Roland Zini

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.

Curcumin induces the mitochondrial permeability transition pore mediated by membrane protein thiol oxidation

Curcumin is a natural compound showing antiproliferative properties. Recent studies suggest that these properties might be due to its ability to induce apoptosis in tumor cells. As mitochondria play a pivotal role in the induction of the apoptotic process, we analyzed the effect of curcumin on mitochondrial function. Curcumin induced an increase in rat liver mitochondrial membrane permeability, resulting in swelling, loss of membrane potential and inhibition of ATP synthesis. These effects were mediated by the opening of the permeability transition pore. Curcumin pore induction involved the oxidation of membrane thiol functions and required the presence of low Ca2+ concentrations. These data suggest that mitochondria might be a target by which curcumin induces apoptosis of tumor cells.

Myocardial ischemic postconditioning against ischemia-reperfusion is impaired in ob/ob mice

Ischemic postconditioning (IPCD) significantly reduces infarct size in healthy animals and protects the human heart. Because obesity is a major risk factor of cardiovascular diseases, the effects of IPCD were investigated in 8- to 10-wk-old leptin-deficient obese (ob/ob) mice and compared with wild-type C57BL/6J (WT) mice. All animals underwent 30 min of coronary artery occlusion followed by 24 h of reperfusion associated or not with IPCD (6 cycles of 10-s occlusion, 10-s reperfusion). Additional mice were killed at 10 min of reperfusion for Western blotting. IPCD reduced infarct size by 58% in WT mice (33+/-1% vs. 14+/-3% for control and IPCD, respectively, P<0.05) but failed to induce cardioprotection in ob/ob mice (53+/-4% vs. 56+/-5% for control and IPCD, respectively). In WT mice, IPCD significantly increased the phosphorylation of Akt (+77%), ERK1/2 (+41%), and their common target p70S6K1 (+153% at Thr389 and +57% at Thr421/Ser424). In addition, the phosphorylated AMP-activated protein kinase (AMPK)-to-total AMPK ratio was also increased by IPCD in WT mice (+64%, P<0.05). This was accompanied by decreases in phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MAP kinase phosphatase (MKP)-3, and protein phosphatase (PP)2C levels. In contrast, IPCD failed to increase the phosphorylation state of all these kinases in ob/ob mice, and the level of the three phosphatases was significantly increased. Thus, although IPCD reduces myocardial infarct size in healthy animals, its cardioprotective effect vanishes with obesity. The lack of enhanced phosphorylation by IPCD of Akt, ERK1/2, p70S6K1, and AMPK might partly explain the loss of cardioprotection in this experimental model of obese mice.

Separation procedures used to reveal and follow drug-protein binding

The review gives a critical evaluation of the different separation procedures used to study drug-protein interactions and describes their various fields of application. For pharmacological studies, the most widely used methods are dialysis and ultrafiltration, because they allow measurements with solutions of high protein concentrations, such as those found in therapeutic conditions. Both techniques use membrane devices, which may induce additional binding effects. Another drawback of these techniques is the need for radiolabelled compounds. Chromatographic methods, which now take advantage of the technology of high-performance liquid chromatography, are generally faster and do not use drug labelling because of the higher sensitivities of the detectors. Two different approaches are possible: either all the interacting species (protein and drug) are dissolved in the mobile phase, or one of them (protein or drug) is immobilized on the support. Several chromatographic methods are available for studies in solution that differ according to the sample injection mode (frontal or zonal elution) and the nature of the mobile phase used. They include quantitation of the drug-protein complex by zonal elution, the Hummel and Dreyer method, frontal elution, the vacancy peak method, and retention analysis by zonal elution. Frontal elution is the most rigorous method since all the species at equilibrium are present in the mobile phase with known and constant concentrations. The most promising one is the Hummel and Dreyer method, because of the very small amount of protein injected in the mobile phase containing the drug. Drug-protein interactions may be studied by affinity chromatography by immobilizing one of the interacting species on the support. Comparison of the constants obtained with methods when both the drug and the protein are in solution is questionable, since the immobilized species in affinity separations differ in their physical properties from those in solution. The main advantage with studies on immobilized proteins is the easy comparison of the binding properties of various drugs, especially when they are enantiomeric. The results of the binding constants measured by different separation methods are given for the albumin-phenylbutazone and albumin-warfarin systems. Good agreement is generally obtained, which proves the validity of using chromatography as a tool to study drug-protein interactions.

Cardioprotective Effect of Morphine and a Blocker of Glycogen Synthase Kinase 3β, SB216763 [3-(2,4-Dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], via Inhibition of the Mitochondrial Permeability Transition Pore

Morphine has been shown to protect the myocardium against ischemia-reperfusion injury through inhibition of glycogen synthase kinase-3β (GSK-3β). Given that GSK-3β is known to modulate the mitochondrial permeability transition pore (mPTP), we investigated the role of mPTP in the cardioprotective effect of morphine and the GSK-3β inhibitor SB216763 [SB; 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] during ischemia-reperfusion. Both morphine (0.3 mg/kg) and SB (0.6 mg/kg) reduced infarct size in a model of regional myocardial ischemia-reperfusion in rats (13 ± 1 and 14 ± 3% of the area at risk versus 33 ± 4% in controls; p < 0.05). Morphine and SB protected the ischemic myocardium against Ca2+-induced mPTP opening as demonstrated by the increased capacity of mitochondria to retain Ca2+ when they were isolated from the ischemic zone 10 min after the onset of reperfusion (59 ± 8 and 66 ± 3 versus 29.5 ± 6 nmol Ca2+/mg · protein, respectively; p < 0.05). This was associated with a restoration of mitochondrial oxidative phosphorylation parameters. In isolated adult rat cardiomyocytes subjected to anoxia-reoxygenation, morphine (2 μM), SB (3 μM), and the direct mPTP inhibitor cyclosporine A (3 μM) delayed mPTP opening as assessed by the calcein loading Co2+-quenching technique. This was accompanied by an increase in cell survival as measured by nuclear staining with propidium iodide. These in vitro effects of morphine on inhibition of mPTP opening during anoxia-reoxygenation were suppressed by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin (0.1 μM). These data indicate that the infarct-limiting effect of morphine and SB is linked by a cause-effect relationship, which leads to an increased mitochondrial resistance and inhibition of mPTP opening through the PI3-kinase pathway and subsequent inactivation of GSK-3β.

Disease-induced variations in plasma protein levels. Implications for drug dosage regimens (Part I).

SummaryMany diseases appear to lead to a decrease of drug plasma binding due either to hypoalbuminaemia or to a modification of albumin structure. In other diseases, the binding of a drug may increase due to elevated concentrations of α1-acid glycoprotein or lipoproteins. However that may be, the free fraction of a drug may vary in different pathologies. But an increase or decrease of the drug free fraction does not automatically mean an increase or decrease of the free drug concentration. Whatever the drug, a variation in the volume of distribution more or less proportional to the variation in the plasma free fraction can be expected. With respect to the clearance, the problem is much more complex and depends on the hepatic extraction ratio of drug. If the extraction is related to the free fraction (fu) of drug, a variation in fu will lead to a variation in the total drug concentration but no variation in the free drug concentration and no change in the pharmacological effect. If the extraction of a drug is dependent on hepatic flow, a variation in fu will lead to a change in the free drug concentration (with no change in the total drug concentration) and hence changes in the pharmacological effect.The aim of this article is to review the literature concerning disease-induced variations in plasma protein levels during the past 10 years. Finally, possible implications for drug dosage regimens are discussed generally from examples studied in the literature.

In vitro effects of nicotine on mitochondrial respiration and superoxide anion generation

In this study, we investigated the effects of nicotine on rat brain mitochondria. The polarographic studies determined the effects on the respiratory chain, whereas enzymatic assays and [3H]-nicotine binding allowed us to precisely identify its target and site of action. The measurements of oxygen consumption showed a significantly concentration-dependent inhibition by nicotine (EC50 was 4.95x10(-11) M), and a maximal decrease of 23.90% at 10(-7) M. Nicotine bound to complex I of the respiratory chain and inhibited the NADH-Ubiquinone reductase activity. We also showed that nicotine and NADH were competitive on complex I. Effects of cotinine, the main nicotine metabolite, and nornicotine, were also investigated: nornicotine inhibited the mitochondrial respiration whereas cotinine did not. Because the complex I generates superoxide anion, we investigated the effects of nicotine, following NBT oxidation, and showed that nicotine was able to inhibit this reactive oxygen species (ROS) generation by 15.74% with an EC50 of 2.02x10(-11) M. In conclusion, the present study shows that nicotine interacts with the complex I of brain mitochondrial respiratory chain and decreases ROS generation. This may explain a part of the beneficial and protective effects of nicotine in few neurodegenerative diseases, as suggested by many epidemiological studies.

A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1-20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.

Peripheral Benzodiazepine Receptor-Induced Myocardial Protection is Mediated by Inhibition of Mitochondrial Membrane Permeabilization

Opening of the permeability transition pore (PTP) is a key event in ischemia-reperfusion injury and several ligands of the peripheral benzodiazepine receptor (PBR), a mitochondrial outer membrane protein possibly associated with PTP, have been demonstrated as potent cardioprotective agents. Here, we investigated the mechanisms by which the specific PBR ligand 4′-chlorodiazepam (CDZ) protected the myocardium against ischemia-reperfusion. In either global or regional models of myocardial ischemia-reperfusion in rats, CDZ reduced infarct size in a dose-dependent manner (e.g., 11 ± 1% of the area at risk at 10 mg/kg versus 31 ± 3% in control; p < 0.05) and to a similar extent as ischemic or diazoxide-induced preconditioning. CDZ (10 mg/kg) reduced apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining), restored mitochondrial recovery, improved oxidative phosphorylation parameters, and reduced mitochondrial membrane permeabilization with inhibition of cytochrome c and apoptosis-inducing factor releases. CDZ increased the resistance of mitochondria to Ca2+-induced PTP opening. All these cardioprotective effects of CDZ were associated with an improved stabilization of the association of Bcl-2 with the mitochondrial membrane and inhibition of the association of a cytosolic fragment of Bax, occurring during ischemia-reperfusion, with the outer mitochondrial membrane. In addition, the PTP opener atractyloside (20 μM) and the Bcl-2 inhibitor ethyl-2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1) (20 μM) abrogated CDZ-induced reduction of infarct size. These results demonstrate that PBR occupancy by CDZ renders the heart more resistant to ischemia-reperfusion injury by limiting mitochondrial membrane permeabilization. This is due to a reorganization of the balance between pro- and antiapoptotic proteins of the Bcl-2 family proteins at the level of mitochondrial membranes.

Evidence for binding of certain acidic drugs to α1-acid glycoprotein

The binding of some acidic drugs to alpha 1-AGP was studied by equilibrium dialysis at 37 degrees, pH 7.4. Certain acidic drugs bound to alpha 1-AGP at one binding site with a high affinity. Though the alpha 1-AGP plasma concentration is far lower than the HSA concentration, the association constants of some acidic drugs with alpha 1-AGP are high enough to suggest that binding to alpha 1-AGP will contribute significantly to the total plasma binding of these drugs.