Estimation of kinetic parameters in the inactivation of an enzyme by a suicide substrate.

A method was developed to estimate the extended Michaelis constant and maximum velocity of a suicide substrate from the time-course of remaining enzyme activity with the use of simulation data calculated from the representative kinetic model for a suicide substrate proposed by Walsh et al. (Walsh, C., Cromartie, T., Marcotte, P. and Spencer, R. (1978) Methods Enzymol. 53, 437-448). For this purpose an analytical equation for the time-course of remaining enzyme activity, based on the suicide kinetic model, was derived by the steady-state method reported by Tatsunami et al. (Tatsunami, S., Yago, N. and Hosoe, M. (1981) Biochim. Biophys. Acta 662, 226-235). The accuracy of this analytical solution was proved by comparing the result with the exact solution obtained by numerical computation. A method was also developed to estimate the most important factor for a suicide substrate, the partition ratio, from the time-course of remaining enzyme activity.

The behavior of remaining enzyme activity in a suicidal enzyme system.

We derived an equation which describes the plot of the remaining enzyme activity versus ratio of initial concentration of suicide substrate to that of enzyme to obtain a partition ratio from the time-course of remaining enzyme activity. The simulation data calculated from the representative kinetic model for a suicide substrate were used to verify this equation, which approximated steady state kinetics. Although the time-dependent loss of enzyme activity is usually characterized by pseudo-first-order kinetics, the present results show that pseudo-first-order kinetics are followed only when the ratio of initial concentration of suicide substrate to that of enzyme is greater than the partition ratio. Our results also show that the present method can be used to obtain the partition ratio of a suicide substrate from the time-course of the remaining enzyme activity when the suicide substrate is given an arbitrary concentration of one, where the ratio of initial concentration of suicide substrate to that of enzyme is less than the partition ratio. The theoretically verified equation was also checked against reported experimental data for a microsomal enzyme system.

The dithiane Ro 44-5912 enhances vinblastine sensitivity of drug resistant and parental KB lines in vivo.

The multidrug resistance modifying activity of a dithiane analogue of tiapamil, Ro 44-5912, was examined in vivo. Results of acute toxicity studies in mice indicated that lethal toxicity occurred with doses greater than 1 mmol/kg of body weight. In a preliminary pharmacokinetic investigation, Ro 44-5912 appeared to have a longer half-life in mice than did its (R) enantiomer Ro 44-5911 (3.15 +/- 0.02 h versus 2.15 +/- 0.14 h) as measured by total radiolabel in plasma. In non-tumour bearing mice, Ro 44-5912 enhanced the toxicity of vinblastine in a manner that was dependent on the dose of both drugs. Vinblastine did not have a significant effect on tumour growth when given to nude mice bearing the parental cell line KB-3-1 at a dose of 1.5 mg/kg once per week for 3 weeks. Combination treatment with Ro 44-5912 markedly enhanced the antitumour activity of vinblastine. Similar results were seen when KB-3-1 tumours were treated with the combination of vinblastine plus cyclosporin A. Another tiapamil analogue, Ro 11-2933, had no enhancing activity with this tumour when used at an equitoxic combination dose. Ro 44-5912 also significantly enhanced vinblastine activity with P-glycoprotein-expressing KB-8-5 tumours. In three independent experiments, Ro 44-5912 enhanced the growth inhibiting activity of vinblastine by a mean of approximately 40%. Neither Ro-11-2933 nor cyclosporin A, at the maximal tolerated doses in combination with vinblastine, led to significant inhibition of KB-8-5 tumour growth compared to treatment with the two vehicles alone. These results show that Ro 44-5912 is an active modulator of drug resistance in vivo.

Distribution and metabolism of L-3-O-methyldopa in rats.

1. After intraperitoneal administration of L-2-(14)C-3-O-methyldopa ((14)C-O-methyldopa) to rats, the amino-acid was distributed evenly in blood, brain, heart, adipose tissue and liver, whereas it accumulated more in the kidney and the pancreas. (14)C-O-methyldopa showed a biological half-life of about 12-13 h in blood, brain and heart.2. The concentration curve of (14)C-O-methyldopa in brain (after increasing doses of the amino-acid) was linear if measured 2 h after administration, but seemed to reach a plateau at the higher doses if determined after 16 h.3. The concentrations of (14)C-O-methyldopa metabolites (mainly homovanillic acid and 4-hydroxy-3-methoxyphenyllactic acid) were low, except in the kidney, and varied according to the tissue.4. Twenty-four hours after administration of (14)C-O-methyldopa, 33% of the injected radioactivity appeared in the urine. This radioactivity consisted of about 95% of metabolites (probably in the main (14)C-homovanillic acid and (14)C-4-hydroxy-3-methoxyphenyllactic acid) and of 5% of unchanged (14)C-O-methyldopa. In the faeces, 10% of the radioactivity appeared, mainly as metabolic end-products.5. It is concluded that (14)C-O-methyldopa easily penetrates from the blood into various tissues, including brain, and that the majority of the amino-acid undergoes a slow metabolism. The different shape of the concentration curves for (14)C-O-methyldopa in the brain after 2 and 16 h might indicate the presence of two tissue pools of the amino-acid.

A possible intraneuronal site of action of thymoleptics.

The uptake of catecholamines (CAs) into crude mitochondria preparations (P2 fractions) and vesicle preparations from rat hypothalamus and striatum were compared in terms of the inhibition by thymoleptics and other drugs. Thymoleptics preferentially inhibited the uptake of CAs into hypothalamic P2 fractions, while ATPase inhibitors preferentially inhibited the uptake of dopamine into striatal P2 fractions. When the preparation obtained from rats pretreated with reserpine was used, the preferential inhibition of hypothalamic uptake by thymoleptics was entirely abolished. When P2 fractions from both regions were incubated with 10(-6) M 14C-imipramine, the intrasynaptosomal distribution of labeled imipramine revealed its affinity not only to the synaptosomal membrane, but also to the synaptic vesicles. Accumulated 3H-norepinephrine (NE) could be released by a hypoosomotic shock from striatal P2 fractions, but not from hypothalamic P2 fractions. The ATP-Mg2+-dependent uptake of 3H-NE into the synaptic vesicles from rat brain stem was inhibited by desipramine. These results indicate that the inhibition of CA uptake by thymoleptics in the hypothalamus is predominantly due to the inhibition at the synpatic vesicle, while in the striatum the uptake at the synaptosomal membrane is predominantly inhibited.

Determination of endogenous GABA released from the cerebral cortex slices of the rat by high-performance liquid chromatography with a series-dual electrochemical detector.

A new, simple, rapid and sensitive method for the determination of ?-aminobutylic acid (GABA) has been developed by high-performance liquid chromatography with electrochemical detection (LCEC). A new and unique technique for LCEC by using the reductive-oxidative mode of a dual electrochemical detector provided a simple and sensitive assay method for GABA. The standard curve was linear over a concentration range of 1-1000 ng. The detection limit for GABA was less than 0.5 ng. This new method was adapted to the assay of the transmitter released endogenously from the cerebral cortical slices of the rat. Endogenous GABA release evoked by high K(+) was reduced when superfusion was performed in the presence of 100 ?M forskolin.

Dopamine-sensitive adenylate cyclase in the rat kidney particulate preparation.

Dopamine, apomorphine, isoproterenol and norepinephrine all increased the concentration of adenosine 3',5'-monophosphate in the rat kidney particulate preparation, which was composed of tubules, glomeruli and blood vessels. The concentrations of dopamine, apomorphine, isoproterenol and norepinephrine causing a half-maximal increase were 50, 83, 0.1 and 10 muM, respectively. The alpha-blocker, phentolamine, at a concentration as high as 1 mM, did not significantly reduce the effect of these drugs on the adenosine 3',5'-monophosphate concentration. The beta-blocker, propranolol (50 muM), blocked the effect of isoproterenol and norepinephrine, but not that of dopamine. The effect of dopamine was selectively blocked by spiroperidol (50 muM), a dopamine receptor antagonist, whereas the effects of isoproterenol and norepinephrine was not blocked by spiroperidol. These results suggest that in the rat kidney particulate preparation there is a specific dopamine receptor which can lead to the increase in the concentration of adenosine 3',5'-monophosphate.

Elevation of adenosine 3',5'-monophosphate in the perfusate of rat kidney after addition of dopamine.

Dopamine (10-4 M) and vasopressin (1 mU/ml) were found to increase the level of cyclic AMP in the perfusate of rat kidney. There were some differences in the mode of action of these two drugs. Firstly, the effect of dopamine, but not of vasopressin, was completely antagonized by spiroperidol. Secondly, the maximal response was attained within 1 min after dopamine perfusion, but 8 min after vasopressin perfusion. These results suggest that a specific dopamine receptor which acts to increase the concentration of cyclic AMP is located in the vascular tissue of rat kidney.

Simultaneous determination of a new anticancer drug galocitabine and its metabolites in blood by high-performance liquid chromatography.

A relatively simple and sensitive high-performance liquid chromatographic (HPLC) method is described for measuring galocitabine (Ro 09-1390) and its meatbolites, i.e. 5'-deoxy-fluorocytidine (5'-DFCR), 5'-deoxy-fluorouridine (5'-DFUR) and 5-fluorouracil (5-FU), in blood for the purpose of studying pharmacokinetics and toxicokinetics in small animals. The procedure for blood includes deproteinization with acetonitrile. Blood components were separated on a reversed-phase C18 column with a linear gradient of acetonitrile and water and detected at a wavelength of 270 nm. The between-day relative standard deviation (RSD) was less than 10% for all compounds at concentrations of 10-100 micrograms ml-1. The calibration curves obtained from the analysis of blood samples were linear and the correlation coefficients ranged from 0.997 to 0.999. The calculated determination limits were 6.9 micrograms ml-1 for galocitabine, 3.0 micrograms ml-1 for 5'-DFCR, 4.0 micrograms ml-1 for 5'-DFUR and 3.7 micrograms ml-1 for 5-FU.

The disposition of the tolcapone 3-O-methylated metabolite is affected by the route of administration in rats.

Catechol-O-methyltransferase (COMT) catalyses the transfer of the methyl group from S-adenyl-L-methionine (SAM) to one of the hydroxy groups of a catechol, usually the hydroxy group in position 3. COMT is present mainly in a soluble form (S-COMT) in the cytosol, but a small fraction is bound to cell membranes (MB-COMT). MB-COMT has higher affinity for the catechol substrate than does S-COMT by a factor of > 10, and high MB-COMT activity is observed in the intestinal muscle layer. The present study investigates the effect of the administration route on the disposition of the tolcapone 3-O-methylated metabolite following intravenous and oral tolcapone administration in rats. Tolcapone is a substrate for COMT although the 3-O-methylated metabolite produced has no pharmacological actions. The 3-O-methylated metabolite was eliminated very slowly following oral administration of tolcapone, and its concentration approached a plateau level, which was in contrast to the situation following intravenous administration of tolcapone. It is thought that the oral dose of tolcapone receives a high exposure to MB-COMT in the intestinal muscle layer during its absorption, and tolcapone seems to form a complex with MB-COMT having a high affinity constant (i.e. a very low Ki). The fraction of the intravenous dose of tolcapone metabolized to the 3-O-methylated metabolite at 10 mg kg-1 was 2.6%, whereas those of the oral doses, which were corrected by the bioavailability, were 5.4% for 20 mg kg-1 and 2.7% for 40 mg kg-1.

Lack of an effect of Madopar on the disposition of tolcapone and its 3-O-methylated metabolite in rats.

The effect of Madopar (benserazide and L-dopa, 1:4) on the disposition of the new selective inhibitor of catechol-O-methyltransferase, tolcapone, in rats was investigated. There was no statistically significant difference in the pharmacokinetic parameters of tolcapone in the presence or absence of Madopar except for a change in the mean residence time after oral administration. Thus, we rejected the hypothesis that the consumption of S-adenyl-L-methionine by Madopar would change the disposition of tolcapone. There were no statistically significant differences in the cumulative amount absorbed of drug and the absorption rate in the presence or absence of Madopar. We concluded that there was no interaction between tolcapone and Madopar.