Inhibition of mammalian tumour thymidylate synthetase by 5-alkylated 2'-deoxyuridine 5'-phosphates

Improved syntheses, based on Lewis acid-catalyzed nucleosidation, are described for the preparation of 5-alkyl-2'-deoxyuridines. These were converted to their 5'-phosphates with the use of wheat shoot phosphotransferase. The dUMP analogues, 5-ethyl-dUMP and 5-propyl-dUMP, were competitive vs dUMP inhibitors of thymidylate synthetase purified from mouse L1210, Ehrlich ascites and HeLa cells, the former being the stronger inhibitor. Both analogues were shown to bind cooperatively to each of the mouse tumour enzymes, two molecules of inhibitor interacting with a single enzyme molecule, as reflected by the parabolic character of the replots of the slope vs inhibitor concentrations. dTMP was a stronger inhibitor of the mouse tumour enzymes than its higher alkyl homologues.     

Pharmacokinetics of intact cisplatin in plasma. Infusion versus bolus dosing

Plasma levels of total platinum, total filterable platinum and intact cisplatin were monitored in 4 patients who received cisplatin in a regimen consisting of 20 mg/m² by i.v. bolus followed immediately by 80 mg/m² by 6 h infusion. Baseline pharmacokinetic parameters were obtained from a previous study in which 100 mg/m² of cisplatin was administered by a single i.v. bolus. These baseline pharmacokinetic parameters were used in an attempt to predict the pharmacokinetic behavior of cisplatin in the present study. The results demonstrated close agreement between observed and predicted plasma level-time profiles and the area under the plasma concentration-time profiles for cisplatin. The ratios of the various platinum species in plasma over the time course of the study were also consistent with those previously reported. These findings suggest that at a dose of 100 mg/m², the pharmacokinetics of cisplatin and its conversion to other species in plasma are independent of dosage schedule. Since 100 mg/m² is a relatively high dose of cisplatin, it is likely that this approach is applicable to other doses and schedules, and ultimately might prove useful in designing optimum cisplatin dosage regimens.     

5-substituted 2′-deoxyuridines: Correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase

A large variety of 5-substituted 2′-deoxyuridines (dUrds) and 2′-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their K_i/K_m values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP > 5-formyl-dUMP > 5-fluro-dUMP > 5-oxime of 5-formyl-dUMP > 5-azidomethyl-dUMP > (E)-5-(2-bromovinyl)-dUMP. The k_i/K_m values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37°). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5′-monophosphates followed by an inhibition of dTMP synthetase.     

Toxicity-distribution relationships among 3-alkylfurans in the mouse lung

The present study was designed to investigate the extent to which a homologous series of 3-alkylfurans, 3-methylfuran, 3-ethylfuran, and 3-pentylfuran, may cause lung injury in mice in order to determine whether the chemical properties of these compounds are related to their toxic potential. The pulmonary concentration and pneumotoxicity of these and various other furan derivatives were also measured to determine if a correlation existed between the magnitude of pneumotoxicity produced by these toxins and their concentration in the lung. Along with the 3-alkylfurans, the other furan derivatives investigated were furan, 2-ethylfuran, 2-furamide, and 3-methylthiophene. The absence or presence of lung damage was evaluated by light microscopy. The quantitative index used to assess and rank the compounds as pneumotoxins was the incorporation of [14C]thymidine into DNA. The results obtained with the 3-alkylfurans showed that 3-methylfuran and 3-ethylfuran were toxic to the lung whereas 3-pentylfuran did not produce pneumotoxicity. 2-Ethylfuran, furan, and 2-furamide also caused lung damage but 3-methylthiophene did not. All the compounds, with the exception of furan, reach the lung in comparable concentrations; therefore, there does not seem to be a correlation between pneumotoxicity and concentration of the toxin in the lung. From these studies, it is also apparent that the pneumotoxicity of the 3-alkylfurans extends beyond the methyl group but that the toxicity decreases with increasing chain length.     

In vivo inhibition of hepatic lipogenesis in the rat by cyclandelate (3, 3′, 5-trimethylcyclohexanylmandelate)

Rates of hepatic lipogenesis were measured in vivo in rats by incorporation into lipids of [³H] from injected [³H]H₂O 17 hr after a single oral dose of cyclandelate (3, 3′,5-trimethylcyclohexanylmandelate, a vasoactive substance). Cyclandelate administration resulted in a significant inhibition (40–60%) of both sterol and fatty acid synthesis in the livers which was independent of the 3.2-fold diurnal variation in the rates of hepatic sterol and fatty acid synthesis. The inhibition of accumulation of newly synthesized fatty acid in intestine also reached statistical significance. The accumulation of newly synthesized sterol was significantly depressed in serum but did not result in any change in the concentration of serum total cholesterol. These results are interpreted in terms of the inhibitory effect of cyclandelate on hepatic 3-hydroxy-3-methylglutaryl-CoA reductase previously reported by us (Biochem. Pharmac. 32, 649, 1983).     

Phospholipid-sensitive Ca2+-dependent protein kinase inhibition by R-24571, A calmodulin antagonist

R-24571 (calmidazolium), a derivative of the antimycotic agent miconazole, inhibited phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK), with an ic₅₀ (the concentration causing 50% inhibition) of 5.3 μM. It also inhibited the calmodulin/Ca²⁺-stimulated enzymes, with ic₅₀ values of 1.6 and 0.1 μM for myosin light chain kinase (MLCK) and phosphodiesterase respectively. Analysis of inhibition by R-24571 of PL-Ca-PK and MLCK revealed complex kinetics, suggesting that the agent interacted with the cofactors, the enzyme, and/or the cofactor-enzyme complexes. At saturating concentrations of the cofactors, R-24571 inhibited PL-Ca-PK and MLCK noncompetitively with their respective cofactors. Inhibition of MLCK by R-24571 was completely overcome by phosphatidylserine, indicating a strong hydrophobic interaction between R-24571 and the phospholipid in the presence of calmodulin. R-24571 also inhibited phosphorylation of various endogenous proteins in brain stimulated specifically by phosphatidylserine/Ca²⁺ or calmodulin/Ca²⁺. The present findings inducated that R-24571 has little specificity in inhibiting two types of Ca²⁺-dependent protein kinases sensitive to phospholipid or calmodulin.     

Hydralazine inhibition of the post-translational hydroxylation of deoxyhypusine, a polyamine-derived amino acid

Logarithmically growing Chinese hamster ovary cells, cultured in the presence of [1,4-14C]putrescine, synthesize a protein(s) containing the unusual amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This protein was separated and identified by two-dimensional gel electrophoresis and fluorography. The labeled hypusine isolated from an acid hydrolysate of the cell protein by ion exchange chromatography was identified by oxidative degradation and analyses of the products. Hydralazine, one of the most frequently prescribed drugs for the treatment of moderate to severe hypertension, added to the culture, resulted in the accumulation of a protein(s) containing the precursor amino acid deoxyhypusine [N epsilon-(4-aminobutyl)lysine]. Demonstration of this intermediate and its subsequent conversion to hypusine suggests that the synthesis occurs in several steps, one of these involving a hydroxylation reaction which can be inhibited by hydralazine.     

Enzyme inhibition and the toxic action of moniliformin and other vinylogous α-ketoacids

The inhibition of two thiamine-requiring enzymes by the potent mycotoxin, moniliformin (1-hydroxycyclobutene-3,4-dione), was investigated. Rat brain transketolase and pyruvate dehydrogenase were inhibited 25 percent by 10^?9 M moniliformin. Studies carried out to determine if moniliformin causes enzyme inhibition by reaction with thiamine were negative. Varying the hydroxycyclobutenedione structure by substitution or ring expansion resulted in loss of toxicity and inhibition.     

Selective inhibition of rat pulmonary monooxygenase by O, O, S-trimethyl phosphorothioate treatment

The effects of oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in widely used organophosphorus insecticides, were studied using pulmonary and hepatic microsomal enzymes of rats. The animals were treated with OOS at 10, 20 and 40 mg/kg, and were killed on day 3 after treatment. Their relative lung weights increased markedly at 20 and 40 mg/kg, increasing 94% at the highest dose, whereas the weight of liver decreased. At 20 mg/kg OOS, the cytochrome P-450 content of the lung and liver decreased to 83 and 80% of the control levels respectively. Pulmonary microsomal 7-ethoxycoumarin (7-Ec) O-deethylase decreased in a dose-dependent manner; activities were less than 10% of control at the 40 mg/kg dose. The activity of pulmonary coumarin hydroxylase also decreased following OOS treatment, but the decrease was not dose-dependent since no activity was detectable at doses over 10 mg/kg. In contrast, the effect of OOS treatment on hepatic monooxygenase activity was moderate. 7-Ec deethylase activity was not affected by OOS treatment at any dose level, while p-nitroanisole (p-NA) demethylase activity was decreased only at the 40 mg/kg dose of OOS. Pulmonary malathion carboxylesterase activity was not affected by OOS treatment. In contrast, a dose-dependent decrease was observed in the liver carboxylesterase. Time course effects of OOS treatment on these parameters were examined by treating rats at 20 mg/kg. The animals were killed 0.5, 1, 3 and 7 days after the treatment. The 7-Ec deethylase activity of pulmonary microsomes was decreased on days 0.5, 1 and 3 after treatment, the maximum decrease being observed on day 1. Significant decreases were not observed in hepatic microsomal activities of 7-Ec deethylase or p-nitroanisole demethylase throughout the experimental period; rather, these activities were higher on day 7. Hepatic microsomal malathion carboxylesterase was lower on days 0.5, 1 and 3 after OOS treatment.     

Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (K_i = 0.2–1.2 μM) of any compound reported to date and V_max = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (K_i = 10–30 μM) and greatly decreased substrate activity (V_max < 1 per cent relative to inosine). The K_i of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a K_i value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (V_max < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.     

Detection and determination of active metabolites of 1-(2-o-chlorobenzoyl-4-chlorophenyl)-5-glycyl-aminomethyl-3-dimethy l-carbamoyl-1H-1,2,4-triazole hydrochloride dihydrate, (450191-S), in rat tissues, using a radioreceptor assay for benzodiazepines

1-(2-o-Chlorobenzoyl-4-chlorophenyl)-5-glycyl-aminomethyl-3- dimethylcarbamoyl -1H-1,2,4-triazole hydrochloride dihydrate, (450191-S), exhibits pronounced central nervous system (CNS) activities similar to those of benzodiazepines, but it has only low affinity for benzodiazepine receptors. However, when 450191-S was administered to rats at a dose of 10 mg/kg, brain extracts markedly inhibited [3H]diazepam binding to the receptors. Thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), and radioreceptor assay (RRA) were used to isolate three metabolites that could inhibit [3H]diazepam binding prominently. These were identified by gas chromatography-mass spectrometry (GC/MS) as compounds having the triazolo-benzodiazepine skeleton. They showed high affinities for benzodiazepine receptors (Ki = 0.9 to 2.1 nM) and exerted potent pharmacological effects similar to those of 450191-S. In addition, their levels in the brain were sufficient to explain the pharmacological activity of 450191-S, which could not be detected in tissue extracts 15 min after administration. These results indicate that the pharmacological activity of 450191-S is largely due to the action of active metabolites, although some points remain to be elucidated to fully account for the large attenuation of the side effect (ataxia) compared with the major effects (anti-convulsant and hypnotic). We also determined the brain levels of metabolites following the administration of 450191-S and evaluated the extent to which each active metabolite contributes to the pharmacological activities of this drug.     

Cell cycle specific fluctuations of adenosine 3',5' -monophosphate and prostaglandin binding in synchronized mastocytoma P-815 cells

Endogenous adenosine 3',5' -monophosphate (cAMP) levels in mastocytoma P-815 cells, synchronized either at the G1/S transition by amethopterin- or double thymidine-block or in mitosis by colcemid block, were highest during late S and early G2 phases and lowest during mitosis. These cell cycle-dependent changes in cAMP levels were largely accounted for by changes in adenylate cyclase and phosphodiesterase activities. Similar fluctuations occurred simultaneously with specific prostaglandin E1 (PGE1) binding, histidine decarboxylase activity, histamine content, and [35S]SO-2(4) incorporation into glycosaminoglycans of the cells. In addition, endogenous levels of the E group of prostaglandins (PGEs) and "14C]carachiodonic acid incorporations into PGE, phosphatidylcholine and phosphatidylinositol also exhibited fluctuation patterns similar to that of cAMP levels. Since cAMP levels still fluctuated in a serum-depleted medium where DNA synthesis and cell division were inhibited, endogeneous levels of prostaglandin and cAMP appeared not to be regulated solely by serum factor(s). Exposure of cells at G1/S transition to 1-methyl-3-isobutylxanthine (MIX) resulted in 10-fold elevation of cAMP levels throughout the cell cycle without affecting DNA synthesis. On the other hand, PGE1 and/or MIX added at late S phase elevated cAMP levels, prolonged C2 phase and retarded the cell division, but these agents added at the beginning of mitosis elevated cAMP levels without affecting the cell division. These results suggest that prostaglandin newly synthesized by the increased metabolism of phospholipids promote the cAMP synthesis via their binding to the receptors and thereby control the division and phenotypic expression of mastocytoma P-815 cells.     

Effects of vinca alkaloids on calcium-calmodulin regulated cyclic adenosine 3' ,5'-monophosphatase phosphodiesterase activity from brain

The effects of a series of vinca alkaloids on calcium-calmodulin regulated brain cyclic adenosine 3',5'-monophosphate phosphodiesterase (PDE) activity were examined. The alkaloids tested included the dimeric indole alkaloids, vinblastine, vincristine and desacetylvinblastine amide, and the monomeric alkaloids, catharanthine and vindoline. The order of magnitude of the inhibitory effects on the calcium-calmodulin stimulated activity of phosphodiesterase was vinblastine > desacetylvinblastine amide s > vincristine = catharanthine > vindoline. In contrast, both catharanthine and vindoline were more potent inhibitors than the dimeric vinca alkaloids of the basal unstimulated phosphodiesterase activity. Vinblastine and vincristine inhibition of calcium-calmodulin activated partially purified PDE was non-competitive with substrate. In contrast, the inhibitory actions of vinblastine, desacetylvinblastine amide, vincristine and catharanthine, but not of vindoline, were competitive with calcium-calmodulin. Colchicine inhibited both activated and basal phosphodiesterase activity. The inhibitory effect of colchicine was not reversed by the addition of either calmodulin or calcium. These results suggest that the calcium-calmodulin dependent inhibition of PDE activity by the dimeric vinca alkaloids had the greater specificity, and that this inhibitory action required the catharanthine moiety. In view of these results, dimeric vinca alkaloids may provide a useful tool for the elucidation of the physiological role of calcium-calmodulin.