Role of daunorubicinol in daunorubicin-induced cardiotoxicity as evaluated with the model of isolated perfused rat heart.

Cardiotoxicity is the major side-effect and limits the clinical use of the anthracyclines, doxorubicin and daunorubicin. A special problem is raised by the metabolites of these drugs, the amount of which may vary according to drug combinations and formulations. Doxorubicinol, the 13-dihydroderivative of doxorubicin, has been shown to be more cardiotoxic than unchanged doxorubicin. Daunorubicinol has been assumed also to be more cardiotoxic than unchanged daunorubicin but its direct effect on cardiac function has never been evaluated in preclinical models. We have compared the cardiac effects (developed pressure, contractility and relaxation of the left ventricle) induced by daunorubicinol to those induced by daunorubicin, using the model of the isolated perfused rat heart. After treatment of rats with 6 doses of 3 mg/kg intraperitoneally, daunorubicin strongly decreased the cardiac functional parameters, while daunorubicinol did not induce cardiotoxicity. Both treatments induced a similar accumulation of daunorubicinol in the myocardium, while daunorubicin was only found in the hearts of rats treated with this drug. Direct perfusion of the hearts of untreated rats with both drugs at 10 microM induced a depression in heart function, but daunorubicin induced a progressive increase in diastolic pressure, reflecting the difficulties encountered by the heart to maintain its activity in the presence of this drug, while daunorubicinol did not. We conclude that daunorubicinol is not responsible for the important cardiac toxicity of daunorubicin.

Preclinical evaluation of the cardiotoxicity of taxane-anthracycline combinations using the model of isolated perfused rat heart.

Paclitaxel strongly potentiates the cardiotoxicity of doxorubicin in the clinical setting. In this study, we aimed (1) to determine whether this potentiation could be reproduced in an ex vivo model and, if so, (2) to select drugs and protocols that did not cause this potentiation. The effect of paclitaxel and docetaxel on the cardiotoxicity induced by doxorubicin and epirubicin was studied using the model of isolated perfused rat heart. Cardiac performances were evaluated after several combination protocols administered every 2 days over a period of 12 days, and anthracycline concentrations in the heart and liver were determined on Day 12. When administered simultaneously, paclitaxel strongly potentiated the cardiotoxicity of doxorubicin ex vivo, and this effect was not due to Cremophor EL, the solvent used in the formulation of paclitaxel. The potentiation of anthracycline cardiotoxicity could be avoided by the replacement of doxorubicin by epirubicin, and/or of paclitaxel by docetaxel. Cardiotoxic potentiation was also avoided by the introduction of a 24-h lag time between the repetitive injections of doxorubicin and docetaxel. The concentration of doxorubicin and its cardiotoxic metabolite, doxorubicinol, in the heart and liver was not significantly altered by the taxanes, but that of epirubicin was increased twofold both in the heart and the liver. These results show that the potentiation of doxorubicin-induced cardiotoxicity by paclitaxel can be reproduced with an ex vivo model, and that it is not related to an increase in tissue concentration of the drug or active metabolite. Our model, therefore, may be useful for the selection of anthracycline-containing protocols with no increased risk of cardiotoxicity for the patients.

Comparative cardiotoxicity of idarubicin and doxorubicin using the isolated perfused rat heart model.

Attempts to reduce the incidence of congestive heart failure following anthracycline therapy include the replacement of the parent compounds (especially doxorubicin) by less cardiotoxic analogs. Among these analogs, idarubicin (4-demethoxy-daunorubicin) was shown to be less cardiotoxic than doxorubicin in phase II clinical trials, but its actual cardiotoxicity has never been evaluated in large series and has never been compared to that of doxorubicin in relevant experimental models. Using the isolated perfused rat heart model, we compared the cardiac effects (developed pressure, contractility and relaxation of the left ventricle) induced by idarubicin to those induced by doxorubicin. Drugs were administered i.v. every other day for 11 days at doses of 1, 2, 2.5 and 3 mg/kg per injection for doxorubicin and 0.5, 0.75 and 1 mg/kg per injection for idarubicin. We confirmed that similar general toxicity symptoms were obtained for a dose ratio of 1:4 (idarubicin:doxorubicin). However, at the maximum tolerated doses of both drugs (3 mg/kg per injection for doxorubicin and 0.75 mg/kg per injection for idarubicin), the cardiac toxicity of idarubicin remained significantly lower than that of doxorubicin. Anthracycline cardiac accumulation was evaluated in parallel and revealed a lower cardiac accumulation of idarubicin, which could explain the reduced cardiac toxicity of this analog. Direct perfusion of the drugs in the isolated hearts of untreated animals revealed that idarubicin was taken up more readily than doxorubicin in the cardiac tissue, despite the fact that it had less deleterious effects on cardiac function. This indicates that idarubicin also had less intrinsic cardiotoxicity than doxorubicin in this model.

Preclinical evaluation of the cardiac toxicity of HMR-1826, a novel prodrug of doxorubicin.

Cardiotoxicity represents the major side-effect limiting the clinical use of anthracyclines, especially doxorubicin, in cancer chemotherapy. The use of non-toxic prodrugs, or of liposome-encapsulated drugs, allows a better targeting of the tumours and may, therefore, improve the tolerance to the treatment. Using the model of isolated perfused rat heart, we have evaluated the cardiotoxicity of a novel prodrug of doxorubicin, HMR-1826, which consists of the association of doxorubicin to glucuronic acid. We have compared the cardiac effects (developed pressure, contractility and relaxation of the left ventricle) induced by HMR-1826 to those induced by doxorubicin and Doxil, a liposomal form of doxorubicin. HMR-1826 was administered intravenously every other day for 11 days at doses of 50-200 mg kg(-1) per injection while doxorubicin was administered according to the same protocol at doses of 1-3 mg kg(-1) per injection. Doxorubicin strongly decreased the cardiac functional parameters at the doses of 2.5 and 3 mg kg(-1) per injection. Doxil (3 mg kg(-1) and HMR-1826 (50-150 mg kg(-1)) were largely devoid of cardiotoxicity. HMR-1826 only induced significant alterations of the cardiac function at the highest dose used (200 mg kg(-1) per injection). These alterations were much lower than those of doxorubicin at 2.5 mg kg(-1) per injection, despite similar general toxicity symptoms (weight loss, nose bleeding and diarrhoea) at these respective doses. Thus, HMR-1826 appeared about 100-fold less cardiotoxic than doxorubicin.

Role of glutathione in the detoxification of ferriprotoporphyrin IX in chloroquine resistant Plasmodium berghei.

The reduction in hemozoin content is a well known feature of chloroquine-resistant Plasmodium berghei. Using NK65-derived lines displaying increasing resistance levels, we observed an inverse relationship between the hemozoin content, and the glutathione (GSH) and glutathione S-transferase (GST) levels. Treatment of highly chloroquine-resistant-infected mice with buthionine sulfoximine (BSO), which has previously been shown to partially reverse this chloroquine resistance, led to a significant increase in hemozoin production. In vitro studies on the polymerization of ferriprotoporphirin IX (FPIX) at pH 5.0 showed that GSH partially inhibited beta-hematin synthesis, while GST had a trivial and non specific effect. Furthermore, chloroquine-sensitive parasites invading reticulocytes displayed higher GSH level and GST activity, and reduced hemozoin synthesis and susceptibility to chloroquine. We conclude that, in chloroquine resistant P.berghei, GSH can detoxify hemin within the food vacuole, thus precluding its polymerization and preventing the activity of chloroquine and other quinoline-containing drugs. It is proposed that vacuolar GSH could be ascribed to an erythrocytic origin, since the resistant lines invade reticulocytes, which contain higher levels of GSH and GST than normocytes.

High-level chloroquine resistance of Plasmodium berghei is associated with multiple drug resistance and loss of reversal by calcium antagonists.

The chloroquine resistance of Plasmodium falciparum is reversed in vitro by numerous compounds, including calcium antagonists, which could enhance the accumulation of the drug in the parasite food vacuole. However, this mechanism of resistance could be insufficient when the resistance level increases. Using in vitro drug trials on strains of Plasmodium berghei displaying various chloroquine-resistance levels, we confirmed previous results obtained in vivo in the chloroquine-resistant strains of P. berghei are cross-resistant to related drugs (amodiaquine, quinine and mefloquine), the resistance levels to these drugs being related to their analogy to chloroquine. Furthermore, we showed that high-level resistant lines were associated with a loss of drug potentiation by verapamil and nicardipine in vivo, but that the reversal rates obtained in vitro are of low significance. We conclude that the parasite is able to escape the activity of these reversing agents.

Interleukin 6 upregulates TNF-alpha-dependent C3-stimulating activity through enhancement of TNF-alpha specific binding on rat liver epithelial cells.

The authors investigated the role of tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6), two major pro-inflammatory cytokines, in the stimulation of the third component of complement (C3) production by rat liver epithelial cells. Though often considered as the most potent inflammatory cytokine, IL-6 alone displayed no activity, whereas TNF-alpha upregulated C3 production in a dose-dependent manner. However, IL-6 was shown to synergistically stimulate C3 production in the presence of TNF-alpha. To account for this interaction, it was postulated that IL-6 modulates the binding of TNF-alpha on liver target cells. That IL-6 increased the binding of TNF-alpha on the surface of the hepatic cells, whereas TNF-alpha alone downregulated its own specific binding capacity is reported. Furthermore, this upregulatory effect of IL-6 was mainly attributable to an increase in the number of plasma membrane TNF-alpha specific receptors, with little change in their affinity. These results suggest that the synergistic IL-6 activity on C3 production may occur, at least partially, through its capacity to upregulate the number of TNF-alpha receptors on the surface of the rat liver epithelial cells.

Plasmodium berghei: implication of intracellular glutathione and its related enzyme in chloroquine resistance in vivo.

Glutathione (GSH) plays a critical role in the detoxication and the protection of cells against oxidative stress. In the present study we examined the relationship between the intracellular GSH levels as well as glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx) activities and how they relate to Plasmodium berghei resistance to chloroquine. Resistant strains (CQR30 and CQR60) were selected in vivo from a sensitive strain (NK65). Marked increases in GSH levels and GST activity within resistant parasites were observed, compared to sensitive parasites. On the other hand, GR and GPx activities were similar in sensitive and resistant parasites. Treatment with chloroquine did not influence the intracellular level of GSH, but it was found to significantly decrease GR activity. Intracellular depletion of GSH, by a nontoxic concentration of buthionine sulfoximine (BSO), significantly sensitized the resistant parasites to chloroquine. These results suggest that the P. berghei resistance results from altered GSH and GST levels and activity, respectively, which enable the detoxification of chloroquine in resistant parasites.

Human interleukin-6 acts as a co-factor for the up-regulation of C3 production by rat liver epithelial cells.

We investigated the role of human interleukin-6 (IL-6) in the regulation of the third component of complement (C3) biosynthesis by cultured rat liver epithelial cells. A natural human IL-6 (nh IL-6) preparation was shown to up-regulate C3 production, whereas an Escherichia coli-derived recombinant human IL-6 (rh IL-6) displayed no activity on C3 biosynthesis. However, the C3-stimulating activity of the nh IL-6 preparation was only partially reduced when treated with an antihuman IL-6 monoclonal antibody. Binding studies indicated that although it was devoid of any C3 stimulating activity, rh IL-6 specifically bound to hepatic cell receptors (Kd = 0.38 nM) and possessed the same binding affinity as nh IL-6. Furthermore, the substitution of natural IL-6 molecules for the recombinant IL-6 led to the recovery of the initial C3-stimulating activity. These studies demonstrated that human IL-6 alone does not stimulate rat liver epithelial cell C3 production but is able to accentuate the C3-stimulating activity of unrecognized components which are present in the nh IL-6 preparation. Human IL-6 thus appears to act as a co-factor for the up-regulation of hepatic C3 production.

Virus inactivation of fresh frozen plasma by a solvent detergent procedure: biological results.

In order to increase the safety of blood products, we have developed a procedure for the virus inactivation of fresh frozen plasma. Several batches have been prepared and with the first 10 batches, each of them composed of 60 litres of plasma, we have determined a set of biological parameters. Virus inactivation was realised using TnBP (1%) and Octoxynol 9 (1%). After their elimination with castor oil using chromatography on insolubilized C18 resin, glycine was added and the pH of the plasma was adjusted to 7.4. Plastic bags were aseptically filled with a mean volume of 200 ml of plasma. The mean levels of coagulation factors were all over 0.7 U/ml and their recovery from initial plasma was nearly the same as total protein except for factor VIII:C. The net loss in factor VIII:C was 16%, when including the dilution of plasma. In vivo and in vitro tests demonstrated that in the final product there were no activated factors. As in fresh frozen plasma, the protein concentration was over 50 g/l and the potassium level lower than 5 mmol/l. According to these results, virus-inactivated plasma has the same qualities of fresh frozen plasma and could now replace it.

[Isolation from a pregnant woman of a serum protein responsible for inhibiting an in vitro cytotoxicity reaction]. / Isolement chez la femme enceinte d'une protéine sérique responsable de l'inhibition d'une réaction de cytotoxicité in vitro.

A non specific immunosupressive factor able to block an in vitro cytotoxicity reaction is demonstrated in the serum of pregnant women. A solution containing this blocking factor is obtained by gel filtration and precipitation of plasma by polyethylene glycol 4 000. Then after immunisation of rabbits the immune serum can be used for affinity chromatography. An alpha 2 glycoprotein has been separated which inhibits the in vitro cytotoxicity reaction and whose molecular weight determined by gel filtration and polyacrylamide gel is about 200 000 daltons.