Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review

BIOCHIMIE. 1984, 66, 333-352 Revue

Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review.

Genevieve AUBEL-SADRON and Danielle LONDOS-GAGLIARDI*.

CNRS : Centre de Biophysique Moldculaire, 1A, avenue de la Recherche Scientifique, 45045 Orldans Cedex, France.

(Refu ie 9-2-1984, acceptd le 11-4-1984).

Cette revue est dddi6e ?t la mdmoire du Dr. Rend MARAL

R~sum~ - - La daunorubicine et la doxorubicine, deux antibiotiques du groupe des anthracyclines, sent tr~s utilisdes dans la chimiothdrapie du cancer. Leur intercalation entre les paires de bases du DNA natif serait la cause principale de leur activitd. La formation de complexes entre la daunorubicine et la doxorubicine avec des polyddsoxyribonucldotides, des acides ddsoxyribonucldi- ques de diverses compositions en bases et en sdquence ou des chromatines, a dtd dtudide d'une fa¢on approfondie par un grand nombre de techniques. Beaucoup d'auteurs ont essayd de relier l'activitd biologique ou thdrapeutique ?t l'affinitd pour le DNA ou pour des sdquences particuli~res du DNA. Ces anthracyclines causent dgalement in vivo des dommages au DNA tels que des fragmentations de la moldcule ou des coupures sur Fun ou l'autre brin. Flies inhibent la synth~se du RNA et du DNA. Leur emploi comme agent antitumoral est iimitd par une cardiotoxicitd, chronique ou aigiie et par une rdsistance spontande ou acquise. Dans ies deux cas, il y a probablement une action au niveau de la membrane. En effet, la daunorubicine et la doxorubicine ont une affinitd particuli&e pour ies phospholipides et !'a.~parition de la rdsistance est !ie'e h un certain hombre d'altdrations de la membrane.

Mots-cl~s : anthracycline / daunorubicine / doxorubicine / interaction / cardiotoxicit~ / r~sistance.

Summary - - Daunorubicin and doxorubicin, two antibiotics belonging to the anthracycline group, are widely used in human cancer chemotherapy. Their activity has been attributed mainly to their intercalation between the base pairs of native DNA. Complex formation between daunorubicin or doxorubicin with polydeoxyribonucleotides and DNAs of various base composition or chromatins has been investigated by numerous techniques. Many authors have tried to correlate biological and therapeutic activities with the affinity o f the drugs for DNA or some specific sequences o f DNA. In vivo these ~Tnthracycline drugs cause DNA damage such as fragmentation and single-strand breaks. The mechanism of action of anthracyclines involves the inhibition oJ RNA and DNA syntheses. There exists two limiting factors in the use of anthracyclines as antitumoral agents : a chronic or acute cardiotoxicity and a spontaneous or acquired resistance. In both cases, there is probably an action at the membrane level. It has to be noted that daunorubicin and doxorubicin have a particular affinity for phospholipids and that the development o f resistance is linked to some membrane alterations.

Key-words : anthracycline / daunorubicin / doxorubicin / interaction / cardiotoxicity / resistance.

* Present address : INSERM, U 117 et Fondation Bergonid, 229, cours de I'Argonne, 33076 Bordeaux Cedex, France.

334 G. Aubel-Sadron and D. Londos-Gagliardi

I n t r o d u c t i o n

In 1962, an antibiotic was isolated from strains of Streptomyces caeroleorubidus in France [1]. At about the same period, the same substance was isolated from strains of Streptomyces peucetius in Italy and in the Soviet Union [2, 3]. This antibiotic was called rubidomycin [1], daunomycin [2], ru- bomycin [3]. It is now known as daunorubicin (DNR). Quickly it was unders tood that this glycoside antibiotic belonging to the group of anthracyclines has a very powerful anti leukemic

action. It was the first drug giving long remission and what could be considered as a complete recovery in some acute leukemias which, until the discovery of anthracyclines, were always lethal.

A related d r u g : adr iamycin (doxorubicin, D O X ) has later on been isolated from a mutant of Streptomyces peucetius [4]. This new antibiotic has an enlarged spectrum and a better efficiency against solid tumors. In Table I, we present the chemical formulae of D N R , D O X and related compounds .

T A B L E I

, o o .

3 8

H3C-O 0 OH

R 2

Compound Rj R2 Activity*

(%)

Daunorubicin

Daunorubicinol

Doxorubicin

Detorubicin RP 19920

RP 38422

RP 21080

RP 32885 RP 32886 RP 33365 RP 33366

N-L-leucyldaunorubicin

N-tri fl uoroacetyl-adriamyci n- 14-valerate (AD 32)

-- COCH3

-- CHOHCH3

-- CO-CH2OH __ C ' f ) ~ 1 4 . O F N P I4 [ f ~ ( ' _ I-I_ L

-- C(CH3)= NNHC(= NH)NH~

- - C O C H 2 S C H 2 C O O C H 3

-- COCH3

-- COCH3

-- COCH2OCOCH2CH2CH2CH3

C~H NH2

N H 2

t-leucyl daunosamine

HaC NH ~ OH j:O.CF 3

180

160

190 1 of~ 1 7 u

150

100

180

190 176 132 140

163

n.d.

Derivative RP 21080 (Mw : 523.5) is composed of four stereoisomers which have been isolated : the two trans isomers (RP 33365 and 33366) and the two cis isomers (RP 32885 and 32886). All-these drugs stabilize the double helical structure of native DNA. All daunorubicin derivatives are semisynthetic.

* Activity is expressed by the following ratio : 100 (mean survival time (treated mice)/mean survival time (control mice)). Mice are treated at the maximal tolerated dose on days 0, I, 2, 3 and 4. The activity is significant at 150 %. From Jolles et aL [5]

Mechanism of action of daunorubicin and doxorubicin

TABLE l (suite)

335

Nogal amycin

o 1 coocH,

OH O OH O

5" I CH30/-- o,, I

~ H 3 / I 3"I 2"

OCH 3 OCH 3

Cinerubin A VII Cinerubin B VIII

HO O CO2CH3 13

~ y , ~ ~ lO . - ,4 ~ 8 "'OH / II

OH 0 I R

6 w 5' / 1' H3 C ~ 0 % 4 ' ~ 2"

J N(CH3) 2 6" O C. 5" / 1" "s~---oJ 4, t~.~, . . . . .X2, CH

I OH O

, ¢ ~ " - " - " 2 " 1 O O

CH 3

I N(CH3)

O

336 G. AubeI-Sadron and D. Londos-Gagliardi

In this review, we will not discuss the therapeutic action of anthracycline drugs: we only will review the results obtained at the molecular level.

In the first part, we will explain the interaction of these drugs with the molecular components, DNA and chromatin, since it is admitted that DNR and DOX give an intercalation complex with DNA. Together with the formation of complexes, some DNA damages can be induced. Afterwards we will show that anthracyclines inhibit RNA and DNA synthesis. The successful use of these drugs has been limited by their toxicity (acute dose-limiting toxicity is myelosup- pression and chronic cumulative dose-limiting toxicity is irreversible cardiomyopathy). This problem has stimulated many researches on the relation of structure and activity of these molecules. Now hundreds of structural analogs have been synthesized and studied.

Finally, the most important cause of treatment failure is the development of resistance of malignant cells against the chemotherapeutic agents. This problem will be discussed in the last part of this review.

Interaction with nuclear components

A. D N A

1. Complexation

Since ~Imost 20 years, it is admitted that DNR gives an intercalation complex with DNA. In 1965, Calendi et aL [6] described an increase of the relative viscosity of the complex DNA-DNR when the dose of DNR is increased. Simulta- neously, the sedimentation constant tends to decrease. DNR shifts the melting temperature of DNA towards higher values. After addition of DNA to DNR in solution, changes in visible and UV spectra of DNR can be observed. Addition of increasing levels of DNA decreases the absor- bancy at 475 nm followed by a shift towards higher wavelength. For a D N A / D N R molar ration of about 7/1 the maximum absorption of DNR is at 505 nm.

In UV, DNR spectrum shows two maxima: 233 and 255 nm. The spectrum of a mixture of DNA-DNR is different from the spectra of the two substances separately. The mixture of the two components shows a single maximum at 257 nm.

Finally, DNR has a typical fluorescence spectrum with an excitation at 485 nm and an emis-

sion at 580 nm. The addition of DNA is accompanied by a quenching of the fluorescence. Two chemical groups: the hydroxyl group of the chromophore and the amino group of the sugar (daunosamine) are responsible for the linkage of DNR to DNA. Two different bonds can be postulated : one involves the intercalation of the chromophore between adjacent base pairs of the DNA and the other the amino group of the sugar. The latter is evidenced by the viscosity changes induced by DNR and not by N-acetylderivative [6, 7].

When studying DNR and two other anthracycline antibiotics (cinerubin and nogalamycin) Kersten et al. [8, 9] showed that the three drugs increase the viscosity, decrease the sedimentation constant and stabilize the native DNA to thermal denaturation. The stability of the DNA-anthracycline complexes under high ionic strength contri- butes to the progressive decrea_~e of the buoyant density of the complexes sedimented in CsCl and Cs.,SO4 density gradients. The most pronounced density I"'" " s,, ',, ~s obtained with nogalamycin, a lesser effect is observed with cinerubin and DNR.

Intercalation of drug between the DNA base pairs alters the conformation of nucleic acids [10] and can induce changes in the circular dichroism spectrum. The CD spectrum of the complexes DNR-DNA and nogalamycin-DNA is altered as a function of the ratio of bound drug to nucleotide. For DNR (and perhaps nogalamycin), it is neces- sary to postulate the existence of two types of complexes to fit the CD spectra. Waring [l l] studying the interiacation ol IDNK with a closed circular DNA ( 0 X 174-replicative form) has shown, by analytical centrifugation, changes in the sedimentation coefficient corresponding to a local uncoiling of the double helix.

By X-ray diffraction study of a film of a DNA-DNR complex, Pigram et al. [12] showed teat the pitch of the helix increases with the amount of drug fixed to the DNA. The increase of the pitch is in good agreement with an unt- wisting of the helix of 12 ° per molecule of drug.

To give a good representation of the experi- mental data, they built a model where the daunosamine is in the large groove and at the side of the groove close to a sugar phosphate chain, allowing an interaction.

The behaviour of DNR on DNA-cellulose column chromatography [13] is in agreement with the spectrophotometric titration and the Scat- chard's plot indicates more than one binding site. Using sonicated DNA it is demonstrated, by low

Mechanism of action of daunorubicin and doxorubicin 337

shear viscosity measurements that there is an increase of the length of the DNA chain and possibly a decrease of the flexibility. By visco- metric measurements it was proved that the binding of DNR and DOX induces stiffening, bending and elongation of the DNA molecule [14].

In order to improve the knowledge of the stoichiometry of the complex DNA-DNR and a postulated specificity of interaction, Barthe- lemy-Clavey et al. [15] used natural or synthetic DNAs with different base composition and native and heat denatured DNAs. The complex formation has been investigated by equilibrium dialysis, visible and UV spectrophotometry, CD and thermal denaturation.

The equilibrium dialysis~ using Scatchard's plot, shows that two types of complexes are formed with both native and heat denatured D N A : a strong binding corresponding to the intercalation between the base pairs for relatively low concentration of drug (r ~< 0.2, r : ratio of the concentration of complexed DNR and the DNA concentration), and a weak binding corresponding to a ionic complex for higher values of r. The thermal denaturation does not affect the equilibrium constant for the strong binding but the apparent number of fixation sites increases by 20 per cent.

The analysis of the results of the melting curves of DNAs with different base composition (Ta- ble II) in the presence of DNR and competition experiments using equilibrium dialysis do not give any evidence of specificity of the antibiotic for base composition or base sequence.

However, Chaires et al. [16] also by competitive dialysis showed that at low P/D (1.57) (P: DNA concentration, moles of nucleotide per liter, D : drug concentration) GC base pairs are slightly preferred as binding sites for DNR. Using fluorescence and absorbance methods, the interaction of DNR with four deoxypolynucleotides of defi- ned sequences has been studied. The results show that DNR binds better to alternating AT sequences than to non alternating AT sequences. On the other hand, there is the same affinity for alternating and non alternating GC sequences. The intrinsic binding constants decrease in the order: poly(d(AT)).poly(d(AT)), poly(d(GC)). poly(d(GC)), poly(dG).poly(dC), poly(dA). poly(dT). The determination of the sedimentation constants of DNR-deoxynucleotide complexes shown an unusual non linear dependence of Sapp with binding ratio, except for poly(dG).poly(dC). Binding of the drug to GC polydeoxyribonucleotides results in an almost complete quenching of the DNR fluorescence, but with AT polymers the quenching is not so important [17].

TABLE II Change of the melting temperature of DNAs

with different base composition after complexation by DNR

DNA G + C (%) T~ (°C) P/D ATm AT AT'

Micrococcus luteus 72 88.6 ! 1.2 7.0 -- -- E. Coil 50 76.7 ! !.35 10.0 7.5 10.0 Chicken erythrocytes 44 73.2 I !.3 10.3 9.0 14.5 Calf thymus 44 72.4 12.0 I0.6 9.25 ! 3.75 Proteus vulgaris 39 71.5 I 1.2 10.5 8.0 12.5 * Cancer pagurus • poly d(AT) 3 48.4 ! 1.3 5.4 4.5 6.25

main component 43 70.9 ! !.3 10.0 ! i.0 12.0 Synthetic poly d(AT) 0 45.6 I 1.3 ! 3.4 2.0 12.0

Tm in 0.015 M NaCI, 0.0015 M trisodium citrate P : DNA concentration, moles of nucleotides per liter (mean molecular weight of nucleotide : 326) D : DNR concentration ATm (°C) increase of the melting temperature after complexation by DNR AT and AT' (°C) width of the transition between 17 and 83 % of hyperchromicity, respectively for

native DNA and DNA complexed with DNR *']'he two components of the DNA from the crab Cancer pagurus have been isolated by a

chromatography on hydroxyapatite at 70 °C. Therefore the poly d(AT) satellite is no longer in native form but is partially renatured.

C1 In all cases, DNR stabilizes the polydeoxynu-

cleotides. Finally, the data obtained with calf thymus DNA and other natural DNAs represent average binding properties and mask specific binding at specific regions or sequences of DNAs.

Raman and resonance Raman spectroscopy show that the chromophore of DOX is intercalated in the GC sequences of calf thymus DNA. The authors suggest that the phenolic groups of the chromophore are involved in the drug-DNA intercalation and in addition to n - n, hydroxyl and amino group interactions [18]. Using fluorescence quenching, Vedaldi et al. [19] concluded that the GC base pair regions bind DOX and DNR stronger than the AT base pair regions. But the binding parameters of the complexes show that the drugs have a higher affinity for polynu- cleotides with an alternate sequence of purines and pyrimidines in each strand. However in their conclusions the authors did not consider the possibility of differences in the conformations of natural DNAs and synthetic polydeoxynucleoti- des such as poly(dAT).(dAT), poly(dA).(dT) and poly(dGC).(dGC). For example DNR and DOX do not intercalate into DNAs in A conformation [201. Anyway there is no significant difference either in the number of binding sites or in the association constants of DNR for DNAs extracted from human normal or leukemic leukocytes and for calf thymus DNA [211.

The results obtained by high resolution proton nuclear magnetic resonance suggest a sequence specificity of DNR for alternating purine-pyrimidine synthetic DNAs in solution [22, 23]. Rings B and C of DNR (Table I) overlap with adjacent base pairs [24]. The crystal structure of DNR with a DNA fragment d(CpGpTpApCpG) has been determined by X-ray diffraction methods [25] (Fig. 1, 2). This DNA fragment is a six-base-pair right-handed double helix with two molecules of DNR intercalated in the d(CpG) sequence. The distortion of the DNA double-helix due to DNR intercalation is more complex than the changes postulated by Pigram et al. [12]. There is a net dislocation of the double helix with an unwinding of 8 ° per DNR. The positively charged amino group is not interacting with the phosphates [12, 26] but is sitting in the minor groove. The non-planar substituents in ring A interact through hydrogen bonding with the double-helix. One can see that there are three different parts in the DNR molecule: the intercalators (rings B-D), the an- choring function associated with ring A, and the amino sugar. These results, obtained by cristallo-

FIG. I. -- View of the intercalator perpendicular to the base plane.

The DNR ring system is stippled. The adjacent G2-G5* base pair closer to the reader is shown by thick lines and the CI-G6* base pair further away is shown by thin lines. The two nucleotide backbones are different. Also note that the center of the G2-C5" base pair has moved up, toward the major groove relative to the CI-G6* pair.

* Designates the complementary sequence. From Quigley et al. [251.

G 6+ CI

338 G. A u b e l - S a d r o n a n d D. Londos -Gagl iard i

FIG 2. -- Diagram of DNR intercalated into d(CpGpTpApCpG), showing intermolecular attractions.

Note two hydrogen bonds between 09 of DNR and N2 and N3 of G2. In" addition, water forms a hydrogen-bond bridge between 013 of DNR and 02 of CI. Oxygen atoms are shown as ellipses : nitrogen atoms are shown as squares.

From Quigley et ai. [251.

graphic data, can be extended to studies in solution.

DNR is an inhibitor of the B ~ Z transition of poly(dGC). DNR binds preferentially to alterna- ted purine-pyrimidine sequences which are able to induce the B ~ Z transition. At high ratios of DNR, the B---~Z transition is inhibited [271. Addition of DOX to poly (dGmSdC) .po ly

Mechanism of action o f daunorubicin and doxorubicin 339

(dGmSdC) in the Z form converts the Z form to the B form [28]. DOX inhibits the Z-DNA formation in poly(dG-dC), total inhibition is obtained with l DOX for 9 base pairs and in calf thymus DNA with l DOX for l l.5 base pairs. In cells in culture, there is, for the same DOX-DNA concentrations, an inhibition of DNA synthesis [29]. These effects could explain, at least in part, the mechanism of the antineoplastic action of DNR and DOX.

It could be tempting to try to correlate the affinity for DNA of various anthracycline drugs and their antitumoral activity [30,31]. Molinier- Jumel et al. [30] have developed electrochemical techniques which allowed a direct determination of the number of free molecules and which avoided most of the disadvantages of equilibrium dialysis (use of large quantities of product, ad- sorption on the dialysis membrane). It is possible to obtain rapid and reproducible results with small quantities of compound. The curves dis- plays the well-known two kinds of complex. The values of the constant are independent of the molecular weight of the DNA (calf thymus DNA, molecular weight 8.106 or sonicated calf thymus DNA, molecular weight 0.5.106) and are not in- fluenced by DNA concentration up to 30 gg/ml. Using DNR and some of its analogues (Table I) they have determined their binding parameters (Table III). There is no striking differences in the binding parameters of all the tested derivatives; it is therefore not possible to establish a correlation between the affinity of the drugs for DNA and their more or less important antitumoral activity.

To elucidate the influence of the structure and composition of the anthracyclines on their binding affinity for DNA, several DNR and DOX derivatives have been synthesized [32-39]. Modi- fications were introduced either on the chromophore or on the sugar moiety. The inverted configuration at position 7 and 9 of the agiycone ring decreases the DNA binding, but the removal of the methoxy group at position 4 slightly increases the binding affinity. The methylation at position 6 and I I leads to inactive products, with an almost complete loss of affinity for DNA.

The binding is more sensitive to modifications in the sugar moiety than in the C9 substituents. The stereochemistry of the amino sugar ring as well as the presence of NH: at the C3' are essential for the formation of the intercalation complex. The cytotoxic activity against HeLa cells of new derivatives modified on the C4' is linearly related with their ability to bind to DNA. Ho- wever, antitumor activity is not related to these parameters (Fig. 3).

Studying some DNR analogues, Londos-Ga- gliardi et al. [40] have shown that, whatever their biological activity, under conditions where the intercalation complex is preponderant, these derivatives give an intercalation complex between the base pairs of native DNA. The results obtained with some of these drugs (RP 21080 and its four isomers) confirm the major importance, for intercalation, of the quinone system and of the presence of a basic group in an appropriate geometric position [5].

TABLE I I I Binding parameters for the interaction of daunorubicin and some of its derivatives with calf thymus

sonicated DNA in SSC* buffer, according to Scatchard representation

Compound Technique Kap p M -~ x 10 -S n Kn x M -1 10 _4

Daunorubicin ac polarography 6 0.13 7.8 Daunorubicinol ac polarography 3.1 0.127 3.9 Daunorubicin voltametry 6 0.125 7.5 Detorubicin voltametry 5.6 0.197 I ! .03 RP 21080 voltametry 4.4 0.12 5.3 RP 32885 voltametry 7 0.143 ! 0 RP 32886 voltametry 5.8 0.105 6. ! RP 33365 voitametry 6.5 0.094 6. ! RP 33366 voltametry 7.2 0.088 6.3

K, pp and n were determined from Scatchard's plots. * SSC • Standard saline citrate" 0.15 M Nacl, 0.015 M tri-sodium citrate. From Molinier-Jumel

et al. [301.


24

t ° 20

' 2 1 . o \

4

o ~o 2'o 3'o 4'0 go I DS0 ( n 9/m[ )

FIG. 3. -- Relatiottship between cvtutoxtcity and abili 0" to bind to DNA qf anthracycline derivatives.

Abscissa : IDso on HeLa cells (ng/ml); ordinate : 2 nK~pp 10 -5, e, DNR aad its derivatives; o, DX and its derivatives; X, 4"-epi-4'-C-methyl DNR and 4"-epi-4'-C-methyl-4'-O- methyl DNR, which were not considered for the calculations of the linear regression. The correlation coefficient was = --0.76 : intercept = 17.1; slope = --0.302.

From Bargiotti et al. [38].

Crooke et aL [41] divided anthracyclines into two groups: class I such as DNR, DOX, etc. inhibit nucleolar RNA synthesis and DNA syn-

thesis at the same concentration; class II inhibit preferentially nucleolar RNA synthesis at concentrations 200 to 1,500-fold lower than the concentrations required to inhibit DNA synthesis. In this class II one can find anthracycline molecules with a carbomethoxy group at the position 10 of the aglycone and a glycosidic chain with two or three sugars (aclacinomycin, marcellomycin, rudolfomycin, etc.) [42, 44] (Table IV). Removal of the carbomethoxy group at the position 10 (marcellomycin, rudolfomycin) decreases the inhibition of nucleolar RNA synthesis and simultaneously the cytotoxicity in vivo and in vitro. Binding parameters of these two anthracyclines with four natural DNAs of different base composition show the importance of this carbomethoxy group and that its removal decreases the binding ability of the drugs (Table V). There is also a correlation between the length of the glycosidic chain and the affinity for DNA : musettamycin is less active than rudolfomycin and marcellomycin in the inhibition of nucleolar RNA synthesis. These drugs have no sequence specificity.

In a recent work [45] using phase partition techniques which permit the utilization of very

TABLE IV Structures of the class I and class Ii anthracyclines and the descarbomethoxy-analogs of marcellomycin and rudolfomycin

0 OH 0 OH 0 COOCH 3

- o . - C,ass I nt,,rac,c,,nes

OR 1 0 OH b P,t R.~ OH 0 OH 0

H 3 C ~ Adriamycin CH3 CH2OH H3C ~

OH NH2 Carminomycin OH CH3 01~ NICH3)2

Pyrromycin Class II Anthracyclines

R1 0 R2 HO ~ Anthracycline R, R2 R~

Musettamycin OH COOCH3 Cinerulose Rudolfomycin OH COOCH3

OH 0 OH 0 H3C ~ H3C ~T.~ 0...,~ Aclacinomycin H COOCH3

Marcellomycin OH COOCH3

() N(CH3) 2 OH Descarbomethoxy- marcellomycin OH H

H3 C C . ~ 2-DeoxyfucOSeo CH 3 Descarbomethoxy-

~ . . . Z _ j O ~ rudolfomycin OH H OR 3 ~--

Rednosamine

H Rednosamine

Cinerulose 2-Deoxyfucose

2-Deoxyfucose

Rednosamine

Nl'12 From du Vernay and Crooke [44]

Mechanism of action o f daunorubicin and doxorubicin 341

TABLE V

Binding parameters of marcellomycin, descarbomethoxy-marcellomvcin, rudolfomycin and descarbomethoxy-rudolfomycin for salmon sperm DNA, thymus DNA, Micrococcus iuteus DNA and Clostridium perfringens DNA

Anth:acycline

CI. perfringens Salmon sperm Calf thymus M. luteus DNA DNA(28%GC) DNA(41%GC) DNA(43%GC) (72%GC)

Kappb c napp Kapp napp K~pp napp Kapp n~p~

Marceliomycin Descarbomethoxy-marcellomycin Rudolfomycin Dt:scarbomethoxy-rudolfomycin

6.05 0.130 9.51 0.169 5.03 0.194 5.25 0.229 1.26 0. ! 66 ! .28 0.303 2.14 0.224 1.21 0.264 2.44 0.164 3.11 0.219 1.98 0.218 2.02 0.286 0.96 0.295 1.54 0.377 1.42 0.321 0.42 0.607

a) p values were determined from tables of significance limits for correlation coefficients. All p values obtained were less than 0.001. The number of averaged values used to construct composite Scatchard's curves was usually 12, but no le3s than 10.

b) Kapp apparent association constant, in units of l0 b M ~. Values were: obtained by linear regression analyses of composite Scatchard's curves obtained from two or more separate experiments, each of which contained duplicate or triplicate values at each DNA concentration.

c) napp apparent number of binding sites per nucleotide. Values are obtained as for Kapp. From Du Vernay et ai. [43].

low quantity of bound drug (< 0.01), Graves and Krugh have shown that DNR and DOX exhibit a cooperative binding in 0.1 M NaCl. This coope- raive binding is dependent on the ionic strength and can be related to a more or less flexibility of the molecule of DNA. At ionic concentration of 0.01 M and l M NaCI, tnese drugs interact with DNA in a non-cooperative manner [16, 45]. DOX binds to DNA wi'~h a higher degree of coopera- tivity than DNR.

However, it must be pointed out, as did Neidle and Taylor [46] according to conformationai studies, that the existence of high affinity for DNA is not necessarily followed by an antitumoral activity. For example, an anthracycline (RP38 422, Table I) completely devoid 9f an oncostatic activity has nevertheless for DNA an affinity of the same order of magnitude than DNR or DOX [47] and the same intracellular localisation than other products with a high antitumorai activity [48].

2. DNA integrity

In the last few years a number of authors have attributed the cytotoxic action of anthracyclines to DNA damage, such as single strand breaks or alkaline labile regions.

Using neutral and alkaline sucrose gradient centrifugation [49] or hydroxyapatite column chromatography at 60°C [50] it was shown that

DOX and related compounds induce DNA strand scissions.

Using leukemia cells from several patients, DNA damages were different depending on the population of cells whereas uptake and retention of drug were characteristic of each drug and uniform for the different cell populations [51]. By use of alkaline sucrose gradient centrifugation, DOX was shown to induce both single- and double-strand breaks [52]. DOX and DNR induce the formation, in nuclear DNA, of regions which can be susceptible to hydrolysis by Neurospora crassa endonuclease, and which can be demonstrated by changes in the sedimentation properties of nuclear DNA in neutral sucrose gradients [53]. DOX [54], DNR [54, 55] and aclacir~omycin A [56] induce single strand scissions in the presence of reducing agents.

The in vivo effects of anthracycline upon the integrity of DNA has been studied using cell lysate~ or nuclear structures of various kinds of cells: human lymphoblastic cells [49], leukemia cells [59], HeLa, Ll:10 leukemia and Me-180 cells and a rat excision repair-deficient line [52] and mouse fibroblasts [53]. Lown et al. [54, 55] and Someya and Tanaka [56] followed the cleavage of a superhelical DNA. No degradation of Ehrlich ascites tumor cell DNA was shown by treatment of the animals with DNR or DOX [57], in contras: to the damage observed with LI2~O tumor cells.


In order to study the possibility of a correlation between the number of single-strand breaks induced by an anthracycline in vivo and its therapeutic activity, Londos-Gagliardi and Aubel-Sa- dron reported the results obtained with DNR and some of its derivatives after administation of the drugs to mice bearing an Ehrlich ascites tumor [511], The use of a technique based on the iso- pycnic centrifugation of nuclei extracted from Ehrlich ascites tumor cells followed by a CsC! gradient enables the separation of undegraded DNA [59]. The sedimentation contant of this DNA has been determined in an analytical ultracentrifuge, before and after alkaline degradation and subsequently the number of single strand breaks has been calculated [60].

In vitro, no single-strand breaks can be detected with the whole set of compounds. It is not the same in vivo after treatment of mice bearing an Ehrlich ascites tumor by the same anthracycline derivatives. These results confirm that the anthracyclines need the cellular environment to induce single-strand breaks in the DNA [49, 50] and that no correlation can be established between the number of single-strand breaks induced by an anthracycline and its therapeutic efficiency.

With a covently closed circular DNA (PM-2 DNA) and at low concentration of the drugs no breaks can be detected, without the addition of a reducing agent. But the studied anthracyclines induce superhelical conformational changes [61].

In another way, without isolation of the DNA, DNR and DOX induce interstrand DNA cross- links in HeLa $3 cells [62]. The importance of these cross-links for biological activity remains to be established.

The DNA binding of anthracyclines does not explain how these anthraquinone drugs cause DNA damage such as fragmentation or single- strand breaks. As it was previously established DNA strand breakages require cellular activity. Bachur et al. [63-66] propose that quinone anticancer agents are activated intracellularly to site-specific free radicals (Fig. 4). These free radicals enter the nucleus and bind (through intercalation or other mechanism) to nuclear DNA. The drug free radicals may direct their reactive energy to the complexed DNA or may generate reactive oxygen radicals such as superoxide or hydroxyl radical which react with DNA to produce the specific destructive effects to DNA. In good agreement with this hypothetical mechanism, Maral [67] describes a decrease of the toxicity of DNR when this drug was injected into

Microsmnal activation o f quinone anticancer agents

NADPH

NADP ~ ( ~

~+ tDNA

, , ~ , i ~ o "° Letc

O

CLASSES

BENZANTHRAQUINONE

N-HETEROCYCLIC QUINONE

o( NAPHTHOQUINONE

O

O O O

0 0 0

cO 0

FIG. 4. -- Proposed tnechanism o f microsonml activation oj quinone anticancer agents to .free radicals.

The hypothetical reaction of anticancer agents derived site-specific free radicals, and nucleic acid targets are indicated. In the lower half of the chart, the classes of quinone anticancer drugs are represented as nuclear structures.

From Bachur et al. [641.

mice in combination with superoxide dismutase which destroys the free radicals.

This mechanism of action may provide an explanation for the mode of action of AD 32 (N-trifluoroacetyladriamycin-14-valerate) : it is well known that AD 32, although it does not intercalate in DNA, has similar effects on ma- cromolecular synthesis and chromosomal damages [68, 69]. NADPH-cytochrome P450 reductase, xanthine oxidase, nitrate reductase, NADH and NADPH cytochrome C reductase catalyse the reductive cleavage of anthracyclines and the formation of free radicals [70-72].

B. C H R O M A T I N

Complexation of DNR to chromatin results, as for the complexation of DNR to DNA, in a red shift and a decrease of the intensity of the


~- ~d 4

20

@ ®

° e

0"t 0'2 ,"

FIG. 5. - - Scatchard3 plots.[or the binding of daunonthicin to chromatin and DNA.

Ascites c h r o m a t i n f rom sensi t ive cells : • f luorescence, a absorp t ion . Ascites D N A : • f luorescence, o absorpt ion .

F r o m Sabeur et al. [73].

absorption maximum. DNR interacts with chromatin with at least two types of binding. There is only a slight change in the binding constant but the number of high affinity sites decreases from 0.17 for DNA to 0.08 for chromatin (Fig. 5) [73]. Studying the influence of the ionic strength, Zunino et al. [74] had shown that at low ionic strength DNR binds to chromatin at two different sets of sites and the binding constants are similar to those found for the circular and linear DNA, respectively. The curvature of the Scatchard's plot has not been found for the interaction of DOX with chromatin. This result can be consistent with the view that DOX binding is less sensitive to changes in DNA conformation than is DNR.

In vitro DOX is capable of inducing a com- paction of isolated chromatin which becomes more resistant to cleavage by Micrococcal nuclease [75].

These results are in contradiction with those obtained by Grimmond and Beerman [76] who showed that the binding of DNR and DOX to chromatin DNA induces two structural changes : an unfolding of chromatin structure and a disrup- tion of the core particle. The effects are stronger for DNR than for DOX. This unfolding of chromatin structure renders more nuclease sensitive sites available.

To obtain a better knowledge of the importance of proteins in the action of anthracyclinc drugs to DNA, the interaction of anthracyclines

with nucleosomes has been studied [77-79]. The binding affinity of DNR and DOX is reduced with mononucleosomes. With H, depleted nucleosomes containing DNA of 146 and 175 base pairs, DNR causes an unfolding of nucleosomes followed by a trend to aggregate. The sedimentation coefficient decreases to a minimum value for r = 0.174 and afterwards increases for higher xihlues of r. In nuclei, it is postulated that the drugs prefer regions with less nucleosomal structures. The antitumoral activity of anthracyclines can be related to their special affinity for linear free DNA.

Enzymatic inhibition

The mechanism of action of anthracyclines may involve the inhibition of RNA and DNA synthesis. DNR seems to inhibit preferentially RNA synthesis, the inhibition of DNA synthesis requiring higher concentrations of drug [80]. However this early result has not been confirmed and it is generally admitted than in vivo RNA and DNA synthesis are inhibited to the same extent [81-83] either by DNR or DOX. In vitro DNA polymerase seems to be more sensitive to DOX or DNR than RNA polymerase [82, 83].

But, as we previously mentioned, this is not the case for all the anthracyclines, since it is possible to divide them in two classes, according to their ~ff~etc n ~ l-~bJA aria DT~IA c,~eh~c;c r A l l l

The inhibition is due to the interaction of the drug with the DNA template [1t4] and a mechanism of inhibition of RNA synthesis has been proposed by Barthelemy-Ciavey et al. [85] using RNA polymerase I and II and the DNA extracted from Ehrlich ascites tumor cells. DNR does not inhibit the binding of the enzymes to DNA, but prevents the transformation of the DNA-DNR- RNA polymerase unstable complex into the highly stable one. Since incubation of DNA with RNA polymerases does not prevent inhibition by DNR, it appears that DNR can bind to the DNA-enzyme complex. The resulting inactive ternary complex DNA-DNR-RNA polymerase has a faster dissociation rate than that of the stable complex formed without DNR. Anyway it is well known that DNR induces changes in the DNA conformation which can explain the important inhibition of the initiation step. After formation of the first phosphodiester bond, which initiates the synthesis of the RNA chain, the dissociation rate of the DNA-DNR complex is

344 G. AubeI-Sadron and D. Londos-Gagliardi

fast enough not to inhibit the elongation of the synthesized molecules.

The same type of mechanism has been described for the inhibition of the DNA degradation by acid DNase [86].

In a recent work [87] using RNA polymerase II from LI210 cells, it has been found that DOX induces two types of inhibition according to the drug/DNA molar ratios. At low drug /DNA ratios, DOX acts as an uncompetitive inhibitor, at high ratios, it inhibits RNA synthesis compe- titively.

In the case of an anthracycline which does not bind to DNA (AD 32), the inhibition of RNA synthesis may involve an interaction between the drug and the RNA polymerase [88,89]. In the case of DNA polymerases, one can see that DOX and DNR preferentially inhibit the DNA polymerase extracted from rat liver or from calf thymus [90,91]. DNR is the most active inhibitor of thymidine and uridine incorporation, DOX is the second one [92]. At low concentrations of DNR, the drug preferentially affects the initiation of replicating units. At high concentrations, there is also a decrease of the rate of chain growth [93]. No correlation can be established between the inhibitory action of different anthracyclines on DNA synthesis and their biological efficiency [941.

DOX and DNR also inhibit DNA polymerase from RNA tumor viruses [95-99] : Rauscher and Moloney routine leukemia virus, murine sarcoma virus and AKR murine leukemia virus. The inhibition of DNA polymerases from RNA tumor virus by some DNR derivatives is dependent on the polynucleotide template [95]. DOX and DNR have a greater inhibitory activity towards viral DNA polymerases than towards bacterial DNA polymerases [85]. The mechanism of inhibition of the avian myeloblastosis virus DNA polymerase by DOX has been thoroughly studied [100]. DOX have no effect on initial enzyme-DNA interaction. The presence of the drug which is bound to DNA weakens the affinity of the reverse transcriptase for 3'-termini. Therefore, the initiation is slower. In addition, there is a premature dissociation of DNA polymerase.

Cardiotoxicity

Cardiomyopathy occurs in some patients (2.2 %) after cumulative doses of DNR or DOX higher than 650 mg/m 2 or 550 mg/m 2, respecti-

vely. Cardiac effects of these drugs are expressed by changes in the electrocardiogram which are generally reversible or by a congestive heart failure with a lethality of 60 % [101].

Histologic changes have been observed in the hearts of necropsied patients, consisting of dama- ged, degenerate and atrophic muscle cells. In l0 % of muscle cardiac cell cytoplasm, one can observe the swelling of mitochondria, the lysis of mitochondrial membranes and cristae and in the nuclei, the transformation of chromatin into thick fibers, which can be related to an uncoiling of chromatin [102-104]. At the level of mitochondria from myocardial tissue degenerating alterations have been observed [105].

Chronic cardiotoxicity has been studied in different animals. In the rabbits, Jaenke [106] has shown mitochondrial and myofilamentous dege- neration, which can be secondary to DOX-induced alterations in myocardial electrolyte metabolism. At the end of the treatment by DOX, there is an increase of the Ca 2+ and Na + content of the myocardium. The same myocardial lesions have been observed in rats [107, 108] together with an increase of the ventricular tissue Ca 2+ [109].

Using mice cultured heart cells which retain their morphological integrity and which aie spon- taneously beating, Dasdia et al. [I 10] showed that DOX reduces the exchangeable cellular calcium without alteration of the total calcium concentration. There is probably an action at the rnernhrana level, which may be due *'~ o k;,,a;.. . of the druB to a component of the membrane. Low concentrations of DOX inhibit the Na+/Ca 2÷ exchange in isolated dog heart sarco- lemmal vesicles [111]. These results can be related to the particular affinity of DOX and DNR to negatively charged phospholipids such as cardiolipin (cardiolipin is a component of the mitochondrial membrane and mitochondria are very numerous in the heart muscle) phosphatidyi- serine, phosphatidylinositol and phosphatidic acid [112-115]. A complex is formed, stabilized by electrostatic interactions. The association constant of the DOX-cardiolipin complex is ef the same order ot magnitude than the association constant for the DOX-DNA complex. Therefore cardiolipin can be a competitive target for the drug. To demonstrate a possible correlation between cardiotoxicity and affinity for cardiolipin, the study of DOX derivatives in the presence of model membranes containing cardiolipin is very useful. A good correlation exists between mitochondrial toxicity of each drug and its affinity for cardiolipin. DOX inhibits the formation


of non-bilayer lipid structures and the uptake of Ca 2÷ [116] into an organic phase. The formation of a complex between DOX and cardiolipin stimulates the activity of NADH-coenzyme Q- oxidoreductase and coenzyme Q-cytochrome C-oxidoreductase. The transfer of electrons through DOX induces the formation of a very strong association of DOX and cardiolipin and a modification of the mitochondrial membrane fluidity.

The formation of free radicals is enhanced in the heart [117-119] because this organ presents a very low level of superoxide dismutase [118]. These free radicals are metabolized in the presence of superoxide dismutase to hydrogen pe- roxide which is in turn detoxified by selenium dependent glutathione peroxidase [121] to water and molecular oxygen.

The inhibition of coenzyme Q~0 enzymes succi- noxidase and NADH-oxidase [122-125] can be prevented by pretreatment with coenzyme QI0 which reduces the acute toxicity of DOX given in mice [126], rabbits [127] and rats [1281.

It has been reported that the administration of coenzyme Q~0 improves the cardiac function of cancer patients treated with DOX. In addition, coenzyme Ql0 does not modify the antitumor activity of the drug [129].

The cardiac toxicity of anthracyclines is associated with peroxidation of cardiac lipids. It has been suggested to use simultaneously coenzyme Qi0 and vitamine B2-butyrate which reduce lipid peroxidation [130].

Cardiac toxicity and peroxidation of cardiac lipids can be reduced by treatment of animals by ot-tocopherol. Vitamin E alleviates the cardiotoxic effects of DOX without affecting the oncostatic action of the drug. However, with or without concomitant administration of selenium (DOX decreases the activity of glutathione-peroxidase because the drug decreases the concentration of selenium which is required for the activity of the enzyme [131]), it seems that vitamin E delays rather than prevents the lethal toxicity of anthracycline and gives only a partial protection against chronic cardiotoxicity [132-139].

Resistance

The most important cause of treatment failure in cancer chemotherapy is due to the development of resistance of malignant cells against the chemotherapeutric agents. In 1976, DANO has

published an exhaustive review of the results obtained on the study of resistance to DNR developed in cells [140]. The conclusions are the following ones :

In vivo, development of resistance in Ehrlich ascites tumor cells can be obtained by treatment of mice with the drug [141l. The resistance remains stable without treatment by the drug for a minimum of 32 to 138 cell generations. The loss of resistance is probably due to an overgrowth of the wild type cells in a population which consists mainly of resistant cells with a rate of division lower than that of wild type. DOX resistance was maintained without DOX treatment through at least 225 cell generations. One line of DOX-resistant tumor was maintained in 50 weekly passages in mice not treated with DOX and without loss of resistance. In this case it may be that no wild type cells had remained among the resistant cell population.

There exists a cross resistance between DNR and its analogue DOX and the vinca alkaloids [142, 143].

The mechanism of resistance seems to be composed of several factors :

-- a decreased uptake of the drug; -- an altered sensitivity of the RNA and DNA

synthesizing enzymes and preferentially an inhibition of rRNA synthesis [144],

-- the most important cause could be at the level of the transport of the drug through the cell membrane : an active outward transport of DNR exists in resistant Ehrlich ascites tumor cells [145].

Several authors have confirmed that there is always a reduced uptake of DNR or DOX in resistant cells from Ehrlich ascites tumor or P388 leukemia, and a markedly enhanced efflux of the drug from the resistant cells [146-152]. It is possible to enhance the uptake of anthracyclines in Ehrlich ascites tumor or P388 leukemia resistant cells [146-150] by use of inhibitors of oxidative phosphorylation (sodi~;l az!.de or 2,4-dinitrophe- nol) and by omission of glucose. A further addition of glucose induces drug efflux, the effect being more pronounced in resistant than in wild type cells (Fig. 6) [153]. These results are consistent with an energy-dependent mechanism of drug extrusion, localized at the level of the plasmic membrane or perhaps of the nuclear membrane. There is also a small reduction of the nuclear binding of DNR [148, 153] or DOX [147] in resistant cells. But the reduction is too small to explain the marked reduction of drug in intact resistant cells.


80C

"~ 60C

5o0 ~ 40(

-6 30C E

20O

r,, 100

O [Control (o) .[Glucose (A)

wi ld-type tumor" cells

,b 20 A , o sb 6~

® JControl (o)

DNR-resistanl" tumor cells i i i

;o 2'0 3'0 ,o so 60 time (rntn)

FIG. 6. -- Efflux of daunorubicin (DNR) induced by glucose in wild-type (A) and resistant Ehrlich ascites tumor cells (B). The cell suspension (0.5 % v/v) was incubated in phosphate buffer without glucose containing 10 mM sodium azide ( o / e ) . The cells were loaded with 5 I~M daunorubicin at zero time. At the point indicated by the arrow 10 mM glucose was added to the suspension (a /A) .

In controls a corresponding volume of 0.9% NaCI was added (Skovsgaard, T.; unpublished).

From Skovsgaard and Nissen [153].

At the molecular level it has not been possible to observe differences in the affinity of the drug for DNAs or chromatins extracted from sensitive and resistant cells [73].

A comparison of the inhibitory action of DNR towards RNA polymerases extracted from sensitive or resistant Ehrlich ascites tumor cells could have been interesting" no significant difference has been found between sensitive and resistant cells. E_n!ymes extracted from the two different kinds of cells have the same properties. The relative proportions of RNA polymerases A and B are the same whatever their cellular origin. However, the cellular concentrations of the RNA polymerases do not always reflect the cellular rate of RNA synthesis [85].

No modifications in the subcellular distribu- tion of DNR can be observed • the largest part of DNR which has penetrated into the cells is recovered in the nuclei. Since a 2-4 times decreased uptake of DNR is observed in resistant cells, it is obvious that 2-4 times less DNR is found associated with nuclear DNA in resistant cells. These results are consistent with a mechanism of cell resistance to DNR relying on an increased outward transport mechanism of DNR localized on the plasma membrane [148]. No significant differences in the metabolism of DNR which in all cases is very low (at most 5 % of daunorubicinol) has been observed between the subcellular fractions of Ehrlich ascites tumor sensitive and resistant cells [154].

The membrane transport of anthracyclines has been throughly reviewed by Skovsgaard and Nissen [153]. The change in the cell membrane which results in an enhanced efflux of the drug is not yet known, but it has been shown that development of resistance is linked to some membrane alterations: a greater rate of membrane glycoprotein synthesis, an increase of the cell surface electronegativity, a decrease of glycoprotein degradation and a higher level of glyco- syltransferase activity. The higher level of membrane glycoproteins could alter the membrane fluidity [155]. Progressive resistance to DOX is associated with a progressive content of sialic acid at the cell surface [156] (the level of sialic acid can be considered as an index of cell surface carbohydrates).

In Ehrlich ascites tumor cells [157] as well as in chinese hamster lung cells [158] and in chinese hamster ovary cells [159], the development of resistance is accompanied by the synthesis of a cell-surface glycoprotein of higher molecular weight (150,000 to 170,000) (for wild-type cells the major glycoprotein synthesized has a molecular weight of about 1002000 whereas for the resistant cells the major glycoprotein has a molecular weight of about 150,000). When resistant, chinese hamster lung cells are incubated with N-ethyl- maleimide there is an increase of the cellular accumulation of DOX. When these cells are labeled with (32Pi) before treatment by N-ethyima- leimide there is a super phosphorylation of the 180K plasma membrane giycoprotein. With DOX sensitive cells there is only minor levels of this protein. When ~'esistant cells reverted to sensitivity a correlative loss of the phosphorylated P-180 was observed [160].

In addition, control cells synthesize gangliosi- des from hematosides to disialogangliosides, whereas resistant cells are blocked at the level of hematosides [156]. Both changes seem to be correlated with the development of resistance.

Using Friend leukemia cells, Tapiero et al. [161] suggest that resistance is the consequence of changes induced in the components of the plasma membrane. These changes could differ with the tested drug DNR or DOX.

In an attempt to overcome the resistance of cells to DNR or DOX, the use of calcium antogonists and calmodulin inhibitors has been considered. For example, verapamil is a coronary vasodilator the primary target of which is the membrane. Verapamil which inhibits the slow channel of Ca 2+ transport, enhances the cellular

Mechanism o f action o f daunorubicin and doxorubicin 347

uptake of DOX in P38s/DOX resistant cells and restores the sensitivity to DNR in resistant ascites carcinomas [162-166]. Trifluoroperazine which is an inhibitor of calmodulin improves the sensitivity to DOX of P38s resitant cells [167]. Perhexiline maleate and triparanol increase the sensitivity of P38s resistant cells to DOX and DNR, without an effect on sensitive cells. This effect might be associated to alterations of the structural order of plasma membrane lipids [168, 169].

C o n c l u s i o n

Number of works have been devoted to the study of interactions between anthracylines and DNAs and chromatins. It seems to us that the very recent works on the inhibition of the B _.l. Z transition and of the conversion of the Z form DNA to B form may be very important for the understanding of the mode of action of anthracycline drugs.As it is suggested by Nordheim et ai. [170, 171] if Z-DNA may play an important role in biological systems for example in the regulation of gene expression, it is tempting to attribute the inhibition of transcription and replication by anthracycline, to the inhibition of the B --+ Z transition.

From this review, one can see that the use of anthracyclines in cancer treatment set three kinds of problems : cardiotoxicity, development of resistance in the malignant cells and lack of selectivity of action towards these cells.

In the case of cardiotoxicity the peculiar affinity of DOX and DNR towards negatively- charged phospholipids such as cardiolipin which are present in the membrane may explain, at least in part, the cardiotoxic effect of the drug. Cardiac- toxicity which is associated with peroxidation of cardiac lipids can be reduced by the use of tt- tocopherol and coenzyme Qi0.

The development of resistance seems to be related to modifications of the cell membrane. Some results has been obtained with the use of calcium antagonists and calmodulin inhibitors (verapamil and trifluoroperazine).

In both cases, one has invested a great amount of hope in the synthesis of analogues of DNR and DOX. In spite of their abundance none of the new anthracycline analogues tested has demonstrated, simultaneously, a better therapeutic efficiency and a drastic reduction of the cardiac toxicity. Few of them are used in chemotherapy [172]. Nevertheless, some interesting results were

obtained with 4-epiadriamycin which has a reduced cardiac toxicity [173].

It is well known that anticancer drugs are toxic not only for malignant cells but also for normal proliferating cells. Therefore, the selective targe- ting of drugs has been thoroughly studied by means of binding to carders such as DNA, dextrans, proteins, lectins, immunoglobulins and antibodies.

This carder has to be • -- stable in the blood stream, - - non immunogenic, -- able to cross the plasma membrane, -- able to be degraded in the cellular com-

partment. I t~i.g the high net,--:.,, ,.¢ DNR "~"~ DO.'( ~..~ ~*** flllalllLj va o . i l~, . l

toward DNA, Trouet et aL have used DNA-DNR complexes in cancer chemotherapy [174-175] in order to improve selectivity (the complexed drug penetrates the cell by pinocytosis and free drug is released by lysosomal DNase) and to, reduce cardiotoxicity [176-1781.

Another approach is the binding or DNR or DOX to protein. A covalent binding between DNR or DOX and protein has been described by Hurwitz et al. [179]. Later on, conjugates of DNR to antitumor antibodies or immunoglobulins have been investigated in order to increase the specificity of action of the drug. Rudling et al. [180], have shown an increased intracellular accumulation of aclacinomycin A when glioma cells were L I ~ * ~ t b q ~ J l J IVgltit f f ILP~ ([P. ~ I I I J J I ~ A ~LV~'lflV~lilV~Jlil~ v~| l l A -low density lipoprotein, the cellular drug accumulation being dependent on the low density lipoprotein receptor activity of the cells. Very soon the importance of the link between the anthracycline and the protein was demonstrated [179, 181, 182]. Using a dextran bridge, Tsukada et al. [183, lg4] have shown that in vivo conjugates

o f DNR with monoclonal antibody to a-fetoprotein are considerably more effective against an tt-fetoprotein secreting hepatoma.

Trouet et al. and Baurain et al. [185, 186] have described albumin-DNR conjugates with an oli- gopeptide arm ~.:t,~,alate0 b~:tween the drug and the carder. With a tri- or tetrapeptide arm the bond between the drug and the protein is stable in serum but easily digested by lysosomal hydrolases, releasing the free drug. These conjugates are more active against the intraperitoneal form of Li210 leukemia than free DNR, thus opening promising prospects in cancer chemotherapy.

Other important carders for drugs can be liposomes [187]. In vivo macrophages of the


reticuloendothelial system take up liposomes of the blood stream. Although large multilamellar vesicles do not seem to interact with parenchymal liver cells, small unilamellar vesicles reach the hepatocytes to a large extent [188]. To more selectively target iiposomes to the target cells, one can incoporate antibodies against the malignant cells into the surface of the drug containing liposomes [189].

After i.v. injection of DOX encapsulated in liposomes, half-life is longer than that of free drug and its uptake is increased in tissues rich in reticuloendothelial cells (spleen, liver) and decreased in heart [190]. Rahman et al. [191] described a protection against DOX-induced chronic cardiotoxicity by liposomal adminitration of DOX, but these authors have a better effect with positively charged liposomes.

Forssen and Tokes [192, 193] observed a reduced cardiac toxicity of DOX, with anionic iiposome, accompanied by an increased action against Lewis lung carcinoma. This increased antineoplastic activity was not proportional to an increased association of drug with tumor cells. It seems that the improved therapeutic effect of DOX entrapped in iiposomes may be due to different mechanisms :

-- an altered disposition of drug which reduces cardiotoxicity;

-- an increased exposure of the tumor cells to the drug;

-- a reduction of the immunosuppressive res- ponse to DOX.

In the case of metastatic liver cells Gabizon et ai. [194] described a significant increase of DOX level in J~56 lymphoma cells from mice injected with iiposome-entrapped DOX. These results indicate that liposome delivery may provide an efficient mean of improving the therapeutic efficiency of DOX in certain forms of metastatic liver disease.

A critical review is given by Arndt et al. [195] for the composition and charge of lipid vesicles containing DNR. It seems that variation of the lipid composition, charge and size do not alter the therapeutic effectiveness.

Numerous fields have not been considered in this review : for example, the metabolism of the various drugs, the fate of the metabolites in organs and cells, the therapeutic efficiency of numerous derivatives, the results of cancer chemotherapy. But the aim of this work was to show that results at the molecular level can sometimes be useful for

research of new drugs and for the understanding of their mode of action.

Acknowledgements

Dr. R. Maral and Dr. J. Robert kindly read this manuscript and made a number of useful suggestions which are gratefully acknowledged.

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