Adriamycin associated cardiotoxicity: Research on prevention with coenzyme Q

Pharmacological Research Communications, V d . 9, No. 3, 7977

aDRIAMYCIN ASSOCIATED C M D I O ~ X I C I T Y : RESEARCH ON PREVENTION WITH COENZYME Q (*> Bertazzoli C, and M e Ghione ("1

-

qFarmitalia9 Viale E e BezzF, 24 20146 Milano Italy

-

-

Received 27 April 7976

Drug associated cardiotoxic effects can occur in humans treated with Adriamycin (ADM) and have been reproduced in some experimental models in vitro and in vivo. At l e a s t two

types of cardiotoxic effects have been observed: early transient ECG alterations and delayed cumulative dose related cardiomyopathy which can be followed by congestive heart fail

-

ure. The early reactions are traced to the action of ADM on cardiac mitochondria (either direct or c e l l nucleus mediated). The delayed reactions are attributed to the concurrence of myocytolysis and humoral and cell mediated antiheart autoimmune phenomena whose onset was observed in ADM chronically treated animals. In rabbits submitted to a prolonged ADM treat

-

ment as well as in mice or in isolated and perfused rabbit heart preparations surviving in vitro the administration of (*) The antitumor activity was studied by CASAZZA A.M.,

CIULIANI F. and PRATES1 B. Division of Experimental Oncology B Istituto Nazionale per lo Studio e la Cura dei 'kunori Milana. To whom the requests for reprints should be addressed.

-

(O)

-

Pharmacological Research Cormnunications, Vol. 9, No. 3, 1977

236

Coenzymes QI0 or Q9 prevents the appearance of ADM associated cardiotoxic phenomena. The antitumor activity of ADM on some experimental tumors in mice is not modified by COQlo treatment. INTRODUCTION ADM is an anthracycline antibiotic active against a wide spectrum of experimental and human tumors /-DI MARCO, 1972; BONADONNA et al.~

19757.

Its use is restricted by the onset

of early and delayed cardiotoxic effects /-LEFRAK et al., 1973; PRAGA et al.,

1975_7.

The early dose-independent ECG changes are sporadically monitored the first few days after the ADM injection. The incidence of delayed reactions (characterized by insidious onset of tachycardia followed by congestive heart failure) is low (1.2 %) but abruptly increases at a cumulative dose zn e x c e s s

o f 4 5 0 - 5 0 0 mg/m 2 . T h e s e r e a c t i o n s

occur a long

time (weeks or months) after treatment has ended. Histologic ~BERTAZZOLI et al., 1974; JAENKE, 1974; YOUNG, 19757 and biochemical evidence /-CARGILL et al., 1974; GOSALVEZ et al., 1974; BACHMANN et al., 1975; GAJA et al., 1975; FERRERO et al,,

1976_7stress

the crucial rSle played by the mitochon-

drial impairment in the pathogenesis of the ADM associated syndrome. Experiments have been carried out to study the antidotic activity exerted by the administration of some mitochondrial essential constituents whose synthesis and/or funorion was supposed to be directly or indirectly altered by ADM. Coenzyme %

and its higher homolog % 0

( C o % , C o % 0 ) , ac-

ting as physiological components common to different electron transport chain in mammalian mitochondria _/-FOLKERS~ 1969_7 were particularly studied.

Pharmacological Research Communications, VoL 9, No. 3, 1977

237

MATERIALS AND METHODS In vitro Different grm~_ps of five rabbit hearts (Langendorff's aration) were perfused at 37°C with glucosated

prep-

(0.I %) and

aerated (95 % 02 + 5 % C02) Tyrode solution either plain or medicated with ADM (5 /ug/ml) for two hours. Afterwards

the

different heart groups were perfused with Tyrode solution either plain or medicated with COQlo (150 ng/ml) or with COQlo solvents (glycofurol 0.015 % + DMSO 0,135 Q/~ finai concentrations), The experimental design is shown in Table I. In vivo J

Mice: different groups of I0 SPF Swiss juvenile male mice (Charles River) received i,v. either saline solution or ADM 2.0 or 3.5 mg/kg/day for three consecutive days, In the lower dose groups the course was repeated after a three day rest period and C o ~

(500 mg/kg/day)

in olive o i l solution (i0 mg/

/kg/day) or plain olive oil was administered by garage contemporaneously to the ADMtreatment

and for four days thereafter.

In the higher dose groups only one course of treatment was given and the C o ~

or olive oil administration

(at the above

mentioned doses) started four days after the completion of ADM treatment and was prolonged for seven days thereafter. The body weight was determined daily. Aliquots of animals were sacrificed at different times and the heart weight was determined. The designs of these experiments are shown in Tables 2 and 3. The interference of CoQI 0 administration on the an t i tumor activity of ADM was studied in mice bearing transplanted Gross leukemia or L 1210 leukemia, Adult C3H mice (I0 per group) inoculated i.v, with 2.5 x 106 spleen cells taken from mice bearing Gross virus induced leukemia were treated i.v. at days I s 2 s 3 after tumor inoculation with ADM (dissolved in distilled water)

238

Pharmacological Research Communications, VoL 9, No. 3, 1977

or CoQI 0 (microcrlstal suspension in saline) or both, Adults BDF I mice inoculated with 105 L 1210 cells taken from mice bearing ascitic L 1210 leukemia were treated i,p, on day I with ADM dissolved in distilled water or with C O C o I0 % Tween 80) or both, C o % 0

(suspension in

was injected in the morning and

ADM solution in the afternoon, The doses are shown in Tables 4 and 5, Rabb its : different groups of 20 adult male NZW rabbits reii

l|

ceived i®v, every morning O,I or 0,4 mg/kg/day of C O C o

(micro-

cristalllne saline suspension) or plain saline solution. All the animals received ADM i,v. (0,8 mg/kg/day)

on three consecutive

afternoons q 1 or q 2 weeks for 7, 12 or 15 courses, The ECG tracing was periodically recorded, Eventually the animals were sacrificed,

some urinary and haematologic parameters were deter-

mined and histologic and immunologic /-FIORETTI et

al., 19767

analysis were carried out.

RESULTS In_ vitro The isolated rabbit heart perfused with Tyrode solution can survive for more than five hours, The heart rate and contractility maintain a steady state for the first two hours and decline slowly afterwards

(Table 1). The presence of ADM deter-

mines a progressive decrease of the pulse rate and even if the drug containing solution is substituted after two hours with Tyrode solution either plain (Table 1 -

Group 2) or containing

the solvents (Table 1 - Group 3) the cardiac function lapses, the pulse rate drops and eventually the heart dies. This illfated course is stopped by the addition of COQlo (Table 1 Group 4). The ECG pattern worsened by the presence of ADM in the perfuslng solution is restored by the C O C o and the blood flow decrement is counteracted,

supplement

Pharmacological Research Communications, VoL 9, No. 3, 1977 TABLE

i

Experimental Groups

i

I

- -

.

-

_)n

__

I

HR.%

II

-

HR%

l

PT .n

2

I

I .l.

1

I

239

:t00 r

ii

l

TA

PT i

i

i

72 + 6

.

50 + 10

FT l

1

l

'l

5 + 5 l l

l

1

1

l

3

TA

50 + I0

TS

18 + 8

4

TA

50 + I0

TQ

55 + 8

--

l

Mean values + SE of the heart rate as percent of initial values (HR %) o~ the different groups of isolated rabbit hearts perfused during the first period (I = hours 0-2) and the last period (II = hours 2-5) of observation with Tyrode solution either plain (PT) or containing ADM (TA) or solvents (TS) or CoQIo (TQ),

In vivo Mice: In juvenile mice the physiological steady body growth increase stops coincidentally with the ADM administration period(s) and resumes immediately afterwards, This phenomenon is not modified, either by the C o ~

or by the olive oil

treatment, The heart weight to body weight ratio (heart index) is siE nificantly decreased in the ADM treated animals. The decrement is not modified by olive oil administration but is reversed by C o ~

treatment

(Table 2 - Group 3)

(Table 2 - Group 4) as can

be deduced by the heart index values determined in the animals sacrificed two days after the end of the ADM administration (Table 2 - column A), The activity of C o %

is observed even when the compound is

administered only after the ADM treatment (Table 3),

Pharmacological Research Communications, Vol. 9, IVo. 3, 1977

240

TABLE

Experimental Groups

Treatment

1

SS + O0

2

HI

%

A .430 + 14

.421 + I0 ,,

SS + CQ

2

,

3 4 i

lil

.,

i

®420 +

9

m

||,

m

i

i

.

m.

.

,

..

. 4 3 2 + 15

|

im

i

Hi

I --

--

ADM + O0

o385 + i0

,405 +

5

A D M + CQ

,410 +

8

.411 +

9

.

,

.

|

i

Mean values + SE of heart weight to body weight ratio (HI = heart index) in juvenile mice treated i.v, with saline solution (SS) or ADM 2.5 mg/kg/day (days 1-3, 7-9) and orally with olive oil (O0) or CoQ9 (CQ) at the days I-3~ 7-9~ 13-16 and sacrificed at the llth day (column A) or at the 23rd day (column B).

TABLE i

Experimental

i

mpl

mn _ _

3

HI

Treatment

Groups -

um

I

1

•

n

,

•

=%

|,

ADM

O0

.387

+

7

+

6

i

ADM

2

cQ

I

.410 ill

Mean values +_ SE of the heart index (HI %) of juvenile mice treated i.v. with ADM 3.5 mg/kg/day (day 1-3) and orally with either olive oll (OO) or CoQ 9 (CQ) at the days 6-I0, 13-14 sacrificed at the end of the treatment.

The data reported in the Tables 4 and 5 show that the antltumor activity of ADM in experimental murine leukemias is not antagonized by C O C o

administration and that COQlo itself is devoid

of antitumor effect.

Pharmacological Research Communications, VoL 9, No. 3, 1977 TABLE

4 .

mg/kg/day

ADM

.

.

.

.

.

.

.

.

T/C %

LTS

0.4

loo

o

-

109 I00 0

0.2

99

-

156

-

.

I

1/10

-

0 181 137 0

0 m

0.0

.

.

.

.

.

.

.

T/C %

LTS I II

-

.

.

.

.

0 o0

TIc %

o.6

241

- 163

I0

-

i I~ II

-

162

-

-

162

-

-

175

-

I I/ 1/9

3/i0

~

....

I00 i00 0

LTS

•

0

0 163 144 1 / 1 0

L.

0

Activity of ADM and/or CoQl O treatment on C3H mice bearing Gross leukemia. The percent variation of the median survival time in days over controls (T/C %) and the number of long term survivors (LTS) out of the number of mice per group are given for two sets of experiments (I and II). - = not done. TABLE

mg/kg

ADM

5

0.0

5.0

I0,0

| m

C°Qlo 3.O

TIC %

LTS

I00

0

TIc % 139

0 m

1.5

0

I00

Tic %

LTS

133

i

0

166

IO0

LTS

(*)

0 i

•

0

155 (.)

i

0.0

.

m

0

139

0

lS3 (*)

3/10

Activity of ADM and/or CoQl 0 treatment on BDFI mice bearing L 1210 leukemia. The percent variation of the median survival time in days over controls (T/C %) and the number of long-term survivors (LTS) out of the number of mice per group are given. (*) = not slgnifically different. ( P > O . O S ~ Student's t test).

Rabbits: In long term ADM treated adult rabbits the heart weight is not modified but the heart frequence is significantly increased

(mean values + SE: 284 + 19.8 beats/mln, mmmB

vs. 225 +

.

[

-Pharmacological Research Communications, Vol. 9, No. 3, 1977

242

19.8 of the control group).~ the ECG pattern is altered by T wave flattening or inversion and QRS voltage decreased. Light and electronmicroscopic

evidence of cardiomyocyt e in-

juries consistently observed in these animals are represented by the appearance of nucleolar derangements~

cell vacuoles~ myelin

figures associated with swelling or degenerating mltochondria~ myofibrillar damage~ myocytolysis with replacement fibrosis, The findings are in keeping with the data of BERTAZZOLI et al.~ 1974 a~ b~ c; JAENKE~ 1974; ROSENOFF et al.~ 1975; YOUNG~ 1975; LAMBERTENGHI-DELILIERS In ADM plus C O C o

et al,~ 1975.

treated animals the heart rate (277 + ~2

beats/min.) is significantly (P< 0.05 - Student's t test) decreased vs. ADM treated animals. The occurrence of ECG changes and histologic alterations is significantly lower. Besides the heart function and morphology changes only some of the many symptoms associated with the ADM long term treatment in rabbits are antagonized by C O C o

treatment. The

increased creatinine blood levels are depressed but the increased cholesterol and fibrinogen blood levels

the bone

marrow suppression and the proteinuria are not modified.

DISCUSSION The cumulative dose-dependant anthracycline associated cardiomyopathy has been observed to occur in humans as the major dose-llmlting side-effect of these antibiotics. A number of studies has been carried out by testing the action of anthracycllnes in many experimental models such as newborn mouse heart cell explants I-NECCO et al.,

19767~

chick embryo heart in ovo i-PANNUTI, 1972 a and bT, isolated and perfused heart of rabbit /_'ARENA et al., 197_27, rat ~-LANGSLET et al,, 19757, dog ~-HERMAN et al., 1972_7 or intact animal: mice I'ROSENOFF et al.,

1975_7,hamster

~HERMAN

Pharmacological Research Communications, Vol. 9, No. 3, 1977

et al.,

19717, rats

/-CARGILL et al.,

TAZZOLI et al.~ 1974; JAENKE,

19727

19747,

1974_7,rabbits

243

[-BER-

dogs I-ARENA et al.,

and monkeys _/-DENINE & SMITH, 19757.

The congestive heart failure observed in humans treated with ADM /-LENAZ & PAGE,

19767

can be reproduced only by pro-

longed treatment of rats, rabbits or monkeys but substantial information can be gathered even from other experimental models to enlight the pathogenesis of the toxic syndrome and to define the activity of potential antidote(s). The data of this paper have been collected aiming at this purpose. A first rel evant point emerging from them is the long-lasting character of the ADM toxic effect. The ADM induced function derangement of the rabbit heart surviving in vitro is self perpetuating even after the drug discontinuation. This after-effect is reversed by supplement ing the perfusing solution with COQl O, The finding can be correlated with the statistically significant depletion of heart C o C o

content induced in rabbits by prolonged ADM

treatment ~-BERTAZZOLI et al., 19767 and with the antidote activity exerted by COQl O administration in long-term drug treated animals. The heart weight to body weight ratio (heart index) is depressed by ADM treatment only in juvenile mice. In this species the myocardiocytes can actively multiply for a 4 week period after birth and at a lower rate for 3 months thereafter ~-PETERSEN & BASERGA~

1975_7.The

heart index de-

crease can be ascribed both to myocytolysis and inhibition of cell multipllcation,

in keeping with the data of ARENA

et al., 1974-1975; YOUNG et al., 1975; ROSENOFF et al., 1974; ZEDECK et al.~ 1976 on the heart DNA function impairment induced by ADM. The heart atrophy is counteracted by C o ~

treatment. CoQ9

Pharmacological Research Communications, Voi. 9, No. 3, 1977

244

is the physiologic coenzyme Q component in rodents and possesses the same characteristics of its higher homolog C°QIo, prevalent in other mammalians. The COQl 0 administration antagonizes the cardiotoxic effects induced in rabbits by long-term ADM treatment~ but does not reverse some of the symptoms of the drug associated toxic syndrome (e.g. hypercolesterolemia,

hyperfibrinogenemia,

bone

marrow suppression) or the peculiar antitumor activity of ADM in mice experimental tumors. On isolated beef heart mitochondria in vivo the COQlo activity is blocked by relatively high concentrations of ADM /-IWAMOTO et al.,

1974_7.These

findings and the share

of quinoid structure by CoQ and ADM have been worked out by KISHI & FOLKERS, 1976 to hypotize that a selective and direct activity can be displayed by the antibiotic upon the enzyme function(s)

in the electron transport system(s) of

mitochondria. Bes ides the above quoted biochemical and his tologic data additional evidence pointing toward the mitochondria as a target of ADM actions

is

given by the obser-

vation of DUARTE-KARIM et al., 1976

on

the drug aptitude to

form complexes with cardiolipin, a characterist ic pho s pholipid component of mitochondria. GOSALVEZ & BLANCO~ 1975; V ~

ROSSUM & GOSALVEZ,

1976 em-

phaslze the ADM inhibitory actlvlty on Na+-K+-dependant ATPase. On the other hand the time sequence of the ultrastructural lesion outflow in myocardiocyte of ADM intoxicated animals locates the most precocious drug-induced injury at the nuclear or nucleolar level ~-LAMBERTENGHI-DELILIERS

et al.,

19757 where the instructions for the synthesis of cytoplasmic and mitochondrlal components are encoded and transcribed. The mltochondrial les ions become manifest after a definite lag period. The earliness of the nuclear and/or nucleolar lesions

Pharmacological Research Communications, Vol. 9, No. 3, 1977 are coherent with the generally

245

accepted basic mechanism of

action of ADM based on intercalation within DNA helices and DNA function inhibition ~-DI MARCO,

19727.

In any case owing to the directly and/or indirectly ADM in duced impairment of the mitochondrial

structure and function

a bioenergetic crisis strikes the cell. The most affected are the myocardiocytes whose function heavily depends on the bioenergetic supply. The clinical symptomatology sustained by these early reactions occurring in humans few hours after the ADM administration is quite scarce and limited chiefly to ECG chang es. The myocardiocyte death which can eventually follow not on ly deprives the cardiac muscle of unreplaceable elements which are essential for its pump function but also releases myocardial antigen(s) able to elicit autoimmune reactions with cell mediated cytotoxic (CMC) phenomena /-FIORETTI

et al., 19767.

Analogous mechanisms play a rSle in the genesis of many iatro genic s parasitic and even so called idiopathic cardiomyopathies /-LAUFER,

1974; GHIONE,

19757.

The severe delayed cardiomyopathic side-effects whose sudden occurrence can be observed in humanss rats s rabbits and monkeys chronically treated with ADM is ascribable to the concomitant progress of myocytolysis and antiheart autoimmune cell mediated reaction(s). The humoral antibodies

(reaching the peak at the 7th course

and declining afterwards probably owing to the formation of an tigen-antibody complexes> react by immunofluorescence

(IF) test

with heart (and kidney or muscle) rabbit embryonic cells s are devoid of cytotoxic effect s even in presence of complement s but can induce in otherwise harmless spleen cells of untreated anlmals an antibody dependant cell mediated cytotoxic effect (ADCC) specifically directed towards the rabbit myocardlocytes /-FIORETTI

et al.,

19767.

Pharmacological Research Communications, VoL 9, No. 3, 1977

246

The IF is enhanced when the target cells are pretreated with subinhibiting concentrations of ADM~ but no anti-ADM antibodies were detected. This finding is ascribed to the enhancement by ADM of cell susceptibility towards antibodies /'SEGERLING et

al., 1975_7.The

flow of these immune humoral and cell mediated

reactions is probably slowed down to a certain extent~ but not stopped~ by the selective immunosuppressant

properties of ADM

/-ISETTA et al.~ 1971; CASAZZA et al. 9 1975; DELLA BRUNA & SAN FILIPPO,

197!7.

Some relevant conclusions can be drawn from these considerations, The early transient mild cardiotoxic effects can be co~ sidered as the forerunners of the delayed severe cardiopathic phenomena, To break the pathogenetic vicious circle which is rapidly established a suitable antidote should be timely administered, This is particularly true in the case of C o ~

and COQl 0

whose antidote effect is attributed to their interference on to the early reaction phase, Their administration should be started contemporaneously or even before the ADM treatment to exploit their potential activity. Obviously the usefulness of these compounds will be very reduced if they are applied once the autoimmune antiheart reaction has been already primed. The reason why the ADM antitumor effect is not modified by CoCo

treatment is attributed to the fact that the malignant

cells contain a very low amount of COQio !-SCHICHIRI et al., 19687 and a limited number of COQl 0 loci. On the contrary the heart myocells contain a relevant number of mitochondrla and a high COQIo concentration. The exogenous C O C o

administration can

concur to protect the heart myocells from the unwanted side-effect of drugs acting (directly or indirectly) upon the mitochon dria without modifying their antitumor property. The selectivity of C O C o

antidote action alleged by these

data is a point of obvious interest~ but more research is needed

Pharmacological Research Communications, VoL 9, No. 3, 1077

247

to find out if this property is more apparent than real, being supposedly contingent on the forcibly limited dose range explored in these trials or on some other factor(s) connected with experimental conditions ° The margin of selectivity can be narrow and dependent upon the schedule of treatment but in dealing with drugs endowed J

with a limited therapeutic index like the antitumor compounds even a possible circumscribed gain is worthy of exploration.

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_

m ,

,,

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Pharmacological Research Communications, Vol. 9, No. 3, 1977

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