Measurement and disposition of ciclosporin and its metabolites.
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Bile↗
Biomedical subjects
K F Sewing
Publications and source records attributed to K F Sewing.
Liquid-chromatographic measurement of cyclosporin A and its metabolites in blood, bile, and urine.
Using solid-phase extraction columns and "high-performance" liquid-chromatographic (HPLC) analysis, we could determine cyclosporin A and nine of its metabolites in blood, bile, and urine. To facilitate calculations of concentrations of cyclosporin A and its metabolites from the chromatograms, we used cyclosporin D as internal standard. For the HPLC analysis we used two sequential 250-mm analytical columns filled with reversed-phase octyl (C8) sorbent, eluting with a concave gradient of water, adjusted to pH 3.0 with phosphoric acid, and acetonitrile. Peaks were detected at 205 nm. For characterization of the chromatographic peaks, we isolated, by semi-preparative HPLC, 32 fractions representing peaks potentially related to cyclosporin A metabolites and re-injected them into the HPLC system under the same conditions as authentic cyclosporin A metabolites. Analytical recovery was 70-80%. The inter-assay CV for bile was 7.2%, for urine 12.3%. The method was used for routine monitoring of cyclosporin A and its metabolites.
Bile↗
Effect of the application form of cyclosporine on blood levels: comparison of oral solution and capsules.
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Administration, Oral↗
Protein kinase C and parietal cell function.
Ca2+-phospholipid dependent protein kinase activity and the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on H+ secretion was studied in guinea-pig parietal cells. In resting parietal cells PK-C activity was distributed almost equally in both the cytosolic and particulate cell fraction. Exposure of the cells to TPA resulted in a loss of cytosolic PK-C activity which was not accompanied by a concomitant increase of particulate activity. Furthermore TPA inhibits the K+/H+-ATPase and dissipates H+ gradient in intact gastric vesicles by a protonophoric action. The role of these biochemical events for the antisecretory action of TPA found in the intact parietal cell are discussed.
Adenosine Triphosphatases↗
SCH 28080 is a more selective inhibitor than SCH 32651 at the K+ site of gastric K+/H+-ATPase.
The anti-secretory agents SCH 32651 and SCH 28080 were compared for their potency to interact with the K+ site of guinea-pig parietal cell K+/H+-ATPase and dog kidney Na+/K+-ATPase. SCH 32651 and SCH 28080 had an inhibition constant of 9.0 and 0.02 mumol/l, respectively, for the K+/H+-ATPase. The Ki values for the Na+/K+-ATPase were 140 and 220 mumol/l. The data show that both drugs have a higher affinity to the K+ site of the K+/H+-ATPase than to that of the Na+/K+-ATPase and that the affinity ratio of SCH 28080 in favour of K+/H+-ATPase is much greater (11,000) than that of SCH 32651 (15).
Adenosine Triphosphatases↗
Measurement of cyclosporin A and of four metabolites in whole blood by high-performance liquid chromatography.
A high-performance liquid chromatographic procedure using cyclosporin D as internal standard for the routine measurement of cyclosporin A and four of its metabolites is described. Whole-blood samples were purified on refillable solid-phase glass extraction columns. The chromatographic method includes a gradient elution using acetonitrile and water (pH 3.0) as eluents and an RP-8 analytical column. More than 1000 samples have already been analysed without any loss. The inter-assay variation was 6.3% and the intra-assay variation 4.9%. A linear correlation was found over a range of 0-3000 ng cyclosporin A per ml whole blood. The detection limit was 20 ng and the recovery was found to be 80-90%. Metabolites 1, 17, 18 and 21 could be characterized.
Biotransformation↗
Inhibition of gastric K+/H+-ATPase by acid-activated 2-((2-pyridylmethyl)sulphinyl)benzimidazole products.
The inhibitory effects of timoprazole- and omeprazole-derived metabolites were studied in different in vitro test systems in order to characterize the metabolites of substituted benzimidazoles originating from acid activation. Acidification of timoprazole and omeprazole to pH 1.0 markedly increased the inhibitory potency on gastric K+/H+-ATPase. The timoprazole-derived tetracyclic thiol and radical were found to be equally or more potent on the K+/H+-ATPase than the mother compounds dissolved at pH 1.0. Kinetic studies with omeprazole sulphide revealed a competitive inhibition of the K+/H+-ATPase with respect to K+. The mercaptan dithiothreitol reversed the inhibitory effect of omeprazole, acidified timoprazole and the timoprazole-derived radical in the parietal cell and K+/H+-ATPase preparation. In contrast, the inhibitory effect of omeprazole sulphide and the timoprazole-derived thiol could not be reversed by dithiothreitol. Wash-out experiments indicated that acidified timoprazole and the tetracyclic compounds interact irreversibly with the K+/H+-ATPase, which contrasts with the properties of timoprazole in the parietal cell preparation. It is concluded from these data that neither the tetracyclic compounds nor the sulphide act as the 'active principle' of substituted benzimidazoles in the parietal cell preparion.
2-Pyridinylmethylsulfinylbenzimidazoles↗
Uptake kinetics of 14C L-leucine and 14C L- and 14C D-methionine in rat brain and incorporation into protein.
Because the amino acids 11C L-leucine, 11C L-methionine, and 11C D-methionine are used for examinations of brain tumors with positron emission tomography (PET), the uptake of the corresponding 14C substances and their incorporation into protein was studied in the rat brain. The uptake of all three substances from the plasma, across the blood-brain barrier, and into the brain took place quite quickly; return to the plasma seemed negligible. Incorporation into protein took place much more slowly.
Animals↗
The gastric proton pump, a target for neuroleptics and antidepressant drugs?
The antisecretory action of the antidepressant drugs trimipramine, doxepin and nortriptyline was studied in two different in-vitro test systems; the isolated and enriched guinea-pig parietal cell and the purified H+/K(+)-ATPase preparation. The effect of the antidepressants was compared with that of the neuroleptic agents chlorpromazine, triflupromazine, trifluperazine, haloperidol, fluspirilene and with that of the tricyclic anticholinergic agent pirenzepine. All neuroleptics and antidepressants inhibited acid formation in intact parietal cells with IC50 values in the nanomolar range. The inhibitory potency for each compound was identical regardless of whether histamine or db-cAMP was used as stimulant. Isolated H+/K(+)-ATPase, measured in the presence of 5 mmol litre-1 KCl, was inhibited by all psychotropic drugs with IC50 values in the micromolar range. EGTA did not affect the inhibitory potency at the H+/K(+)-ATPase, indicating that the action of the drugs does not depend on their calmodulin blocking activity. Pirenzepine was ineffective in both test systems. Kinetic studies done with nortriptyline, chlorpromazine and haloperidol showed a competitive type of inhibition with respect to K+ at low inhibitor concentrations. This competitive type was changed to a mixed type of inhibition with increasing inhibitor concentrations, demonstrating cooperative effects between drug binding and K+ activation of the enzyme. From these data it is suggested that antidepressants and neuroleptics act by an allosteric mechanism of action, and that the lipid solubility is a significant factor to establish enzyme inhibition.
Animals↗
Effect of omeprazole on eicosanoid formation in and release from guinea-pig gastric mucosal cells.
Guinea-pig gastric mucosal cells isolated by collagenase and pronase digestion were used to study the release of prostanoids prostaglandin I2 (PGI2; measured as 6-keto PGF1 alpha), PGE2, PGF2 alpha and thromboxane A2 (TXA2; measured as TXB2). Lysophosphatide acyltransferase (LAT) and phospholipase A2 (PLA2) were measured in the microsomal fraction of isolated but not separated gastric cells and isolated and enriched parietal and mucous cells. In all cell preparations PLA2 activity was approximately 5 times higher than that of LAT. Acid-activated omeprazole inhibited LAT in a concentration-dependent manner with similar IC50 values in gastric, parietal and mucous cells. It had no effect on PLA2. Gastric cells constantly produced PGI2, PGE2, PGF2 alpha and TXA2. The main prostaglandins released were PGI2 and PGE2. PGF2 alpha and TXA2 were released in smaller quantities. Omeprazole dissolved in polyethylene glycol 400 (PEG) pH 2 inhibited spontaneous PGI2 release in a concentration-dependent manner with an IC50 of 14.3 +/- 4.8 microM. Only concentrations as high as 100 microM produced a significant reduction in PGE2 release by 60%. No significant changes could be detected in the spontaneous release of PGF2 alpha and TXA2. Omeprazole dissolved in PEG pH 7 had no effect on PGI2 release except at 100 microM which led to an insignificant decrease by 40%. These data suggest that omeprazole beyond its inhibitory effect on parietal cell K+/H+-ATPase also affects gastric mucosal prostanoid formation and release. The inhibitory effect on PGI2 does not support the view that omeprazole protects the gastric mucosa by increasing prostanoid formation.
Acyltransferases↗
Immunosuppressive activity of cyclosporine metabolites in vitro.
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Antigen-Antibody Reactions↗
[Effect of benzydamine on release of prostaglandin E2 and prostacyclin from rabbit gastric mucosa cells].
The effect of the antiinflammatory drug benzydamine (Tantum) on prostaglandin E2 (PGE2) release from rabbit gastric mucosal cells was investigated. Up to 1 mmol/l benzydamine had no significant effect on PGE2 and prostacyclin (PGI2) formation and release. The lack of interference with gastric mucosal prostaglandin synthesis might explain the non-ulcerogenicity of this compound compared to other non-steroidal antiinflammatory drugs (e.g. indomethacin).
Animals↗
Mechanism of gastric antisecretory effect of SCH 28080.
The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea-pig and guinea-pig gastric membranes purified and enriched with K+/H+-ATPase. In guinea-pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. SCH 28080 inhibited the purified K+/H+-ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 microM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+-ATPase showed that this enzyme was only slightly depressed by SCH 28080. It is concluded that SCH 28080 acts with high selectivity on the parietal cell K%/H+-ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+-site of the gastric ATPase.
4-Nitrophenylphosphatase↗
The sulphoxide moiety of substituted benzimidazoles is essential for inhibition of parietal cell K+/H+-ATPase.
The antisecretory action of the benzimidazole sulphoxide derivative B 823-10, 2[(4-methoxy-3-methyl-2-pyridylmethyl)-sulphinyl]- 5-trifluoromethyl(1H)-benzimidazole, was compared with the effect of the corresponding sulphide B 823-08 in several in vivo and in vitro and in vitro test systems. The sulphide B 823-08 and the sulphoxide B 823-10 were found to be equipotent in the Shay rat. The sulphide was found to inhibit H+ secretion in intact rabbit gastric glands and enriched guinea-pig parietal cells with lower potency than the corresponding sulphoxide. The relative potency in antisecretory activity (sulphide/sulphoxide) decreased in the following rank order: Shay rat: gastric glands: parietal cells. Purified K+/H+-ATPase was not blocked by the sulphide, whereas the sulphoxide inhibited the overall as well as the partial reactions of this enzyme. In all in vitro systems tested, inhibition of H+ secretion and enzyme activity by the sulphoxide, but not by the sulphide, was antagonized by SH-compounds such as dithiothreitol. It is concluded that in vivo sulphoxidation of the sulphide plays an important role in acid inhibition. In vitro an additional inhibitory mechanism of the sulphide has to be considered.
2-Pyridinylmethylsulfinylbenzimidazoles↗
Interaction of ranitidine and famotidine with guinea pig-isolated parietal cells.
The inhibitory effects of the histamine H2-receptor antagonists, ranitidine and famotidine on histamine-stimulated gastric acid secretion have been studied in guinea pig isolated, enriched parietal cells using the 14C-aminopyrine accumulation technique. The 14C-aminopyrine accumulation curves in response to histamine were shifted towards the right in a parallel fashion by ranitidine, and in a nonparallel fashion by famotidine. The inhibitory effect of ranitidine, but not that of famotidine, was readily reversed by washing the parietal cells. It is concluded that the histamine H2-receptors in guinea pig parietal cells are blocked competitively and reversibly by ranitidine, but noncompetitively and partially reversibly by famotidine.
Animals↗
[Prostaglandins in ulcer?].
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Gastric Acidity Determination↗
The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa.
In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H+ secretion, as measured by 14C-aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10(6) cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H+ secretion, but that the acid response elicited by histamine requires another intracellular pathway.
Adenylyl Cyclases↗
Indometacin-induced gastrointestinal lesions in relation to tissue concentration, food intake and bacterial invasion in the rat.
The effect of food intake and gut bacterial flora on gastrointestinal lesions caused by oral indometacin (IND) was studied in rats. A dose of 10 mg/kg IND caused no intestinal lesions when the animals were starved before and after treatment; it produced moderate lesions when the animals were continuously fed and maximal lesions when the animals were fed in the postdrug period after starvation in the predrug period. Under germ-free conditions, 15 mg/kg IND induced significantly less intestinal lesions than under specific pathogen-free conditions. The differences in the magnitude of intestinal lesions under the varying feeding and maintenance conditions were not associated with different IND concentrations in the jejunal mucosa. The dose of 10 mg/kg IND produced most gastric lesions when the animals were previously starved for 24 h and subsequently fed, medium lesions in continuously starved animals and only a few lesions in animals fed before and after IND. The disposition of IND from the gastric mucosa did not differ under the different feeding conditions. As the dose of IND is high enough to inhibit prostaglandin synthesis, it was concluded that additional factors are important for the development of gastrointestinal lesions caused by IND. Secondary bile acids in conjunction with IND are important for the development of intestinal lesions, while gastric acid influences the intensity of gastric lesions.
Animals↗