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.

Quantitative and qualitative control of cytotoxic preparations by HPLC-UV in a centralized parenteral preparations unit.

The constantly growing incidence of cancer and long-term treatment are leading to an increasing number of cytotoxic preparations in hospital pharmacies. Security and quality standards of cytotoxic preparations are essential to assure treatment efficiency and limit iatrogenic toxicity. In order to secure the process of cytotoxic preparations; we decided to install a quantitative and qualitative High Performance Liquid Chromatography (HPLC) control of cytotoxic preparations carried inside our pharmacotechnic unit. A 100 microl sample of each preparation was assayed by HPLC with ultraviolet/visible-diode array detection, which enabled the identification of all cytotoxic agents thanks to their characteristic UV spectra. We developed rapid and specific HPLC assays that determined qualitatively and quantitatively the presence of 21 different cytotoxic agents in less than 3.5 min. A fifteen per cent tolerance from the theoretical concentration was chosen in agreement with preparation and dosage bias, and a first period control of more than 4400 preparations revealed that around 7.7% preparations did not conform. The main objective of these controls was to avoid the administration of defective chemotherapies to patients and finally to use their results to identify error factors; as a result we will take corrective measures in order to reduce error frequency.

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.

Characterization of a 5'-aldehyde terminus resulting from the oxidative attack at C5' of a 2-deoxyribose on DNA.

The 5'-aldehyde terminus is a DNA oxidative damage resulting from attack at C5' of 2-deoxyriboses by some potent natural or chemical DNA cleavers. To offer a fast and specific method for characterization of this type of damage, we used on-line electrospray ionization mass spectrometry (ESI-MS) detection during liquid chromatography analyses. The intrinsic reactivity of 5'-aldehyde terminus with nucleophiles (formation of hydrate with water, of a Tris adduct with Tris buffer) or through beta-elimination reaction resulted in complex LC profiles and MS data. We showed that derivatization of the aldehyde function as an oxime ether gives a stable derivative easy to characterize during on-line ESI-MS analyses. Complete structural characterization of the Tris adduct and the oxime ether derivative were obtained from MS and detailed NMR studies performed on derivatized 5'-aldehyde thymidine models.

A fast and efficient metal-mediated oxidation of isoniazid and identification of isoniazid-NAD(H) adducts.

It is currently believed that isoniazid (INH) is oxidised inside Mycobacterium tuberculosis to generate, by covalent attachment to the nicotinamide ring of NAD(H) (beta-nicotinamide adenine dinucleotide), a strong inhibitor of InhA, an enzyme essential for mycolic acid biosynthesis. This work was carried out to characterise the InhA inhibitors (named INH-NAD(H) adducts) which are generated, in the presence of the nicotinamide coenzyme NAD+, by oxidation of INH with manganese(III) pyrophosphate, a nonenzymatic and efficient oxidant used to mimic INH activation by the catalase-peroxidase KatG inside M. tuberculosis. The oxidation process is almost complete in less than 15 minutes (in comparison to the slow activation obtained in the KatG-dependent process (2.5 hours) or in the nonenzymatic O2/Mn(II)-dependent activation (5 hours)). The alkylation of NAD+ by the postulated isonicotinoyl radical generates, in solution, a family of INH-NAD(H) adducts. Analyses with liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) and experiments performed with 18O- and 2H-labelled substrates allowed us to propose two open and four hemiamidal cyclised dihydropyridine structures as the main forms present in solution; these result from the combination of the isonicotinoyl radical and the nicotinamide part of NAD+. A small amount of a secondary oxidation product was also detected. Structural data on the forms present in solution should help in the design of inhibitors of enzymes involved in the biosynthesis of mycolic acids to act as potential antituberculosis drugs.

[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.

Nuclease activity and binding characteristics of a cationic "manganese porphyrin-bis(benzimidazole) dye (Hoechst 33258)" conjugate.

To increase the binding affinity and/or the sequence selectivity of the chemical nuclease manganese-(III) tetrakis(4-N-methylpyridiniumyl)porphyrin, we synthesized a conjugate molecule by associating a tris(4-N-methylpyridiniumyl)metalloporphyrin motif to Hoechst 33258 (H33258), a DNA minor groove binding dye known for its selective affinity for A-T tracts. Selected double-stranded (ds) 35-mer oligodeoxyribonucleotides have been used to probe DNA chain breakages induced by the managanese derivative of the conjugate after activation by potassium monopersulfate. Gel electrophoresis analyses show that DNA cuts were generated by the metalloporphyrin moiety of the hybrid molecule, with the H33258 entity interacting in two different possible orientations, upstream or downstream, with its preferred affinity site inside the minor groove. Also studied was the cleavage of a ds 29-mer oligodesoxyribonucleotide containing two stretches of A.T basepair (bp) which clearly showed that the hybrid can occupy the binding region at least in four preferred ways. These cleavage experiments support the strong and selective interaction of the metalloporphyrin-dye hybrid with DNA and allow the estimate of 10 bp as an average size for the affinity site of an isolated conjugate molecule. Further studies by UV-visible spectroscopy, DNA melting temperature determinations, and DNase I footprinting showed, for higher concentrations of H33258 conjugate, a preferential interaction of only the H33258 moiety with DNA (estimated binding site size 6-7 bp) with the porphyrin entity pushed out of the groove and, for the highest concentrations, self-aggregation of the H33258 conjugate all along the DNA strand in a nonselective mode.

Model systems for oxidative drug metabolism studies. Catalytic behavior of water-soluble metalloporphyrins depends on both the intrinsic robustness of the catalyst and the nature of substrates.

Sulfonated manganese and iron porphyrins have been used as catalysts in attempts to mimick the oxidation of acetaminophen and two ellipticine derivatives by horseradish peroxidase. Cofactors were potassium monopersulfate for the synthetic catalyst and hydrogen peroxide for the natural enzyme. Hindered metalloporphyrins, i.e. with ortho positions of the meso-phenyl rings substituted with methyl groups [iron(III) and manganese(III) derivatives of octasodium mesotetrakis(3,5-disulfonatomesityl)porphyrin], were shown to be at least 10 times more robust than unsubstituted derivatives [iron(III) and manganese(III) derivatives of tetrasodium meso-tetrakis(4-sulfonatophenyl)porphyrin] when activated in the absence of substrate. The catalytic activity depends on the nature of the substrate as shown by a decrease or an increase in reactivity observed, respectively, in the oxidation of acetaminophen or ellipticine derivatives catalyzed by hindered metalloporphyrins compared with nonhindered ones. Only sterically hindered metalloporphyrins, even in the case of lowered reactivity, were allowed to mimick the behavior of horseradish peroxidase when activated in the absence of substrate (stability toward autodegradation) and in the course of repeated infusion of substrate (retained catalytic activity as time advances).

Preferential hydroxylation by the chemical nuclease meso-tetrakis-(4-N-methylpyridiniumyl)porphyrinatomanganeseIII pentaacetate/KHSO5 at the 5' carbon of deoxyriboses on both 3' sides of three contiguous A.T base pairs in short double-stranded oligonucleotides.

Selected double-stranded oligodeoxyribonucleotides have been used to probe, at the molecular level, DNA chain breakages induced by the chemical nuclease mesotetrakis(4-N- methylpyridiniumyl)porphyrinatomanganeseIII pentaacetate/KHSO5. The results show that cleavage selectively occurs on the two 3' sides of three contiguous A.T base pairs (an A.T triplet). Hydroxylation at 5' carbon of the deoxyribose targets represents the initial damage on the sugar-phosphodiester backbone and leaves a 3' phosphate and a 5' aldehyde at the ends. The fragments were separated by HPLC and unambiguously identified through chemical and biochemical reactions and/or sequencing after enzymatic conversion to mononucleosides. Also studied was the degradation of a 22-nucleotide DNA molecule containing two A.T triplets. Gel electrophoresis analyses on the corresponding 5'-32P-end-labeled substrate supported the above cleavage specificity and mechanism.

Mechanism of DNA cleavage by cationic manganese porphyrins: hydroxylations at the 1'-carbon and 5'-carbon atoms of deoxyriboses as initial damages.

Cationic manganese-porphyrin complexes, free or targetted with an intercalating agent, are able to cleave DNA using oxygen atom donors like potassium monopersulfate or magnesium monoperphthalate as coreactants. Detailed studies of the cleavage of calf thymus DNA, before and after a heating step, show that free bases and 5-methylene-2-furanone are the main reaction products, indicating that hydroxylation at the 1'-carbon atom is the main target of these chemical agents. These data confirm that metalloporphyrin derivatives interact with the minor groove of double-stranded DNA. Hydroxylation of one of the two C-H bonds at position-5' is another initial DNA damage, characterized by the formation of furfural as sugar degradation product. Besides these two main initial damage sites, a low contribution of a hydroxylation reaction at C4' can not be definitively discounted, while an hydroperoxidation route at C4' can be excluded.

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.

Oxidative degradation of cationic metalloporphyrins in the presence of nucleic acids: a way to binding constants?

Mn(III) and Fe(III) complexes of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (M-TMePyP) and related hybrid molecules ("metalloporphyrin-ellipticine") were activated by potassium monopersulfate in the presence of variable calf thymus (CT) DNA and NaCl concentrations. Monitored by visible spectroscopy (Soret band), fast degradation of the free metalloprophyrin was observed while the DNA-bound form appeared protected. This direct quantitation of free versus bound metalloporphyrin ratios allowed determination of binding constants: Mn- and Fe-TMePyP respectively bind to CT DNA (5 mM phosphate buffer, 0.1 M NaCl, pH 7) with K = 3 X 10(4) and 1.2 X 10(4) M-1. Mn-TMePyP showed a greater affinity for poly[d(A-T)] (K = 1.2 X 10(5) M-1) than for poly[d(G-C)] (K = 0.2 X 10(4) M-1). This method allowed us access to the intrinsic DNA affinity of the metalloporphyrin moiety of the hybrid molecules "metalloporphyrin-ellipticine".

31P NMR characterization of terminal phosphates induced on DNA by the artificial nuclease 'Mn-TMPyP/KHSO5' in comparison with DNases I and II.

Phosphorus-31 NMR has been applied to the characterization of terminal phosphates on fragments of calf thymus DNA induced by three different nuclease systems: DNase I, DNase II and the artificial nuclease 'Mn-TMPyP/KHSO5'. In this last case, the oxidative damage to deoxyribose leads to two monophosphates esters (at the 3' and 5' ends) on both sides of the cleavage site. This method constitutes a promising approach to visualise the phosphate termini generated in DNA or RNA cleavage by cytotoxic drugs or chemical nucleases and provides a novel insight into the molecular aspects of their mechanism of action.

Model systems for metabolism studies. Biomimetic oxidation of acetaminophen and ellipticine derivatives with water-soluble metalloporphyrins associated to potassium monopersulfate.

Some original water-soluble metalloporphyrins/KHSO5 systems were developed to mimic the metabolic biooxidation of drugs. Oxidation of acetaminophen and various ellipticine derivatives were used as model reactions. Oxidative products (mainly quinone-imine structures) were obtained in good yield after 2 min of reaction, for a catalyst/substrate ratio of 0.04. Iron(III) derivative of tetrasodium meso-tetrakis(p-sulfonatophenyl)porphyrin and manganese(III) derivative of tetraacetate meso-tetrakis(4-N-methyl-pyridiniumyl)-porphyrin were the best catalysts for the oxidation of acetaminophen and ellipticine compounds, respectively. At low catalyst concentration, initial turnover rates could rise up to 8 catalytic cycles/sec. In some conditions, these catalytic systems are nearly as efficient as horseradish peroxidase/H2O2. They might have a real future as oxidation catalysts, in complement to the use of purified monooxygenase and peroxidases, to predict the possible in vivo oxidative metabolite pathways.

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.

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Potassium monopersulfate and a water-soluble manganese porphyrin complex, [Mn(TMPyP)](OAc)5, as an efficient reagent for the oxidative cleavage of DNA.

Reported studies indicate that the association of potassium monopersulfate with [Mn(TMPyP)](OAc)5, a water-soluble manganese porphyrin complex, leads to an efficient reagent for the oxidative cleavage of DNA. Single-strand breaks (SSBs) are observed on double-stranded DNA at manganese porphyrin concentrations as low as 0.5 nM with a short incubation time of 1 min. The number of SSBs linearly varies with the concentration of the manganese complex, and potassium monopersulfate is at least 3 orders of magnitude more efficient as oxygen source than hydrogen peroxide. Cleavage efficiency is optimal in the pH range 7.5-9.0 for a NaCl concentration between 80 and 150 mM or for a MgCl2 concentration of 10 mM. At very low manganese porphyrin concentration and by increasing the incubation time a catalytic cleavage activity of the complex is evidenced: up to 5 SSBs per manganese porphyrin are observed. The high cleavage activity of the monopersulfate-manganese porphyrin system makes it a good candidate for DNA-footprinting experiments.