Structure-property relationships of trimetazidine derivatives and model compounds as potential antioxidants.

Twenty-five compounds (trimetazidine derivatives and other compounds, mostly having a free phenolic group) were examined for their radical scavenging and antioxidant properties. Their reaction with DPPH (2,2-diphenyl-1-picrylhydrazyl) as a measure of radical scavenging capacity was assessed by two parameters, namely EC50 (the concentration of antioxidant decreasing DPPH by 50%), and log Z, a kinetic parameter proposed here and derived from initial second-order rate constants and antioxidant/DPPH ratios. Antioxidant activities were determined by the inhibition of lipid peroxidation and albumin oxidation. The most active compounds were derivatives having a trolox or hydroquinone moiety. Physicochemical and structural properties were determined by molecular modeling as lipophilicity (virtual log P calculations) and H-Surf (solvent-accessible surface of hydroxyl hydrogen) and by quantum mechanical calculations (deltaH(ox) = oxidation enthalpy; deltaH(abs) = enthalpy of hydrogen abstraction). QSAR models were derived to identify molecular mechanisms responsible for the reactivity toward the DPPH radical and for the inhibition of lipid peroxidation. A useful prediction of antioxidant capacity could be achieved from calculated molecular properties and the kinetic parameter developed here.

[Interaction of thiamine diphosphate with magnesium ion]. / Etude de l'interaction de la thiamine diphosphate avec l'ion magnésium.

The action of magnesium ion on the exchange rate of the proton in C2 of thiamine and thiamine diphosphate is studied at different values of pD. Above pD 5 the ion Mg2+ increases this exchange rate. The phenomenon is markedly enhanced for TDP rather than thiamine and increases with pD. Below pD 5 magnesium decreases the exchange rate. This decrease is greater for TDP than for thiamine. The maximum effect is reached at a magnesium concentration of 0.5/1 for thiamine and of 1/1 for TDP. T1 measurements are made for different pH values with and without magnesium ion. Results seem to prove that an increase in pD values from 3.9 to 5.9 leads to an accentuation of the molecules "folded" form. Nevertheless for a given pD value the TDP-Mg complex seems to have a more "folded" form than TDP.

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation.

1. The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV-3P) on Ca2+-induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria. 2. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV-3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC50 = 3.0 +/- 0.9 microM). 3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 microM) totally inhibited the rate of swelling. 4. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV-3P on mitochondrial swelling. Indeed, the IC50 value of ZDV-3P increased from 3.0 to 17.4, 93.6 and 66.5 microM, in the presence of 20 microM, ADP, ATP or atractyloside, respectively. 5. ZDV-3P did not displace [3H]-ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV-3P and [3H]-ATP do not share the same binding sites. 6. ZDV-3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca2+-dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV-3P).

The in vitro effect of some nitroimidazoles on microtubule formation.

The in vitro effects of some nitroimidazoles (metronidazole, ornidazole) and their metabolites on microtubule formation have been tested. Cyclic metabolites are without effect. Metabolites proceeding from cleavage of the imidazole ring inhibit microtubule formation and reduce the polymerization rate of tubulin. This inhibitory effect might be correlated to some of the side-effects of these drugs. Isaxonine phosphate corrects this effect.

The restoration of ATP synthesis may explain the protective effect of calcium antagonists against cyclosporine A nephrotoxicity.

Cyclosporine A at pharmacological doses decreases the rate and yield of ATP synthesis in rat mitochondria. This action seems to be due to the mitochondrial calcium storage induced by the drug. If such an effect occurs in vivo, the ATP deficit will affect calcium extrusion pumps, so triggering vasoconstriction which is the major side effect of Cyclosporine A. Calcium antagonists (Nifedipine and Verapamil) at least partially correct this effect on ATP synthesis: this finding may be related with the beneficial clinical effect conferred on Cyclosporine A toxicity by calcium antagonists. This effect of calcium antagonists may be due to an interaction with Cyclosporine A at the level of mitochondrial calcium efflux.

1H nuclear magnetic resonance and clinical studies of interaction of calcium antagonists and hypoglycemic sulfonylureas.

The hypoglycemic effect of gliclazide is mainly due to its action on ATP stimulated K+ channels, but the calcium ionophoretic effect of this drug may also be involved in its physiological properties. Using 1H NMR we demonstrated the antiionophoretic effect of nifedipine and diltiazem. We attempted to verify whether this in vitro interaction also occurs in vivo. A clinical trial, was performed on patients treated concomitantly with gliclazide and nifedipine or diltiazem. Results showed that no in vivo interaction occurred. The discrepancy between in vivo and in vitro results may be explained by a too weak plasma concentration in the case of nifedipine and by a large plasma protein binding in the case of diltiazem.

Lack of pharmacodynamic interaction between trimetazidine and cyclosporin A in human lymphoproliferative and mouse delayed hypersensitivity response models.

The hypothesis of an interaction between trimetazidine and the immunosuppressive effect of cyclosporin A was investigated in two models: a) ex vivo, the lymphoproliferative response of normal human lymphocytes to phytohemagglutinin and a murine monoclonal antibody against the CD3 T-lymphocyte membrane complex; b) in vivo, the delayed hypersensitivity response model in mouse. The uptake of methyl-3H-thymidine was measured in both models. For the lymphoproliferative response, statistical analysis showed that there was a significant inhibitory effect of cyclosporin A on cell proliferation (P < 0.001) and confidence intervals obtained by ANOVA showed the equivalence of the results when trimetazidine was combined with cyclosporin A (all CI95% < or = 10). In the delayed hypersensitivity model, cyclosporin A was also found to be very effective in inhibiting the immune response (P < 0.001), while trimetazidine did not interfere with cyclosporin A's effect. It was concluded that trimetazidine exerted neither an immunostimulatory nor an immunosuppressive effect in the two models, suggesting of the absence of interaction between trimetazidine and cyclosporin A's effectiveness when both drugs are given in combination.

Comparison of the effects of cyclosporine A and trimetazidine on Ca(2+)-dependent mitochondrial swelling.

Cyclosporine A (CsA) is a known potent inhibitor of pro-oxidant-induced mitochondrial swelling. In the present study we show that CsA's effect is only transient when the liver mitochondrial swelling in induced by Ca2+ plus tert-butylhydroperoxide (t-BH). After an initial inhibition, swelling is worsened by CsA as evidenced by an extent of mitochondrial swelling that exceeds that of the control. Unlike CsA, trimetazidine (TMZ), an anti-ischemic drug decreases both the extent and the rate of the swelling with an IC50 value of 214 +/- 24 microM. Its inhibition effect on the initial swelling rate mimicks that of CsA but the mechanism may be independent. During long-term swelling. TMZ counteracts the worsening effect of CsA. The inhibition of swelling induced by TMZ is assessed by the fact that TMZ significantly increases the EC50 of Ca(2+)-induced mitochondrial swelling (46.6 +/- 6.0 to 85 +/- 10 microM, P < 0.01), without affecting its cooperativity. Apparently, TMZ seems to behave like trifluoperazine (TFP), a phospholipase A2 inhibitor that, under our experimental conditions, inhibits the mitochondrial swelling induced by Ca2+ and t-BH with an IC50 value of 25 +/- 10 microM. Both drugs are able to protect mitochondria from both phases (early and late) of the swelling, especially the late, which is enhanced in the presence of CsA. TFP and other phospholipase A2 inhibitors were able to displace [3H]TMZ from its mitochondrial binding sites whereas CsA was ineffective. We suggest that TMZ, like TFP, inhibits the CsA insensitive mechanism involved in the swelling process which is responsible for the worsening effect observed in the presence of CsA when the swelling is generated by Ca2+ and t-BH.

1H-NMR study of suloctidil-A23187 calcium ionophore interaction.

Suloctidil is a molecule with calcium antagonist properties, whose anti-ionophoretic effect has previously been reported. In the presence of A23187 calcium ionophore free acid (A+), the NMR spectra of suloctidil (S +/-) are modified at the level of H-1 protons and to a lesser degree in the CH3-3 and aromatic regions. Experiments with one of the enantiomers of suloctidil and decoupling investigations led us to postulate the existence of diastereoisomers S+/A+, S-/A+ in the suloctidil +/-/A23187 + mixture. Moreover our results allow the hypothesis that suloctidil and calcium compete for the same binding site of the ionophore molecule.

Calcium anti-ionophoretic effect of some calcium channel blockers in rat liver mitochondria.

Beyond their classical action on calcium channels, some calcium channel blockers also exhibit a calcium anti-ionophoretic effect. We studied this effect on respiratory control and Ca2+ fluxes in a mitochondrial model to compare calcium antagonists chosen among three clinical classes: vascular, cardiac, and mixed effects. Synthetic calcium ionophore A23187 decreases respiratory control and modifies Ca2+ fluxes. We show that calcium antagonists partially restore the parameters altered by A23187. By calculating the percentage of restoration, we found that vascular drugs exhibit a strong anti-ionophoretic effect, cardiac drugs exert no significant effect, and mixed calcium antagonists exert an intermediate effect. Thus, it appears possible to link the intensity of calcium anti-ionophoretic effect with the clinical interest of a calcium antagonist.

Inhibition in vitro of the polymerization of tubulin by uremic middle molecules: corrective effect of isaxonine.

The polymerization of tubulin leads to the formation of microtubules which are one of the components of the axons of nerve cells. This reaction is the limiting factor in the growth of axons. Uremic middle molecules inhibit in vitro the polymerization of tubulin in a dose dependent way. It is possible that a similar phenomenon could occur in vivo in uremic patients, and this might be involved in the development of neuropathy. In addition, isaxonine phosphate counteracts the inhibitory effect of uremic middle molecules on the polymerization of tubulin.

Protective effect of some calcium antagonists against mitochondrial calcium injury.

Ischaemia induces an increase in calcium cytosolic concentration, leading to a mitochondrial Ca2+ overload. As Ca2+ uptake and storage into mitochondria are the alternative route to oxidative phosphorylation, the Ca2+ overload induces a decrease in ATP synthesis. We have tested in vitro the ability of certain calcium antagonists to restore the ATP synthesis inhibited by mitochondrial calcium overload. Our results showed that diltiazem and bepridil, clinically used as antiischaemic agents, each can restore the ATP synthesis. It is suggested that our in-vitro results might serve to explain the antiischaemic properties of some calcium antagonists.

Counteraction by nicardipine of the ionophoretic effect of gliclazide: a mitochondrial study.

Using mitochondria, we demonstrate that gliclazide exhibits a calcium ionophoretic activity. Indeed, gliclazide induces a decrease in the respiratory control of mitochondria; this effect is increased by addition of Ca2+ and corrected by ruthenium red, all characteristics of a calcium ionophoretic compound. Our results, on a biological membrane, confirm previous findings in vitro. Moreover, we show that nicardipine counteracts the action of gliclazide. Besides the effect on ATP-stimulated K+ channels, the ionophoretic effect of gliclazide may play a role in its hypoglycaemic effect, thus this counteracting action of nicardipine might induce drug interaction during the concomitant clinical administration of gliclazide and nicardipine.

Microcalorimetric study of magnesium-adenosine triphosphate ternary complex.

Using an original microcalorimetric method, the existence of the Mg2ATP ternary chelate has been studied. The thermodynamic parameters of this complex are delta H = 7.2 +/- 0.5 kJ mole-1 and K = 49 +/- 9 M-1. These values are compared with those previously obtained for binary chelate Mg ATP2-. A possible regulation role of Mg2ATP is discussed.

In vitro inhibition of microtubule assembly by disulfiram.

Disulfiram (Tetraethylthiuram disulfide, DSF) inhibits in vitro tubulin polymerization in a dose-dependent manner after a preliminary incubation time. Electron micrographs show that microtubules are shorter and less numerous. This inhibition may be correlated to blockade of protein SH groups. The linkage of DSF-tubulin hinders the vinca alkaloids-tubulin binding as shown using radioactive assay and microcalorimetric measurements. These results could be responsible for the occurrence of some side effects observed during the therapeutic use of this drug.

Is the beneficial effect of calcium channel blockers against cyclosporine A toxicity related to a restoration of ATP synthesis?

ATP synthesis inhibited by Cyclosporine A is restored by calcium channel blockers: nifedipine, verapamil, bepridil, diltiazem. ATP synthesis was estimated using liver mitochondria by measuring the rate of respiration during state 3 and a measure of the yield of ATP synthesis, the P/O ratio. The study of calcium fluxes through mitochondrial membrane indicates that calcium channel blockers counteract the mitochondrial calcium storage induced by cyclosporine A. If the restoration of ATP synthesis observed in vitro also occurred in vivo, the increase in ATP pool might contribute to a better functioning of the Ca2+ extrusion pumps of the cells, thereby maintaining the cytosolic calcium concentration (Cai), in the normal range. The nephrotoxicity of cyclosporine A appears to be due to a vasoconstrictive effect related to an increased Cai. This result may account for the reduction of clinical cyclosporine A toxicity by calcium channel blockers. Verapamil appears to be the most efficient in restoring ATP synthesis.

Trimetazidine reverses calcium accumulation and impairment of phosphorylation induced by cyclosporine A in isolated rat liver mitochondria.

When applied to rat liver mitochondria in contact with Ca++, cyclosporine A (CsA) induced both an accumulation of this ion and a decrease in oxidative phosphorylation. Trimetazidine (TMZ) reversed both phenomena in a dose-dependent manner. These two effects were demonstrated in separate experiments. A decrease in oxidative phosphorylation was observed with succinate as substrate. V3 and P/O (ratio corresponds to the number of ADP molecules added in the medium per oxygen atom consumed during phosphorylation and represents the yield of ATP synthesis) were simultaneously decreased by CsA (1 microM) and restored by TMZ. Ca++ accumulation in mitochondria was observed when it was added to the mitochondrial suspension; its uptake was followed by a new equilibrium. CsA prolonged its duration, whereas TMZ reduced it in a dose-dependent manner. The same phenomenon was observed when ADP was used instead of CsA. Ca++ efflux from mitochondria could be induced by TMZ without the addition of CsA. It was immediate and always partial and followed by a reuptake process only observed at concentrations of TMZ of >1 microM. Compared with ruthenium red, which blocks Ca++ uniporter, TMZ seemed to act on Ca++ efflux mechanisms. Interestingly, low TMZ doses promote a Ca++ efflux process without activating reentry mechanism, which may explain the correction of deleterious effect of CsA on V3 and P/O. As nephrotoxicity observed in humans after CsA chronic administration is considered to be related, at least in part, to an alteration of Ca++ intracellular homeostasis, TMZ seems to be a candidate for alleviation of CsA nephrotoxic effects in humans.

Action of hydrolyzed cisplatin and some analogs on microtubule protein polymerization in vitro.

The inhibition of tubulin microtubule polymerization by cisplatin was investigated. The interaction was monitored by turbidity measurements and electron microscopy. For a 2.5 x 10(-5) M concentration of cisplatin, the inhibition at 37 degrees C was nearly 50% after 1 hour of incubation and 80% after 2 hours. A similar but lesser effect was observed after incubation at 4 degrees C. One analog of cisplatin, sulfato-aqua(1,2)diamminocyclohexane platinum II, showed the same inhibition effect.