Effect of (E)-5-(2-bromovinyl)uracil on the catabolism and antitumor activity of 5-fluorouracil in rats and leukemic mice.

In contrast to thymine and 5-fluorouracil (FUra) which were cleared from the bloodstream within 2-4 h after their i.p. administration (200 mumol/kg) to rat, (E)-5-(2-bromovinyl)uracil (BVUra) maintained a concentration of 50-70 microM for at least 6 h and was still present in the plasma 24 h after its administration. In vitro experiments with rat liver extracts indicated that BVUra was not a substrate but an inhibitor for the reductive step in pyrimidine degradation catalyzed by dihydrothymine dehydrogenase. Kinetic and dialysis experiments suggested that BVUra was an irreversible inhibitor of this enzyme. The binding of BVUra to the enzyme depended on the presence of reduced nicotinamide adenine dinucleotide phosphate in the reaction mixture. Dihydrothymine dehydrogenase activity was also inhibited in the dialysed 105,000 X g supernatant fraction of livers from rats that had previously been treated with BVUra. Such inhibitory effects also occurred in vivo; previous administration of BVUra increased the plasma half-lives of thymine and FUra by 10- and 5-fold and their area under the curve by 9- and 8-fold, respectively. The effect of BVUra on the antitumor activity of FUra was evaluated in DBA/2 mice inoculated with 10(6) P388 leukemia cells. The mean survival times for the control and FUra-treated mice (5 mg/kg at 1, 3, 5, and 7 days after tumor cell inoculation) were 9.7 and 12.4 days, respectively. When BVUra (200 mumol/kg) was administered 1 h before each injection of FUra, the mean survival time was extended to 17.1 days. BVUra alone did not affect the mean survival time. When the dose of FUra was increased to 20 mg/kg, the mean survival time was 15.3 days; upon a preceding injection of BVUra the mean survival time decreased to 9.2 days. The latter effect probably resulted from an increased toxicity of FUra. Similar results were obtained if FUra was replaced by 5-fluoro-2'-deoxyuridine and BVUra by (E)-5-(2-bromovinyl)-2'-deoxyuridine. The enhancement of both the antitumor and toxic effects of FUra by BVUra were most probably due to an inhibition of FUra degradation, since, like in rats, BVUra increased the plasma half-life of FUra in DBA/2 mice. Hence BVUra appears to be an interesting compound, increasing the potency of FUra by decreasing its degradation.

Catabolism of thymidine in human blood platelets: purification and properties of thymidine phosphorylase.

A pyrimidine nucleoside phosphorylase was partially purified from human blood platelets. The purified enzyme, as well as crude enzyme preparations, catalyses the phosphorolysis of thymidine and deoxyuridine, but not of uridine, and is able to catalyse direct pentosyl transfer from these deoxyribonucleosides to uracil or thymine; this enzyme has the properties of a thymidine phosphorylase. It has a molecular weight of about 110,000 and is composed of two identical subunits; it is phosphate dependent, has a maximal activity at a pH value of 5.7, and an isoelectric point of 4.4. This enzyme was mainly of cytoplasmic origin. Although platelet thymidine phosphorylase could promote the degradation or synthesis of thymidine, intact platelets degraded thymidine but were not able to synthesize thymidine from thymine. Blood platelets may play an important role in the degradation of plasma thymidine.

Influence of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on cell cycle progression and proliferation of cultured arterial smooth muscle cells.

8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative inhibitor of intracellular calcium mobilization, causes a dose-dependent inhibition of serum-induced proliferation of arterial smooth muscle cells in culture. Neither early rise in cytosolic calcium concentration nor induction of early induced cell cycle dependent genes (c-fos, ornithine decarboxylase) are inhibited after serum stimulation in presence of 100 microM TMB-8. In contrast, expression of thymidine kinase, a gene normally induced in late-G1 phase, is entirely inhibited by TMB-8. Taken together with flow cytometry studies, these results indicate that TMB-8 blocks cell cycle progression in mid- or late-G1 phase by a mechanism not directly related to early responses to serum stimulation since TMB-8 is also effective when introduced several hours after serum stimulation.

The human blood platelet: a cellular model to study the degradation of thymidine and its inhibition.

Intact platelets catabolize extracellular thymidine into thymine. Studies of the concentration dependent degradation of thymidine by intact platelets indicate a Michaelis mechanism with an apparent Km of about 0.12 mM and a Vmax of 2.5 nmoles/min for 3 X 10(8) platelets. This degradation process is inhibited by various nucleosides, pyrimidine bases and C-5 or C-6 substituted uracils. Cytidine, deoxycytidine, adenosine and deoxyadenosine seem to inhibit thymidine degradation by reducing the intracellular transport of thymidine. Uridine inhibits both the thymidine transport and the activity of the phosphorolytic enzyme, thymidine phosphorylase (EC 2.4.2.4). Some substituted uracils are specific inhibitors of thymidine phosphorylase activity. 6-Amino-5-bromouracil, the most active of them, either with acellular extracts or purified thymidine phosphorylase, is also the best inhibitor of thymidine degradation in intact human platelets. Platelets constitute a new model to study the efficiency of specific inhibitors on thymidine catabolism in an 'human intact cell' which contains only one pyrimidine nucleoside phosphorylase, the thymidine phosphorylase.

Phosphorolysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted-2'-deoxyuridines by purified human thymidine phosphorylase and intact blood platelets.

Various 5-substituted-2'-deoxyuridines (dUrd), including 5-ethyl,5-propyl-, 5-trifluoromethyl-, 5-hydroxymethyl-, 5-formyl-, 5-vinyl-, (E)-5-(2-chlorovinyl)-, (E)-5-(2-bromovinyl)-, 5-fluoro-, 5-chloro-, 5-bromo-, 5-iodo-, 5-cyano-, 5-thiocyano-, 5-nitro- and 5-amino-dUrd, were shown to be effective substrates for the thymidine (dThd) phosphorylase isolated from human blood platelets. Some of dUrd analogs, i.e. the highly potent and selective antiherpes agent (E)-5-(2-bromovinyl)-dUrd, were degraded more rapidly than the natural substrates, dUrd and dThd. All dUrd analogs were also readily catabolised by intact human blood platelets. The potent inhibitors of thymidine phosphorylase, 6-amino-thymine and 6-amino-5-bromo-uracil, strongly inhibited the phosphorolysis of (E)-5-(2-bromovinyl)-dUrd by both purified enzyme and intact platelets.

Deoxyribosyl exchange reactions leading to the in vivo generation and regeneration of the antiviral agents (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine and 5-(2-chloroethyl)-2'-deoxyuridine.

In the rat, the highly potent anti-herpes drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd) is rapidly converted to its base (E)-5-(2-bromovinyl)uracil (BVUra) through the action of pyrimidine nucleoside phosphorylases. However, BVdUrd can be regenerated or even generated de novo from BVUra by a pentosyl transfer reaction upon the administration of 2'-deoxythymidine (dThd), 2'-deoxyuridine (dUrd) or 5-ethyl-2'-deoxyuridine (EtdUrd). The antiherpetic drugs EtdUrd and 5-(2-chloroethyl)-2'-deoxyuridine (ClEtdUrd) can also be regenerated or generated de novo from their respective bases 5-ethyluracil (EtUra) and 5-(2-chloroethyl)uracil (ClEtUra), by a pentosyl transfer mediated by the administration of dThd or dUrd as deoxyribosyl donor. The generation or regeneration of BVdUrd, EtdUrd and ClEtdUrd from their bases (BVUra, EtUra and ClEtUra, respectively) is readily achieved because the latter have long half-lifes. Thus, the active anti-herpes drugs can be (re)generated repeatedly after a single administration of these nucleosides or their bases, followed by repeated administrations of dUrd.

[Study of the extracellular macromolecular meshwork elaborated by rat aortic cells in secondary culture]. / Etude de la trame macromoléculaire extracellulaire élaborée par des cellules aortiques de rat en culture secondaire

Rat aortic smooth muscle cells isolated by digestion of the vessels by elastase and trypsin and grown in subculture, are examinated by phase, optic and electron microscopy for their ability to synthesize connective tissue components. Large amounts of extracellular material accumulates within the spaces between the cell; it consists of amorphous substance identified histochemically as elastin, of 110 A microfibrils and of periodic fibrils (430-490 A); the chemical nature of these two last components is discussed.

[Enzyme alterations of the arterial wall following the combination of a lathyrogenic treatment and of moderate hypercholesterolemia in the young rabbit]. / Altérations enzymatiques de la paroi artérielle consécutives à l'association d'un traitement lathyrogène et d'une hypercholestérolémie modérée chez le lapin

To determinate the part of humoural and parietal factors in atherosclerotic injury genesis, metabolism alteration study is realised on aortic cell wall of rabbits which are submitted to a chronical lathyritic intoxication alone, or simultaneously or alternatively associated with a cholesterolemic diet. Catabolic activity increase of beta-glucuronidase occurs in hypercholesterolemic rabbits. Beta-aminoproprionitrile, lathyrogenic drug used, stimulates biosynthetic pathways: increase of soluble proteins, energetic enzyme activities (lacticodeshydrogenase, malicodeshydrogenase), conjonctival protein metabolism (procollagen lysyl hydroxylase); in the same time, cell wall disturbances and lipidic deposits are facilitate when rabbits are submitted to cholesterolemic diet.

Regeneration of the antiviral drug (E)-5-(2-bromovinyl)-2'-deoxyuridine in vivo.

The highly potent and selective antiherpes drug BVdUrd [(E)-5-(2-bromovinyl)-2'-deoxyuridine] is cleared within 2-3 hours from the bloodstream upon intraperitoneal administration to rats. It is degraded to BVUra [(E)-5-(2-bromovinyl)uracil] and this inactive metabolite is cleared very slowly from the bloodstream so that 24 hours after the administration of BVdUrd, BVUra is still detectable in the plasma. This contrasts with several other 5-substituted uracils, i.e. 5-fluorouracil, 5-iodouracil, 5-trifluorothymine and thymine itself, which are, like their 2'-deoxyuridine counterparts FdUrd, IdUrd, F3dThd and dThd, cleared from the plasma within 2-3 hours. The injection of dThd or any of the other 5-substituted 2'-deoxyuridines at 3 hours after the injection of BVdUrd, that is at a time when BVdUrd has disappeared completely from the circulation, results in the re-apparition of BVdUrd in the plasma. Apparently, BVdUrd is regenerated from BVUra following the reaction catalyzed by pyrimidine nucleoside phosphorylases : BVUra + dThd----BVdUrd + Thy. BVdUrd can even be generated de novo if dThd (or FdUrd, IdUrd or F3dThd) are administered 3 hours after a preceding injection of BVUra. These findings represent a unique example of the (re)generation of an active drug from its inactive metabolite in vivo.

[Influence of a lathyric agent and of hypercholesterolemic serum on cell cultures of fetal rabbit aorta]. / Influence d'un agent lathyrique et d'un sérum hypercholestérolémique sur des cultures cellulaires d'aorte de foetus de lapin

Cell cultures of foetal rabbit aorta are cultivated with a lathyric agent (beta-amino-propio-nitrile) or with an hypercholesterolemic serum; if morphological features, in these two cases, correspond with modifications observed, in vivo, when adult rabbits are respectively submitted to the same treatment, enzymatic activities of collagen metabolism vary in opposite way. Therefore, the influence of different parameters to be studied on vascular cell functions become easier.

Influence of cell density on collagen biosynthesis in fibroblast cultures.

Characteristic features of collagen metabolism in human skin fibroblasts were studied in relation to cell density. Measuring peptide-bound hydroxyproline we found that collagen synthesis per cell decreased when cultures approached confluency. On the other hand, the relative rate of collagen synthesis (collagen/total protein) was higher in quiescent than in proliferating cultures. With increasing cell density the proportion of type III collagen in comparison with type I was found to be slightly increased. In addition, in low-density cultures [alpha I(I)]3 collagen trimers were produced in considerable amounts, whereas they were no longer detected in cultures with a high cell density. Although hydroxylation of proline residues was normal in all cell stages, conversion of procollagen into collagen was found to depend strongly on the density at which the cells were investigated. Almost no cleavage of procollagen peptides was observed in rapidly growing cells, whereas highly confluent cell cultures converted most of the newly synthesized procollagen molecules.

Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells.

In addition to the well established cyclooxygenase pathway, cultured aortic smooth muscle cells convert arachidonic acid to several polar metabolites identified by high performance liquid chromatography and gaz chromatography-mass spectrometry. 15-Hydroxyeicosatetraenoic acid, 12-Hydroxyeicosatetraenoic acid and 5-Hydroxyeicosatetraenoic acid are the major products formed. These observations indicate that the rabbit aortic smooth muscle cells are a potential source of lipoxygenase products and raise the possibility that this pathway of arachidonic acid metabolism can influence the biological functions of arterial myocytes under normal and pathological conditions.

[Partial purification and characterization of thymidine phosphorylase in human platelets]. / Purification partielle et caractérisation de la thymidine phosphorylase des plaquettes humaines.

An enzyme which catalyzes the phosphorolytic cleavage of thymidine, and whose behaviour is characteristic of thymidine phosphorylase, was purified 130 times from human blood platelets. The results obtained by chromatography and electrophoresis, enable us to consider the existence of a dimeric form of this enzyme.

[Biosynthesis in vitro of extracellular matrix in arteriosclerotic areas of rabbit aortas. Autoradiographic study of tritiated proline incorporation kinetics]. / Biosynthèse de la matrice extracellulaire dans les aires athéroscléreuses d'aortes de lapins in vitro. Etude autoradiographique de la cinétique d'incorporation de proline tritiée.

The elaboration of the extracellular matrix was investigated by radio-autoradiography after incubation of aortic strips, isolated from thoracic aortas of healthy and atherosclerotic rabbits, with tritium labeled proline. Time course experiments indicated that there was a delay of about one hour before significant amounts (25%) of tritiated macromolecules were released from the intact smooth muscle cells. On the contrary, both, the level of incorporation and the rate of excretion of macromolecular components by modified smooth muscle cells of the atherosclerotic area from injured strips were increased. The results indicate that, under the conditions of incubation in vitro, the modified smooth muscle cells of atherosclerotic plaque exhibit a particular behaviour for both, quantitative and qualitative features of macro-molecular synthesis of the extracellular matrix.