[CANCER RESEARCH 39, 2269-2276, June 1979]0008-5472/79/0039-0000$02.0O

Interactions of Hyperthermia and Chemotherapy in Animals'

Jane B. Marmor

Department of Radiology and Medicine (Oncology), Stanford University School of Medicine, Stanford, California 94305

Abstract

In tissue culture, the cytotoxicity of a number of commonlyused chemotherapeutic drugs is greatly enhanced at elevatedtemperatures. However, pharmacokinetics, drug concentrations, oxygen tension, and pH in tumors in animals can all varywidely from those in cell cultures. In addition, tissue culturestudies do not give vital information on the effect of combinations of drugs and hyperthermia on normal tissues or metastases. Available studies of drugs and hyperthermia in animalsare reviewed, and they yield clinically useful information. Onestudy indicated that the activity of methotrexate was not enhanced by hyperthermia in vivo. Results for alkylating agentswere not conclusive. Antitumor effects of 1,3-bis(2-chloroethyl)-1 -nitrosourea, bleomycin, and cis-diamminedichloroplatinum, however, were significantly potentiated by local hyperthermia. 1,3-bis(2-chloroethyl)-1 -nitrosourea effects were enhanced between 41 and 42°C,and thus, it is potentially of usein the setting of systemic hyperthermia. Bleomycin, however,was enhanced significantly only near 43°,suggesting that itsclinical use is more appropriate with local hyperthermia. Adriamycin and S-(2-aminoethyl)isothiouronium dihydrobromide, although potentiated in vitro, were not potentiated in vivo indoses which were clinically tolerable. Discrepancies betweenthe in vitro and in vivo findings are likely due, at least in part,to differences in drug concentration. The combination of localprimary tumor hyperthermia and chemotherapy did not adversely affect the incidence or severity of spontaneous lungmetastases in KHT tumor-bearing mice.

Very few studies of the pharmacology of drugs in vivo in thepresence of hyperthermia have been reported. Uptake of chemotherapeutic drugs may be enhanced in hyperthermic tissue.Further in vivo studies both of effectiveness and drug pharmacology for combined hyperthermia and chemotherapy areadvisable to define optimum time-dose schedules for clinicaltrials.

Introduction

Studies on a variety of cells in vitro have shown that thecytotoxic efficiency of a number of commonly used chemotherapeutic drugs is greatly enhanced at elevated temperatures(8, 13). These findings have raised the possibility that hyperthermia can be used clinically to increase the effectiveness ofsystemically administered chemotherapeutic agents. However,relatively few studies have attempted to see whether the interaction between chemotherapy and hyperthermia is useful intumor-bearing animals treated systemically with drugs. Evenfewer clinical studies have attempted to take advantage of thepotential interaction (13).

, These studies were supported by Grant CA-i 9386, NatIonal Cancer Institute,Department of Health, Education, and Welfare. Presented at the Conference onHyperthermia In Cancer Treatment, September 15 and 16, 1978, San Diego,Calif.

There are several reasons why results from studies of combinations of hypothermia and chemotherapy in vivo may differfrom the in vitro results. The first of these is that drug doses towhich cells in a tumor (in vivo) are exposed may differ fromthose to which cells in vitro are exposed. Drug levels in tissuecultures are likely to remain relatively stable during the experimental period (often only 1 hr), and drug doses are easier toquantitate, usually being the concentration times the time. Drugdoses to which cells in tumors in vivo are exposed are muchmore complex and are a function of the distribution, metabolism, and excretion of the drugs by the animals being tested aswell as the tumor blood supply. The situation in animals is madeeven more complex by the probability that hyperthermia itselfaffects the distribution, metabolism, and excretion of the drugand could markedly affect drug levels to which cells in thetumor are exposed (1, 17). Even local hyperthermia may affectblood flow (and thus the drug delivered to the tumor), localdrug transport, and metabolism. Unfortunately, very few studiesare available on the effect of hyperthermia on the pharmacology of antineoplastic drugs. Finally, the effects of heat ontissues or organs may differ in some respects from those onisolated cells in vitro. Although mechanisms of cell killing dueto hyperthermia may be the same in vitro as in vivo, the situationin vivo is more complex because of the interaction of structuralcomponents of a given organ or tissue. For example, recentstudies have shown that hyperthermia has a profound effecton the vasculature of an experimental tumor in vivo and thatclinical effects observed in the tumor following hyperthermiaare in part secondary to the effect on blood vessels and notdirect effects of the heat on tumor cells.2 If circulation to atumor were dramatically changed due to hyperthermia, druglevels would be affected as well as conditions under whichtumor cells are exposed to drugs, such as pH and oxygentension. Both pH and oxygen tension have been shown to alterthe sensitivity of cells to hyperthermia (6, 7). Finally, studies ofthe effects of combined hyperthermia on cells in tissue culturefail to give vital information on the effect of combined therapyon normal tissue.

For all these reasons, it seems imperative that the interactionof hyperthermia with chemotherapeutic agents be studied invivo as well as in vitro to aid in the design of appropriate clinicaltrials. In this paper, I will review previous studies of combinedeffects of hyperthermia and chemotherapy in animals, includingrecent data from our own laboratory.

Studies of Combined Chemotherapy and Hyperthermia InTumor-bearing Animals

Relatively few well-documented studies on effects of combined hyperthermia and chemotherapy in animal systems havebeen reported in the past. Only studies where exposure to heat

2 L. F. Fajardo, B. Egbert, J. B. Marmor, and G. M. Hahn. Effects of hyper

thermia in a malignant tumor, submitted for publicatIon.

JUNE 1979 2269

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

J.B.Marmor

and drugs was in vivo are included in this review; studies wherecells were exposed in vitro but assayed in vivo are omitted. Inaddition, only published work which included details of experimental protocol is included.

Antlmetabolites

In 1973, Muckle and Dickson (18) reported on combinedmethotrexate and heating of VX-2 carcinomas in rabbits. Hostanimals were given 6 daily injections of methotrexate (0.4 mg/kg), and 4 days after starting the course, tumors were heated

to 42°C for 1 hr by water bath. Response was analyzed byvolume change and by rate of respiration and glycolysis oftumors. The effect of the combined therapy was not significantly different from that for methotrexate alone by eitherassay.

Alkylating Agents

Suzuki (24) studied the effect of the combination of Nitromin(mechlorethamine N-oxide) and hyperthermia on the Yoshidasolid sarcoma in rats. The combination of 42°Clocal waterbathhyperthermia with Nitromin (5 mg/kg) caused local tumorregression, whereas either of these treatments alone onlycaused slowing of tumor growth. At higher doses of Nitromin(10 mg/kg), tumor regression occurred with or without heat,and the difference between them was not significant. Localtoxicity in terms of swelling and loss of feet was worse in thegroup which received combined treatment. In a later study,Dickson and Suzanger (5) examined the effects of 42°Chyperthermia in conjunction with the alkylating agent MDMS3 onYoshidasarcomasin rats.Thisstudyshowedno improvementin clinical results from the combination of drug and heat. Infact, tumors treated with heat and MDMS regressed moreslowly than did tumors treated with MDMS alone. When theauthors assayed for inhibition of glycolysis, the 2 treatmentswere additive at 24 hr after treatment, but later the effect ofheat and drug on tumor glycolysis was the same as that fordrug alone. There were several flaws in the design of thisexperiment, however. In the first place, curative doses ofMDMSalonewere utilized.Secondly,drug and hyperthermiawere not administered simultaneously; drug was followed 20hr later by 42°Chyperthermia. Thus, a synergistic effect thatrequired the 2 modalities to be given close together in timewould have been missed.

Antitumor Antibiotics

Adriamycin. Hahn et a!. (10) examined the combination ofADR and 43°Chyperthermiafor 30 mm in BALB/c micebearing the EMT6 sarcoma. Analysis of the effects was by invitro cloning of cells from tumors treated in situ. Effects ontumor growth or cure rates were not reported. In doses between10 and 25 mg/kg, the combined cytotoxicity of ADR and 43°Chyperthermia was clearly superior to either modality alone.

Clinical observations of effects on tumor growth of the com

3 The abbreviations used are: MDMS. methylenedimethanesulfonate; ADR,

Adriamycin; BLEO. bleomycin; BCNU. 1,3-bis(2-chloroethyl)-1 -nitrosourea; cisDDP, cis-diamminedichloroplatinum; AET, S-(2-aminoethyl)isothiouronium dihydrobromide.

bination of ADR and heat in mammary carcinoma bearing C3Hx DBA/2 F1(hereaftercalled C3D2F1/BOM)mice were reported by Overgaard (19). Local hyperthermia was administered 5 mm after drug at 40.5°Cfor 2 hr or 42.5°Cfor 1 hr;one group of animals underwent systemic hyperthermia at40.5°Cfor 2 hr. Unfortunately, ADR was utilized in a dose thatwas not clinically tolerable (25 mg/kg). As a result, 18 of 20control animals which received ADR alone died approximately7 days after treatment. Both 40.5 and 42.5°Ccombined withADR gave a higher proportion of cures than heat alone. How

ever, since only 2 animals treated with ADR alone survived, itis not possible to tell whether this effect is greater than onewould expect from ADA alone or from an additive effect of the2 modalities. All animals treated with systemic hyperthermia

and ADA died a toxic death. Surprisingly, the author noted adecreased number of toxic deaths in the animals given bothlocal hyperthermia and ADA. This study might have been moredefinitive if it had been done at a practical clinical dose level ofADA.

Our laboratory has studied the combination of ADA and43°Clocal hyperthermia in 2 experimental mouse tumor systems (1 6). The EMT6 tumor in BALB/c mice (22) utilized tostudy the effects of local tumor hyperthermia and i.v. administered ADA on tumor cell survival. The KHT mammary carcinomain C3H mice (14) was utilized to study effects of single andcombined therapies on growth delay, tumor cure, and mcidence of lung metastases. Heating was by radio-frequencyfields as described previously (15) and was for 30 mm at 43°Cbegun immediately after injection of drug.

Results of cell survival studies on EMT6 tumors excised 2 hrafter i.v. injection of graded doses of ADA with or without localheating of the tumor are shown in Chart 1. At the highest dose(10 mg/kg), there appeared to be significant enhancement ofADAcytotoxicityby 43°C.However,nosignificantpotentiationwas seenat dosesof 5 mgor 2.5 mg/kg.

The results of experiments in which combinations of ADRand heat were administered to KHT tumor-bearing C3H miceare shown in Chart 2 and Table 1 (16). In these and subsequentexperiments, effects on tumor growth were expressed as themedian time (in days) which it took for tumors to double theirmean tumor diameter (defined as the geometric mean of 3orthogonal measurements). Doses of ADA (10 mg/kg) whencombined with hyperthermia resulted in 14 of 25 deaths about5 days after treatment. The same dose of drug alone caused 3of 16 deaths. Thus, in contrast to result of Overgaard (19),combination treatment was more toxic than was either treatment alone. Clinically tolerable doses of ADA (2.5 and 5 mg/kg) were not significantly increased in effectiveness by simultaneous local hyperthermia (open circles) over that expectedfrom simple additive effects of the 2 modalities (closed circles).In addition, tumor cures were not seen as a result of combinedtreatment (Table 1). Thus, despite the fact that the cytotoxiceffects of ADA in tissue culture are markedly potentiated bylocal hyperthermia (10, 12), such potentiation was not demonstrated in vivo.

There are several possible explanations for the apparentdiscrepancy between the in vivo and in vitro results for ADAand for the apparent discrepancy between Overgaard's resultsand ours. The most probable is that suggested by Chart 1;drug doses may be critical, and sufficiently high concentrations

2270 CANCERRESEARCHVOL. 39

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Interactions of Hyperthermia and Chemotherapy in Animals

high toxicity of drug alone, he did believe some potentiationoccurred at this high drug dose. In any case, if hyperthermicpotentiation can only be demonstrated at doses which are toxicto the host it can be expected to be of little clinical use.

A second possible explanation for the discrepancy betweenin vivo and in vitro findings for ADA and heat is that timing ofthe drug and heat dose may not have been optimum in thesestudies. Studies on cells in vitro indicate that heating for toolong a period or prior to drug can actually make cells resistantto ADA (12). It is possible, therefore, that a different heatfractionation might demonstrate sensitization.

BLEO. In vitro cell killing by BLEO is markedly potentiatedby heat (3). Animal studies from our laboratory indicate that

antitumor effects of BLEO are also potentiated by local heating(16). Chart 3 and Table 1 show the effect of combined BLEOand 43°Con KHT tumor-growth delay and cure rate. Cureswere seen with combined BLEO and hyperthermia given together, although no cures were seen for the 2 modalities given24 hr apart. BLEO and hyperthermia given together alsocaused a significantly greater growth delay than did eithermodality alone or than both heat and BLEO given 24 hr apart.The fact that the 2 modalities had to be given close together intime to achieve a synergistic effect suggests that there is a true‘‘interaction.‘‘It also has implications for the clinical use ofcombinations of heat and BLEO. Further definition ofthe criticaltime interval between BLEO and heat administration in vivo isdesirable.

The combination of BLEO and heat was more toxic than waseither alone. Although BLEO (15 mg/kg) could be given 5times to C3H mice without toxicity, 3 administrations of this

20

15 -

10 -

0

I-

wQ.

U)-J-JwU

U

zLuID0z0-IU

Chart 1 . Survival of cells from EMT6 tumors treated in situ with combinationsof Adriamycin and 43°Chyperthermla. EMT6 tumor-bearing mice were treatedwith graded doses of Adriamycin lv., and tumors were then immediately eitherheated to 43°for 30 mm or “sham―heated. Tumors were excised 2 hr afterdrug administration and cell survival was assayed by cloning (22). •,43°Cfor30 mm (no drug); A, 37°C(no drug); t@,ADR wIthout heat; 0, ADR plus 43°Cfor30 mm. Shaded area, range of expected “additive―effect based on the productsof the upper and lower limIts of survival after drug alone and heat alone. 43°Calonepoint,meanandS.E.of 26 experImentalpoints.Theuntreatedcontrolisthe mean and SE. of 12 experIments. Other points represent individual experiments based on pooling 2 treated tumors for analysis. The line connects thegeometric means of these points.

of drug cannot be maintained in vivo for long enough periodsof time to show potentiation. The in vitro data indicate thatsignificant potentiation occurs after 3 hr incubation at a concentration of 0.5 gig/mI (10). While this concentration can beachieved in vivo, the serum half-life of drug is very short (17).Thus exposure on the average will be to a much lower dose invivo. Indeed, drug dose experiments (Chart 1) suggest that, athigher drug doses which are too toxic to be utilized clinically,some potentiation of ADA by heat does occur in vivo. Althoughthe studies of Overgaard were inconclusive because of the

(A)ADRIAMYCIN2.5me/kg

..@..

5 10ADRIAMYCIN DOSE (mg/kg)

(B) ADRIAMYCIN 5 mg/kg15

10

S

2@0

NUMBER OF TREATMENTS

Chart 2. Mean tumor diameter doubling times for KHT tumors in C3H micetreated with combinations of heat and Adriamycin. Points, the medians for 6 to15 animals. Shaded area, control range (mean ±2 S.D.). 0, 43°Cfor 30 mm;& Adriamyclnalone;•,Adriamycinand43°CgIven24hrapart;0, Adrlamycinand 43°CgIven simultaneously.

JUNE1979 2271

U,>-

00I-

LU-J

20I-

LU

I-

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

TreatmentIncidence

of tumorcures1

treatment23

treatments treatmentsTotal,1—3

treatmentsControl0/2243°for30min0/150/12

0/120/39ADR(2.5mg/kg)

ADR(2.5mg/kg)+43°C(24hrapart)ADR(2.5mg/kg)+43°C(together)0/12

0/60/60/6

0/61/5 0/80/5 0/60/24

1/190/17ADR(5mg/kg)

ADR(5mg/kg)+43°C(24hrapart)ADR (5 mg/kg) + 43°C(together)0/6

0/80/70/6

0/20/80/1 10/14

0/160/18BLEO(7mg/kg)

BLEO(7mg/kg)+43°C(24hrapart)BLEO(7mg/kg)+43°C(together)0/8

0/80/70/8

0/80/7 0/70/8 0/70/24

0/220/22BLEO(i5mg/kg)

BLEO(15 mg/kg) + 43°C(24 hr apart)BLEO(15 m9/kg)+ 43°C(together)0/6

0/71/120/6

0/60/63/140/18

0/134/26BCNU(lOmg/kg)

BCNU(lOmg/kg)+43°c(24hrapartrBCNU (10 mg/kg) + 43°C(together)0/6

0/140/150/6

0/60/13 0/133/15 2/170/18

0/405/47cis-DDP(2mg/kg)

cis-DDP(2mg/kg)+43°C(24hrapart)cis-DDP(2mg/kg)+43°C(together)0/6

0/60/60/6

0/60/7 0/30/6 0/60/18

0/160/18a

BCNU given 24 hr before heat in 8 animals each group and24 hr after heat in 8 animalseach group.

DrugBLEO

(15 mg/kg)

Ratio3.45.0100.0

BCNU (5 mg/kg) 41450019002.442400010040.0434001040.0

J.B.Marmor

Table1Incidence of KHT flank tumor cure

Table2Ratio of expected/observed survival values for BLEO and BCNU with

heatThe ‘‘expected'‘valueis the productof cell survivalfor heatalone

and drug alone and is the theoretic “additive―effect of the twomodalitiesValuesarederivedfromFigs 3 and5.

ExpectedObservedTernsurvivalsurvivalpera

(clonogenic(clonogenlcture(°C)cells/tumor)cells/tumor)41170050042150030043900.9

U,>-400I-

LU-J

20I-

Ui

dose of BLEO together with heat caused 5 of 6 deaths intreated animals. Autopsies on animals that died following heatand BLEO showed edema, necrosis, and perforation of bowelnear the tumor (i.e. , in an area that probably received someheating). The finding of increased normal tissue toxicity fromthe combination also has important clinical implications.

In cell survival studies with EMT6 tumors, the cytotoxicity ofBLEO combined with 43°C hyperthermia was 1 0- to 1 00-fold

greater than that expected from simple additive effects. Effectsat lower temperatures, however, were less pronounced. At41 °C, cell survival after the combination treatment was within

the expected additive range. At 42°C,survival was lower thanexpected from additive effects but much less dramatically sothan at 43°C.Aatios of ‘‘observed'‘to ‘‘expected'‘survivalsfor the 3 temperatures are shown in Table 2. There is atemperature ‘‘threshold'‘for the combined effect of BLEO andhyperthermia in vivo between 42 and 43°C.This is consistent

NUMBER OF TREATMENTS

Chart 3. Mean tumor diameter doubling times for KHT tumors In C3H micetreated with combinations of heat and BLEO. Points, medians for 7 to 15 anImals.Shaded area, control range (mean ±2 S.D.). 0, 43°Cfor 30 mm; Is, BLEOalone; •.BLEO and 43°C given 24 hr apart; 0, BLEO and 43°Cgiven simultaneously.

2272 CANCERRESEARCHVOL. 39

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Interactions of Hyperthermia and Chemotherapy in Animals

with the finding of a similar temperature ‘‘threshold'‘in vitro(10). Since safe levels of systemic hyperthermia are limited to41 .8°(20), this finding has important implications for clinicaltherapy. To obtain significant therapeutic benefit from thecombination of heat and BLEO, it appears necessary to utilizetemperatures above those that can be tolerated systemically.Thus, BLEO is not an appropriate drug to combine with systemic hyperthermia. A more appropriate use is combined withlocal hyperthermia where temperatures in the range of 43°Ccan safely be utilized.

Actinomycin D. Yerushalmi(26) studied the effect of actinomycin D and local hyperthermia on a solid SV10 fibrosarcoma in BALB/c x C57BL/6 F1 mice. A marked increase inmedian survival time was noted following combined treatmentcompared to actinomycin alone. Unfortunately, the studylacked information on the effect of heat alone, so one cannottell if the effect was more than additive.

Nitrosoureas

BCNU. Twentymanet a!. (25) studiedBCNU and hyperthermia in the EMT6 mouse-tumor system. Animals bearing flankor leg tumors were treated with BCNU (20 mg/kg) immediatelyprior to hyperthermia by water bath at an average tumortemperature of 41 .6°Cfor 1 hr. Both tumor regrowth and invitro assay for survival of tumor cells were reported. In bothassays, the effect of the combination treatment was clearlysuperior to that of either treatment alone. By the cell survivalassay, the effect of the combination appeared to be greaterthan that expected from additive effects of the 2 modalities.

We have also studied the combined effects of BCNU andhyperthermia on both the EMT6 and KHT tumor systems andour results agree substantially with those of Twentyman.4 Considerable potentiation of the in vivo cytotoxic effects of BCNUwas seen at both 42 and 43°C,although the effect was lesssignificant at 41 °C(Table 2). These results agree with thetemperature response reported in vitro for BCNU (8). Since thepotentiation threshold, if any, for BCNU is between 41 and42°C,this drug is an appropriateone to considerfor adjuvantuse with systemic hyperthermia.

The effects of BCNU with or without heat on tumor growthdelay and cure of the KHT carcinoma are shown in Chart 4 andTable 1. Considerable increase in the antitumor activity ofBCNU was demonstrated both in growth delay and incidence

of tumor@ ‘cures'â€w̃hen heat was used with the drug. Heat andBCNU had to be given close together in time (open circles) to

observe this effect; when exposure to the 2 modalities wasseparated by 24 hr, the effects were much less (solid circles).For exposures separated by 24 hr, results were the samewhether heat or drug was given first.

OtherAgentsEnhancedInVitro

Cls-DDP. The enhancementof cytotoxicityin vitroofcis-DDPat elevated temperatures was described by Hahn (9). Studiesin our laboratory have also shown that the antitumor effects ofcis-DDP were also potentiated in vivo.4The combined effect ofhyperthermia and cis-DDP on KHT tumor growth delay is shownin Chart 5. For a single combined treatment, no increase in

4 J. B. Marmor, and G. M. Hahn. Combined effects of three nitrosoureas and

hyperthermia on solid tumors in mice, manuscript In preparation.

NUMBER OF TREATMENTS

U,>-400

LU-I

000I—

LU

Chart 4. Mean tumor diameter doubling times for KHT tumors In C3H micetreated with combinations of heat and BCNU (10 mg/kg). Points, medians for 7to 16 anImals. Shaded area, control range (mean ±2 S.D.). 0, 43°Cfor 30 mm;& BCNU(10mg/kg);•.BCNU(10mg/kg)and43°gIven24hrapart;0, BCNU(10mg/kg)and43°Cgivensimultaneously.

U,

400I-

LU-J

000I-LU

I.

Chart 5. Mean tumor diameter doubling times for KHT tumors In C3H micetreated with combinations of heat and cis-DDP (2 mg/kg i.p.). Points, mediansfor 6 to 15 animals. Shaded area, control range (mean ±2 S.D.). 0, 43°Cfor 30mm; t@,cis-DDP (2 mg/kg); •.cis-DDP (2 mg/kg) and 43°CgIven 24 hr apart;0, cis-DDP (2 mg/kg) and 43°CgIven together.

NUMBER OF TREATMENTS

effect was noted; however, when 2 or 3 treatments were given,the combination was clearly superior to either modality aloneor to combined therapy given at 24-hr intervals.

Noncytotoxic Agents. Some drugs which are not toxic tocells at normal temperatures become highly cytotoxic at elevated temperatures (2, 11). Bleehan et aI. (2) studied theinteraction of hyperthermia and the hypoxic cell sensitizer RO07-0582 on the EMT6 mouse tumor using a cell survival assay.No significant cytotoxic effect of the drug was observed at 37°.However, at tumor temperatures above 42.5°, the drug becamemarkedly cytotoxic. No tumor growth measurements were reported in this study.

In the preceding paper, Hahn (9) described studies on thecytotoxicity of the thiol radioprotective agent AET when com

2273JUNE1979

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

J.B.Marmor

bined with heat in vitro. This compound was nontoxic to cellsat 37°Cbut became markedly cytotoxic at 43°C.We haveattempted to see whether this cytotoxicity at elevated temperatures was useful against animal tumors in vivo. Chart 6 showsthe results of cell survival studies from tumors excised aftertreatment with AET in the maximum tolerated dose with orwithout 43°Chyperthermia. No cytotoxicity was demonstrated.If anything, a small amount of protection from the effects of

heat occurred. Studies of the effects on tumor growth of thecombination confirmed that there was no antitumor effect ofAET at elevated temperatures (Chart 7). Thus, in the case of

AET, the in vitro studies did not predict accurately the effect

observed in animals, at least in these systems.

Effects of Local Hyperthermia With or Without Chemotherapy on Spontaneous KHT Lung Metastases

There has been considerable concern about the possibilitythat local hyperthermia to a primary tumor might increase therate of metastasis. Suzuki (24), and Muckle and Dickson (18)noted that, with combinations of drug and local hyperthermiato a primary tumor, there was no survival advantage despiteregression of the primary tumor because animals died at approximately the same rate as controls from lung metastases.Dickson and Ellis (4), in a later study, suggested that localhyperthermia at 42°Cincreased the rate of metastasis of theYoshida tumor in rats. However, this study is not conclusivesince the animals so treated had significant systemic hyperthermia and a high death rate from hyperthermia alone. Indeed,151 of 167 animals died during treatment or within 24 hr andanother 10 died 3 to 12 days later leaving only 6 survivors for

@0

0I-

LU-J

000I-

LU

I-

2

NUMBEROF TREATMENTSChart 7. Mean tumor diameter doubling times for KHT tumors in C3H mice

treated with combinations of heat and AET (300 mg/kg i.p.). Points, medians for6 to 15 animals. Shaded area, control range (mean ±2 S.D.). 0, 43°Cfor 30mm; & AET (300 mg/kg); •,AET (300 mg/kg) and 43°given 24 hr apart; 0,AET (300 mg/kg) and 43°Cgiven together.

pathological examination. Clearly the survivors of such a toxicregimen may have been systemically debilitated, which mayhave affected growth of metastases.

We have considered this problem in KHT tumor-bearinganimals treated with local hyperthermia alone or with chemotherapy. This tumor metastasized spontaneously to the lungsin close to 100% of control mice. Metastasis first occurredbetween 10 and 13 days after implantation of intradermal flanktumors (16). Since the usual day of treatment for tumors was11 days after implantation, it was possible to assess the effectsof local hyperthermia on the incidence and severity of lungmetastases. Lungs were examined for metastasis 23 to 25days after treatment, and the severity of metastasis was gradedfrom 0 (macroscopically free of metastases) to 4. The averagegrades for control and treated animals are shown in Table 3(1@ Heat alone did not appear to increase the averageseverity of metastases over control. For ADR and AET, treatments not potentiated by heat, the average grade for animalsgiven heat with drug was similar to that for drug alone. Heatdecreased the severity of lung metastasis when used withBLEO or BCNU. The most dramatic effect was seen with BCNU.Thirty-two of 47 lungs were negative for tumor following BCNUand heat given together compared to 2 of 18 negative withBCNU alone and 2 of 38 when BCNU and heat were given 24

hr apart (16). These data do not support the contention thatlocal hyperthermia increases metastasis. In fact, it appears thatthose treatments which are most effective against the primarytumor decrease metastasis whether or not they include hyperthermia.

Studies of Pharmacokinetics of Drugs In the Presence ofHyperthermia

The plasma clearance, distribution, biotransformation, andexcretion of drugs may be affected by hyperthermia eitherdirectly or as a result of changes in blood flow due to thehyperthermia (1, 17). However, few published studies address

5 J. B. Marmor, and G. M. Hahn. Enhanced antitumor activity of cis-diam

minedichloroplatinum when combined with local tumor hyperthermia, manuscriptin preparation.

AET

@- —4. 0

: 8

10@

(06

0

IL)a- id'

Co-I-ILU0

0zLU 4

@ 10z0-I0

310

0 300AET DOSE (mg/kg)

Chart 6. Survival of cells from EMT6 tumors treated in situ with combinationsof AET and 43°Chyperthermia. Procedures were as described under Fig. 1.•,43°Cfor30mm(nodrug);A, 37°control;t@,AET(300mg/kg);0, AET(300mg/kg) plus 43°Cfor 30 mm. Shaded area, expected ‘‘additive―range for the2 modalities.

2274 CANCERRESEARCHVOL. 39

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Interactions of Hyperthermia and Chemotherapy in Animals

the problem of alterations in drug pharmacokinetics in thepresence of hyperthermia.

Aochlin et al. (21) reported on the rate of binding of 32P-tagged thiotepa in isolated perfused dog limbs held at differenttemperatures. Binding of drug was increased at increasingtemperatures. Unfortunately, most of his observations were inthe hypothermic temperature range; only 3 observations in thehyperthermic range were made, and no temperature greaterthan 38.9°Cwas studied.

Shingleton et al. (23) described uptake of 14C-Iabelednitrogen mustard under conditions of regional normothermia, hypothermia, and hyperthermia in normal dog legs and in VX-2carcinomas in rabbits; 42°Chyperthermia was produced byeither regional perfusion (abdomen) or by radio-frequency inductive heating (limbs). In both tumors and normal tissue, theuptake of drug was greatest under conditions of local hyperthermia. Local toxicity of the nitrogen mustard was also increased at the elevated temperature.

Finally, Mimnaugh et aI. (17) studied the disposition andmetabolism of ADA in normothermic and hyperthermic rabbits.In general, distribution of drug was to the same tissues atsimilar concentrations in both normothermic and hyperthermicanimals. However, the authors did note increased concentrations of ADA in skeletal muscle and increased total ADA in thehearts of hyperthermic animals. There was no significant difference in urinary or biliary excretion of ADA or its metabolites.Plasma clearance was similar in both groups although the datasuggested an expanded ‘‘peripheral'‘drug compartment in thehyperthermic animals. They stated that, theoretically at least,

Table3Effect of hyperthermia and drugs on the incidence and severity of

spontaneous KHT lung metastasesLungswereexcised23 to 25 daysaftertreatmentandexaminedfor

macroscopiclungmetastases.Eachsetof lungswasgradedonascaleof 0 (no macroscopicmetastases)to 4+ dependingon severityofmetastasesAveragegradefor eachtreatmentgroupwascalculated.

tumor would be included in the expanded peripheral compartment and, therefore, increased distribution of drug to tumormight occur under conditions of systemic hyperthermia. However, whether this actually happens is dependent in part uponwhether blood is shunted around tumor under conditions ofhigh cardiac output during systemic hyperthermia.

Drug transport may be affected at the cellular level by hyperthermia, and this would not be detected by large animalpharmacological studies. For instance, Hahn et al. (10) reported that HAl cells incubated in vitro with high concentrations of ADA exhibited greater fluorescence of ADA at 43 thanat 37°C.He suggested that cellular uptake of ADA was greaterat 43°Cbecause of alterations in the cell membrane at thistemperature.

Conclusions

Tissue culture studies have raised the possibility that theeffectiveness of a number of chemotherapeutic agents may begreatly enhanced by systemic or regional hyperthermia. Theanimal studies reviewed here suggest that some, but not all,drugs which are potentiated by heat in vitro also show clinicallyuseful potentiation in vivo. Clinically useful data, which will beimportant in designing clinical trials of hyperthermia and chemotherapy, can be obtained from animal studies.

The results of the animal studies reviewed in this paper areshown in Table 4. Significant potentiation occurs with BCNU,BLEO, and cis-DPP. BCNU appears to be one of the mostpromising drugs for clinical trials. Its effect was potentiatedsignificantly at doses well below the toxic range. Potentiationwas near maximal between 41 and 42°C.Thus, BCNU is anappropriate drug to consider with systemic hyperthermia.BLEO, in contrast, was maximallypotentiatedonly at temperatures between 42 and 43°C. Since systemic temperatures inthis range cannot be safely achieved, BLEO is more likely tobe useful as an adjunct to local hyperthermia.

Incidence of Not all drugs that are potentiated in vitro show clinically

Iungrnetastasese neg:tive useful potentiation in vivo. ADA failed to show significant po

tentiation by heat in clinically tolerable doses. Studies at high@@ g:@ g@ dose, however, did suggest potentiation. Thus, ADA does not

appear to be a good candidate for hyperthermic potentiation of@@@ ?@ systemically administered drug. It might be appropriate, how

2.6 ±0:33 0/1 1 ever, to utilize hyperthermia and ADA in regional perfusion

where higher local concentration of drug could be achieved@@@@ without systemic toxicity. AET is another example of a drug

1.6 ±0.27 2/14 that is potentiated in vitro but did not show clinically usefulpotentiation in vivo.

@@@ g@ In general, toxicity to normal tissues and death rates were2.6 ±0.23 0/22 increased in animals receiving combination therapy.

Treatment

2 0 ±0 29 1 12 Local hyperthermia to primary tumors did not appear to

2.8 ±0:32 0/1 2 adversely affect the incidence or extent of spontaneous lung1.9 ±0.24 3/26 metastases. In fact, combinations that were very effective

2 0 ±0 26 2/18 against primary tumors, such as heat and BCNU, appeared to

2:3 ±0:26 1/1 8 decrease the incidence of lung metastases.

2.2 ±0.29 1/20 Few studies of the effects of hyperthermia on pharmacolog

0.6 ±0.15 32/47 ical disposition of drug were available. Some of these indicated

3.0 ±0.22 0/18 that tissue binding of alkylating agents is enhanced by local

2.9 ±0.28 0/16 hyperthermia. More studies of the interaction of hyperthermia

2.2 ±0.26 1/18 and drug metabolism are desirable, as such information will be

extremely useful in designing appropriate clinical trials of hyperthermia and chemotherapy.

Control43°for30 mm

ADR (2.5mg/kg)ADR (2.5 mg/kg) + 43°C(24 hr apart)―ADR (2.5 mg/kg) + 43°C(together)

ADR (5 mg/kg)ADR (5 mg/kg) + 43°C(24 hr apart)―ADR (5 mg/kg) + 43°C(together)

BLEO (7mg/kg)BLEO(7 mg/kg) + 43°C(24 hr apartYBLEO (7 mg/kg) + 43°C(together)

BLEO (15 mg/kg)BLEO (15 mg/kg) + 43°C(24 hr apart@BLEO (15 mg/kg) + 43°C(together)

BCNU(10 mg/kg)BCNU (10 mg/kg) + 43°C(24 hr apart)―BCNU (10 mg/kg) + 43°C(24 hr apart)cBCNU (10 mg/kg) + 43°C(together)

cis-DDP (2 mg/kg)cis-DDP (2 mg/kg) + 43°C(24 hr apart)ccis-DDP (2 mg/kg) + 43°C(together)

a Mean ± SE.

b Drug gIven first.

C Heat given first.

JUNE 1979 2275

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Combined chemotherapy and hyperthermia in animaltumorsDrug/classTemperature(°C)AssayEffectInvestigators andreferenceAntimetabolite

Methotrexate42Tumor growthNot enhancedMuckle and Dickson(18)Alkylating

agentsNitrominMDMS42 42Tumor

sizeTumor sizeGlycolysisAdditive

or synergisticLess than additiveAdditiveSuzuki

(24)Dickson and Suzanger(5)Antibiotics

AdriamycinAdriamycinAdriamycin

Actinomycin DBLEO43

40.5 and 42.543

42.3 and 43.643Tumor

cell survivalTumor cure and growth delayTumor cure and growth delayTumor cell survivalSurvival timeTumor cure and growth delayTumor cell survivalSynergistic°

Additive or synergisticNot enhancedSynergistic at high doseaAdditive or synergisticSynergisticSynergisticHahn

et a!. (10)Overgaard (19)Marmor et al.(16)

Yerushalmi (26)Marmor et al.(16)Nitrosoureas

BCNU

BCNU41

.6

42 and 43Tumor

regrowthCell survivalTumor cure and regrowthCell survivalAdditive

or synergisticSynergisticSynergisticSynergisticTwentyman

et al. (25)

Marmor andHahn4Miscellaneous

cis-DDP

AET43 43Tumor

cure and regrowthCell survivalTumor cure and regrowthCell survivalAdditive

or synergisticAdditive or synergisticNot enhancedNot enhancedMarmor

and Hahn―

Marmor―

J. B. Marmor

Table4

a At doses greater than 1 0 mg/kg.

b Unpublished observations.

Advances in Radiation Biology, pp. 229—266.New York: Academic Press,Inc., 1976.

14. Kallman, R. F., Silini, G., and van Putten, L. M. Factors Influencing thequantitative estimation of the in vivo survival of cells from solid tumors. J.NatI. Cancer Inst., 39: 539-549, 1967.

15. Marmor, J. B., Hahn, N., and Hahn, G. M. Tumor cure and cell survival afterlocalized radiofrequency heating. Cancer Res., 37: 879—883,1977.

16. Marmor, J. B., Kozak, D., and Hahn, G. M. Effects of systemic bleomycin orAdnamycin with local hyperthermia on primary tumor and lung metastases.Cancer Treat. Rep., in press, 1979.

17. Mimnaugh, E. G.. Waring, A. W., Sikic, B. I., Magin, R. L., Drew, R., Litterst,C. L., Gram, T. E., and Guarino, A. M. Effect of whole-body hyperthermia onthe disposition and metabolism of Adriamycin in rabbits. Cancer Res., 38:1420—1425,1978.

18. Muckle, D. S. and Dickson, J. A. Hyperthermia (42°C)as an adjuvant toradiotherapy and chemotherapy in the treatment of the allogeneic vx2carcinoma in the rabbit. Br. J. Cancer, 27: 307—315, 1973.

19. Overgaard, J. Combined Adriamycin and hyperthermia treatment of a murinemammary carcinoma in vivo. Cancer Res., 36: 3077-3081 , 1976.

20. Pettigrew, A. T., Gait, J. M., Ludgate, C. M., Horn, D. B., and Smith, A. N.Circulatory and biochemical effects of whole body hyperthermia. Br. J.Surg.,61:727—730,1974.

21 . Rochlin, D. B., Thaxter, T. H., Dickerson, A. G., and Shiner, J. The effect oftissue temperature on the binding of alkylating agents in the isolationperfusion treatment of cancer. Surg. Gynecol. Obst., 113: 555—561. 1961.

22. Rockwell, S., and Kallman, A. F. Cellular radiosensitivity and tumor radiationresponse in the EMT6 tumor cell system. Radiat. Res., 53: 281—294,1973.

23. Shingleton, W. W., Bryan, F., O'Quinn, W., and Krueger, L. C. Selectiveheating and cooling of tissue in cancer chemotherapy. Ann. Surg., 156:408-416,1962.

24. Suzuki, K. Application of heat to cancer chemotherapy. Nagoya J. Med.Sci., 30: 1—21,1967.

25. Twentyman, P. R., Morgan, J. E., and Donaldson, J. Enhancement byhyperthermia of the effect of BCNU against the EMT6 mouse tumor. CancerTreat. Rep., 62: 439—443,1978.

26. Yerushalmi, A. Combined treatment of a solid tumour by local hyperthermiaand actinomycin D. Br. J. Cancer, 37: 827—832,1978.

2276 CANCERRESEARCHVOL. 39

References

1. Ballard, B. E. Pharmacokinetics and temperature. J. Pharm. Sci., 63: 1345—1358, 1974.

2. Bleehan, N. M., Honess, D. J., and Morgan, J. E. Interaction of hyperthermiaand the hypoxic cell sensitizer Ro-07-0582 on the EMT6 mouse tumor. Br.J. Cancer, 35: 299—306,1977.

3. Braun, J.. and Hahn, G. M. Enhanced cell killing by bleomycin and 43°hyperthermia and the inhibition of recovery from potentially lethal damage.Cancer Res., 35: 2921—2927,1975.

4. Dickson, J. A., and Ellis, H. A. Stimulation of tumour cell dissemination byraised temperature in rats with transplanted Yoshida tumours. Nature, 248:354-358, 1974.

5. Dickson, J. A., and Suzanger, M. In vitro-in vivo studies on the susceptibilityof the solid Yoshida sarcoma to drugs and hyperthermia (42°).Cancer Res.,34: 1263—1274,1974.

6. Gerwick, L., and Rottinger, E. Enhancement of mammaliancell sensitivity tohyperthermia by pH alteration. Radiat. Res., 67: 508—511, 1976.

7. Hahn, G. M. Metabolic aspects of the role of hyperthermia in mammaliancell inactivation and their possible relevance to cancer treatment. CancerRes., 34: 3117-3123, 1974.

8. Hahn, G. M. Interactions of drugs and hyperthermia in vitro and in vivo. In:C. Streffer (ed), Proc. Int. Sym. on Hyperthermia in Cancer Treatment, pp.72—79.Baltimore-Munich: Urban and Schwarzenberg, 1978.

9. Hahn, G. M. Potential for therapy of drugs and hyperthermia. Cancer Res.,39: 2264-2260, 1979.

10. Hahn, G. M., Braun, J., and Har-Kedar, I. Thermochemotherapy: synergismbetween hyperthermia and Adriamycin or bleomycin in mammalian cellinactivation. Proc. NatI. Acad. Sci. U. S. A., 72: 937—940,1975.

11. Hahn, G. M., Li, G. C., and Shiu, E. Interaction of amphotericin B and 43°hyperthermia. Cancer Res.. 37: 761-764, 1977.

12. Hahn, G. M., and Strande, D. P. Cytotoxic effects of hyperthermia andAdriamycin on Chinese hamster cells. J. NatI. Cancer Inst., 57: 1063—1067,1976.

13. Har-Kedar, I., and Bleehan, N. Experimental and clinical aspects of hyperthermia applied to the treatment of cancer. In: J. T. Left and H. Adler (eds.),

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1979;39:2269-2276. Cancer Res   Jane B. Marmor  Interactions of Hyperthermia and Chemotherapy in Animals

  Updated version

  http://cancerres.aacrjournals.org/content/39/6_Part_2/2269

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/39/6_Part_2/2269To request permission to re-use all or part of this article, use this link

on May 17, 2020. © 1979 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from