TOXICOLOGY AND APPLIED PHARMACOLOGY lm,9-23 (1989)

Structural Requirements for Anthracycline-Induced Cardiotoxicity and Antitumor Effects

YOGENDRA SINGH, LINDAULRICH,DONNAKATZ, PATRICKBOWEN,AND GOPALKRISHNA’

Section on Drug Tissue Interaction, Laborat0r.v @Chemical Pharmacology, National Heart. Lung. and Blood Institute. National Institutes ofHealth. Bethesda, Maryland 20892

Received August 29, 1988; accepted April 22. 1989

Structural Requirements for Anthracycline-Induced Cardiotoxicity and Antitumor Effects. SINGH, Y., ULRICH, L.. KATZ, D.. BOWEN, P., AND KRISHNA, G. (1989). Toxicol. Appl. Phar- macol. 100,9-23. By employing rat cardiac myocytes in culture and mouse L-1210 leukemia cells, we have compared different anthracycline analogs with respect to their ability to kill cardiac myocytes and tumor cells. Anthracyclines induced a decrease in cellular ATP and glutathione from both cardiac myocytes and L- 12 10 cells in a time- and concentration-dependent fashion. Moreover, the decrease in ATP in cardiac myocytes was followed by release of the cytoplasmic enzyme lactic acid dehydrogenase and of adenine nucleotides after anthracycline treatment. At very low concentrations of anthracyclines, at which ATP and glutathione were not affected, the drugs induced complete cessation of the growth of L- I2 10 cells. Some structural alterations in the anthracycline molecule resulted in parallel changes in antitumor activity and in cardiotoxic- ity. But other structural alterations resulted in dissimilar changes in antitumor activity and car- diotoxicity. Although the results indicate that the structural requirements for inducing cardio- toxicity and antitumor activity may be different, they also indicate that the mechanisms by which anthracycline causes cell death in tumor cells and cardiac myocytes may be the same. t 1989 Academic Press. Inc

The anthracyclines adriamycin and dauno- mycin are potentially among the most valu- able antitumor agents currently available be- cause of their spectrum of activity (Blum, 1975). Although several drugs are employed in cancer chemotherapy. in general, only an- thracycline antibiotics are known to cause cardiotoxicity (Unverferth et al., 1982: Shir- hatti et al., 1986) and cardiac myopathy, which limits their use as antitumor agents (Myers, 1984; Pence et al., 1983). Several mechanisms of toxicity have been proposed, including the release of histamine and cate- cholamines from damaged heart (Bristow et al., 1978) free radical generation and lipid peroxidation (Myers et al., 1977; Bachur et

’ To whom reprint requests should be addressed.

al., 1978). mitochondrial damage (Ferrer0 et al., 1976) excess calcium influx (Olson et al.. 1974), interference in the Na’-K’ ATPase pump (Gosalvez et al., 1979) effect on nucleic acid and protein synthesis (Buja et ul.. 1973; Lewis et al., 1983) depletion of cellular ATP and GSH (Shirhatti et ul., 1986). and in- hibition of uptake of adenine, amino acids. and glucose (Reese et al., 1987). By contrast. anthracyclines are thought to elicit their anti- tumor effects through direct binding to DNA (DiMarco et ~1.. 1975: Schwartz and Kanter. 1979).

Several analogs have been developed to re- duce cardiotoxicity and increase or retain an- titumor activity. For example, a doxorubicin analog, 3’-deamino-3’-(3 cyano4-morpholi- nyl)doxorubicin recently has been found to

9 0041-008X/89 $3.00 Copyright 1~. 1989 by Academic Press. Inc All rqhts of’rrproduct~n in ~“y for”, rr\ev et,

10 SINGH ET AL.

be approximately 1000 times more potent than parent compound but noncardiotoxic at therapeutic dose levels (Sikic et al., 1985). However, there has been no systematic study to elucidate the structural requirements for inducing cardiotoxicity and antitumor activ- ity at clinically relevant concentrations.

Several in vivo models are available for studying toxicity of anthracyclines (Mettler et al., 1977; Doroshow et al., 1979), but prob- lems of drug distribution, elimination, and metabolism have clouded the picture regard- ing the structural requirements of anthracy- clines for evoking effects (Campeneere et al., 1982). Cultured cardiac myocytes from neo- natal rats (Acosta et al., 1978; Miletich et al.. 1983) and adult rats (Lowe and Smallwood, 1980) have been used to overcome some of these problems. A number of events occur- ring prior to cell death induced by anthracy- clines have been reported using cultured beating cardiac myocytes. These include slowing and cessation of beating of myocytes, depletion of cellular ATP, leakage of cellular glutathione and cytoplasmic enzymes, and inhibition of uptake of adenine, amino acids, and glucose (Shirhatti et al., 1986; Reese et al., 1987); however, most of the studies have not been performed at clinically relevant con- centrations. In the present study, we have used beating cultured cardiac myocytes to in- vestigate the cardiotoxicity of several anthra- cycline analogs at clinically relevant concen- trations. For comparison, a peak plasma con- centration of 0.36-0.50 pg/ml adriamycin was obtained when administered to humans at a dose of 30 mg/m2 with a terminal half-life of 34-44 hr (Benjamin et al., 1974; Bachur et al., 1977). Similar peak plasma concentra- tions of 0.40-0.65 p&/ml daunomycin were also obtained after administration at a dose of 1 OO- 120 mg/m* with a terminal half-life of 55 hr (Alberts et al., 1971). A terminal con- centration of 0.05-o. 1 pg/ml adriamycin was attained when administered at a dose of 75 mg/m* to patients with breast cancer (Rossi et al.. 1987). From these studies, it is possible

to infer that clinically relevant concentra- tions of adriamycin and daunomycin range between 0.1 and 1 pg/ml. Since these drugs do not induce cardiotoxicity until a com- bined dose of 500 mg/m* has been reached, we have employed a concentration range of 0. l- 10 pg/ml for these drugs. Cardiotoxicity was also compared with antitumor activity at these concentrations by employing murine L-12 10 leukemia cells, which have been widely used to study the antitumor activity of drugs.

MATERIALS AND METHODS

Cardiac myocytes were isolated from 2- to 3-day-old neonatal Sprague-Dawley rats as described previously (Shirhatti et al., 1986). The cells were cultured in 25-cm2 Primaria tissue culture flasks at a seeding density of 1 million cells per flask and incubated at 37’C in an atmo- sphere of 95% air and 5% CO? with Williams’ medium E supplemented with 10% Nu serum, 4 mM glutamine, 0.02 IU/ml insulin, 1 pM epinephrine, streptomycin (100 U/ml), and penicillin ( 100 pg/ml). Medium was replaced with fresh medium after 24 hr when cells had attached and then every 48 hr.

On Day 8, culture medium was replaced with 5 ml of Williams’ medium E containing various concentrations of the anthracycline analogs under study. Most of the drugs were hydrochloride salts and were thus water solu- ble. Analogs that were water insoluble were dissolved in 0.01 N HCl or 0.01 N NaOH and added to the medium in a volume 25-50 ~1 with no significant change in the pH of the medium.

Cardiotoxicity was evaluated by measuring leakage of lactic acid dehydrogenase and adenine nucleotides, as de- scribed earlier (Shirhatti and Krishna, I985b). Cells were incubated with [14C]adenine (0.04 rCi/ml, sp act 53 mCi/mol; NEN) in Williams’ medium E for 2 hr. The medium was removed and fresh medium containing the analogs was added to the plate. Leakage ofadenine nucle- otide was monitored by determining the radioactivity in aliquots of medium at different times. LDH was deter- mined using pyruvate as a substrate and NADH as the cofactor. The rate of change in absorbance of NADH at 354 nm was measured every 2 set for 1 min with an HP- 8450 spectrophotometer. Total LDH (in cells and me- dium) was determined after solubilization of cells with 1% Triton X- 100, and results were expressed as percent- ages ofthe total: 100% LDH leakage represents 100% cell death.

The medium was removed and the total cellular gluta- thione (GSH and GSSG) was assayed spectrophotomet-

ANTHRACYCLINE TOXICITY II

rically by measuring the rate of formation ofpnitrothio- phenolate anion during the cyclic enzymatic reduction ofGSSG and oxidation ofGSH by DTNB (Tietze, 1969). The reaction mixture contained 100 yl of sample and 1.1 ml oEO.1 M potassium phosphate buffer, pH 7.5, contain- ing 5 mM EDTA, 1 unit ofglutathione reductase, 0.4 mM DTNB. and 0.4 mM NADPH. The rate of change of ab- sorbance at 4 I2 nm was measured every 2 set for 1 min with an HP-8450 spectrophotometer.

ATP was extracted from the cells with perchloric acid and was determined by a method based on the luciferin- luciferase reaction with DuPont Biometer (Shirhatti and Krishna. 1985a).

Murine L- 12 10 leukemia cells were obtained from Dr. S. Ahmad (NCI. Bethesda. MD) and grown in RPMI- 1630 medium containing 16% heat-inactivated fetal bo- vine serum, 50 FM fi-mercaptoethanol, streptomycin ( 100 U/ml), and penicillin (100 pg/ml). Cell cultures were split every 2 to 3 days until they reached the late logarithmic phase of growth (1 .O X IOh to I .4 X IOh cells/ ml). The cells were incubated in media containing different concentrations of anthracycline. At different time intervals, the cells excluding trypan blue were enu- merated under a microscope using standard counting chambers. The cultures were centrifuged and the cells as- sayed for cellular glutathione as described by Ahmad cl 111. ( 1986). ATP was extracted from the cells by perchloric acid and estimated by the method of Shirhatti and Krishna. 1985a).

The reagents used in this study were analytical grade. [‘“C]Adenine was obtained from NEN (Boston, MA). Williams’ E. RPMI-1630. and fetal bovine serum were purchased from Gibco (Grand Island. NY). Nu serum was obtained from Collaborative Res. Inc. (Lexington, MA) and collagenase was obtained from Cooper Bio- medical (Malvern. PA). Adriamycin and daunomycin analogs were gifts from Dr. M. Suffness (NCI. Bethesda, MD) and Dr. F. Arcamone (Farmitalia. Milan, Italy). The purity of these analogs was determined by reverse- phase high-performance liquid chromatography (HPLC) analysis under conditions described by Casazza LV ~1. t 1979) and is reported in Table I

RESULTS

Cardiac cell damage induced by anthracy- cline analogs was studied by measuring the release of the cytosolic enzyme lactic acid de- hydrogenase (LDH) and r4C-labeled adenine nucleotides into medium and the loss of ATP and cellular glutathione from cardiac myo- cytes. Antitumor activity of anthracyclines was calculated from their capacity to inhibit

growth and to promote the killing of mouse L- 12 10 leukemia cells. A concentration-de- pendent release of LDH and adenine nucleo- tides was observed in 48 hr. Figure I illus- trates leakage of [14C]adenine nucleotides and LDH by cardiac myocytes after 48 hr of incubation with different anthracycline ana- logs at various concentrations. In general, an- thracyclines that were toxic induced more ad- enine nucleotide leakage than LDH; in fact. the leakage of adenine nucleotides afforded a better measure of cell damage: 4-demethoxy- daunomycin was most toxic and 1 l-deoxy- adriamycin was least toxic (Fig. 1). Table i shows the concentrations of daunomycin and adriamycin analogs required to induce 50% release of LDH and ‘“C-adenine nucleotide\ from cardiac myocytes and to inhibit the growth or kill 50% of mouse L- 12 10 leukemi,) cells in 48 hr.

Daunomycin (X) appears to be more car- diotoxic than adriamycin (I) on the basis of‘ LDH release (Fig. 1 and Table 1). Among adriamycin analogs, 11 -deoxyadriamycin (IV) was least cardiotoxic, and, among dau- nomycin analogs, XIV appears to be least car- diotoxic (Table 1). Nevertheless, 1 l-deoxy- adriamycin (IV) and 4-demethoxydauno- mycin (XII) were not very potent in killing L- 12 10 cells among the analogs listed in the present study. Daunomycin was at least two times more potent than adriamycin in inhih- iting growth of mouse L- 12 10 cells (Fig. 2). 3- Demethoxydaunomycin (XII) was a very PO.- tent antitumor agent and, at 0.03 pg/mi. completely inhibited the growth of L- 1211) cells in 48 hr. Among all the analogs tested. cyanomorpholinoadriamycin ( IX) and. among daunomycin analogs, 4-demethox! - daunomycin (XII) were the most potent antr- tumor agents. but they were also the most po-

tent in inducing cardiotoxicity (Table 1). Anthracycline analogs also induced in 24 111

a marked loss of 4TP and cellular glutathr- one before leakage of LDH from cardiac myocytes, which occurs at 48 hr. The loss of ATP and cellular glutathione was greater

12 SINGH ET AL.

TABLE 1

STRUCTURE, PURITY, GROWTH INHIBITORY CONCENTRATIONS (IC50). LETHAL CONCENTRATION-50 (LC50) BASED ON GROWTH INHIBITION AND CELL DEATH IN L- I2 10 CELLS AND LDH AND [‘4C]A~~~~~~ NUCLEOTIDE RELEASE FROM CARDIAC MYOCYTES, OFADRIAMYCIN, DAUNOMYCIN, AND THEIR ANALOGS’

Rat cardiac myocytes cell death

NO structure (Purity) MW

CAS registry number

Mouse leukemia cells Based 011 adenine

Inhibition of Cell death nucleotides Based on LDH cell growth LGO leakage leakage L& Go (ww (ww LGO (PM (kw (48 hr.) (48 hr.) (48 hr.) (48 hr.)

Adriamycin and Analogs

I 0 580 253 16-40-9 0.12 >I.72

NH, (96)

0.3 1 17.24

706 5 1898-39-6 0.02 >o. 14 2.83 3.85

ND

522 75363-85-S 0.04 70.2 5.75 9.58

564 7 1800-89-o 0.16 >5.32 17.73 62.06

(80)

580 56390-09-l 0.02 0.17 0.52 6.90

ANTHRACYCLINE TOXICITY I3

TABLE 1 -Continued

Rat cardiac myocytes cell death

NO Structure (Purity) MW

CAS registry number

Mouse leukemia cells Based on adenine

Inhibition of Cell death nucleotides Based on LDH cell growth LCSO leakage leakage IX .,,

Go WJ) t&t) KS,, (P‘W I p,w I (48 hr.) (48 hr.) (4X hr.) (48 hr.)

Adriamycin and Analogs

550 64363-63-9 0.005 2-0.18 0.18 0.64

688 - 0.003 (1.07 0.15 3.Y1

0.004 0. I5 -,() , < 675 89146107-6

(S) 10.00 I

89 196-08-7

Daunomycin and Analogs

564 23541-50-6 0.07 >I.77 0.32

563 67324-99-6 0.016 L-O.18 5.33 p .: :,

0.004 0.4 I 0.04 I?. il

0 OH

XIII 0 548 63568-77-4 ND ND ND ND

NH1 (95)

_- .-_

14 SINGH ET AL.

TABLE l--Continued

Rat cardiac myocytes

cell death

NO Structure (Purity) MW

CAS registry number

Mouse leukemia cells Based on adenine

Inhibition of Cell death nucleotides Based on LDH cell growth LGO leakage leakage LC&

G ( ww (Pm LG, ( ww (df) (48 hr.) (48 hr.) (48 hr.) (48 hr.)

Daunomycin and Analogs

XIV 579 346 1 O-60- I 0.017 0.17 >17.27 >17.27

722 66996-56-3 0.055 20.14 2.49 13.85

70095-83-9 0.005 0.08 4.85 >8.08

ND

744 70878-5 l-2 0.027 >0.13 > 13.44 > 13.44

Analogs Not Related to Adriamycin or Daunomycin

818 796 17-46-2 0.009 >0.12 0.11 > 12.44

>0.23 3.38 >22.52 m 444 65271-80-9 0.038

541 7 I628-96- i 0.054 >0.19 I 1.09 > 18.48

193)

’ N.D., not determined.

ANTHRACYCLlNE TOXICITY 15

B

FIG. 1. Concentration response of adriamycin. daunomycin, and their analogs on LDH (0) and [‘“Cl- adenine nucleotide (a) leakage from cardiac myocytes. Cardiac myocytes ( I X IO’ cells) were cultured on &cm’ Primaria flasks for 8 days and then incubated in 5 ml of Williams’ E medium containing [‘“Cl- adenine (0.04 &I/ml. sp act 53 mCi/mmol) for 2 hr. The medium was removed and fresh medium contain- ing the analogs was added to the plate. At the end of 48 hr. leakage of [“Cladenine was measured by counting the radioactivity in the medium. LDH was determined by measuring amount of LDH leaked into the medium. Each point is the average of four values that did not deviate by more than 5%. (A) Adriamycin (I). (B) daunomycin (X), (C) 4-demethoxydaunomycin (XII). (D) rubidazone (XV). (E) I I- deoxyadriamycin (IV).

with daunomycin than with adriamycin (Fig. Anthracycline also depleted ATP and cel- 3). 1 1-Deoxyadriamycin (IV) had less effect lular glutathione from mouse L- 12 10 leuke- on ATP and glutathione than adriamycin (I) mia cells (Fig. 4). Adriamycin and daunomy- in cardiac myocytes (Fig. 3). Daunomycin tin depleted 50% of ATP and GSH at a con- (X) and 4-demethoxydaunomycin (XII) were centration above 0.3 pg/ml medium in 24 hr almost equally effective in depleting ATP and (Fig. 4). 4-Demethoxydaunomycin (XII) was glutathione from cardiac myocytes. very effective in depleting ATP and glutathi-

16 SINGH ET AL.

wo /’

al P p b BEa i z d

/ :40 0 z

L!! /I //

22 /I P’ 0 0 0.001 0.010.03 0.1 1.0

A

DAUNOMVCIN l~lml, AORIAMVCIN Qdg/mll

C

COEMETHOXV OAUNOMYCIN ,~,ml, RUBIOAZONE lpa’glmll

0 LA a/

/ I I I ,

0 0.001 0.01 0.1 1.0 10.0

II-DEOXY AOAlAMVClN ,,,,,,“I,

FIG. 2. Concentration response of anthracyclines on growth inhibition of L- 1210 cells. Murine L-12 10 cells were grown in RPMI- 1630 medium containing 16% heat-inactivated fetal bovine serum, 50 PM p-

mercaptoethanol, streptomycin (100 U/ml), and penicillin (100 pg/ml). Cells were split when they reached logarithmic phase of growth and were incubated in medium containing different concentrations of adria- mycin, daunomycin, and their analogs. At the end of 48 hr (0) and 72 hr (A), the cells excluding trypan blue were enumerated under a microscope using a standard counting chamber. Each value is the average of four values that did not deviate by more than 10%. (A-E) Same as in Fig. 1.

one from L- 12 10 cells even at low concentra- the compounds. Esterification of the hydroxy- tion. 11 -Deoxyadriamycin (IV) and rubida- methyl group (II) and replacement of the side zone (XV) were less effective in depleting chain by hydrogen (III) increased the cardio- ATP and GSH from L- 12 10 cells. toxicity, based on LDH leakage, and the anti-

Table 1 shows how the structural modifica- tumor activity, based on inhibition of cell tions of adriamycin and daunomycin altered growth and cell death of L12 10 cells. Re- the cardiotoxicity and antitumor activity of moval of the OH group from the 11 th position

ANTHRACYCLINE TOXICITY

Irn r

FIG. 3. Concentration response of adriamycin, daunomycin, and their analogs on ATP and glutathione depletion in cardiac myocytes. Cardiac cells were grown for 8 days as described in Fig. 1. On Day 8. medium containing different concentrations of adriamycin, daunomycin, and their analogs were added to the plate. At the end of 24 hr, total cellular glutathione (A) and ATP (0) was measured by the methods described under Materials and Methods. Each point is the average of four values that did not deviate by more than 5Yo. (A-E) Same as in Fig. 1.

(IV) reduced cardiotoxicity without markedly increased cardiotoxicity and antitumor acti v-

changing the antitumor activity, based on in- ity. Replacement of the hydroxymethyl group hibition of cell growth. Any modification at by a methyl group at the C-9 side chain of the 4’ position of the sugar moiety, such as adriamycin resulted in the other parent com- epimerization of the OH group (V) or replace- pound, daunomycin (X), which was more car- ment of the OH group by hydrogen (VI) or diotoxic and had more antitumor activity. iodine (VIII), increased both the antitumor Loss of ATP and glutathione was greater with effect and the cardiotoxicity of adriamycin. daunomycin (X) than with adriamycin. Con- Removal of methoxy group at the 4 position version of the quinone moiety of imidoqui- of the aglycone by hydrogen (VII) markedly none as in XI, which has a reduced capacity to

18 SINGH ET AL.

D

FIG. 4. Concentration response of adriamycin, daunomycin, and their analogs on glutathione and ATP depletion in L- 12 10 cells. Cells were grown in RPMI- 1630 medium as described in Fig. 2. Adriamycin, daunomycin, and their analogs were added in the medium when cells were split and then incubated for 24 hr. At the end of 24 hr, cells were centrifuged and processed for ATP (0) and total cellular glutathione (A) estimation as described under Materials and Methods. Each point is the average of four values that did not deviate by more than 5%. (A-E) Same as in Fig. 1.

generate radicals (Lown et al., 1979), resulted in less cardiotoxicity and more antitumor ac- tivity. Replacement of methoxy in the 4-posi- tion of the aglycone XII significantly in- creased the cardiotoxicity and also increased antitumor activity. Modification of the keto group of the C-9 side chain in daunomycin, as in XIV, XV, and XVI, led to a decrease in

cardiotoxicity and an increase in antitumor activity. Substitution of two hydrogens of the amino group of the sugar moiety by two ben- zyl groups (XVII) also led to a decrease in car- diotoxicity and an increase in antitumor ac- tivity.

The three compounds not related to either adriamycin or daunomycin (XVIII, XIX,

ANTHRACYCLINE TOXICITY

.xX lam

STRUCTURE NO. (THERAPEUTIC INDEX)

ANTITUMOR ACTIVITY (INHIBITION OF GROWTH-ICw ktM))

FIG. 5. Correlation between anthracycline-induced cardiotoxicity and antitumor activity (therapeutic index). Therapeutic index (TI) was calculated by dividing the IC50 for cardiotoxicity. as measured by leakage of [??]adenine. by the IC50 for tumor cell growth.

XX) were less cardiotoxic than daunomycin and were potent antitumor agents.

Figure 5 shows the correlation between car- diotoxicity and antitumor activity of the an- thracycline analogs. The therapeutic index (TI) was calculated by dividing the IC50 for cardiotoxicity, as measured by leakage of [14C]adenine nucleotides. by the IC50 for tu- mor cell growth. With certain modifications, as in XVI, XI, III, and VII, the antitumor ac- tivity was altered, but cardiotoxicity was not changed. Many analogs (i.e., XX, III, XIX, XV, V, VI, VII, XII) fall into a group where the modifications alter both the cardiotoxic- ity and the antitumor activity. The XVI ana- log had the highest TI, but many of the ana- logs were less cardiotoxic and more potent antitumor agents than adriamycin or dauno- mycin.

DISCUSSION

In the present study, we have compared the effects of anthracycline analogs at clinically

relevant submicromolar concentrations in elicitation of toxic reactions in cardiac myo- cytes in culture and in capacity to kill or in-

hibit the growth of a mouse L- 12 10 leukemia cells. Anthracycline-induced death in cardiac myocytes correlates well with depletion of cellular ATP and glutathione. Cell death in L-1210 cells also correlates with ATP and glutathione depletion, but their effect on the inhibition of growth of L-l 2 10 may not be correlated with their effect on ATP and gluta- thione. The mechanism of anthracyclinc- induced cardiotoxicity is not yet known. Several mechanisms have been proposed. According to the different mechanisms, an- thracyclines (1) intercalate with DNA. (2) in- teract with iron in the cell and thereby gener- ate free radicals, (3) interfere with oxidative phosphorylation, (4) inhibit uptake of nutri- ents, or (5) deplete cells of glutathione and ATP. However, it is not clear which of these mechanisms plays the leading role or if all ot them act in concert to induce cardiotoxicit)

20 SINGH ET AL.

and antineoplastic activity. Intercalation with DNA and inhibition of DNA synthesis may be the primary reasons for the cessation of growth or killing of tumor cells, since gen- eration of new cells is important for the via- bility of a tumor (Hopkins et al., 1978); how- ever, these probably are not the primary causes of cardiotoxicity because cardiac cells do not proliferate (Zak, 1973). Anthracy- clines also interfere with RNA synthesis and indirectly with protein synthesis (Momparler et al., 1976) which may be important for cel- lular repair. It has been reported that anthra- cyclines evoke more severe effects on DNA than on RNA and protein synthesis (Momp- arler et al., 1976).

With some structural changes, an increase in antitumor effects correlated with an in- crease in cardiotoxicity, e.g., 4-demethoxy- daunomycin (XII) and 4-demethoxyadria- mycin (VII). With other changes in the an- thracycline structure, however, the changes in antitumor activity did not correspond with changes in cardiotoxicity. For example, mod- ification of the keto group in the side chain (XVI) resulted in increased antitumor activ- ity but decreased cardiotoxicity. As shown in Fig. 5, the imido analog of daunomycin (XI) and a daunomycin analog (XVI) appeared to have a higher therapeutic index than the other analogs. The quinone moiety in the an- thracycline structure may be reduced to radi- cals, which in turn may undergo redox cy- cling to produce reactive oxygen metabolites and superoxide radicals. Indeed, lipid peroxi- dation by superoxide has been proposed as a factor contributing to cardiotoxicity (Bachur et al., 1978). Although the finding that imido- daunomycin (XI), which has a reduced ca- pacity to generate radicals, was less cardio- toxic both in our study and in that reported by Lown et al. (1979), 4’-deoxyadriamycin (VI), which does not form radicals in cell-free systems (Dickinson et al., 1984) was very cardiotoxic. The antitumor effects, however, of these two analogs were similar, suggesting

that radical formation may not be related to cardiotoxicity and antitumor activity.

4’ - Iodo - 4’ - deoxyadriamycin (XIII) was found to be 25-30 times more toxic than adriamycin as an antitumor agent (Table 1). This is in agreement with the recent studies reported by Barbieri et al. ( 1987) on the effect of this compound on various human tumor cell lines. These authors also show that this iodo derivative did not induce cardiotoxicity in mice when administered either intrave- nously or orally at doses up to 3 mg/kg day twice a week for 5 weeks (Barbieri et al., 1987). However, the results from the present study indicate that the iodo derivative of adriamycin was about twice as toxic as adria- mycin based on adenine nucleotide leakage. This discrepancy is probably due to the phar- macokinetic differences attributed to changes in the lipophilic character, as well as p&, of the compound by replacement of the 4’-hy- droxyl group of iodine in adriamycin (Bar- bieri et al., 1987).

3’-Deamino-3’-(3 cyano-4-morpholinyl)- adriamycin (IX) was found to be the most po- tent antitumor agent in this study (Table 1). This compound was more than 120 times as toxic as adriamycin to L-12 10 cells. Recent studies by Sikic et al. indicated that this com- pound is 100-2000 times more potent than adriamycin against a number of tumor cell lines (Sikic et al., 1985). The present study in- dicates that the cyanomorpholino analog was twice as toxic as adriamycin to neonatal rat cardiac myocytes when toxicity was evalu- ated as leakage of adenine nucleotides, whereas Sikic et al. ( 1985) showed that there was no difference in cardiotoxicity when esti- mated as the percentage of residual LDH ac- tivity in cultured fetal mouse hearts after drug exposure. The major differences in these studies are the use of fetal mouse organ cul- ture versus neonatal rat cardiac cell culture and the utilization of loss of LDH versus leak- age of adenine nucleotides for measurement of cell variability. Moreover, the variability in the studies by Sikic et al. was such that it

ANTHRACYCLINE TOXICITY 71 -?

would have been difficult to monitor twofold differences in potency.

4’-Epi-adriamycin (V) was shown to be 6- 10 times more potent than adriamycin as an antitumor agent in this study (Table 1). It is about I .7 times less cardiotoxic based on ade- nine nucleotide leakage in neonatal rat car- diac myocytes (Table 1). This compound, which is an epimer of adriamycin, evokes an- titumor activity against a variety of tumors such as breast carcinoma, soft-tissue sarco- mas, lymphomas. melanomas, leukemias. ovarian, gastric and rectal cancers, suggesting a broad spectrum of activity. Only two cases of mild-to-moderate reversible conjestive heart failures have so far been observed in about 700 treated patients (Ganzina, 1983).

4’-Demethoxydaunomycin (XII) is 17 times more potent than daunomycin as an antitumor agent. In various experimental tu- mor models, it has been found to be more po- tent than the parent drug (Casazza, 1979; Ca- sazza et al., 1980). This compound has been shown to be less cardiotoxic in various ani- mal models (Casazza et al., 1979). In the pres- ent study, however, 4’-demethoxydauno- mycin was found to be 4-5 times as toxic as daunomycin in inducing cardiotoxicity. This discrepancy between in viva and in vitro stud- ies of cardiotoxicity may be explained by the difference in the pharmacokinetic parame- ters of these two compounds. The terminal half-life of the demethoxy analog is about 27 hr, whereas that for daunomycin is about 55 hr(Lueral., 1986:Albertsetal., 1971).Clear- ante of the demethoxy analog after intrave- nous administration was 21 liters kg-’ hr~-’ based on an average of three studies (Lu et al., 19861, which was not very different from the clearance of daunomycin reported earlier (Riggs, 1984). There was, however, a high variation in clearance of the demethoxy ana- log after oral administration. indicating a great variation in the bioavailability of this drug (Lu et al., 1986).

It is possible that various factors other than bioavailability and drug kinetics may mark-

edly alter the findings obtained in in vitro cell culture to in vivo results. The fact remains that in vitro testing offers a better understand- ing of the structure and activity of adriamy- tin and daunomycin and their analogs as an- titumor agents and predicts their ability to cause cardiac damage. From the studies rc- ported here, it is possible to postulate that the mechanisms by which anthracyclines cause cell death in tumor cells and cardiac myo- cytes may be similar, but the mechanisms of inhibition of tumor cell proliferation may be different. We have projected the loss of cell ATP as an ultimate mechanism involved in cell death. We have utilized leakage of [‘“C]i- adenine nucleotides to monitor loss of ATP and also as a marker for cell injury. This type of screening system would greatly help in dis- covering new analogs of anthracyclines that have highest antitumor potency with the least effect on cardiac myocytes.

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