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Recently, microdosing has been introduced in clinical studies to minimize the risk of harmful events to human subjects. This can also reduce the influence of low-affinity, high-capacity enzymes. To date, there are only a few reports estimating in vivo CYP450 isoform (1A2, 2C9, 2C19, 2D6, 2E1 and 3A) activities using low-dose probes for drug–drug interactions. While microdosing is beneficial in terms of side effects and possible interactions, it requires extremely sensitive analytical tools that are able to detect and quantitate both probes and their metabolites. The present report describes a highly sensitive LC-MSn method that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites. After administration of a mixture of six probes (cocktail approach: caffeine 100 μ g · kg - 1 , tolbutamide 100 μ g · kg - 1 , omeprazole 500 μ g · kg - 1 , dextromethorphan 500 μ g · kg - 1 , chlorzoxazone 50 μ g · kg - 1 and midazolam 100 μ g · kg - 1 ) to SD rats, plasma samples were analyzed by LC-20A & 5500Qtrap ESI-MSn in MRM mode. The MS/MS reaction selected ions (with good linearity in the range of) 181.2/124.0 m/z (0.2–200 ng · mL - 1 ) ; 195.2/138.2 m/z (0.1–25 ng · mL - 1 ) ; 269.1/170.0 m/z (0.01–40 ng · mL - 1 ) ; 285.1/186.0 m/z (0.02–12.5 ng · mL - 1 ) ; 346.1/198.1 m/z (0.05–100 ng · mL - 1 ) ; 362.2/214.2 m/z (0.01–25 ng · mL - 1 ) ; 272.3/147.1 m/z (0.1–200 ng · mL - 1 ) ; 258.2/157.0 m/z (0.05–12.5 ng · mL - 1 ) ; 168.1/132.1 m/z (0.01–200 ng · mL - 1 ) ; 326.1/291.2 m/z (0.2–200 ng · mL - 1 ) ; and 342.1/324.2 m/z (0.2–25 ng · mL - 1 ) for caffeine, paraxanthine, tolbutamide, 4-hydroxytolbutamide, omeprazole, 5-hydroxyomeprazole, dextromethorphan, dextrorphan, chlorzoxazone, midazolam, and 1 ′ -hydroxymidazolam, respectively. The stability %RSD for all probes was less than 15%. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses. The established LC-MSn method was suitable for the pharmacokinetic study of this mixed cocktail probe group and could be applied to CYP phenotyping research. Acknowledgements Project supported by National Natural Science Foundation of China 81473579 81273654 81102879, 7173267 and 2013ZX09103002-022.

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K + channels (K ATP channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al. , pages 1685–1694, this issue). To elucidate the underlying mechanisms, K ATP channel activity, plasma membrane potential and the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 μM each) K ATP channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 μM) induced a marked reduction of K ATP channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 μM) or tolbutamide (500 μM) had no lasting effect on K ATP channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca 2+ ] i normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca 2+ ] i increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca 2+ ] i elicited by a K + depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K ATP channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca 2+ ] i regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca 2+ ] i (in the case of tolbutamide).

The sulfonylurea derivative, tolbutamide, and the phenylalanine derivative, N-benzoyl-D-phenylalanine (NBDP), both of which stimulate insulin secretion through interaction with the sulfonylurea receptor (SUR1), were studied for their ability to increase the [Ca(2+)](i) and to interact with the glucose-induced slow large amplitude [Ca(2+)](i) oscillations in isolated mouse pancreatic islets. Tolbutamide as well as NBDP induced [Ca(2+)](i) oscillations of extremely slow frequency. Both compounds also lowered the threshold for the glucose-induced slow large amplitude [Ca(2+)](i) oscillations and significantly reduced their frequency in intact islets as well as in single pancreatic beta cells. These [Ca(2+)](i) oscillations apparently require a glucokinase-mediated glycolytic flux. This conclusion is supported by the observations that KIC, a mitochondrial fuel, cannot replace glucose in this synergism and that mannoheptulose, an inhibitor of glucokinase and glucose-induced insulin secretion, abolishes these slow [Ca(2+)](i) oscillations. In conclusion, these compounds potentiate the effect of glucose. This additive effect is the likely result of a synergistic closing action upon the ATP-sensitive K(+) (K(ATP)) channel, mediated in the case of glucose through an action upon the channel protein itself of ATP generated in glucose catabolism and in the case of tolbutamide and NBDP upon the sulfonylurea receptor (SUR1) associated with this channel.

Functional effects of prolonged exposure to the sulfonylurea, tolbutamide, were examined in the clonal electrofusion-derived BRIN-BD11 cell line. In acute 20-min incubations, 50-400 microM tolbutamide stimulated a dose-dependent increase (P < 0.01) in insulin release at both non-stimulatory (1.1 mM) and stimulatory (8.4 mM) glucose. Culture with 100 microM tolbutamide (18 hr) caused a marked (67%) decrease in subsequent insulin-secretory responsiveness to acute challenge with 200 microM tolbutamide, though notably, tolbutamide culture exerted no influence on 200 microM efaroxan-induced insulin secretion. Duration of exposure (3-18 hr) to 100 microM tolbutamide in culture also time-dependently influenced subsequent responsiveness to acute tolbutamide challenge, with progressive 47-58% decreases from 6-18 hr (P < 0.001). Similarly, 6- to 18-hr culture with 100 microM efaroxan specifically desensitized efaroxan-induced insulin release. Tolbutamide- and efaroxan-induced desensitization exhibited a time-dependent reversibility, with a sustained return to full insulin-secretory responsiveness by 12 hr. Notably, 18-hr culture with tolbutamide or efaroxan did not significantly affect insulinotropic responses to 16.7 mM glucose, 10 mM 2-ketoisocaproic acid, 10 mM alanine, 10 mM arginine, or 30 mM KCl. Diverse inhibitory actions of tolbutamide or efaroxan culture on late events in stimulus-secretion coupling reveal that drug desensitization is both a specific and important phenomenon. As such, the model system described could prove an important tool in determining the complex modes of action of established and novel clinically useful insulinotropic compounds.

58C80 [2-(4- t -butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone] is an experimental naphthoquinone antimalarial drug which undergoes extensive alkyl hydroxylation to a single t -butylhydroxy metabolite in man in vivo and also in human liver microsomes, where this is catalysed primarily by a 54 kDa CYP2C9 form of cytochrome P450, P450hB 20–27 . Microsomal 58C80 hydroxylation (58OH) activity showed a marked inter-individual variation in a bank of 39 individual human livers but did not correlate with the immunoquantified levels of either of two microsomal proteins (54 and 50 kDa, respectively) recognised by a polyclonal antibody against CYP2C9 (P450hB 20–27 ). Neither 58OH activity nor the concentrations of the CYP2C9-immunorelated proteins showed any relationship with the individuals' age, sex, cigarette smoking habit, alcohol consumption or clinical drug treatment, including long term antiepileptic therapy with phenobarbitone or phenytoin. 58OH activity did not correlate with either TBOH (tolbutamide hydroxylation) or MPOH (S-mephenytoin 4′-hydroxylation) activities, while 58C80 inhibited both TBOH and MPOH in human liver microsomes non-competitively ( K i = 30 and 175 μ M for TBOH and MPOH, respectively). 58C80 could be a useful model substrate for measuring human CYP2C activity in vitro .

Polyphosphoinositide synthesis in isolated islets of the rat was determined by the phosphorylation of endogenous phosphatidylinositol (PtdIns) by PtdIns kinase and [gamma-32P]ATP to form [32P]-phosphatidylinositol 4-phosphate (PtdInsP) in cell homogenates. Glucose stimulation of intact islets resulted in a time- and concentration-dependent reduction in PtdInsP synthesis. Similarly, the stimulation of intact islets with carbachol (CCh), cholecystokinin (CCK-8S), or tolbutamide for 15 min reduced PtdInsP production in a concentration-dependent manner. The effects of glucose, tolbutamide and CCh were reversible. PtdInsP hydrolysis did not account for the reduction in PtdInsP recovery. The addition of exogenous PtdIns to the PtdIns kinase assay significantly increased basal PtdInsP levels. In addition, exogenous PtdIns completely reversed the inhibitory effects of glucose and increased PtdIns kinase activity in homogenates of glucose-stimulated islets to levels found in control homogenate with PtdIns. Exogenous PtdIns also increased PtdIns kinase activity in CCK-8S-treated islets, although exogenous PtdIns did not overcome the tolbutamide-induced inhibition of PtdIns kinase. The Vmax of PtdIns kinase in homogenates of islets treated with tolbutamide was reduced significantly, although glucose did not affect the Vmax. In addition, the Km values for ATP and PtdIns were not altered by exposure of the islets to cell stimuli. The results suggest that the level of PtdIns in islet cell membranes is rate limiting for PtdInsP synthesis, and that tolbutamide is a noncompetitive inhibitor of PtdIns kinase.

The metabolism of metronidazole was studied in microsomes isolated from livers of human kidney donors. The formation of the major in vivo metabolite, hydroxymetronidazole, proceeded according to biphasic kinetics, suggesting the involvement of at least two enzymatic sites. The affinity constant (Km) of the high affinity site ranged from 140 to 320 microM and metabolism at this site contributed more than 75% of the intrinsic clearance. Thus, at therapeutic doses of metronidazole most of the hydroxylation in vivo should be associated with this site. Antipyrine, cimetidine, alpha-naphthoflavone, caffeine, theophylline, mephenytoin, tolbutamide, quinidine, acetone and nifedipine were poor inhibitors of the formation of hydroxymetronidazole by human liver microsomes. Propranolol (500 microM) inhibited the hydroxylation rate by 70%. Phenacetin inhibited metronidazole hydroxylation with a competitive inhibition constant (Ki) of 4-5 microM. However, metronidazole did not inhibit the O-deethylation of phenacetin. It is concluded that cytochromes P450 IA2, IIC9, IIC10, IID6, IIE1 and IIIA3 do not contribute significantly to the high affinity hydroxylation of metronidazole in man.

Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K(ATP) channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5mM). Only when the glucose concentration was stimulatory by itself (10mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K(ATP) channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100microM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5mM, whereas other K(ATP) channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500microM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca(2+)](i)). In the presence of 5mM glucose gatifloxacin and moxifloxacin at 500microM but not at 100microM elevated [Ca(2+)](i). It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K(ATP) channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.

The effects of the oral hypoglycaemic drugs, phenformin and tolbutamide, and insulin, alone and in combination, on steroid metabolism in hepatocytes isolated from control and streptozotocin-diabetic male rats has been studied. Both phenformin and tolbutamide mimic the action of insulin in stimulating hepatic steroid metabolism in a dose-dependent manner in control cells. Unlike insulin, however, both drugs give a similar effect in cells derived from diabetic animals although to a lesser extent. Both drugs can partially restore the effect of insulin in cells derived from diabetic animals. Biguanides and sulphonylureas, therefore, have a direct effect on liver cells to mimic insulin action and can still have an effect under conditions where insulin is inactive. Both types of oral hypoglycaemics can also affect insulin-insensitive cells isolated from diabetic rat liver to restore to a certain extent their response to insulin.

Pretreatment with various doses of phenobarbital (PB) has been used to create a pool of rats with a wide range of hepatic microsomal monooxygenase activity to systematically examine relationships between and within in vivo and in vitro markers. The in vivo clearance of tolbutamide (TOL), theophylline (TH), antipyrine (AP) and its metabolites were determined in the same rats used for hepatic microsome preparation and assessment of P450 content and activities (via 7-ethoxycoumarin O-deethylase (ECOD), 7 ethoxyresorufin O-deethylase, 7-methoxycoumarin O-demethylase (MCOD) and aldrin epoxidase determinations). A graded dose-response relationship was found between PB treatment and most but not all parameters. The need for careful selection of in vivo and as well as in vitro markers is apparent from these studies. The most responsive parameters--TOL and AP clearances, MCOD and ECOD activities--were also those producing the strongest in vivo-in vitro correlations. Despite the diffuse nature of the PB induced response in P450 complement, good predictive relationships were apparent between ECOD and TOL clearance (r2 = 0.88).

Taurine has been found to inhibit ATP-sensitive K+ (KATP) channels in rat pancreatic beta-cells [Park et al., Biochem Pharmacol 2004;67:1089-1096] which could be due to its interaction with a benzamido-binding site on SUR1. In present study, we further evaluated the mechanism of taurine action on the KATP-channel inhibition, using cloned KATP-channels with different types of SUR subunit expressed in Xenopus laevis oocytes. The oocytes were coinjected with Kir6.2 mRNA, and mRNA encoding SUR1, SUR2A or SUR2B. Macroscopic currents were recorded from giant excised inside-out patches. The binding of glibenclamide to SUR1 was assessed by using a glibenclamide-fluorescent probe. Intracellular taurine inhibited all three types of KATP-channels to a similar extent. They were fit to the Hill equation, showing IC50 of 11.0 mM for Kir6.2/SUR1, 10.9 mM for Kir6.2/SUR2A, and 9.0 mM for Kir6.2/SUR2B currents. Taurine at the concentration of 10 mM enhanced the high-affinity bindings of glibenclamide and repaglinide on all types of SUR, whereas the low-affinity binding on Kir6.2 was not affected. The intensity of glibenclamide fluorescence was higher in the plasma membrane of taurine-pretreated oocytes. The high-affinity binding of tolbutamide or gliclazide on SUR was not modified by taurine. These results suggest that the taurine inhibition of KATP-channels is mediated by an interaction with the site on SUR where the benzamido group is bound. Therefore, intracellular concentrations of taurine in different tissues may be more important in determining taurine modulation of the KATP-channel rather than distinct types of SUR subunit.

A prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100microM each), the sulfonylurea tolbutamide (500microM), or the alkaloid quinine (100microM) resulted in morphological damage of 4-18% of beta-cells compared to less than 2% in controls. Thus, the question arose whether K(ATP) channel-blocking insulin secretagogues are beta-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24h with idazoxan reduced MTT conversion by 50% at 100microM and by 98% at 1000microM. Phentolamine and quinine reduced viability comparably at 1000microM, but were less toxic at 100microM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000microM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100microM of idazoxan and phentolamine, whereas at 1000microM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca(2+) influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for beta-cells in intact mouse islets. In summary, beta-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca(2+) influx via L-type Ca(2+) channels. Thus, secretagogue-mediated closure of K(ATP) channels is apparently not per se beta-cell toxic.

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K(+) channels (K(ATP) channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685-1694, this issue). To elucidate the underlying mechanisms, K(ATP) channel activity, plasma membrane potential and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 microM each) K(ATP) channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 microM) induced a marked reduction of K(ATP) channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 microM) or tolbutamide (500 microM) had no lasting effect on K(ATP) channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca(2+)](i) normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca(2+)](i) increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca(2+)](i) elicited by a K(+) depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K(ATP) channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca(2+)](i) regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca(2+)](i) (in the case of tolbutamide).

The desensitization of pancreatic B-cells against stimulation by insulin secretagogues that inhibit ATP-dependent K(+) channels (K(ATP) channels) was investigated by measuring insulin secretion of perifused pancreatic islets. Additionally, the islet insulin content and the number of secretory granules per B-cell were determined. Prior to the measurement of secretion, islets were cultured for 18 h in the presence or absence of the test agents in a cell-culture medium containing 5 mM glucose. The effects of three imidazolines, phentolamine, alinidine, and idazoxan (100 microM each) were compared with those of the well-characterized sulfonylurea, tolbutamide (500 microM), and those of the ion channel-blocking alkaloid, quinine (100 microM). Insulin secretion was strongly reduced upon re-exposure to phentolamine, alinidine, tolbutamide, and quinine, whereas idazoxan, which stimulated secretion only weakly, had no significant effect. The imidazoline secretagogues phentolamine and alinidine induced a cross-desensitization against the stimulatory effect of tolbutamide and quinine. A long-term depolarization with 40 mM KCl was also able to induce a significant reduction of the secretory response to all of the above secretagogues. The insulin content of cultured islets was moderately, but significantly reduced by alinidine, whereas the reduction by phentolamine, tolbutamide, and quinine was not significant. In contrast to these observations, the ultrastructural examination revealed that tolbutamide-treated B-cells had a high degree of degranulation, whereas the other test agents and 40 mM KCl produced only a partial degranulation, except for phentolamine, which produced no significant degranulation at all. These results suggest that the desensitization of insulin secretion is a common property of all agents that stimulate insulin secretion by depolarisation of the plasma membrane. Depending on the specific secretagogue, additional mechanisms, proximal and distal to Ca(2+) influx, appear to contribute to the desensitization (see Rustenbeck et al., pages 1695-1703, this issue).

The sulfonylurea derivative, tolbutamide, and the phenylalanine derivative, N-benzoyl-D-phenylalanine (NBDP), both of which stimulate insulin secretion through interaction with the sulfonylurea receptor (SUR1), were studied for their ability to increase the [Ca(2+)](i) and to interact with the glucose-induced slow large amplitude [Ca(2+)](i) oscillations in isolated mouse pancreatic islets. Tolbutamide as well as NBDP induced [Ca(2+)](i) oscillations of extremely slow frequency. Both compounds also lowered the threshold for the glucose-induced slow large amplitude [Ca(2+)](i) oscillations and significantly reduced their frequency in intact islets as well as in single pancreatic beta cells. These [Ca(2+)](i) oscillations apparently require a glucokinase-mediated glycolytic flux. This conclusion is supported by the observations that KIC, a mitochondrial fuel, cannot replace glucose in this synergism and that mannoheptulose, an inhibitor of glucokinase and glucose-induced insulin secretion, abolishes these slow [Ca(2+)](i) oscillations. In conclusion, these compounds potentiate the effect of glucose. This additive effect is the likely result of a synergistic closing action upon the ATP-sensitive K(+) (K(ATP)) channel, mediated in the case of glucose through an action upon the channel protein itself of ATP generated in glucose catabolism and in the case of tolbutamide and NBDP upon the sulfonylurea receptor (SUR1) associated with this channel.

Human liver microsomes had about 8-fold higher 7-hydroxylation activities for S-warfarin than for R-warfarin. Activities of racemic warfarin 7-hydroxylation by liver microsomes of 35 human samples correlated more closely with those of S-warfarin 7-hydroxylation (r = 0.95) than with those of R-warfarin 7-hydroxylation (r = 0.69). The correlation coefficient between R-warfarin 7-hydroxylation and 7-ethoxyresorufin O-deethylation activities was 0.73 in these human samples, suggesting that R- and S-warfarin enantiomers are catalyzed by different forms of human cytochrome P450 (P450 or CYP) enzymes. Anti-CYP2C9 antibodies inhibited completely the 7-hydroxylation of S-warfarin, but not R-warfarin, catalyzed by human liver microsomes, while anti-CYP1A2 inhibited R-warfarin 7-hydroxylation by about 70%. Interestingly, the racemic warfarin 7-hydroxylation activities (turnover numbers of 1.6 +/- 1.0 pmol/min/mg protein in 35 human samples) were found to be low compared with the S-warfarin 7-hydroxylation activities (4.1 +/- 2.5 pmol/min/mg protein), indicating that R-warfarin may have affected the CYP2C9-dependent S-warfarin 7-hydroxylation activities when racemic warfarin was used as a substrate. Several P450 inhibitors, as well as R-warfarin, were examined for their abilities to inhibit S-warfarin 7-hydroxylation; we found that R-warfarin was a non-competitive inhibitor with a Ki value of about 150 microM, whereas both tolbutamide and sulfaphenazole were competitive inhibitors with Ki values of about 100 and 0.5 microM, respectively, for S-warfarin 7-hydroxylation activities. These results suggest that R- and S-warfarin enantiomers are catalyzed principally by CYP1A2 and CYP2C9, respectively, in human liver microsomes, and that the pharmacokinetic properties of S-warfarin may be altered by R-warfarin in vivo when racemic warfarin is administered clinically to humans.

Using intact rat islets, we previously observed that GTP depletion (achieved through the use of mycophenolic acid or other synthesis inhibitors) impedes nutrient- but not K + -induced insulin secretion. It was concluded that a proximal nutrient-dependent step in stimulus-secretion coupling (but not the process of Ca 2+ -induced exocytosis itself) is modulated by ambient GTP levels. To examine Ca 2+ -dependent steps further in intact β cells, INS-1 cells (which synthesize GTP and ATP similarly to rat islets) and HIT-T15 cells (whose synthesis of purine nucleotides is different) were studied following cell culture for 1–18 hr in various concentrations of mycophenolic acid (MPA) or mizoribine (MZ). Both agents profoundly reduced GTP content (mean: −78%) and lowered the GTP/GDP ratio by an average of −73%; concomitantly, MPA or MZ reduced insulin secretion induced by 10 mM glucose, 30 or 40 mM KCl, or 100 μM tolbutamide, independent of any changes in cell viability, insulin content, ATP content, the ATP/ADP ratio, or cytosolic free Ca 2+ concentrations. In INS-1 cells (which appear to have normal nucleobase transport and “salvage” pathway activities), guanine (but not adenine) restored GTP content, the GTP/GDP ratio, and Ca 2+ -induced secretion. In HIT cells, the phosphoribosylation of exogenous guanine or hypoxanthine is defective; however, provision of 500 μM guanosine (but not adenosine) reversed the effects of MPA. We conclude that, at least in certain situations, a requisite role for GTP in the distal step(s) of exocytosis can be demonstrated.

Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4'-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters (Km and Vmax) for the 4'-hydroxylation of (R)- and (S)-flurbiprofen in human liver microsomes, to determine if enantiomeric interactions occur when both enantiomers are present, and to identify the specific P450 form(s) involved in this reaction. In human liver microsomes, the Km and Vmax (mean +/- SD) for (R)-4'-hydroxy-flurbiprofen formation were 3.1 +/- 0.8 microM and 305 +/- 168 pmol.min-1.mg protein)-1, respectively. In comparison, the Km and Vmax (mean +/- SD) for (S)-4'-hydroxy-flurbiprofen formation were 1.9 +/- 0.4 microM and 343 +/- 196 pmol.min-1.mg protein-1, respectively. Enantiomeric interaction studies revealed a decrease in Km and Vmax for both enantiomers and an apparent loss of stereoselectivity. Racemic-warfarin, tolbutamide, alpha-naphthoflavone and erythromycin were studied as potential inhibitors of this process. The estimated Ki values for the inhibition of (R)- and (S)-4'-hydroxy-flurbiprofen formation by racemic-warfarin were 2.2 and 4.7 microM. This reaction was also inhibited by tolbutamide. In contrast, erythromycin and alpha-naphthoflavone had no appreciable effect on 4'-hydroxy-flurbiprofen formation. cDNA-expression of individual forms was used to determine which P450 was involved in 4'-hydroxy-flurbiprofen formation. P450 2C9 and an allelic variant (R144C) readily catalyzed the formation of 4'-hydroxy-flurbiprofen. P450 1A2 was also active albeit with a turnover rate 1/140th that of P450 2C9R144C (P450s 2C8, 2E1 and 3A4 were not active toward either enantiomer). The results of these studies indicate that the enantiomers of flurbiprofen may exhibit stereoselectivity with respect to enzyme affinity but have roughly equal maximum formation velocities. Additionally, these two enantiomers may compete for the enzyme resulting in lower maximum velocities for both enantiomers. Finally, of those P450 forms examined, only P450 2C9 and an allelic variant catalyzed the 4'-hydroxylation of both (R)- and (S)-flurbiprofen.

The metabolism of praziquantel (PZQ) was studied in microsomes isolated from livers of differently pretreated rats and in the presence of various inhibitors of cytochrome P450 (P450) isoforms. Microsomes from phenobarbitone (PB)-pretreated rats metabolised PZQ to its major metabolite 4OH-praziquantel (4OH-PZQ) at a greater rate than those from 20-methylcholanthrene (MC) and saline (SA) pretreated rats. The Vmax for the PB microsomes was 600 nmol 4OH-PZQ formed/mg/min x 10(-3) compared to 91.4 nmol/mg/min x 10(-3) for MC and 238 nmol/mg/min x 10(-3) for SA microsomes. These results indicate that PZQ is metabolised by PB-inducible isoforms of P450. Inhibitor studies were conducted with microsomes from SA-pretreated animals. In these studies, caffeine, disulfuram, and tolbutamide were poor inhibitors of the metabolism of PZQ to 4OH-PZQ, with I50 values not determinable. Quinidine and quinine inhibited the hydroxylation of PZQ but with high Ki values. 17 alpha-Ethynylestradiol, cimetidine and diphenylhydramine were effective inhibitors of the formation of 4OH-PZQ, with 17 alpha-ethynylestradiol being the most potent with a Ki of 0.5 +/- 0.05 microM. From the known specificities of these P450 inhibitors, it is therefore concluded that cytochromes P450 1A2, 2E1, 2C9-10, and 2D6 probably do not contribute significantly to the metabolism of PZQ to its major metabolite in rats. It is likely that cytochromes P450 2B1 and 3A, both inducible by PB, are predominantly responsible for the formation of 4OH-PZQ.

The female dark agouti (DA) rat lacks CYP2D1, the equivalent enzyme in the rat to human CYP2D6 (debrisoquine hydroxylase), and shows impaired metabolism of a number of CYP2D6 substrates. However, from the data available in the literature it is not entirely clear whether the enzyme deficiency in the DA rat is restricted to CYP2D1, and whether factors such as age and substrate concentration are important determinants of interstrain differences in the activity of this enzyme. Given that the female DA rat is used as a model of the human CYP2D6 poor metaboliser phenotype, there is a need for a systematic evaluation of the P450 activities in the DA rat, and of its suitability as a model of the PM phenotype. In the present study metoprolol was used as a probe substrate to investigate CYP2D1 activity since both the alpha-hydroxylation and O-demethylation of this drug are catalysed by CYP2D6 in man. Formation of alpha-hydroxymetoprolol (AHM) and O-demethylmetoprolol (ODM) was 10- and 2.5-fold lower in liver microsomes from female DA rats compared with microsomes from age-matched female Wistar rats, the latter representing the extensive metaboliser strain. Kinetic analysis suggested that in both strains of rat both the alpha-hydroxylation and O-demethylation of metoprolol were catalysed by more than one enzyme. By using quinine as a specific inhibitor of the enzyme, CYP2D1 was identified as an intermediate affinity site in the Wistar strain and was shown to have impaired activity in the DA strain. The activities of lower and higher affinity sites were similar in the two strains. Thus, the only difference between the two strains with respect to both routes of metoprolol metabolism appeared to be in the activity of CYP2D1. Interstrain differences were found to be highly dependent on the choice of substrate concentration, being more marked at lower concentrations. We have also investigated the metabolism of a number of probe compounds for some of the other P450 isoforms commonly involved in drug metabolism to determine the selectivity of the deficiency in the DA strain. p-Nitrophenol hydroxylation and erythromycin N-demethylation were catalysed at higher rates by DA than by Wistar liver microsomes, indicating higher levels of activity of CYP2E1 and CYP3A in the former strain. Felodipine oxidation, tolbutamide hydroxylation and both the hydroxylation and N-demethylation of S-mephenytoin were catalysed at similar rates by microsomes from the two strains, indicating similar activities of enzymes in the CYP2C and CYP3A families.(ABSTRACT TRUNCATED AT 400 WORDS)

Two inhibitors of the nucleotide-sensitive K + (k ATP ) channel, tolbutamide and quinine, were utilized in order to assess the role of this channel in glucose-stimulated insulin release from perifused rat islets. In the absence of these drugs, the addition of 15 mM glucose elicited a marked biphasic stimulation of insulin secretion concomitant with a reduction in the rate of 86 Rb + efflux. In the presence of either 500 μM tolbutamide or 100 μM quinine, a reduced rate of efflux of 86 Rb + was observed together with an elevated rate of insulin release. Under such conditions, the addition of 15 mM glucose retained the ability to stimulate insulin secretion though this was associated with a marked increase in 86 Rb + efflux. It is concluded that a net reduction in β-cell K + permeability is not an obligatory step in glucose-stimulated insulin release. Thus, glucose is likely to exert depolarizing actions on the β-cell in addition to the closure of K + channels.

Pretreatment with various doses of phenobarbital (PB) has been used to create a pool of rats with a wide range of hepatic microsomal monooxygenase activity to systematically examine relationships between and within in vivo and in vitro markers. The in vivo clearance of tolbutamide (TOL), theophylline (TH), antipyrine (AP) and its metabolites were determined in the same rats used for hepatic microsome preparation and assessment of P450 content and activities (via 7-ethoxycoumarin O-deethylase (ECOD), 7 ethoxyresorufin O-deethylase, 7-methoxycoumarin O-demethylase (MCOD) and aldrin epoxidase determinations). A graded dose-response relationship was found between PB treatment and most but not all parameters. The need for careful selection of in vivo and as well as in vitro markers is apparent from these studies. The most responsive parameters--TOL and AP clearances, MCOD and ECOD activities--were also those producing the strongest in vivo-in vitro correlations. Despite the diffuse nature of the PB induced response in P450 complement, good predictive relationships were apparent between ECOD and TOL clearance (r2 = 0.88).

Sulfonylureas inhibit an ATP-dependent K+ channel in the B-cell plasma membrane and thereby initiate insulin release. Diazoxide opens this channel and inhibits insulin release. In mouse pancreatic islets, we have explored whether other targets for these drugs must be postulated to explain their hypo- or hyperglycaemic properties. At non-saturating drug concentrations the rates of increase in insulin secretion declined in the order tolbutamide = meglitinide greater than glipizide greater than glibenclamide. The same rank order was observed when comparing the rates of disappearance of insulin-releasing and K+ channel-blocking effects. The different kinetics of response depend on the lipid solubility of the drugs, which controls their penetration into the intracellular space. Allowing for the different kinetics, the same maximum secretory rates were caused by saturating concentrations of tolbutamide, meglitinide, glipizide and glibenclamide. A close correlation between insulin-releasing and K+ channel-blocking potencies of these drugs was observed. The relative potencies of tolbutamide, meglitinide, glipizide and glibenclamide corresponded well to their relative affinities for binding to islet-cell membranes, suggesting that the binding site represents the sulfonylurea receptor. The biphasic time-course of dissociation of glibenclamide binding indicates a complex receptor-drug interaction. For diazoxide there was no correlation between affinity of binding to the sulfonylurea receptor and potency of inhibition of insulin secretion. Thus, opening or closing of the ATP-dependent K+ channel by diazoxide or sulfonylureas, respectively, appears to be due to interaction with different binding sites in the B-cell plasma membrane. The free concentrations of tolbutamide, glipizide, glibenclamide and diazoxide which are effective on B-cells are in the range of therapeutic plasma concentrations of the free drugs. It is concluded that the hypo- and hyperglycaemic effects of these drugs result from changing the permeability of the ATP-dependent K+ channel in the B-cell plasma membrane.

The binding of diflunisal to human serum albumin and normal human plasma has been studied by equilibrium dialysis at 37°, pH 4. The plasma protein binding data were analysed according to a Scatchard model with two independent classes of binding sites. The number of binding sites and the corresponding association constants have been estimated by nonlinear least-squares regression analysis: N 1 = 2.1, k 1 = 5.28 × 10 5 M −1 , N 2 = 7.7 and K 2 = 0.17 × 10 5 M −1 . At a difluni concentration of 50 μg/ml on average 99.83 per cent of the drug was bound to plasma proteins. The in vitro plasma protein binding of diflunisal was impaired by salicylic acid and phenprocoumon, while diflunisal itself was displaced from its primary binding sites in plasma by salicylic acid and bilirubin. Tolbutamide had no effect on the binding of diflunisal to plasma proteins.

The effects of acetylsalicylic acid, sulphadimidine, phenylbutazone and chlorpromazine on glucose- and tolbutamide-stimulated insulin release by isolated rat islets of Langerhans have been investigated. Acetylsalicylic acid did not influence glucose-stimulated insulin secretion; although at 2.5 mM it potentiated the effect of tolbutamide. Sulphadimidine potentiated the effects of glucose and tolbutamide on insulin secretion, whereas phenylbutazone was found to augment glucose-stimulated insulin release and to antagonize the tolbutamide effect. The effects of glucose and tolbutamide on insulin secretion were inhibited by low concentrations of chlorpromazine (0.005 mM–0.01 mM) and were stimulated by high concentrations (0.5 mM–1 mM). These results are discussed in relation to the possibilities that certain drugs may affect tolbutamide-stimulated insulin release either by altering the binding of tolbutamide to the B-cell or to other proteins, or by affecting the cyclic AMP system of the B-cell.

Glipentide, a second generation sulfonylurea, raised the cellular concentration of fructose 2,6-bisphosphate in isolated rat hepatocytes. Parallel to accumulating this regulatory metabolite, glipentide inhibited basal gluconeogenesis and increased the rate of L-lactate production, as well as the metabolic flux through the 6-phosphofructo 1-kinase reaction. Tolbutamide elicited similar metabolic effects to those reported for glipentide, although the latter sulfonylurea was about 10 times more potent. The biochemical mechanism by which sulfonylureas promote the accumulation of fructose 2,6-bisphosphate in hepatocytes seems to be related to a significant increase of the hexose 6-phosphate pool (glucose 6-phosphate plus fructose 6-phosphate), together with the activation of 6-phosphofructo 2-kinase and inactivation of fructose 2,6-bisphosphatase, enzyme activities responsible, respectively, for the synthesis and degradation of fructose 2,6-bisphosphate.

Tolbutamide (0.2 mg/ml), gliclazide (0.02 mg/ml), glipizide (0.005 mg/ml) and diazoxide (0.2 mg/ml) were found to inhibit glucose-induced proinsulin synthesis in isolated rat islets. The effect of these pharmacological agents was characterized by a preferential inhibition of proinsulin as distinct from other protein synthesis, and was associated with a reduced oxidation of glucose by the islets. The inhibitory effects were more marked at low (1.0 mg/ml) than at high (3.0 mg/ml) glucose concentration. These results indicate that the influence of sulfonylureas upon insular function cannot be ascribed merely to a facilitation of glucose metabolism or a cellular accumulation of adenosine-3′,5′-cyclic monophosphate.

Anti-human NADPH-cytochrome P-450 reductase inhibited all theophylline metabolic path-ways confirming the involvement of cytochrome P-450 isozymes in the metabolism of theophylline. Tolbutamide, debrisoquine, mephenytoin, theobromine, phenylbutazone, sulphaphenazole and sulphinpyrazone did not inhibit theophylline metabolism by human liver microsomes. Verapamil and dextropropoxyphene were non-selective competitive inhibitors of theophylline metabolism. Cimetidine and caffeine selectively inhibited the two demethylations as K i values for these two pathways were lower than for the 8-hydroxylation pathway. The effects of nifedipine, propranolol and alpha-naphthoflavone were atypical. The degree of inhibition by propranolol reached a plateau, which was greater for the two demethylations than for the 8-hydroxylation. Alpha-naphthoflavone (ANF) at low concentrations inhibited the demethylations to a greater extent than the 8-hydroxylation. At higher concentrations ANF activated all pathways, with this effect being most marked for the 8-hydroxylation. Nifedipine inhibited the theophylline demethylations but not the 8-hydroxylation. In some livers the 8-hydroxylation was markedly activated. The results confirm that there are at least two distinct cytochrome P-450 isozymes involved in theophylline metabolism, one isozyme being involved with the demethylations and a different isozyme involved in the 8-hydroxylation pathway. Preliminary correlation studies suggest that the human orthologue to the rabbit polycyclic hydrocarbon inducible P-450 Form 4 may be involved in the N -demethylations of theophylline.

To determine if both the Ca 2+ -calmodulin system and the cyclic AMP system may regulate tyrosine hydroxylase (TH) activity in situ , rat striatal tissue slices that contain all of the components of the TH, cyclic AMP and Ca 2+ -calmodulin systems were subjected to experimental manipulations. Incubation of striatal tissue slices in a medium containing W-7 [ N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], a specific inhibitor of calmodulin, resulted in a dose-dependent decrease of DOPA formation. The concentration of W-7 producing 50% inhibition (3 × 10 −5 M) of DOPA formation was in good agreement with the binding affinity of W-7 to calmodulin. W-7 did not affect TH activity in vitro or cellular cyclic AMP level. A structurally unrelated calmodulin antagonist, trifluoroperazine, also inhibited DOPA formation. On the other hand, incubation of striatal tissue slices in a medium containing dibutyryl cyclic AMP (DBcAMP) increased DOPA formation dose dependently. Kinetic analysis revealed that the enzyme in homogenates of control tissue slices had two different K m values for a cofactor, 6-methyl-5,6,7,8-tetrahydropterin (6MPH 4 ), indicating the presence of two forms in striatal tissue slices: a less active form with a relatively low affinity for the pterin cofactor and a more active form with a relatively high affinity. W-7 produced an increase of the high K m form and a decrease of the low K m form respectively. In contrast, incubation of tissue slices in the presence of DBcAMP resulted in almost complete activation of the enzyme to the low K m form. These results suggest that both the Ca 2+ -calmodulin-dependent system and the cyclic AMP-dependent system may regulate TH activity in the rat striatum in vivo . The different types of kinetic change produced by DBcAMP and W-7 indicate that the two processes may act in different fashions and that the basal level of catechol formation in striatal tissue slices may be dependent upon an activated form of TH.

The effects of tolbutamide and diazoxide on 86Rb+ fluxes, 45Ca2+ uptake, insulin release and B cell membrane potential have been studied in rat or mouse islets. In the presence of 3 mM glucose, tolbutamide rapidly and reversibly decreased Rb+ efflux from perifused islets and depolarised B cells. The effect on Rb+ efflux was paradoxically more marked with 20 than 100 micrograms/ml tolbutamide, at least in the presence of extracellular calcium. Addition of tolbutamide to a medium containing 6 mM glucose and calcium increased Rb+ efflux transiently with 20 micrograms/ml and permanently with 100 micrograms/ml. The drug also inhibited Rb+ influx in islet cells, but had little effect on Rb+ net uptake. Diazoxide rapidly, steadily and reversibly increased Rb+ efflux in a dose-dependent manner (20-100 micrograms/ml). When 20 micrograms/ml tolbutamide and diazoxide were combined in the presence of 3 mM glucose, only a slight decrease in Rb+ efflux was observed. The depolarisation of B cells normally produced by tolbutamide was markedly reduced and the electrical activity completely suppressed by diazoxide. In the presence of 10mM glucose, diazoxide increased Rb+ efflux from the islets and hyperpolarised B cells. Tolbutamide, tetraethylammonium and quinine reversed the increase in Rb+ efflux the inhibition of Ca2+ uptake and the suppression of insulin release produced by diazoxide. Tolbutamide rapidly reversed the hyperpolarisation and restored electrical activity. It is suggested that the stimulation and inhibition of insulin release by tolbutamide and diazoxide are due to their respective ability to decrease and to increase the K permeability of the B cell membrane. This change in K permeability leads either to depolarisation and stimulation of Ca2+ influx or to hyperpolarisation and inhibition of Ca2+ influx.

The prediction of tolbutamide-sulfonamide interaction from in vitro unbound intrinsic clearance was studied by comparing the in vvivo and in vitro total body clearance (CLtot) in rats. The The sulfonamides used were sulfaphenazole (SP), sulfadimethoxine (SDM) and sulfamethoxazole (SMZ). The plasma half-life ( T 1 2 ) of tolbutamine (TB) was increased ssignificantly by SP and SDM, while CLtot was decreased significantly by both drugs; no significant changes were observed in their parameter by SMZ. The in vitro plasma protein binding and microsomal oxidation of TB were competitively inhibited by sulfonamides; the order of inhibitor constantswas SMZ>SP>SDM. Since metabolism is the rate-determining step of the hepatic extraction of TB, the in vivoCLtot can be expressed by the equation; CL tot ⋍ ƒ B CL int , where ƒ B is the blood free fraction and CLint is the hepatic intrinsic clearance of unbound drug. A comparatively good agreement was observed between the in vivoCLtot and that calculated from both in vitro plasma protein binding and metabolic parameters.

l -α-(3,4-Dimethoxyphenethylaminomethyl)-2-hydroxybenzylalcohol 1 2 fumarate (TA-078) is a new hypoglycemic agent structurally different from any existing hypoglycemic drug. It depresses the rise of blood glucose when it is orally administered to glucose-loaded mice, rats and beagle dogs at minimal doses of 1, 10 and 2.5 mg/kg, respectively. In contrast with tolbutamide, TA-078 hardly affected fasting blood glucose levels in rats and dogs and only weakly reduced fasting blood glucose levels in mice. Oral administration of TA-078 to KK mice also improved glucose tolerance, while no improvement was observed in streptozotocin-diabetic rats. TA-078 elevated plasma immunoreactive insulin (IRI) levels in mice and rats soon after its oral administration. In fasted rats, TA-078 caused only a transient increase in plasma IRI but did not affect plasma immunoreactive glucagon (IRG) levels in the early phase after its administration. On the other hand, tolbutamide induced a sustained increase in plasma IRI and a transient but marked decrease in plasma IRG. In perfused rat pancreas, TA-078 stimulated insulin secretion. The stimulation by 10 μg/ml TA-078 in the perfusion liquid required the presence of a normal concn (5.6 mM) of glucose, whereas the same concn of tolbutamide stimulated insulin release even at a low glucose concn (2.8 mM).

The hypoglycemic sulfonylureas chlorpropamide, tolbutamide and carbutamide were found to decrease simultaneously and practically to the same extent, cellular levels of ATP and the incorporation of leucine into the protein of rat diaphragm. Chlorpropamide and tolbutamide also reduced ATP levels in liver slices. When diaphragm was pre-incubated with chlorpropamide or tolbutamide these effects persisted during subsequent incubation in the absence of the drugs. Cell-free mitochondrial and to a smaller extent also microsomal protein synthesis were inhibited by chlorpropamide and mitochondrial ATP was significantly decreased at 10−3 M drug concentration. The oxidation of sugars or Krebs-cycle substrates is not inhibited by sulfonylureas and does not explain the reduced energy stores. In diaphragm and liver the utilization of sugars is even enhanced by chlorpropamide and tolbutamide. Glucose, fructose and mannose relieve to some extent the inhibition of protein synthesis and the decrease of ATP levels by sulfonylureas. Insulin has the same effect and is therefore thought to stimulate energy conservation. Chlorpropamide and tolbutamide but not carbutamide increase oxygen uptake by rat liver mitochondria and abolish respiratory control. The fall in ATP and the inhibition of protein synthesis and other effects of sulfonylureas are supposedly the result of this uncoupling effect. The hypoglycemic activity of these drugs is discussed in the light of their effect upon mitochondrial metabolism.

At low mucosal concentrations the hypoglycaemic sulphonylureas, tolbutamide and glibenclamide, reduced active glucose transfer by sacs of everted rat jejunum. At high drug concentrations glucose metabolism was reduced to levels observed under anaerobic conditions. It is possible that the sulphonylureas disrupt intracellular supplies of ATP, thereby affecting the intestinal transport mechanism. Long-term oral administration of glibenclamide to rats had no effect on weight gain, blood sugar levels or transfer, uptake and metabolism of glucose by gut sacs in vitro . Incubation of an open-ended gut loop, allowing replacement of both mucosal and serosal fluids, demonstrated that response to drug exposure was slow and that recovery from exposure even to high concentrations possible. It therefore seems likely than an oral dose in man under normal conditions would not maintain a sufficiently high intraluminal concentration long enough for a significant change in glucose absorption. Even if some imbalance did occur, removal of the inhibitory influence would allow a return to normal conditions.

The rate of the in vivo oxidation of the antidiabetic drug tolbutamide (N- p -toluenesulfonyl-N'- n -butylurea) to N- p -carboxybenzenesulfonyl-N'- n -butylurea, as measured by the rate of the excretion of the oxidation product, was essentially unaffected by complete deuteration of the site of oxidation.

Induction of the liver microsomal cytochromes, P-450 and b 5 , and mitochondrial delta-aminolaevulinic acid (ALA) synthetase were attempted in mice by treatment with various porphyria-inducing chemicals and several other drugs known to precipitate acute attacks in human hepatic porphyria. On administration of porphyria-inducing chemicals except 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), the level of microsomal cytochromes showed a gradual increase after a transient decrease probably due to disturbed heme metabolism, and activity of ALA synthetase showed a marked increase concomitantly with the decrease of the level of cytochromes. In DDC poisoning, the level of cytochromes showed a persistent decrease, while the increase of ALA synthetase and porphyrin accumulation were most prominent. Treatment with drugs which precipitate acute attacks in human hepatic porphyria resulted in a marked increase of the level of cytochromes without showing a decrease phase, whereas the activity of ALA synthetase remained at a level 1.5–2 times higher than normal without an increase of porphyrin. Incorporation of 14 C-glycine into microsomal heme in vivo and activity of microsomal N -demethylase parallelled the level of cytochrome P-450. An impairment in synthesis of sufficient heme for hepatic hemoproteins in need of them and a positive feedback mechanism for induction of ALA synthetase were suggested as a possible cause of experimental porphyria.

The ATPase (ATP phosphohydrolase, EC 3.6.1.4) stimulating activity of sulfonylureas has been examined. Sulfonylureas stimulate ATPase to variable extents, and this stimulation is oligomycin-sensitive. They are less active than 2,4-dinitrophenol and their relative stimulating activity parallels their uncoupling potency on oxidative phosphorylation. Carbutamide, which in itself does not stimulate ATPase, inhibits ATPase stimulation by other sulfonylureas. Experiments with combinations of chlorpropamide and P594, two active compounds, suggest that P594 penetrates more readily into mitochondria and is a stronger uncoupling agent. All sulfonylureas inhibit ATPase stimulated by optimal concentrations of dinitrophenol. Possible implications of these observations with regard to the metabolic effects of sulfonylureas are discussed.

In isolated fat cells of the rat the in vitro and in vivo effects of tolbutamide on insulin binding and insulin response were studied. 450 mg tolbutamide/kg/day given for 7 days significantly increased the binding of insulin to isolated adipocytes. The binding curves reflected an increase in the number of receptor sites rather than in the affinity. The effect was associated with an enhanced response to insulin of the adipose tissue, since the fat cells obtained from animals treated with tolbutamide converted significantly more glucose to lipids in the presence of insulin than those obtained from the control group. However, the augmentation of insulin binding sites was observed only at a large tolbutamide dosage, which reduced the pancreatic insulin content, the secretory response of the isolated pancreas, and the serum insulin levels. Smaller doses, sufficient to produce metabolic effects via a stimulation of insulin secretion, did not provide additional insulin binding sites. When added in vitro to the binding assay or to adipose tissue incubated for 16 h, tolbutamide failed to increase insulin binding of the fat cells. It is suggested, therefore, that the effects produced by tolbutamide after in vivo treatment reflect an indirect rather than a direct action of the sulphonylurea.

To estimate the functional change occurring in human serum albumin by nonenzymatic glucosylation, glucosylated human serum albumin was prepared by in vitro incubation with glucose. The rate of glucosylation proceeded as a first-order reaction. The binding of sulfonylureas to serum albumin was determined by equilibrium gel filtration. Through this method, it was possible to estimate the binding capacity of a low water solubility drug with a high affinity to protein. The amounts of the sulfonylureas bound to glucosylated HSA decreased by 44% with tolazamide and acetohexamide, 50% with glibenclamide, and 52% with tolbutamide, compared to human serum albumin (HSA). This suggests that a high concentration of glucosylated HSA in diabetic patients may possibly cause an increase in free drug concentration exceeding normal levels. This study shows that the decrease in the binding capacity of sulfonylureas with protein is due to the modification of albumin molecules by the covalent binding of glucose.

The binding of tolbutamide, chlorproramide and glibenclamide to human and bovine serum albumin has been estimated in the presence of a number of acidic drugs. It is shown that agreement between experimental data and that calculated using the competitive binding equation is very poor. The degree of displacement of tolbutamide and chlorpropamide is much greater than that calculated using the equation while displacement of glibenclamide is much less. These findings suggest that displacement is essentially non-competitive and that glibenclamide is less susceptible to displacement by acidic drugs than tolbutamide or chlorpropamide.