Anthracycline glycoside-membrane interactions

Biochimica et Biophysica Acta, 779 (1984) 271-288

271

Elsevier BBA 85263

ANTHRACYCLINE GLYCOSIDE-MEMBRANE INTERACTIONS E. GOORMAGHTIGH and J.M. RUYSSCHAERT Laboratoire de Chimie Physique des Macromolbcules aux Interfaces, Universitb Libre de Bruxelles CP 206 / 2, Boulevard du Triomphe, 1050 Bruxelles (Belgium)

(Received July 13th, 1983)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

271

II. Membrane-anthracyclineglycoside interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Microsome membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Plasmamembrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mitochondrial inner membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cytochrome c oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. CoQ-dependent enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Phosphate carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Calcium transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

272 272 274 275 276 277 280 280 280

III. Physicochemicalstudies of the anthracycline glycoside-lipidinteraction in model membranes . . . . . . . . . . . . . . . . . . . . .

281

IV. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

282

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

284

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction T h e a n t h r a c y c l i n e glycoside a n t i b i o t i c family is o n e of the most effective against several types of cancer (leukemia, solid tumors). A m o n g the m e m bers of this family, a d r i a m y c i n ( N S C 123127), d a u n o m y c i n (NSC 82151) a n d r u b i d a z o n e ( N S C 164011) are the most widely used in clinical therapy. F r o m a chemical p o i n t of view, they constitute an a m i n o - s u g a r linked to a n a n t h r a c y c l i n e ring by a glycoside b o n d . The structure of some of the most widely used drugs of this family is given in Fig. 1. T h e m o d e of i n t e r a c t i o n of a d r i a m y c i n with its n u c l e a r target has b e e n previously re0304-4157/84/$03.00 © 1984 Elsevier Science Publishers B.V.

viewed [1-3]. Both X-ray m e a s u r e m e n t s [4,5] a n d c o n f o r m a t i o n analysis [6] indicate that the p l a n a r moiety of a d r i a m y c i n intercalates between the base-pairs, whereas the sugar moiety fits into the large D N A groove. A n t h r a c y c l i n e glycosides display toxic side-effects against a large variety of cells. Their cardiotoxicity is, however, very specific a n d places a limit on the total dose that m a y be given; the effect is c u m u l a t i v e over several m o n t h s (for a review see Ref. 7). Such a dose-limiting cardiotoxicity is n o t observed with the a d m i n i s t r a t i o n of other a n t i c a n c e r drugs [8]. Interestingly, in a series of related anthracycline glycoside drugs, the

272

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cardiac toxicity can be dissociated from the antitumor activity [9], suggesting a distinct mode of action. Several data permit correlation of this cardiac toxicity with an anthracycline glycosidemembrane interaction. This review is essentially focused on the mode of interaction of anthracycline glycoside drugs with membranes (microsome membranes, plasma membranes and mitochondrial membranes). Since much evidence suggests that the mitochondrial membrane could be the target responsible for the anthracycline glycoside cardiotoxicity, we will discuss the mode of action of these drugs on its essential membrane components (cytochrome c oxidase, N A D H dehydrogenase, succinate dehydrogenase, phosphate carrier and ATPase). We will analyze how the specific adriamycin-cardiolipin complex participates to the mitochondrial toxicity and how a molecular knowledge of its structure could help in the synthesis of new compounds with an increased antitumor activity/cardiotoxicity ratio.

II. Membrane-anthracycline glycoside interactions To reach its nuclear target, adriamycin and derivatives have to cross several membrane bartiers. The mechanism of cellular uptake and re-

R3 2OH 3 2OH 3

-NH 2 -NH 2 -NH-CO-CH 3 -NH-CO-CH 3

lease of these drugs has been extensively reviewed [10] and will not be discussed in this review. We will summarize here the membrane modifications induced by anthracycline glycosides. Effects of these drugs were reported essentially in the microsome membrane, plasma membrane and inner mitochondrial membrane. 11.4. Microsome membrane

In heart sarcosomes and liver microsomes of rats, adriamycin, daunomycin and other derivatives [11-14] dramatically enhance electron flow from N A D P H to molecular oxygen [15]. The process is enzymatic. Most data have been obtained on phenobarbitaMnduced liver microsomes. The K m values for the anthracycline antibiotics tested range from 100 to 900 # M (Table I). Hexobarbital does not decrease the anthracycline effects, although it is a substrate for microsomal hydroxylation at the cytochrome P-450 level [16]. This observation indicates that anthracyclines act at an earlier step in electron transport than hexobarbital hydroxylation. Electron spin resonance studies indicate that the anthraquinone nucleus of anthracyclines is reversibly converted to a free-radical semiquinone which serves to shuttle electrons to

273 TABLE I KINETIC PARAMETERS FOR NADPH CYTOCHROME P-450 O X Y G E N C O N S U M P T I O N S T I M U L A T E D BY A N T H R A C Y C L I N E GLYCOSIDES [15].

Km(~M) Adriamycin 960 N-Dimethyladriamycin 1080 Daunomycin 830 N-Acetyldaunomycin 110 Daunorubicinol 570 Rubidazone 160 Nogalamycin 210 Steffimycin 120 Aclacinomycin A 120 Carminomycin 70

Vrnax(mol/min per mg) (xl07) 26.9 43.3 24.2 3.2 19.2 8.1 5.4 10.7 10.7 13.4

02. During this aerobic process, daunomycin and adriamycin remain unchanged [17] and no metabolites are identified [16]. In anaerobic conditions, a reductive cleavage reaction of these adriamycin, daunomycin [16] and aclacinomycin seems, however, to occur as long as N A D P H is available [18,19]. The published polarographic half-wave potentials for two-electron reduction of the quinone nucleus of adriamycin are of such magnitude that neither N A D P H nor N A D H would be capable of a two-electron reduction in vivo [20]. Goodman and Hochstein [21] suggest that during the reduction-autooxidation cycle of the quinone group, superoxide radicals are formed (destroyed by superoxide dismutase). Hydrogen peroxide is also generated, presumably by non-enzymatic dismutation of superoxide. Peroxidation of lipids by superoxide a n d / o r hydroxyl radicals has also been pointed out [20-33]. According to Mimnaugh et al. [34], superoxide anion 0 2 a n d / o r secondary reactive oxygen species are believed to initiate and propagate the peroxidation of unsaturated membrane lipids, as already described [35-38]. Alternatively [34], electrons could have passed directly from the semiquinone to microsomal lipids, forming lipid radicals that could then undergo peroxidation by oxygen addition. Furthermore, products originating from the peroxidation of microsomal lipids have been demonstrated to bind convalently to rnicrosomal proteins [11] and are likely to damage other biomolecules. When living cells are treated with adriamycin and daunomycin, they

rapidly show signs of DNA damage [39,40] which are never observed in the in vitro adriamycin-DNA complex. Activation of adriamycin in the cell seems to be necessary for damage to be inflicted on the DNA. Since free radicals are highly reactive, they are believed [16] to react either directly with the D N A strands or indirectly in producing oxygen species. Such an explanation still remains difficult to accept. Indeed: the semiquinone radical unstability in normal aerobic conditions prohibits its transport from the reticulum activating site into the nucleus. the generation of free radicals is completely abolished when adriamycin is bound to a nucleic acid [22,23]. Fig. 2 summarizes how adriamycin free-radical formation can damage membrane components or even other biomolecules. From a medical point of view, the formation of semiquinone free radicals activated by the P-450 electron transport system could account for mutagenic properties of adriamycin and daunomycin in man [55]. Indeed, in presence of reducing agents, adriamycin and daunomycin bind covalently to nucleic acids and proteins [51,52]. The exact species that alkylates cellular macromolecules is not known at this time, but a C 7 quinone methide and C 7 free-radical have been proposed [53,54]. The ESR spectrum of the semiquinone radical is indeed time-dependent and progressively changes from a symmetric motionally narrowed spectrum into a completely immobilized spectrum [43,51]. Binding of the semiquinone a n d / o r polymerization of the anthracyclines have been proposed [22,43] to explain this observation. The anticancer properties as well as the cardiotoxicity of the anthracycline glycosides can arise from the semiquinone radical formation. Vitamin E [26,56,57], reduced glutathione or sulfhydryl group [43] and the radical scavenger atocopherol [20,29,58-61] are reported to decrease the cardiotoxicity, although failure of vitamin E to protect against adriamycin-induced cardiotoxicity is sometimes observed [62]. Some selectivity of free radicals towards tumor cells can be expected. Indeed: evidence has been presented that a loss of Mn superoxide dismutase is intimately related to the cancerous phenotype [63]. Therefore, the potential use of O~- in cancer therapy may be eriormous,

274 0 NADPH

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Fig. 2. Possible mechanisms of adriamycin-stimulated damaging reactions in microsomes. Following the formation of adriamycin semiquinone radicals by NADPH-cytochrome P-450 reductase, a single electron may be passed directly to unsaturated membrane lipids forming lipid free radicals or to molecular oxygen forming superoxide anions and other reactive oxygen species that initiate and promote lipid peroxidation and possibly damage other macromoleculesin viva. Alternatively, the adriamycin semiquinone radical can react directly with unspecific biomolecules without reoxidation to the quinone initial form. Products obtained by autoreductive cleavage of the molecule could play the same role. However, the reaction with oxygen seems to predominate over all other reactions. Predominating reactions are tentatively represented by thick arrows. When studied, effects of inhibitors are indicated by a double line. since, if equal a m o u n t s of O f c a n be delivered to b o t h cancer cells a n d n o r m a l cells, then the cancer cell should be preferentially killed because it has lower M n superoxide dismutase activity. O~- prod u c t i o n by cells in presence of anthracycline glycosides is, in this context, of the highest interest. Moreover, t u m o r cells are also deficient in glutathione peroxide [64] a n d catalase [65]. This m a y e n h a n c e again the selective toxicity of the a n t h r a cycline glycosides. Because poorly perfused hypoxic tumors have a m u c h lower level of intracellular oxygen, they are possibly a privileged site for other reactions involving the s e m i q u i n o n e radical directly. Indeed, the free radical m a y be more stable at low oxygen c o n c e n t r a t i o n a n d thus toxic reactions are more likely to be e n h a n c e d u n d e r hypoxic conditions, as observed o n Sarcoma 180 [66]. O n the other hand, it m u s t be m e n t i o n e d that T a n n o c k [67] f o u n d that

hypoxic cells may be more resistant to a d r i a m y c i n t h a n aerobic cells. The reasons for the differences between these results are not clear. I n conclusion, activation of anthracycline drugs by the N A D P H - P - 4 5 0 electron transport system of microsomes is expected to play a role in the destruction of m a l i g n a n t cells and, less probably, in the cardiotoxicity. M u l i a w a n et al. [68] were u n a b l e to d e m o n s t r a t e a significant increase in lipid peroxidation measured as ethane f o r m a t i o n in adriamycin-treated rats. This observation questions whether lipid peroxidation can be considered as a n essential event in a d r i a m y c i n toxicity. l i B . Plasma membrane A d r i a m y c i n effects at the level of the p l a s m a m e m b r a n e are n u m e r o u s b u t their i m p o r t a n c e for the entire cell life a n d functions is unclear. C o n f u -

275

sion arises from the lack of knowledge in the sequence of events. Some of the described events should probably be considered as resulting from toxic effects of adriamycin somewhere else in the cell rather than resulting directly from its interaction with the plasma membrane. Interesting is the finding of Tritton and Yee [69] that adriamycin can exert a cytotoxic action solely by interaction at the cell surface. In their experiments, murine cancer cells (L1210) were exposed to large, insoluble polymeric agarose beads to which adriamycin had been covalently attached. Since the support was larger than the cells, the drug could not penetrate to the cytoplasm or nucleus. The absence of free adriamycin or metabolites inside the cells was demonstrated by high-pressure liquid chromatography [70]. Moreover, it appeared that immobilized adriamycin is 100- to 1000-times more lethal than free adriamycin. The cell surface would therefore be the most sensitive drug target. It is also possible that immobilized adriamycin could lead to the formation of toxic activation oxygen species. Indeed, adriamycin affects redox functions in liver plasma membrane in stimulating NADH oxidase activity with a K m of approx. 1.5/tM [70,71]. This increase in activity is accompanied by increased superoxide formation. Experimental data suggest an action of adriamycin close to the dehydrogenase. Consequences of this adriamycin effect at the plasma membrane level are still unknown, but its importance could be very selective in different cells [72]. An oxidative destruction of erythrocytes ghost membranes catalyzed by an adriamycin-iron complex has also been reported. A decrease in the fluidity of the plasma membrane was measured by ESR spectroscopy in Sarcoma 180 cells [73] after adriamycin treatment. One possible mechanism to explain the membrane rigidification is the formation of free radicals activated by the dehydrogenase as described in microsomes. The mechanism by which adriamycin should activate the dehydrogenase from outside the cell remains to be explained. Other plasma membrane functions were altered by incubation in presence of adriamycin; however, it is not possible to relate them to the prime interaction of adriamycin with the membrane. Modifications of the lipid composition [74-79], of the membrane glycoprotein and glyco-

lipid compositions [80,83] may explain a new susceptibility to immune attack [74-87] and to monoclonal antibody [88]. Unfortunately, the possibility that immune attack due to these surface properties modifications could account for the chronic cardiac toxicity has never been evaluated. Although effects of anthracycline glycosides on the plasma membrane enzymes were only slightly studied, an effect on the Na + transport [89], on the isocitrate dehydrogenase [90], on the membrane potential [91], on the rate of aggregation of lectin receptors [92] and perhaps also on aminoacid [92] and Ca2+ [93] transport was suggested. Among others, inhibition of Na+/K+-stimulated ATPase [94,95] and Na + / C a 2÷-antiporter of heart sarcolemmal vesicles [96] could play a role in cardiotoxicity. Indeed, in heart cells, Ca2+ fluxes are also controlled by two plasma membrane (sarcolemma) pumping systems: a specific ATPase [97] and an Na+/Ca2+-exchange system [98,99]. The Ca2+ disturbance induced by adriamycin could thus conceivably be mediated by the impairment of either one of these two pumping systems, or of both. An effect of adriamycin on these systems is indicated by the finding of reduced exchangeability of Ca2+ in adriamycin-treated isolated cardiac cells [100,101]. Indeed, Caroni et al. [96] demonstrated that adriamycin inhibits the N a + / C a 2+ exchange of dog-heart sarcolemmal vesicles. Gosalvez et al. [102] have claimed that the effect of adriamycin on Ca2+ transport might be related to its CaE+-chelating properties. However, experiments performed in our laboratory indicate that the strength of binding is not large enough for this complex to exist in vivo. The persistence of the inhibition in membranes diluted after preincubation in presence of adriamycin indicates tight binding, but offers no clues as to the nature of the site of interaction. HC. Mitochondrial inner membrane

The development of cardiac failure induced by adriamycin is characterized by a good correlation with the impairment of the mitochondrial functions (O2 consumption and proportional ATP synthesis) [103-105] without perturbation of the sliding of actin and myosin filaments across each other [105]. The rhythmic contractions characteris-

276

tic of myocardiac cells in culture cease with adriamycin treatment [106-109] concomitant with a significant decrease of ATP and phosphocreatin concentrations [110,111]. Histologically, mitochondrial changes are characterized by formation of myelin-like figures [112,113] generally related to the formation of a drug-lipid complex [114-116]. The mitochondrial effect seems to be independent of a nuclear effect, at least at early times [117]. Studies in myocardiac cells in culture show that the inhibition of cell growth and of the rhythmic contractions are due to lack of ATP synthesis [118,119] whereas the A D P / O ratio remains unaltered [103,105,120-122] for adriamycin and daunomycin except at high concentrations [110]. In bovine heart mitochondria, adriamycin and daunomycin inhibit glutamate and pyruvate-malate dependent oxidative phosphorylation. At concentrations much larger than those found in the heart, no inhibition of Ehrlich ascite tumor cells respiration is observed [123]. The effect of adriamycin on cultured heart cells is dose-dependent and observable at concentrations as low as 1 /xM [124]. An inhibitory effect of rubidazone [103] on heart mitochondria has been also reported. Effects of nogalamycin and N-acetyladriamycin were, however, very weak [125,126]. In order to understand the effects of anthracycline glycosides on the whole respiratory chain, we will discuss successively their mode of action on the different membrane components. It was pointed out that adriamycin has a multisite effect on the respiratory chain [127]. As it is unlikely that adriamycin and derivatives interact with each enzyme catalyzing electron transport between N A D H (or succinate) and 02, it was suggested that an interaction of these drugs with a unique phospholipid site could take into account each of the observed effects. Cardiolipin, a phospholipid specific to the inner mitochondrial membrane, was suggested to play this role [128]. Indeed, it was recently demonstrated that most of the enzymes of the respiratory chain require cardiolipin for full enzymatic activity. It is significant that adriamycin is the most cardiotoxic compound and forms the strongest complex with cardiolipin [129]. Rubidazone, which is less toxic than adriamycin at the mitochondrial level [103], binds less effectively to cardiolipin.

Probably because of its high hydrophobicity, rubidazone is also a weak uncoupling agent of mitochondrial respiration [103]. Nogalamycin has no effect on the pigeon heart and rat liver respiration [125,126]. A weak toxicity at this level can again be correlated to a relatively weak affinity for cardiolipin. N-Acetyladriamycin, which does not bind to cardiolipin, does not perturb either rat electrocardiograms or mitochondrial respiration [1261. HC-1. Cytochrome c oxidase

The absolute requirement of cardiolipin for the last oxidation site of the respiratory chain was first suggested by Awasthi et al. [130], then disavowed [131-133] but definitively demonstrated by Fry et al. [134] in 1980. Only the number of cardiolipin molecules associated with the cytochrome c oxidase complex is still under discussion [135-139]. In addition to a few cardiolipin molecules playing a catalytic role, the enzymatic complex must be included in a larger lipid domain (role of dispersion) [134,137,140], which is itself modified in its structure and mobility by the presence of the enzyme [141-145]. Clearly, the lipid-enzyme interactions are of prime importance for the cytochrome c oxidase activity. The inhibition of cytochrome c oxidase activity by seven anthracycline glycosides has been reported [146]; the mechanism of the inhibition was shown to result from the complexation of the enzyme cardiolipin environment rather than from a drug-enzyme direct interaction. Fig. 3 reports the linear relationship found between the affinity of the drug for cardiolipin (a mainly electrostatic complex is formed) and the concentration inhibiting 50% of the cytochrome c oxidase activity on mitochondria extracted from bovine heart. Moreover, the same drug (namely adriamycin) inhibits the enzymatic activity to different extent if purified and lipid-depleted cytochrome c oxidase is reactivated by cardiolipin or phosphatidic acid in proteoliposomes (phosphatidic acid is found in very small amount in mitochondrial membrane but is able in vitro to reactivate cardiolipin-depleted cytochrome c oxidase [147,148]). The affinity of adriamycin for cardiolipin is about 80-times higher than for phosphatidic acid [128] and the adriamycin concentration required to inhibit 50% of the cytochrome c oxidase activity is precisely

277

80-times higher in the phosphatidic-acid-reconstituted system than in the cardiolipin-reconstituted system. Differential scanning calorimetry measurements carried out on mixed DPPCcardiolipin liposomes demonstrated that after addition of adriamycin, the adriamycin-cardiolipin complex segregates in the lipid matrix to form a separate phase. This model was hypothetically extended to the proteoliposome system containing cardiolipin, various phospholipids and cytochrome c oxidase. A schematic representation of the hypothetical enzyme inactivation mechanism is proposed in Fig. 4. Another possibility for explaining the cytochrome c oxidase inhibition due to the cardiolipin-drug complex formation arises from the mechanism of interaction between cytochrome c and cytochrome c oxidase. Cytochrome c is believed to bind to cardiolipin and to induce cardiolipin non-bilayer structures in order to reach a region of the cytochrome c oxidase complex buried in the bilayer [149-152]. 31p-NMR measurements showed that adriamycin inhibits the

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formation of non-bilayer cardiolipin structures normally induced by the presence of cytochrome c [153].

HC-2. CoQ dependent enzymes N A D H dehydrogenase and succinate dehydrogenase were found to be inhibited by adriamycin [154,155]. Reactivation of these enzymes was achieved when various CoQ were added to the system, CoQ10 being more efficient in reactivating these systems than shorter homologs. According to Folkers and co-workers, this indicates a clear specificity of adriamycin for CoQ dependent enzymes. This conclusion is, however, not straightforward, since it is clear that formation of a complex between the CoQ and adriamycin could also explain the inhibition process. Indeed, these experiments were carried out on CoQ10-depleted rnitochondria and the externally added CoQ could be complexed, at least partially by adriamycin. The existence of such a complex has not yet been investigated. It is suggested that CoQm could decrease cardiac toxicity [156-158], but success is not constant [159]. Moreover, CoQ10 pretreatment of mice did not protect against adriamycin cardiac toxicity at high intraperitoneal dose levels. When adriamycin was administrated by the clinically used intravenous route, CoQm did not reduce the adriamycin toxicity, even at moderately lethal doses [160]. A recent study of M u h a m m e d et al. [161] shows that succinate o x i d a t i o n in h e a r t mitochondria was strikingly more sensitive than

_~

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Fig. 3. Relation between the anthracycline glycoside concentration inhibiting 50% of the cytochrome c oxidase activity on extracted bovine heart mitocbondria and the dissociation constant of the cardiolipin-drug complex. 1, Adriamycin; 2, cinerubin; 3, rubidazone; 4, nogalamycin; 5, rhodomycin. Relation between adriamycin concentration inhibiting 50% of cytochrome c oxydase activity in a system containing purified and lipid-depleted cytochrome c oxidase included in pure cardiolipin liposomes (6) or pure phosphatidic acid liposomes (7) and its affinity for respectively cardiolipin and phosphatidic acid.

adriamycin

i

i3El

Fig. 4. Schematic representation of cytochrome c oxidase inactivation mechanism. On the left side of the figure, cardiolipin O is in close contact with the enzyme, permitting its activity. After reaction with adriamycin (right side) the complexed cardiolipin (O) segregates in a separate phase inaccessible to the enzyme, which remains in a lipid environment ([]) incapable of activating it.

278 N A D H dehydrogenase to adriamycin. A significant observation is the tremendous sensitivity to adriamycin of succinate oxidation (C50 ~ = 57/~M) in phosphorylating heart mitochondria in presence of hexokinase (C50 ~ = 5 /~M). The hexokinase effect was lost when membrane integrity was damaged by freeze-thawing or by sonic oscillation [162]. Gosalvez et al. [127] observed that the oxidation of N A D ÷-linked substrates by heart mitochondria was more sensitive to inhibition by adriamycin than was succinate oxidation. In contrast, Zbinden et al. [163] found the opposite trend. For these latter studies, the C50~ is in the range of 200-500 /~M. These discrepancies can be explained by the strong dependence of the inhibition process on the conditions of assay (phosphorylating or non-phosphorylating mitochondria) and on the presence of hexokinase. Whether the greater potency of the drug under these experimental conditions is due to its internalization mechanism has not been investigated. Indeed, one may suppose that the existence of an electrochemical gradient, negative inside the phosphorylating mitochondria, could accelerate the entrance of the cationic drug. Contrary to the inhibition results, adriamycin and derivatives were reported to enhance the N A D H dehydrogenase and cytochrome c reductase activity in some cases [164-167]. This phenomenon was related to the finding that cardiolipin is essential for catalytic activity of these two complexes [168], whereas other phospholipids are also required as dispersing agents [168-170]. Adriamycin was found to have a two-step mode of action on the complex I - I I I segment of the cardiac mitochondrial respiratory chain [164]. (1) A prime interaction with cardiolipin results in the formation of an electrostatic adriamycin-CL complex. This complex is capable to transfer electrons f r o m N A D H to c y t o c h r o m e c in ubiquinone-depleted mitochondria with reversible reduction of the anthraquinone moiety of the adriamycin molecule. Increased activity of the complex I and complex III results from this adriamycin-cardiolipin complex formation in isolated heart submitochondrial particles and N A DH-dehydrogenase-containing proteoliposomes. (2) Transfer of electrons through adriamycin results in enhanced chemical reactivity of

adriamycin which binds covalently to cardiolipin. Indeed, infrared spectra of the adriamycincardiolipin complex after the electron transfer reaction reveal a few new absorption bands, the intensity of which increases as a function of the incubation time in presence of the electron donor N A D H [171]. After a few hours of incubation in presence of N A D H , membrane fluidity is much decreased and the capacity of adriamycin to transfer electrons is abolished. The decrease of membrane fluidity fits well with a lipid peroxidation [172,173], although the fluorescence depolarisation technique used could be inappropriate, since the fluorescence probe seems to be chemically modified by peroxidized lipids [174]. A Schiff base addition could explain the formation of a covalent linkage [175]. After the reaction is ended, a decline in extractable phospholipid is noted for both proteoliposomes [166] and submitochondrial particles [176]. This was correlated to the degree of phospholipid chemical degradation. Phosphatidylethanolamine and cardiolipin, with an average number of double bonds per fatty acid of 1.83 and 1.86 respectively, were degraded at twice the rate observed for phosphatidylcholine (1.07 double bonds per fatty acid). Both step 1 and step 2 are not observed with N-acetyladriamycin [164], which binds neither cardiolipin nor 5-iminodaunorubicin [165], in which the quinone moiety of the daunomycin molecule is stabilized [177,178] by the replacement of C=O with C=NH. It must be pointed out that enhanced N A D H dehydrogenase activity was demonstrated only in the case of submitochondrial particles or proteoliposomes. As we discussed previously [166], interaction of adriamycin with complex I in these two systems is different from the interaction in intact mitochondria. It is indeed known that quinone may interact at different sites inside complex I [179-181] and that sonication can modify the accessibility of the different sites to adriamycin. We demonstrated recently that depending on the sonication time, complex I is first inhibited by adriamycin but is reactivated by adriamycin as the sonication time increases. The pathway of electrons in bovine heart submitochondrial particles was recently investigated by Davies et al. [182]. In presence of rotenone, adriamycin enhanced N A D H oxidation and 02

279

consumption. Succinate can not substitute for N A D H as the electron source. A one-electron reduction of adriamycin by N A D H dehydrogenase was demonstrated by the formation of an adriamycin free-radical observed by electron spin resonance measurements (ESR) ( g = 2.004). The 5-iminodaunorubicin (5-IDAU) radical was observed at a concentration as high as 0.3 mM [182]. Superoxide, hydrogen peroxide and hydroxyl radical evaluation suggests that the drug free-radical reduces molecular oxygen, producing 0 2 which then undergoes dismutation to yield H202. The level of hydroxyl radical seems to us to be overestimated, since the D M P O - O H adduct was used as a measure of the O H concentration [182]. Indeed, the DMPO-O3- adduct is known to be transformed into the DMPO-OH" adduct within less than 1 min and is therefore always detected, even in absence of O H . In the original paper of Thayer [183], 50 # M adriamycin was estimated to be sufficient for obtaining half-maximal stimulation of 03- production. ,Kinetic constants for superoxide production by N A D H dehydrogenase incorporated in liposomes were measured for five adriamycin derivatives by Doroshow [167] (Table II). In intact mitochondria, superimposed on the pattern of inhibition, a one-electron reduction of adriamycin was shown to occur with subsequent superoxide formation [184]. A K m of 454 ttM and a Vmax of 52.6 n m o l / m i n per mg were measured for adriamycin induced 03- formation, independent of the integrity state of the membrane. Every adriamycin derivative in Table II increases 03production except 5-iminodaunorubicin . In contrast to what we observed on liver microsomes, the semiquinone radical is stable even in presence of oxygen. The use of DMPO as spin trapper demonstrates that oxygenated radicals do not appear [185]. However, from recent experience, using a flow technique, we were able to demonstrate the presence of a DMPO-O 2 adduct with a short life-time (unpublished data). This observation must be compared with the work of Brown and Wiathrich [186], who demonstrated that the loss of spin label ESR signal is related to the presence of oxidized lipids, especially cardiolipin, in presence of cytochrome c. The observation that adriamycin can yield free

TA B LE I1 K I N E T I C C O N S TA N TS F O R S U P E R O X I D E F O R M A T I O N BY N A D H D E H Y D R O G E N A S E A F T E R INCUBAT I O N IN P R E S E N C E OF A N T H R A C Y C L I N E G LY C O SIDES From Ref. 167.

Adriamycin Daunomycin Rubidazone Aclacinomycin A 5-lminodaunomycin

K m (gM)

I'm~ ( n m o l / m i n )

167.3 73.3 64.0 47.6 -

8.0 8.1 5.6 2.1 0.0

radical species in cardiac mitochondria is of special importance, since it has been reported that heart sarcosomes are poorly active [15] or not at all active [185] in generating radicals. Mitochondria would remain the main subcellular organite responsible for free-radical formation. Since peroxidized cardiolipin and other phospholipids react covalently with protein [45,46], probably by freeradical chain polymerization [47,48] or by crosslinking induced by malonaldehyde [49], and since lipid peroxidation in the inner mitochondrial membrane, characterized by a decrease of 20--30% in membrane fluidity, inactivates most of the respiratory reactions [187], it was suggested that N A D H dependent semiquinone free-radical formation in the inner mitochondrial membrane could account for the delayed cardiotoxicity of anthracycline glycoside drug. Indeed, the catalytic role of these compounds in electron transport and their high stability at physiological 02 concentrations offer an explanation for the slow but irreversible damages at the mitochondrial level. The site of interaction of adriamycin with complex I in intact mitochondria is unknown. The flavin component of the mitochondrial dehydrogenase is a candidate, since it has already been shown to form a complex with the anthracycline ring of adriamycin [188,189]. One may argue that the concentrations of adriamycin and daunomycin necessary to depress the mitochondrial functions in vitro are much higher than one would expect to find in the heart during therapy (this was evaluated to be in the range of 1-5 # M [190]). A slow, but cumulative process of peroxidation by activated oxygen species

280

could, however, be very effective in damaging the cardiac cell, even at very low concentrations. IIC-3. Phosphate carrier

Very recently, Mende et al. [191,192] were able to demonstrate that the purified mitochondrial phosphate carrier is activated by cardiolipin. The activation is highly specific and could not be obtained with any other applied phospholipid. Interestingly, adriamycin was found to be a potent inhibitor of the Pi carrier incorporated in proteoliposomes [193] with Cs0%inh = 30 ~M. The possibility that the inhibition by adriamycin reflects the binding of the drug to the P~ carrier itself has been tested using the highly reactive adriamycin analog bromodaunomycin which inhibits the Pi uptake to the same extent than adriamycin. When the P~ carrier is isolated from bromo-daunomycin-treated mitochondria, it is free of the inhibitor and is fully active after reconstitution in liposomes [193]. This supports the tentative conclusion that adriamycin and Br-daunomycin react with cardiolipin but not with the Pi carrier itself. IIC-4. A TPase

No absolute requirement of this enzyme for cardiolipin has been shown to date, but anionic phospholipids are probably required [194]. The effect of adriamycin and derivatives on the mitochondrial oligomycin-dependent ATPase activity was tested (unpublished data). Clearly, no inhibitory effects were detected at concentrations up t o l 0 - 3 M . IIC-5. Calcium transport

Mitochondria are believed to play an essential role in activation and in disactivation of contractile systems by the energy-linked Ca 2+ transport. Specifically, in heart muscle [195] (and perhaps in slow skeletal muscle and smooth muscle [196]) sarcoplasmic reticulum is poorly active in Ca 2+ transport, whereas mitochondria are very abundant. Their area represents 87% of the total area of Ca 2+-transporting membranes in heart [197]. They are capable in vitro of extracting Ca 2÷ from troponin. Accumulated Ca 2+ may be rapidly ejected under the influence of a variety of agents [197,198]. Among others, in cardiac cells, Na ÷ is a powerful release-inducing agent [199,200]. Studies

on ion concentrations [201-203] and more specifically on Ca 2+ concentrations and exchangeability [100,127,204,205] pointed out an inhibition of Ca 2+ transport induced by treatment with adriamycin. This result was confirmed for cultured heart cells [205]. As it is known that the mechanism catalyzing the influx of Ca 2+ through the inner mitochondrial membrane is different from the efflux mechanism (for a review see Refs. 101 and 197), a relatively specific action of anthracycline glycosides is expected. Indeed, Moore et al. [206] and Revis and Marusic [207] demonstrated that the Ca 2+ uptake of myocardiac mitochondria is inhibited by the lowest adriamycin concentrations tested (10 ttM). ATP-dependent mitochondrial Ca 2+ uptake is inhibited to the same extent. Interestingly, the lack of significant inhibition of cardiac microsomal Ca 2+ uptake is demonstrated in Refs. 203 and 206. Perturbation of Ca z+ uptake and release from the largest cellular Ca 2÷ reservoir should have immediate dramatic effects. The effect of adriamycin on the myocardiac contractile functions is a continuous dose-dependent prolongation of the time-course of contraction [208,209]. This prolongation may result from a decreased uptake of intracellular Ca 2+ into sarcoplasmic reticulum, mitochondria or both [210]. The mechanism of Ca 2+ uptake (inhibited by anthracycline drugs) requires the ejection of two H ÷ out of the mitochondrial matrix for the entrance of one Ca 2 + [211-215] and is inhibited specifically by Ruthenium red [216,217]. This latter compound does not influence the release system [96,218]. There have been numerous attempts to isolate a (glyco)protein with characteristics consistent with those expected for the Ca 2+ carrier [219-222], but results are not convincing [223-225]. Recently, a cardiolipin-mediated Ca 2÷ transport across the inner mitochondrial membrane through the so-called 'inverted micelles' was proposed. Extensive studies on cardiolipin ionophoretic capabilities [226] as well as microscopic and 31p-NMR experiments [227-231] revealed the existence of inverted micelles. Furthermore, the binding site of Ruthenium red on mitochondria was shown by Raman spectrophotometry to be a phospholipid. Ruthenium red inhibits the formation of cardiolipin non-bilayer structures on model membranes [233]. Strikingly, adriamycin inhibits also the for-

281

-

ADRIAMYCIN

*

T A B L E III

ADRIAMYCIN (ADM/CL:I)

ASSOCIATION CONSTANTS OF A SERIES OF A N T H R A C Y C L I N E G L Y C O S I D E - C A R D I O L I P I N COMPLEXES [129]. M-]

I

- 50

I

~

0

ppm

I

I

I

-50

-50

H -'--"~-

L

I

0

ppm

i

/

. 50

H ~

Fig. 5. Effect of Ca 2+ and adriamycin on the 36.4 M H 2 31p-NMR spectra of phosphatidylcholine/cardiolipin (CL) (1 : 1) muitilameilar liposomes. In absence of Ca 2+ , the spectrum is characteristic of a bilayer phase. Addition of Ca 2+ leads to the formation of a structure characterized by isotropic motion of the phospholipid associated with the occurrence of intrabilayer inverted micelles. Subsequent addition of equimolar adriamycin completely inhibits the formation of these nonbilayer lipid structures.

mation of Ca2+-induced inverted micelles in cardiolipin-containing liposomes and is also capable of inhibiting the uptake of Ca 2÷ by cardiolipin into an organic phase [153]. The effect of Ca 2* and of the subsequent addition of adriamycin on the bilayer/non-bilayer structure of cardiolipin liposomes as determined by 3]p-NMR spectroscopy are reported in Fig. 5. Clearly, adriamycin stabilizes the bilayer structure of cardiolipin and therefore inhibits the Ca 2 ÷ transport through the membrane. This experiment confirms both the inhibitory effect of adriamycin on the cardiolipinmediated Ca 2÷ transport and the cardiolipidic nature of the Ca 2÷ carrier recently proposed. III. Physicochemical studies of the anthracycline glycoside-lipid interaction in model membranes Since the anthracycline glycoside toxicity at the level of the inner mitochondrial membrane, and presumably the cardiac toxicity itself, find their origin in the prime interaction between the considered drug and cardiolipin, it is at the time of writing of first importance to understand better the nature of the drug-cardiolipin complex and the molecular reasons for its stability. First, Hildebrand et al. [234] showed that acidic phospholi-

Adriamycin Cinerubin Rubidazone Nogalamycin Rhodomyein N-Acetyladriamycin Steffimycin

1.0.10 6 0.8.10 6 2.0-10 s 1.7.10 5 1.1- l0 s no charge complexation no charge complexation

pids, and more specifically cardiolipin, redistribute adriamycin into the lipophilic phase of a two-phase solvent system of Folch. Specific effect of adriamycin on the transitions characteristic of cardiolipin-containing liposomes was also pointed out [235,236]. The association constant of adriamycin and related anthracycline glycoside drugs with cardiolipin was determined on cardiolipin-containing monolayers spread at the air/water interface using a surface-potential method recently developed [237,238]. Association constants are reported in Table III. Surface-potential data show clearly that the interaction is mainly electrostatic. Indeed, N-acetyladriamycin (uncharged) does not interact with cardiolipin or other T A B L E IV D I E L E C T R I C C O N S T A N T S O F T H E LIPIDIC M E D I U M S U R R O U N D I N G T H E A N T H R A C Y C L I N E D Y E IN U N I L A M E L L A R LIPOSOMES O F C A R D I O L I P I N (CL), DIP A L M I T O Y L P H O S P H A T I D Y L C H O L I N E (DPPC) A N D E G G P H O S P H A T I D Y L C H O L I N E ( E G G PC) A dielectric constant of 80 is characteristic of the aqueous phase. Dielectric constants lower than 50 indicate a penetration of the chromophore into the hydrophobic part of the membrane [129]. Dielectric constants (c)

Adriamycin N-acetyladriamycin Cinerubin Rubidazone Nogalamycin Rhodomycin Steffimycin

CL

DPPC

Egg PC

50 80 58 43 30 30 21

80 80 80 80 38 65 80

80 80 80 80 58 40 35

282 acidic phospholipids, but adriamycin (positively charged) does not interact with neutral phospholipids [128], even though a slight interaction with DPPC was demonstrated in monolayers spread at very low surface pressure [239]. Since the fluorescence spectrum of the anthracycline drugs is strongly dependent on the dielectric constant, c, of the medium surrounding the dye, penetration of the anthracycline moiety of the drugs in the hydrocarbon chain region of the phospholipid bilayer can be investigated by fluorescence titration of the drugs by small unilamellar cardiolipin liposomes, c values are reported in Table IV [129]. From Table IV, two different kinds of behavior can be distinguished. In a first class, we can include drugs which display the highest association constant for cardiolipin and which are not deeply buried in the lipid bilayers. The latter result is in agreement with quenching of adriamycin fluorescence by iodide, which shows that the bound drug is only partially buried in the liposomal membrane [240]. Other derivatives as daunomycin and adriamycin-14-octanoate are, however, more effective in decreasing the temperature of liposome phase transition [241]. Drugs of class I react specifically with cardiolipin and not with a neutral lipid. Class II includes drugs more weakly associated to cardiolipin and which penetrate without specificity into the lipid bilayer. It is striking to point out that neither the affinity for cardiolipin nor the depth of penetration into the hydrophobic part of the bilayer are related or anticipated from octanol/water partition coefficients evaluated elsewhere [242]. In contrast, a good correlation between the drug-cardiolipin association constant and cardiotoxicity was reported [129]. The specificity of adriamycin towards cardiolipin as compared with other negatively charged phospholipids appears from the association constants with various phospholipids determined by adsorption of tritiated adriamycin on lipid monolayer using a superficial radioactivity counter [128]. K a = 1.6-10 6 M --1 for cardiolipin, 1.8.104 M -1 for phosphatidylserine and phosphatidic acid, and zero for neutral DPPC. The difference in affinity of adriamycin for cardiolipin and for other negatively charged phospholipids can be quantitatively explained by the stacking of neighboring anthraquinonic planes as

revealed by specific changes in its visible absorption spectrum. The stacking phenomena could bring an additional free energy of pair-formation of - 2 . 8 k c a l / m o l exactly sufficient to explain the difference in the association constants [128]. Binding of adriamycin onto cardiolipin should therefore result in a complex including two adriamycin molecules electrostatically bound on the two anionic phosphate groups of the cardiolipin. Since the association constant is 1.6-106 M -1 for the adriamycin-CL complex and is 3.6.106 M -1 for the complex adriamycin-DNA, clearly cardiolipin could be a competitive target for adriamycin. Tritton et al. [236,243] have used proton N M R to ascertain whether adriamycin alters the permeability and fusion characteristics of liposomes. In the presence of the externally added paramagnetic ion Pr 3+, the resonance position of the choline protons is shifted downfield. Since the liposomes are normally impermeable to Pr 3+, only those protons on the outside of the bilayer are accessible to the shift reagent, and hence the choline resonance is split into an inside-outside doublet. If a drug makes the membrane leaky and thereby permeable to Pr 3÷, then the doublet structure will collapse. Addition of adriamycin to such a pr3÷-phos phatidylcholine or cardiolipin-containing phosphatidylcholine liposome system causes no N M R signal change and thus the authors concluded that adriamycin did not make the membrane permeable to ions like Pr 3÷ . Small unilamellar liposomes fusion was monitored by the proton resonance broadening due to the appearance of larger structures. In pure phosphatidylcholine liposomes, addition of adriamycin does not increase the fusion rate. In cardiolipin-containing phosphatidylcholine liposomes, however, adriamycin markedly enhances the rate of vesicle-vesicle fusion.

IV. Perspectives Considerable interest is being devoted in various laboratories to the synthesis of adriamycin derivatives in order to improve the chemotherapeutic index of anthracycline drugs. An alternative approach is to modify the existing drugs to produce derivatives with decreased side-effects. However, this approach requires a detailed knowledge of the nature of the receptor site and the

283

geometry of the complex formed. Several works, mainly based on X-ray and N M R data, report the fine structure of the adriamycinDNA complex. Briefly, in this complex, rings B and C overlap with adjacent pair bases [243-245], whereas rings A and D protrude out the intercalation site. Since ring D does not overlap significantly with the central base pairs of the nucleotides and it extends out from the helix, substituents attached to C(1) and C(2) are not expected to alter significantly the binding to DNA [244]. Even the C(4) methoxy group is of minor importance [246,247]. Modifications at the C(9) position also show a minimal effect (248,249]. However, the C(9) OH group seems to play a key role in interacting with the bases in the minor groove of DNA [250]. For adriamycin, daunomycin and derivatives, the flexible sugar-chromophore system adopts similar conformations, which are probably the biologically active conformations [251]. Modifications of the sugar amino group sterically hinder the full insertion of the anthracycline ring between DNA base-pairs and decrease the stability of the complex. Moreover, structureactivity studies may be of some help in determining structure and conformation requirement for interaction with DNA [252]. Regions of the anthracycline glycoside molecule essential for its interaction with DNA are summarized in Fig. 6. As cardiolipin has been presented as a competitive target for adriamycin responsible, at least partially, for the cytotoxic side-effects, an attempt was made in order to elucidate the geometry for the complex formed and the reasons of its high stability (to be published). Infrared attenuated total

o

reflexion (ATR) spectroscopy (for a review on this method see Ref. 253) permitted the determination of the orientations of the chromophore with respect to the plane of the cardiolipin bilayer as well as changes in the phospholipid conformation resulting from its interaction with adriamycin. More insight into the spatial organization of the complex was obtained from a conformational analysis (for a detailed description of this procedure see Refs. 254 and 255) (also unpublished data) (Fig. 7). A linear crystal-like organization of the complex appears. In this structure, the adriamycin molecules lie parallel, tilted at 36°C with respect to the normal to the bilayer plane in good agreement with the infrared ATR measurements. The total energy of interaction between neighboring adriamycin molecules is favorable to a stacking of the anthraquinone moieties. The mean distance between the positive charges located in the adriamycin molecule in this linear organization is exactly identical to that obtained between the

OH CH 2 OH

OH

/0 CH 3

0

OH

*"0

NH2 Fig. 6. Regions of the adriamycin molecule essential for its interaction with D N A (thick lines).

Fig. 7. Computer picture of two cardiolipin molecules assembled with four adriamycin molecules. This side-view corresponds to a projection on a plane perpendicular to the interface. Black circles represent the P atom of cardiolipin. Open circles refer to oxygen or carbon atoms. Arrows indicate the position of the amine in the adriamycin molecule.

284

negative charges of cardiolipin in the close-packed structure. From the adriamycin-DNA complex structure described, it appears that anthracycline drugs might be modified at each extreme side of the ring system and, from the adriamycin-cardiolipin complex study, it becomes certain that the high stability of the complex arises from intermolecular interactions, more specifically between the plane ring systems of the complexed molecules. Prediction of new structures avoiding the latter stabilization but preserving the interaction with DNA is under study in our laboratory. Acknowledgements One of us (E.G.) is Research Assistant at the National Fund for Scientific Research (Belgium). We thank the "Banque Nationale de Belgique", the National Institute for Health (Bethesda), Prof. A. Trouet, Dr. R. Baurain (Laboratoire de Chimie Physiologique, Universit6 de Louvain, Belgium) and Dr. Hildebrand (Erasme Hospital, Brussels). Their support was essential in several experiments described in this review. References 1 Harteel, J.C., Duarte-Karim, M.M., Karim, D.S. and Arlandini, E. (1975) in Adriamycin Review (Staquet, M. and Tagnon, H., eds.), pp. 27-36, European Press, Medikon, Ghent 2 Calendi, E., Di Marco, A., Reggiani, M., Scarpinato, B. and Valentini, L. (1975) Biochim. Biophys. Acta 103, 25-49 3 Berman, H.M. and Young, P.R. (1981) Annu. Rev. Biophys. Bioeng. 10, 87-114 4 Quigley, G.S., Wang, A., Ughetto, G., Van der Marel, G., Van Boom, J.H. and Rich, A. (1980) Proc. Natl. Acad. Sci. USA 77, 7204-7208 5 Patel, D.J., Kozlowski, S.A. and Rice, J.A. (1981) Proc. Natl. Acad. Sci. USA 78, 3333-3337 6 Neidle, S. and Taylor, G.L. (1979) FEBS Lett. 107, 348-354. 7 Praga, C., Beretta, G., Vigo, P.L., Lenaz, G.R., Pollini, C., Bonadonna, G., Canetta, R., Castellani, R., Villa, E., Gallag,her, C.G., Von Melclmer, H., Hayat, M., Ribaut, P., De Wasch, G., Mattson, W., Heine, R., Waldner, R., Kolaric, K., Buehner, R., Ten Bokkel-Huyninck, W., Perevodchikova, N.I., Manziuk, L.A., Serm, H.J. and Mayr, A.C. (1979) Cancer Treat. Rep. 64, 827-834 8 McGuire, W.P. (1978) Cancer Treat. Rep. 62, 855-863 9 Casazza, A.M. (1979) Cancer Treat. Rep. 63, 835-844

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