Evidence for the importance of 5'-deoxy-5-fluorouridine catabolism in humans from 19F nuclear magnetic resonance spectrometry.

The use of a new methodology, 19F nuclear magnetic resonance, has allowed detection of all the fluorinated metabolites in the biofluids of patients treated with 5'-deoxy-5-fluorouridine (5'-dFUrd) injected i.v. at a dose of 10 g/m2 over 6 h. This technique, which requires no labeled drug, allows a direct study of the biological sample with no need for extraction or derivatization and a simultaneous identification and quantitation of all the different fluorinated metabolites. As well as the already known metabolites, unmetabolized 5'-dFUrd, 5-fluorouracil, and 5,6-dihydro-5-fluorouracil, the presence of alpha-fluoro-beta-ureidopropionic acid, alpha-fluoro-beta-alanine (FBAL), N-carboxy-alpha-fluoro-beta-alanine, and the fluoride anion F- is reported. The catabolic pathway proposed for 5'-dFUrd is analogous to that of 5-fluorouracil, completed with FBAL----F- step, and the plasmatic equilibrium of FBAL with N-carboxy-alpha-fluoro-beta-alanine, its N-carboxy derivative. The quantitative analysis of the different metabolites found in plasma and urine emphasizes the significance of the catabolic pathway. High concentrations of alpha-fluoro-beta ureidopropionic acid and FBAL are recovered in plasma from 3 h after the beginning of the perfusion to 1 h after its end. The global urinary excretion results show that there is a high excretion of 5'-dFUrd and metabolites. Unchanged 5'-dFUrd and FBAL are by far the major excretory products and are at nearly equal rates. The protocol followed in this study produces relatively low but persistent plasmatic concentrations of 5-fluorouracil throughout the perfusion.

omicron-Quinone formation in the biochemical oxidation of the antitumor drug N2-methyl-9-hydroxyellipticinium acetate.

The activation of N2-methyl-9-hydroxyellipticinium acetate (4) by a peroxidase--H2O2 system leads to the formation of an omicron-quinone (7a). This omicron-quinone is not directly generated from the starting material but through a quinone imine intermediate (6) which is subsequently oxidized. This reaction is highly dependent on pH values. The omicron-quinone 7a is easily protonated (7b), gives an addition product with methanol (9), and is reduced by cysteine. The omicron-quinone 7b has a rather low inhibitory effect against L1210 leukemia cell multiplication but acts as an electron carrier and dramatically augments the oxygen consumption in xanthine oxidase-NADH and rat liver microsomes-NADPH systems.

Ribose as the preferential target for the oxidized form of elliptinium acetate in ribonucleos(t)ides. Biological activities of the resulting adducts.

The covalent binding of the oxidized form of elliptinium acetate, an antitumor drug, to various ribonucleos(t)ides is described. In the absence of a strong nucleophile on the bases, e.g., a sulfhydryl group, the main target of this quinone imine derivative is the sugar moiety. With unmodified regular bases, the first electrophilic addition always occurs on the 2'-oxygen of ribose (more slowly for pyrimidine than for purine); in a second step, cyclization of the reoxidized product leads to a spiro derivative: only one stereoisomer is detected with purine nucleoside; the other stereoisomer appears as a minor product (10-20%) with nucleotides and pyrimidine nucleosides. With modified bases, no change is observed except for bases exhibiting an additional strong nucleophilic center: oxidized elliptinium alkylates thioguanine and thioguanosine on the sulfur atom and in this last case not on the ribose moiety. All spiro derivatives are less cytotoxic than the parent compound even if the base is an antimetabolite (azauridine); however, thio-elliptinium adducts maintain high cytotoxicity.

Potential antitumor agents: synthesis and biological properties of aliphatic amino acid 9-hydroxyellipticinium derivatives.

Aliphatic amino acids glycine, alanine, valine, and leucine were conjugated to the antitumor drug N2-methyl-9-hydroxyellipticinium (NMHE) through a peroxidase-catalyzed oxidation reaction. NMR studies of the adducts so obtained have indicated (i) that the amino acids were linked to NMHE between the nitrogen of their primary amine and the C-10 position of the ellipticine ring and (ii) that a double bond was present between the nitrogen and the alpha-carbon of the amino acid moiety. All amino acid-NMHE adducts exhibit a higher lipophilic property than the parent compound (NMHE) directly correlated with the length of the aliphatic chain of the amino acids. The adducts interact with DNA through an intercalating process with apparent binding constant ranging from 2 X 10(5) to 5 X 10(5) M-1 at pH 7.40. The presence of the amino acid moiety linked to NMHE results (i) in a slight decrease of the cytotoxicity on L1210 cells in vitro (ID50 ranged from 0.20 to 0.50 microM) as compared to NMHE (ID50 = 0.05 microM), (ii) in a decrease of the antitumor efficiency in vivo against L1210 leukemia for leucine-NMHE and valine-NMHE (ILS at LD0/2 = 35% and 31%, respectively), (iii) in a suppression of the antitumor activity for alanine-NMHE and glycine-NMHE (ILS less than 25%), (iv) in a strong increase in the bacteriostatic activity on the quaternary ammonium sensitive Escherichia coli BL101 strain and on Salmonella typhimurium TA98 strain. The bacteriostatic effect is directly correlated with the lipophilic property of the drugs. These findings are discussed in terms of a structure-activity relationship.

Peroxidase-catalysed oxidation of N2,N6-dimethyl-9-hydroxyellipticinium acetate. Evidence for the formation of an electrophilic quinone-iminium derivative.

The activation of N2,N6-dimethyl-9-hydroxyellipticinium acetate (DMHE) by a peroxidase-H2O2 system leads to a reactive orthoquinone, or in the presence of a nucleophile like alanine, to adducts with a proposed benzoxazole structure. The stoichiometric and pH metric studies support the generation of a bicationic electrophilic intermediate, namely a quinone-iminium. Since no N6-demethylation occurs during the oxidation process, DMHE is not a prodrug of Celiptium (N2-methyl-9-hydroxy-ellipticinium acetate), but the high electrophilic properties of the species generated might explain its great cytotoxicity and antitumor properties. These results extend the possibility for N6-methyl ellipticine derivatives of a biooxidative activation which can play a role in their cytotoxicity.

Evidence for high-valent iron-oxo species active in the DNA breaks mediated by iron-bleomycin.

The activity of bleomycin is generally attributed to its ability to cleave DNA through the mediation of transition metal salts, molecular oxygen and electrons. Despite the fact that DNA break products are known with great details, the exact nature of the so-called "activated bleomycin", responsible for the activation of C--H bonds of the deoxyriboses, is still a matter of debate. In the present article, we report evidence that high-valent iron-oxo species might be generated by potassium hydrogen persulfate, KHSO5 (a single oxygen atom donor) and are involved in metallobleomycin-mediated DNA breaks.

DNA strand breaks photosensitized by benoxaprofen and other non steroidal antiinflammatory agents.

Benoxaprofen, a non steroidal antiinflammatory drug is known to be highly phototoxic. Upon irradiation at 300 nm, benoxaprofen is shown to enhance the cleavage of phi X 174 DNA in buffered aqueous solution (pH 7.4). A linear relationship between the number of single strand breaks and the irradiation time is found. In deaerated solutions, these breaks are three times greater in the presence than in the absence of benoxaprofen. In both cases the rate of cleavage decreases in the presence of air. The rate of DNA damage increases with the drug per base pair ratio up to approximatively 0.2 and then decreases at higher ratios. Other NSAIDs, naproxen, ketoprofen, diflunisal, sulindac and indomethacin have been tested as photocleavers of DNA by using the same experimental conditions. A comparison of the efficiency of cleavage of all these drugs (including BNP) was obtained at drug concentrations such that the light absorbance was the same. Benoxaprofen, naproxen, ketoprofen and diflunisal induce single strand breaks. Sulindac and indomethacin do not cause breaks, and they can in some conditions even act as screening agents. The most efficient of the series are naproxen and ketoprofen. In the presence of oxygen, at the same concentrations as above, the efficiency of benoxaprofen, ketoprofen and diflunisal is decreased while that of naproxen is increased. This suggests that all these compounds do not interact with DNA by the same mechanism. In the case of BNP, the mechanism of photoinduced DNA cleavage is discussed in detail. It is shown that the photoactive agent is the decarboxylated derivative of benoxaprofen, as the photodecarboxylation of benoxaprofen is much faster than the photocleavage of DNA.

Comparative study of the incorporation of ellipticine-esters into low density lipoprotein (LDL) and selective cell uptake of drug--LDL complex via the LDL receptor pathway in vitro.

Esters of elliptinium with stearic (ST-NME), palmitic (PAL-NME) or oleic (OL-NME) acids, a series of lipophilic derivatives of ellipticine, were synthetized, in order to evaluate their incorporation into Low Density Lipoprotein (LDL). Among the three derivatives, OL-NME shows the most potent incorporation (83 micrograms/mg protein LDL) compared to ST-NME (37 micrograms/mg protein LDL) and PAL-NME (58 micrograms/mg protein LDL). The size of OL-NME-LDL was determined by size distribution particles, showing their homogeneity compared to native LDL. When culture normal human fibroblasts were incubated with [125I]LDL incorporated drug, they bound to the LDL receptor with the same affinity as native LDL and were internalized and degraded intracellularly. The presence of excess native LDL inhibited the cellular uptake and degradation of [125I]drug-LDL. We have used [125I]acetyl-LDL as a probe for a binding site on macrophages that mediated the uptake and degradation of chemically altered or denatured LDL. Mouse peritoneal macrophages were shown to take up and degrade [125I]acetyl-LDL at rates that were greater than those for the uptake and degradation of native [125I]LDL and [125I]drug-LDL. The in vitro cytotoxic test on L1210 murine leukemic cells demonstrated that the complex was cytotoxic and was more effective than the free drug. This cytotoxic activity of the drug-LDL complex depends on the LDL high affinity receptor since the addition of native LDL reduces the killing power. In contrast, methylated LDL, which does not bind to the LDL receptor, has no effect on it. We conclude that it is possible to incorporate a large amount of cytotoxic drug into LDL without modifying their cellular metabolism via the high affinity LDL receptor pathway. It indicates also that the delivery of lipophilic drugs using LDL might provide distinct advantages over the use of synthetic carriers.

Evidence for electrophilic properties of N2-methyl-9-hydroxy ellipticinium acetate (Celiptium) from human biliary metabolites.

The human biliary metabolism of the antitumor agent N2-methyl-9-hydroxyellipticinium acetate is described. Three major compounds have been identified by high-performance liquid chromatography and comparison with synthetic reference derivatives: the unchanged drug, the O-glucuronide conjugate and the cysteinyl-ellipticinium adduct. The latter one is the expected detoxification compound of an intermediate electrophilic quinone-imine derivative generated in vivo. This result provides a further evidence that hydroxylated forms of ellipticine derivatives might be activated by a biooxidation route.

New approach to metabolism of 5'-deoxy-5-fluorouridine in humans with fluorine-19 NMR.

The metabolism of 5'-deoxy-5-fluorouridine (5'dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5'dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5'dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported alpha-fluoro-beta-alanine. The two major metabolites in urine are unmetabolized 5'dFUrd and alpha-fluoro-beta-alanine.

Mechanism of DNA cleavage mediated by photoexcited non-steroidal antiinflammatory drugs.

DNA damage photoinduced by four nonsteroidal antiinflammatory drugs (NSAID) have been investigated by neutral agarose gel electrophoresis. Upon irradiation at 300 nm, in phosphate buffered solution, benoxaprofen, naproxen, ketoprofen, tiaprofenic acid photosensitized the formation of single-strand breaks (SSB) in double stranded supercoiled phi X174 DNA. The efficiency of the cleavage is higher in argon saturated solutions than in aerated solutions and it is not correlated with the quantum yield of photodegradation of the drugs. Simultaneously with the DNA strand breaks, NSAID promote a weak reduction of the electrophoretic mobility of the supercoiled form that may be attributed to the formation of pyrimidine dimers or other DNA unwinding products. These photodimerization processes suggest the involvement of a triplet-triplet energy transfer between NSAID and DNA. Addition of mannitol and superoxide dismutase decreases the efficiency of the cleavage suggesting that HO. and O2.- are involved in the DNA cleavage. Unexpectedly, addition of sodium azide quenches the cleavage both in aerated or in deaerated solutions. Substituting H2O by D2O does not change the number of SSB thus suggesting that 1O2 does not take an important place in the cleavage of DNA. From our data we tentatively assume that the cleavage occurs through a radical mechanism that may involve in a first step an energy or an electron transfer. Gel sequencing on NSAID-photoinduced DNA breakage exhibits no particular specificity except in the case of benoxaprofen where a slight selectivity for cytosine is observed.

Determination of levofloxacin in plasma, bronchoalveolar lavage and bone tissues by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method.

The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of levofloxacin in human plasma, bronchoalveolar lavage and bone tissues. The sample extraction was based on a fully automated liquid-solid extraction with an OASIS cartridge. The method used ultraviolet detection set at a wavelength of 299 nm and a separation with a Supelcosil ABZ+ column. The assay has been found linear over the concentration range 0.25-25 microg/ml for levofloxacin in plasma, 1-6 microg/ml in bronchoalveolar lavage and 0.5-10 microg/g for bone tissues and it provided good validation data for accuracy and precision. The assay will be applied to determine the penetration of levofloxacin in human bronchoalveolar lavage (BAL) and bone tissues during pharmacokinetic steady state.

Excretion of doxifluridine catabolites in human bile assessed by 19F NMR spectrometry.

The biliary excretion of doxifluridine (5'dFUR) catabolites was studied in a patient with external bile derivation using 19F NMR spectrometry, alpha-fluoro-beta-alanine (FBAL) and fluoride ion were detected in patient's bile samples but represented only about 10% of the excreted fluorinated metabolites. The major biliary metabolite (congruent to 90%), whose structure is still unknown, is a conjugate of FBAL. The cumulative biliary excretion of 5'dFUR fluorinated metabolites was low and represented 0.8% of the injected dose.

Hemoglobin-catalyzed transformation of elliptinium acetate into electrophilic species. Evidences for oxidative activation of the drug in human red blood cells.

The anti-tumor drug N2-methyl-9-hydroxyellipticinium acetate (NMHE, Celiptium) after incubation with various N or S containing amino acids (alanine, histidine, aspartic acid, cysteine, glutathione) with hemoglobin and hydrogen peroxide or an organic peroxide (terbutylhydroperoxide) leads to the formation of the corresponding covalent binding adducts, via an oxidative activation. The formation of the covalent adduct glutathione-elliptinium was also demonstrated in human red blood cells. The importance of such process under in vivo conditions is discussed.

Oxidative biotransformation of the antitumour agent elliptinium acetate: Structural characterization of its human and rat urinary metabolites.

The electrophilic properties of the antitumour drug N(2)-methyl-9-hydroxyellipticinium acetate (Celiptium) are revealed by the detection of thiol-conjugate metabolites in man and rat urine. Besides the unchanged drug and its glucuronide, the cysteinyl- (in man) and the N-acetylcysteinyl- (in man and rat) conjugates have been unambiguously characterized using NMR, UV and mass spectral data. The urinary excretion profile exhibits total excreted products of 21% (in man) and 9% (in rat) with respect to the administered dose. The unchanged drug is found to be the major excreted compound from urine in both species (17% in man, 6.3% in rat); whereas the glucuronide (2.6% in man, 1.5% in rat), cysteinyl- (1.3% in man) and N-acetylcysteinyl- (0.2% in man, 1.2% in rat) conjugates represent the minor excreted compounds. The presence of the latter thio-conjugates provides an indirect proof of the in vivo generation of an oxidized intermediate form of the administered drug.

[The mechanism of action of isoniazid. A chemical model of activation]. / Connaissances actuelles sur le mécanisme d'action de l'isoniazide. Apport d'un modèle chimique d'activation.

The antituberculosis drug isoniazid (INH) is quickly oxidized by stoichiometric amounts of manganese(III)-pyrophosphate. In the presence of the nicotinamide coenzyme, the INH oxidation produced the formation of INH-NAD(H) adducts which are potential competitive inhibitors of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway for mycolic acids. Manganese(III)-pyrophosphate is an efficient alternative oxidant to mimick the activity of the Mycobacterium tuberculosis KatG catalase-peroxidase and will be useful for further mechanistic studies of INH activation and for structural investigations on reactive INH species and resulting InhA inhibitors.

Isoniazid: an update on the multiple mechanisms for a singular action.

Isoniazid (INH) is one of the most commonly used drugs in treatment of human tuberculosis and the most efficient. Although it has been 60 years since isoniazid was introduced in anti-tubercular therapy and despite the simplicity of its chemical structure (C6H7N3O) with few functional groups, its exact mechanism of action, which could account for its specificity and exceptional potency against Mycobacterium tuberculosis and justify all profiles of INH-resistance, remains elusive and debatable. This complexity can find an explanation in the high reactivity of INH and also in the possibility that multiple targets and pathways could co-exist for this medicinal agent. Indeed, since the discovery of isoniazid's anti-tubercular potency, several propositions for its mode of action have been reported, including its conversion, by a catalase peroxidase within M. tuberculosis, into an active metabolite able, after reaction with NAD, to inhibit an enzyme (InhA) crucial to M. tuberculosis survival. This represents the most consensual mechanism described to date. Nevertheless, none of the proposed mechanisms considered independently can explain the singular and privileged action of the isoniazid structure on the tubercle bacillus, or all the profiles of resistance. The aim of this paper is to reconsider the literature reporting the different modes of action described for isoniazid in the light of the present and most relevant knowledge, with special attention to understanding the molecular mechanistic aspects of the drug's action.