Targeted colonic release formulations of mesalazine - A clinical pharmaco-scintigraphic proof-of-concept study in healthy subjects and patients with mildly active ulcerative colitis.

Colonic targeting of orally applied therapeutic drugs remains a challenge. Tablet coatings relying on gastrointestinal pH and colonic bacterial enzymes as triggers in association with an inner alkaline layer are expected to improve targeting efficiency. Mesalazine release from three differently coated tablets labelled with 1 MBq 153Sm was characterised in a single centre, open-label, parallel group study in nineteen healthy subjects and seven patients with mildly active ulcerative colitis. Two semi-organic and one aqueous-based outer coating with different ratios of enteric polymer and resistant starch were tested. All coatings showed comparable release lagtimes in biorelevant dissolution media and were not affected by neutron-activation of the samarium tracer. Mesalazine pharmacokinetics and gamma scintigraphy were used to characterise drug release, anatomical site of tablet disintegration and gastrointestinal transit. Initial tablet disintegration occurred at the ileo-caecal junction or beyond in 92 % of the subjects. Time to initial tablet disintegration was inversely correlated with maximal plasma concentrations and systemic mesalazine exposure. Although high inter-subject variability precluded detection of differences between solvent types and different enteric polymer to polysaccharide ratios, the dual pH and enzymatic triggered release system in combination with an inner alkaline layer promoted mesalazine release at the target site with high accuracy.

Effects of collagenase on the release of [3H]-noradrenaline from bovine cultured adrenal chromaffin cells.

Bovine isolated adrenal chromaffin cells maintained in culture at 37 degrees C for 1-7 days become polygonal and bipolar, with typical varicosity-like extensions. Catecholamine levels and dopamine beta-hydroxylase activity decreased after 24-48 h of culture, but recovered to normal levels 3-7 days later. Incubation of 1-7 day-old cells in the presence of increasing concentrations of [3H]-noradrenaline (3.91 to 125 nM) resulted in the retention by the cells of amounts of radioactivity directly proportional to the amine present in the media. One day-old cells took up and retained only one third of the radioactivity found in 2-7 day-old cells. The addition of collagenase to cultured cells caused a decrease in the uptake of tritium. However, the enzyme treatment did not affect the amine taken up by the cell before collagenase treatment. Release of tritium from cultured cells evoked by nicotine, acetylcholine (ACh) or 59 mM K+ was very poor in 24 h-old cells; the secretory response to nicotine, ACh or K+ was dramatically increased after 2-7 days of culture. Bethanecol did not cause any secretory response. When treated with collagenase, cultured cells which had recovered fully their secretory response, lost again the ability to release tritium evoked by ACh or nicotine. However, the responses to high K+, veratridine or ionophore X537A were not affected. The nicotinic response was recovered two days after collagenase treatment. The data suggest that the use of collagenase to disperse the adrenomedullary tissue during the isolation procedure might be responsible for the lost secretory response of young cultured chromaffin cells. Since collagenase specifically impairs the nicotinic cholinoceptor-mediated catecholamine release, it seems likely that the enzyme is exerting its action on the ACh receptor complex. It is unlikely that either voltage-sensitive Na+ or Ca2+ channels are affected by collagenase as the responses induced by high K+ or veratridine were unaffected by this enzyme.

Pharmacological dissection of receptor-associated and voltage-sensitive ionic channels involved in catecholamine release.

The experiments were designed to quantify pharmacologically the degree of participation of channels associated with the nicotinic cholinoceptor compared with voltage-sensitive channels during the evoked release of [3H]noradrenaline from prelabelled 3-7-day old cultured bovine adrenal chromaffin cells. To achieve this purpose we studied (a) the release of [3H]noradrenaline evoked by secretagogues known to trigger the secretory response through activation of receptor-associated channels (acetylcholine, nicotine), voltage-sensitive Na+ (veratridine) and Ca2+ (high [K+] ) channels or direct, channel-independent promotion of Ca2+ entry (ionomycin); and (b) the selective blockade of some of those responses using ionic manipulations (Na+ deprivation, high Mg2+) or drugs known to block the activity of receptor-operated channels (imipramine, cocaine), voltage-dependent Na+ (tetrodotoxin) or Ca2+ (nitrendipine) channels. Inhibition by nitrendipine, a potent Ca2+ antagonist, of the secretory responses to both nicotine and high [K+] indicates a preferential Ca2+ entry through voltage-sensitive channels during the secretory process. Blockade by cocaine and imipramine of the release of [3H]noradrenaline evoked by acetylcholine and nicotine, without alteration of the responses to high [K+], veratridine or ionomycin, speaks in favor of a selective inactivation of the nicotinic receptor-associated channel. Since Na+ deprivation abolished [3H]noradrenaline release produced by nicotine, it seems that Na+ entry through the receptor-linked ionophore might be a primary event in the initiation of the secretory process; the fact that tetrodotoxin did not affect the release favors this view. However, veratridine induced a tetrodotoxin-sensitive secretory response, suggesting the presence of voltage-sensitive Na+ channels which might physiologically be used to propagate action potentials through gap junctions between adjacent chromaffin cells, only in the intact gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the cardiotonic drug ARL-115 on the release of noradrenaline from the cat atrium, the binding of 3H-ouabain to plasma membranes and the movements of calcium in mitochondria.

The cardiotonic pyridine derivative ARL-115 increased the spontaneous and electrically-evoked release of 3H-noradrenaline from the cat right atrium superfused with oxygenated Krebs-bicarbonate solution at 37 degrees C. On the contrary, ouabain inhibited the evoked release while it also enhanced the spontaneous release of the transmitter. Vanadate did not affect either spontaneous or evoked release. Tetraethylammonium chloride (TEA) and 4-aminopyridine (4-AP) greatly potentiated 3H-noradrenaline release induced by electrical stimulation; when applied in addition to each agent, ARL-115 failed to further increase the secretory response. 3H-ouabain specific binding to partially purified bovine adrenal medulla plasma membranes was very efficiently antagonized by cold ouabain, but not by vanadate or ARL-115, even at concentrations as high as 10(-3) mol/l. 45Ca uptake into isolated bovine adrenal medulla mitochondria was prevented by dinitrophenol (DNP) but unchanged in the presence of ARL-115. 45Ca release from preloaded mitochondria was, again, markedly increased by DNP, but not affected by ARL-115. The results suggest that ARL-115 enhances the release of noradrenaline from cardiac sympathetic nerves by a TEA- and 4-AP-like action. In this manner, ARL-115 would inactivate the K+ current in the nerve terminals, thereby prolonging the duration of the action potential, allowing the Ca2+ channels to remain open longer and more Ca2+ to enter the terminal. ARL-115 is not acting like digitalis.

Correlation between catecholamine release and sodium pump inhibition in the perfused adrenal gland of the cat.

1 Ca(2+) reintroduction to retrogradely perfused and ouabain (10(-4) M)-treated cat adrenal glands caused a catecholamine secretory response which was greater the longer the time of exposure to the cardiac glycoside. Such a response was proportional to the external Na(+) concentration [Na(+)](o).2 A qualitatively similar, yet smaller response was observed when glands were perfused with Krebs solution lacking K(+) ions; thus, K(+) deprivation mimicked the secretory effects of ouabain. Catecholamine secretion evoked by Ca(2+) reintroduction in K(+)-free solution (0-K(+)) was also proportional to [Na(+)](o) and greater the longer the time of exposure of the gland to 0-K(+) solution.3 The ionophore X537A also mimicked the ouabain effects, since Ca(2+) reintroduction to glands treated with this agent (25 muM) caused a sharp secretory response. When added together with X537A, ouabain (10(-4) M) did not modify the response to the ionophore.4 N-ethylmaleimide (NEM), another Na(+), K(+)-ATPase inhibitor, did not evoke the release of catecholamines; on the contrary, NEM (10(-4) M) inhibited the catecholamine secretory response to high [K(+)](o), acetylcholine, Ca(2+) reintroduction and ouabain.5 Ouabain (10(-4) M) inhibited the uptake of (86)Rb into adreno-medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30-40 min) to reach its peak. NEM (10(-4) M) blocked (86)Rb uptake in a similar manner.6 The results are further evidence in favour of the presence of a Na(+)-Ca(2+) exchange system in the chromaffin cell membrane, probably involved in the control of [Ca(2+)](i) and in the modulation of catecholamine secretion. This system is activated by increasing [Na(+)](i), either directly (ionophore X537A, increased [Na(+)](o)) or indirectly (Na(+) pump inhibition). However, the simple inhibition of Na(+) pumping does not always lead to a catecholamine secretory response; such is the case for NEM.