Michel Praet

Structure of the adriamycin-cardiolipin complex Role in mitochondrial toxicity

Adriamycin and its derivatives are among the most efficient antimitotics used in clinical therapy. A specific cardiotoxicity places a limit on the total dose of adriamycin that may be administered. The mechanism of cardiac toxicity is complex. Data accumulated from in vitro and in vivo studies indicate a possible common cause for the inhibition of numerous enzymes and tissue degradation by a free radical mechanism: the binding of adriamycin to the inner mitochondrial membrane cardiolipin. The structure of the adriamycin-cardiolipin complex has been investigated by using physico-chemical techniques and via conformational analysis. The results open a rational way to design new structures that are less cardiotoxic.

A new class of free radical scavengers reducing adriamycin mitochondrial toxicity.

Beef heart mitochondria were incubated with ADM and NADH. An adriamycin semiquinone radical was detected using ESR spectroscopy. The semiquinone radical production rate is decreased upon addition of a scavenger (AD 20) in the reaction medium. NMRI mice were treated with AD 20 (70 mg/kg, i.p.) 15 min prior ADM injection (20 mg/kg, i.p.) or with ADM alone. Heart mitochondria were isolated 48 hr later. The enzymatic activities of complex I-III and complex IV of the mitochondrial respiratory chain were strongly depressed in animals receiving ADM alone, whereas these activities were almost completely restored in animals receiving AD 20 and ADM. Fluorescence depolarization measurements indicated that only mice treated with ADM alone presented a decreased fluidity of their cardiac mitochondrial membrane.

Comparison of adriamycin and derivatives uptake into large unilamellar lipid vesicles in response to a membrane potential.

The uptake of adriamycin (ADM) and several derivatives into large unilamellar vesicles (LUV) displaying a transmembrane potential and having a lipid composition close to that of the inner mitochondrial membrane has been measured. Drug association to neutral liposomes, made of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (70:30, w/w) was shown to be potential-dependent: in the absence of potential, accumulation of drug was almost undetectable, whereas between 11 and 50 nmol of drug/mumol phospholipid, depending on the anthracycline used, was associated to LUV exhibiting a membrane potential after 1 h incubation. Association of drugs to LUV with a lipid composition closer to that of the inner mitochondrial (cardiolipin, CL, 20%; PC 50%; PE, 30%, w/w) and displaying a membrane potential is higher than with neutral vesicles (between 40 and 76 nmol of anthracycline/mumol phospholipid after 1 h incubation). Since it is known that ADM and derivatives have a high affinity for CL, a fraction of the associated drug may bind to CL on the outer side of the vesicles. This was confirmed by the fact that, in the absence of potential, between 40 and 56 nmol of anthracycline/mumol phospholipid was still associated to LUV containing CL. In order to discriminate between drug adsorbed at the surface of the LUV and drug accumulated inside the LUV, an anthracycline fluorescence quencher (I-) was used. It was shown on neutral LUV displaying a membrane potential, that between 55 and 81% of the associated drug is actually entrapped inside the vesicles, inaccessible to the quencher. These percentages decreased to between 41 and 68%, respectively, in the presence of LUV containing CL and exhibiting a membrane potential, whereas for LUV of the same composition but displaying no membrane potential almost all the associated drug is adsorbed on the outer face of the LUV, accessible to the quencher, and likely bound to CL. This study brings evidence that antitumour anthracyclines despite important structural homologies do not accumulate to the same extent into vesicles mimicking the lipid composition and the membrane potential of mitoplasts. This ability to reach the matrix compartment of mitochondria could partly explain the differences of cardiotoxicities associated to anthracyclines with closely related molecular structure.

Increasing therapeutic effect and reducing toxicity of doxorubicin by N-acyl dehydroalanines

Doxorubicin toxicity is generally accepted to be free radical-mediated. N-Substituted dehydroalanines (indexed as AD compounds) are capto-dative olefins which react and scavenge free radicals, especially the superoxide anion (O2-) and hydroxyl radical (HO). AD-20, an orthomethoxyphenylacetyl dehydroalanine derivative, decreases the mortality of mice when administered before an acute single dose or multiple non-toxic doses of doxorubicin. Doxorubicin administered to mice induces elevated serum transaminase levels, and the pretreatment of mice with AD-20 decreases significantly these serum enzymatic activities. Preliminary histological examinations suggest that these serum transaminase elevations reflect most likely liver injury. In addition to its cardiotoxicity, doxorubicin induces a severe bone marrow depletion. Although this initial decrease in the peripheral leukocytes induced by doxorubicin is not prevented by the administration of AD-20, it produces a fast recuperation in the white blood cells levels after 1 week, supporting a protective effect at this level. Moreover, the antitumor effect of doxorubicin in L1210 tumor-bearing mice was enhanced when AD-20 was injected before doxorubicin. We postulate that these effects may be related to the free radical scavenging ability of AD-20.

Detection of lytic antimycobacterial antibodies by immune lysis of liposomes sensitized to tuberculin

Two procedures were used in order to incorporate purified protein derivative tuberculin (PPD) from M. tuberculosis, strain H37Rv, into calcein-containing liposomes: formation of multilamellar vesicles (MLV) in a PPD solution or exposure of preformed MLV to this solution. Immune lysis of these PPD-sensitized MLV was studied in the presence of a hyperimmune anti-M. tuberculosis sheep serum using a specific pathogen-free rabbit serum as a source of complement. A 50% release of encapsulated calcein was observed spectrofluorometrically after 30 min and remained unchanged up to 2 h. The release of calcein in the absence of complement or of anti-H37Rv serum or by liposomes which did not contain PPD never exceeded 1-2%. Liposomes formed in PPD solution were more sensitive to anti-H37Rv serum than preformed liposomes exposed to PPD. Trials with human sera from ten tuberculous patients revealed the presence of specific lytic immunoglobulins. In the presence of sera from skin test negative, non-tuberculous subjects, calcein release was significantly lower. This opens the way to a new method for the study of the humoral immunity in tuberculosis.

Reduction of Toxicity and Increase of Antitumor Effect of Adriamycin by N-Acyl Dehydroalanines, a New Family of Free Radical Scavengers

Free radicals are highly reactive chemical species, they abstract hydrogen atoms at sensitive sites, they add to olefinic C-atoms and they are substrates of one-electron exchange reactions. Thus they react easily with miscellaneous molecular targets. One of the main characteristics of free radical reactions is that they produce new free radicals, so that free radical processes propagate through a chain of reactions which cause not only structural but mainly functional disturbances at various cellular levels.

Free radicals formation and membrane damages induced by adriamycin in mitochondria: A study in vivo and in vitro

Adriamicin (ADM) is one of the most effective anthracycline glycoside antibiotic in the treatment of several types of cancer. Its clinical use is however limited by a specific cardiotoxicity. Deviation of electrons from NADPH supplemented cytochrome P-450 to the anthracycline results in the formation of a semiquinone radical which react with O2 to form toxic oxygen species (O2-, OH). These reactions were suggested to be involved in the cardiac toxicity process of ADM till the recent finding that cardiac microsomes do not participate to such reactions (Nohl & Jordan, 1983). Using the spin trapper DMPO combined with a flow technique, we were able to demonstrate that beef heart mitochondria, submitochondrial particles and complex I containing proteoliposomes catalyse the formation of O2- and OH species when incubated in presence of ADM and NADH. The semiquinone radical was also observed by ESR with a characteristic g value of 2.0024 and a line width of 0.4 mT. Interestingly, the 5-iminodaunorubicin analog did not induce the formation of oxygen radical species in good correlation with its weak cardiac toxicity. Membrane damages were further studied on heart mitochondria extracted from ADM-treated mice. A strong rigidifi-cation of the mitochondrial membrane was observed using fluorescence depolarization of a probe (DPH) embedded in the membrane. The rigidification can be considered as resulting from polymerization of peroxidized lipids in presence of O2- and OH species. No rigidification was observed on mitochondrial memmembrane extracted from 5-iminodaunorubicin-tretead mice in good agreement with our ESR results.