-160) for the measurement of fasting and meal-stimulated plasma CCK in the presence and absence of the CCK receptor antagonist loxiglumide. The G-160 CCK antiserum is directed against the C-terminal O-sulfated tyrosine residue of the CCK molecule which is essential for full bioactivity of CCK peptides. For plasma extraction prior to bioassay measurement, hydrophobic reverse-phase chromatography on octadecylsilane cartridges was employed and resulted in simultaneous adsorption and elution of both CCK peptides and loxiglumide with recoveries of 87.5 +/- 9% and 75.0 +/- 5.9%, respectively. In the absence of loxiglumide, fasting and meal-stimulated values for CCK-like bioactivity and CCK-immunoreactivity (IR-CCK) were nearly identical (basal values: 1-2 pmol/l; meal-stimulated plateau levels: 4-6 pmol/l). After intravenous infusion of loxiglumide (30 mg/kg/h for 10 min, 10 mg/kg/h thereafter), resulting in plasma steady state levels of 200-300 mumol/l, meal-stimulated CCK-like bioactivity was undetectable, whereas IR-CCK levels were augmented 6.5-fold. In the bioassay system, standard samples containing 50 mumol/l loxiglumide produced complete inhibition of acinar lipase release in response to 50 pmol/l synthetic CCK-8. We conclude, that postprandial circulating non-CCK-like factors do not contribute significantly to the direct receptor-mediated stimulation of exocrine pancreatic secretion. The good agreement of CCK-like bioactivity and IR-CCK levels in the absence of loxiglumide confirms the sensitive and specific recognition of bioactive CCK peptides by the G-160 antiserum and suggests that this antibody exerts binding characteristics probably similar to a pancreatic acinar receptor.

Cholecystokinin receptor antagonist loxiglumide modulates plasma levels of gastro-entero-pancreatic hormones in man. Feedback control of cholecystokinin and gastrin secretion.

The effect of the potent specific cholecystokinin (CCK) receptor antagonist loxiglumide on meal-stimulated plasma concentrations of CCK, gastrin, pancreatic polypeptide (PP), neurotensin, glucose-dependent insulinotropic polypeptide (GIP), insulin and C peptide was investigated in a placebo-controlled study in 10 healthy male volunteers. Intravenous infusion of loxiglumide (10 mg kg-1 h-1) significantly augmented integrated incremental IR-CCK levels 7.3-fold after stimulation by a standard breakfast (504 +/- 54 vs 3.665 +/- 365 pmol-1 135 min-1, P less than 0.001), as measured by a specific CCK radioimmunoassay. Basal IR-CCK concentrations were not affected by administration of loxiglumide. Oral treatment with bile acids (2 g ursodeoxycholic acid plus 2 g chenodeoxycholic acid) together with the meal abolished this augmentation, whereas high-dose substitution with pancreatic enzymes (4.2 g pancreatin) reduced elevated IR-CCK levels by only 38%. CCK-like bioactivity, determined by a bioassay using rat pancreatic acini, was not detectable in all samples that contained loxiglumide at plasma concentrations of 100-250 micrograms ml-1. Plasma gastrin concentrations in response to the breakfast were elevated 3.2-fold during loxiglumide infusion and not influenced by substitution with bile acids or pancreatic enzymes. Meal-stimulated integrated incremental plasma PP concentrations were significantly suppressed (55-65% inhibition, P less than 0.01) by loxiglumide. Infusion of the CCK receptor antagonist only slightly increased postprandial peak plasma glucose, insulin and C-peptide levels, whereas GIP and neurotensin levels were not significantly influenced. These findings suggest: (i) CCK secretion is under feedback control by intraduodenal bile acids and to a lesser extent by pancreatic enzymes; (ii) simultaneous extraction of CCK and loxiglumide results in circulating plasma CCK-like bioactivity of zero; (iii) gastrin secretion is feedback controlled via an indirect mechanism probably involving CCK-induced somatostatin secretion; (iv) release of PP is under inhibitory control of CCK; (v) CCK does not play a major role as insulinotropic hormone in the entero-insular axis in humans.

Effect of loxiglumide on gallbladder contractile response to cerulein and food in humans.

The present study investigated the effect of loxiglumide, a new selective cholecystokinin-receptor antagonist, on the gallbladder contractile responses to caerulein and to food in humans. In 6 healthy men, the gallbladder emptying driven by intravenous infusion of stepwise increasing doses of cerulein (10-80 ng/kg . h) and that induced by a 550-cal standard meal were monitored by ultrasonography. In both sets of experiments, the effect of loxiglumide was tested at various infusional rates against a control infusion of saline. An infusional rate of 2.5 mg/kg . h of loxiglumide abolished the gallbladder response even to maximal doses of cerulein, whereas a rate of 1.0 mg/kg . h counteracted the cholecystokinetic activity of cerulein up to the dose of 20 ng/kg . h. In postprandial experiments, the cholecystokinin antagonist dose-dependently inhibited the physiologic gallbladder contraction. The maximal gallbladder emptying, which always occurred 85 min after the meal, was 71.1% +/- 3.3% of basal volume in control studies, 39.2% +/- 1.8% during infusion of 2.5 mg/kg . h of loxiglumide, and 17.3% +/- 5.9% when 5.0 mg/kg . h were infused. A dose of 7.5 mg/kg . h of loxiglumide was able to prevent any postprandial emptying of the gallbladder. The present study shows that a selective cholecystokinin receptorial blockade competitively antagonizes cerulein-induced gallbladder contraction and dose-dependently inhibits postprandial gallbladder emptying.

Plasma levels of proglumetacin and its metabolites after intravenous or oral administration in the dog.

The absolute bioavailability of 1H-indole-3-acetic acid, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl 2-[4-[4-[[4-(benzoylamino)-1,5-dioxopentyl]oxy]propyl]-1- piperazinyl]-ethyl ester (+/-) (proglumetacin, CR 604) was studied in 12 dogs, in a triple cross-over experiment with single doses of i.v. proglumetacin diphosphate, oral proglumetacin diphosphate or oral proglumetacin dimaleate. Determined were proglumetacin, 2'-[4-(3-hydroxypropyl)-piperazin-1-yl]-ethyl-(1-p- chlorobenzoyl-5-methoxy-2-methylindol-3-yl)-acetic acid (CR 1015), indometacin and proglumide in plasma. Proglumetacin and CR 1015 were found in plasma only after the i.v. administration. Conversely indometacin and proglumide were found after all administration routes. The areas under the curve of indometacin and of proglumide did not differ significantly after the three treatments, as shown by the analysis of variance.

High-performance liquid chromatographic determination of proglumide in plasma.

A rapid and sensitive method for determining the anticholinergic agent, proglumide, in plasma by high-performance liquid chromatography is described. Samples were acidified with hydrochloric acid and extracted with chloroform. The dried extract was resolved in chloroform and chromatographed on an adsorption chromatographic column using a mobile phase of chloroform-methanol (24:1) on a high-performance liquid chromatograph equipped with a UV absorbance detector (240 nm). The detection limit for proglumide was 0.05 microgram/ml.