The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine.

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.

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.

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.

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.

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 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.

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.

Determination of 5-fluorouracil in plasma and liver after oral administration of 5'-deoxy-5-fluorouridine using gas chromatography-mass spectrometry.

A method for determination of 5-fluorouracil (5-FU) has been established for pharmacokinetic study of 5'-deoxy-5-fluorouridine (5'-DFUR), a newly developed prodrug of 5-FU. 5-FU was extracted with diethyl-ether containing 1-propanol and methylated with CH3I in the presence of (CH3)4NOH. The methylated 5-FU was analyzed by gas chromatography-mass spectrometry using 15N2-5-FU as an internal standard. Quantitation was carried out by selected-ion monitoring of molecular ions of N,N-dimethyl-5-FU (m/z 158) and the internal standard (m/z 160). The sensitivity (greater than 2 ng/g sample), specificity and precision of the method were satisfactory for application to clinical studies of this drug. After an oral administration of 5'-DFUR (800 mg/body) to patients with cancer, 5-FU in plasma peaked within 1 h and eliminated with an apparent half life of about 1 h.

Calmodulin antagonistic action of the cerebral circulation improver 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)-4- ([4-(2-methoxyphenyl)-1-piperazinyl]methyl)isoquinoline.

6,7-Dimethoxy-1-(3,4-dimethoxybenzyl)-4-([4-(2-methoxyphenyl)-1- piperazinyl]methyl)isoquinoline (Ro 22-4839) is a new cerebral circulation improver with vasospasmolytic properties. Preliminarily, Ro 22-4839-induced arterial relaxation was confirmed under the treatment of various constrictors and it was hardly overcome by addition of extra calcium. In this study the mode and site of action of this agent were further explored. Ro 22-4839 was found to more strongly inhibit the superprecipitation of chicken gizzard smooth muscle actomyosin (IC50 = 2.0 mumol/l) than trifluoperazine (38 mumol/l) and W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide) (220 mumol/l), an in vitro model for relaxation-contraction coupling of the smooth muscle in which calmodulin is known to play an important role through phosphorylation of myosin light chain kinase. The calmodulin antagonistic action of Ro 22-4839 was also demonstrated in other calmodulin-related reaction systems such as phosphodiesterase and hydrophobic fluorescent probe, but was very weak in Ca2+, Mg2+-ATPase of rat erythrocyte membrane. Thus, Ro 22-4839 was suggested to have a relative preference for smooth muscle contraction process unlike trifluoperazine and W-7. Moreover, Ro 22-4839 prevented the decrease in erythrocyte deformability induced by hyperosmolarity or intracellular Ca2+ accumulation, like trifluoperazine and W-7. However, Ro 22-4839 itself caused hardly an internal stomatocytic shape of erythrocytes in contrast to known calmodulin antagonists. Further, Ro 22-4839 inhibited erythrocyte membrane rupture, platelet aggregation and lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Polysaccharides in fungi. XIV. Anti-inflammatory effect of the polysaccharides from the fruit bodies of several fungi.

Anti-inflammatory assays on the carrageenin-induced edema and scald-induced hyperalgesia in the hindpaw of rats were studied on polysaccharides obtained from the fruit bodies of various fungi (polysaccharide AC, BC: Tremella fuciformis;MEA, MHA, MCW-A, MCW-N: Auricularia auricula-judae; T-2-HN: Dictophora indusiata;G-A: Ganoderma japonicum). The purified polysaccharides MHA, MCW-A, G-A and T-2-HN exhibited a significant inhibitory effect on carrageenin edema. Among these polysaccharides, T-2-HN (partially O-acetylated alpha-D-mannan) also showed the marked inhibitory effect on scald hyperalgesia. We have found that T-2-HN has more potent anti-inflammatory activity than phenylbutazone in the above two inflammatory models. Since the purified polysaccharide is free from protein and lipid, it is clear that anti-inflammatory effect arises from the polysaccharide itself. The polysaccharides (T-2-HN, locust bean gun, xanthan gum) had little effect on the metabolism of arachidonic acid in canine platelets. The mechanism of the anti-inflammatory activity of the polysaccharide remains obscure.

Ex vivo 3H-spiroperidol binding to rat striatum and the inhibitory effects of neuroleptics.

Neuroleptic drugs were given to rats and at different time intervals the animals were decapitated and 3H-spiroperidol binding to the striatal homogenates was determined in vitro. Two hours after an intraperitoneal administration of spiroperidol, perphenazine, haloperidol, chlorpromazine or thioridazine in doses of 0.03-30 mg/kg, 3H-spiroperidol, binding to the striatal homogenates was inhibited dose-dependently with an ID50 value of 0.11, 0.23, 1.1, 3.7 or 9.4 mg/kg, respectively. After a single intramuscular administration of fluphenazine enanthate (10 mg/kg), a long-acting neuroleptic drug, long-lasting inhibitory effect (over 4 weeks) on the binding was also observed. These results indicate that ex vivo 3H-spiroperidol binding method may be useful to measure the duration and the potency of anti-dopaminergic activities of drugs.

Characterization with 3H-haloperidol of the dopamine receptor in the rat kidney particulate preparation.

The dopamine receptor of rat kidney particulate preparation was identified and characterized by the use of 3H-haloperidol binding. Binding of 3H-haloperidol to the kidney particulate preparation was slow and saturable. The dissociation constants (KD) were 0.41 nM and 5.88 nM, respectively, according to the model of two classes of independent binding sites. Maximal binding of high affinity site was obtained with 166 fmol/mg protein which was about 40% of the total receptor density. A wide variety of neuroleptics at specifically low concentrations in nanomolar range inhibited the 3H-haloperidol binding. There was an excellent correlation between the affinity of numerous neuroleptics for the kidney particulate preparation and that for the brain striatum.

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.

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.