EvrJ Catum, Vol. 28, No. I, pi. b&66,1992. Printed in Cmt hi&

The Effect of Timing of Vincristine Administration During a Course of Fractionated Radiotherapy in a Mouse Carcinoma Giuseppe D. Zanelli, Luigina Rota, Roberto Innocente and Mauro Trovo It has been previously shown that the uptake of 3H-vincristine by the NT carcinoma tumour in CBA mice can vary by a factor of 3 during a course of fractionated radiotherapy (2 Gy day-‘, 5 day week-l, for 5 weeks). Here the effect of therapeutic doses of vincristine given at times of either maximum or minimum uptake has been investigated. The results show that whereas vincristine alone has a dose-related effect on this tumour, when given in combination with fractionated radiation it is only effective if administered during the first few fractions. It does not seem to matter whether it is given at times of maximum or minimum uptake. It is concluded that vincristine either exacerbates the radiation-induced vascular damage so that drug extravasation is reduced or that it causes a shift in the time of appearance of the peaks and troughs of uptake. The possibility that radiationinduced resistance to vincristine may have played a part in the results is also discussed. EurJ Cancer, Vol. 28, No. 1, pp. 64-66,1992. INTRODUCTION IN COMBINED

modality treatment of cancer (CMT) involving radiotherapy and chemotherapy [ 11, the importance of timing of drug administration has been amply demonstrated for both experimental animal tumours and normal tissues [2-41. In general, chemotherapeutic agents have been shown to be more effective when given either shortly before or shortly after the radiation. In all cases the radiation was delivered either as single doses or in a few large fractions. It is well known that radiation alters tissue blood flow, the structure of the capillary endothelium and the rate of extravasation of macromolecules and that these effects are dose dependent [5-71. Clinically small daily fractions are given over a period of several weeks. The vascular changes produced by a clinically relevant fractionation schedule in a mouse carcinoma have been recently investigated by our group using the tumour uptake of tracer amounts of 3H-vincristine as an indicator [8]. We showed that the tumour uptake of tracer quantities of 3Hvincristine varied by a factor of >3 during&e course of radiotherapy, we argued that this could have been due to damage to the turnour vasculature followed by recovery. On this premise it should be possible to show that therapeutic doses of vincristine (a compound which by itself is only moderately effective against the tumour used) given at times of maximum tumour uptake should be more effective in controlling tumour growth than when given at times when the tumour uptake is lowest. The experiment reported herein was designed to determine whether the vascular effects of a realistic course of fractionated radiotherapy could be exploited to improve the results of concurrent radiotherapy and chemotherapy.

Correspondence to G.D. Zanelli. G.D. Zanelli is at the Department of Radiochemistry, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, U.K.; L. Rota is at the Raphael Institute, Calcinato, Italy; and R. Innocente and M. Trovo are at the Centro di Riferimento Oncologico, Aviano, PN, Italy. Revised 8 Aug. 1991; accepted 30 Sept. 1991.

MATERIALSAND METHODS The mice, tumour line and fractionation schedule were essentially as described by us earlier [S]. Briefly, the mice were 10 weeks old, male CBA/He and the tumour was the carcinoma NT fully described by Hewitt et al. [9] implanted intradermally in the flank. The radiation source was a cobalt-60 unit and the dose-rate 9.95 cGy/min. Although this is low by comparison with the doserates used clinically, it was the same dose-rate used in the previous experiments [8] and it was felt imprudent to alter it. In the main experiment seven groups of eight mice had their tumour irradiated daily with 2 Gy/day, 5 days per week for 4 weeks. For irradiation the mice were sedated with mild doses of Hypnorm intraperitoneally (Fentanylcitrate/Fluanisone, Jensen Pharmaceuticals Ltd, Oxford, UK). At the start of the experiment the tumours were 4.6 + 0.28 mm average diameter [D = (a.b.c)“, where a,b,c = perpendicular diameters] and were measured daily for the duration of the experiment. When the average tumour diameter in a group reached a maximum of 11.5-12.5 mm the mice were killed. In the longer lived groups E,F,G and H the tumours were allowed to exceed a diameter of 9 mm before the mice were killed. The time required for the tumour diameter in all the various groups to reach 8.0 mm was then calculated and the differences analysed for statistical significance using a standard log-rank test. The full treatment schedule is shown in Fig. 1. Groups A,B,C and D served as untreated, vincristine only and irradiation only controls. Groups E and G received vincristine at times when the tumour uptake should have been low and groups F and H when the tumour uptake should have been high according to our previously published results [8]. The dose of vincristine (vincristine sulphate, Eli Lilly, Basingstoke, UK) was calculated by scaling down the dose usually given to patients and assuming a surface area of 1.7 m2 for a standard 70 kg man. All injections were given intraperitoneally in a volume of 0.1 ml 4 h after irradiation [8].

RESULTS The results are shown in Fig. 2 and Table 1. In Fig. 2 the error bars have been omitted for the sake of clarity, but the

Combined Modality Treatment:

Table 1. Average number of aizysajier im#antation taken by tumows to attain 8 mm diameter

Group Control (no treatment) A B

mm

C

Da

D

I

E

53

F

1

G H

Group A B

I

C

D E F

I

G

H

55

1112131415 1617181920

FIT fraction no. Fig. 1. The complete treatment schedule for all groups. n = VCR, q =RT.

levels of sign&axe in the regrowth delay between the groups are shown at the bottom of Table 1. Two injections of vincristine

only (group B), on consecutive days, have very little effect in slowing the growth of this tumour, but four injections (group C) spaced as shown in Fig. 1, have a much more pronounced effect and slow down the growth of this tumour nearly to the same extent as 20 x 2 Gy of irradiation alone (group D). The unexpected Gnding is that there is no significant difference in growth delay between groups E,F,G and H. Groups E and G received vincristine twice (group E) or four times (group G) when the tumour uptake should have been low while groups F and H received vincristine 2 (group F) or 4 times (group H) when the tumour uptake should have been optimal. Furthermore, in the combined modality groups E,F,G and H there is no significant difference beween the groups receiving chemotherapy twice (E and F) and groups G and H which were treated 4 times. It would appear that when used by itself the effect of vincristine on this tumour is dose related: the greater the total dose the greater the effect (groups B and C). When administered

Radiotherapy 14rH

Delay relative m group A (days)

19 20 26 31 39 41 44 41

1 7 12 20 22 25 22

D-vs.-E, F, G, H: I’ = 0.013. 6 7 8 910

12345

Days

Log rank test: A-vs-D, E, F, G, H: P = 0.008;

1

I

65

Effect of Timing

c=_l

H

H

concurrently with radiotherapy however, only the vincristine given during the first few fractions (< 10) of radiation is effective while vincristine given during the latter part of the radiation treatment has no effect at all. DISCUSSION In a previous publication [8] we have shown that the pattern of uptake of tracer quantities of vincristine during a fractionated course of radiotherapy is a true and repeatable phenomenon. In 2 separate experiments, using the same dose-rate, the peaks and troughs of uptake occurred on the same days and the tumour content of 3H-vincristine at the peaks was up to 3 times greater than that at the troughs. It was argued therefore that by proper timing of the administration of therapeutic doses of vincristine it should be possible to maximise its effects. The present experiment has shown that this is not the case. In the NT carcinoma vincristine given in conjunction with radiotherapy is very effective but only if given at any time during the first 10 radiation fractions-later treatment appears to be totally ineffective. Two possible factors could be considered as responsible for the results obtained. The first is an interaction between the radiation and the chemotherapy leading to greatly reduced drug entravasation or to a shift in the maxima and minima of drug uptake. Either of these effects would counteract the effects seen when RT and tracer quantities of vincristine were used [8]. The second possibility is the appearance of the phenomenon of radiation-induced multidrug drug resistance (R-MDR) as described by Hill et al. [lo]. Vincristine is a particularly effective drug in this connection and the phenomenon is known to be dependent on radiation dose and fractionation schedule. The present results do not indicate which, if any, of the above two mechanisms may be operating. One conclusion is, however, clear: in the NT carcinoma (at least) vincristine given during a course of fractioned radiotherapy is only effective if administered during the first few fractions of radiation.

1. Steel GG. The search for therapeutic gain in the combination of

2’ 12

I

I

I

I

I

I

1

I

l

16

20

24

28

32

36

40

44

48

Days after tumour transplant Fii. 2. R&ct of the various treatment schedules shown ia Fig. 1 on the growth of the NT carcinoma traasplaated iatradermally.

2.

radiotherapy and chemotherapy. Radio&r Oncol1988,11,31-53. Von der Maasc H. Expcrimcntal studies on interactions of radiation

and cancer chemotherapeutic drugs in normal tissues and a solid tumour. RadbtherOncol1986,?, 47-68. 3. Stephens TC, Adams K, Peacock JH, Steel GG. Temporal interactions in the Lewis lung tumour beetween cytotoxic drugs and acute or fractionated radiotherapy. Rad&&rOncoll986,5,137-146.

G.D. Zanelli et al.

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4. Begg AC, Fu KK, Shrieve DC, Phillips SL. Combination therapy of a solid murine tumour with cyclophosphamide and radiation. Int J Radiat Oncol Biol Phys 1979,5,1433-1439. 5. Zanelli GD, Lucas PB. Effect of X-rays on vascular function in transplanted tumours and normal tissues in the mouse. BrJ Cancer 1976,34,408-416. Eddy HJ. Tumor vascular response following irradiation. Microvasc Res 1980,20,198-211. Krishnan L, Krishnan EC, Jewel WR. Immediate effect of irradiation on the microvasculature. IntJ Radiat Oncol Biol Phys 1989,15,147-150. Zanelli GD, Rota L, Trovo M, Grigoletto E, Roncadin M. The uptake of 3H vincristine by a mouse carcinoma during a course of fractionated radiotherapy. BrJ Cancer 1989,60,310-314.

9. Hewitt HB, Blake ER, Walder AS. A critique of the evidence for the active host defence against cancer, based on personal studies of 27 murine tumours of spontaneous origin. BrJ Cancer 1976,23, 241-263. 10. Hill BT, Whelan RDA, Hosking LK, Shelland SA, Bedford P, Lock RB. Interaction between anti-tumour drugs and radiation in mammalian tumour cell lines; differential drug responses and mechanisms of resistance following fractionated X-irradiation or continuous drug exposure in virro.NCI Monogr 1988,6,177-181. Acknowledgements-One of the authors (R.I.) would like to acknowledge the financial support received from the Italian Cancer Research Society “La Via di Natale”.

EwJ Cancer, Vol. 28, No. I, pi. 6&71,1992. Printed in Great Btirain

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+ a.00

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Potentiation of Etoposide Cytotoxicity against a Human Ovarian Cancer Cell Line by Pretreatment with Non-toxic Concentrations of Methotrexate or Aphidicolin Eugenio Erba, Soumitra Sen, Aurelio Lorico and Maurizio D’hcalci Exposure of human ovarian cancer SW626 cell line to 0.08 pmol/l methotrexate or 25 pmol/l aphidicolin for 24 h caused no cytotoxiciy but enhanced etoposide cytotoxicity. Methotrexate or aphidicolin treatment induced a reversible blockade at the beginning of S phase which was reversed upon drug removal with a consequent wave of synchronisation. The enhancement of etoposide cytotoxicity was not due to higher etoposide intracellular uptake in the methotrexate or aphidicolin-pretreated cells. The topoisomerase II content in methotrexate or aphidicolin pretreated SW626 cells was higher than in control cells assessed by western blotting or flow cytometry. The higher etoposide cytotoxicity observed after synchronization with methotrexate or aphidicolin was apparently unrelated to the number of drug-induced DNA-topoisomerase II complexes evaluated as DNA double strand breaks or DNA-protein crosslinks. These data support the view that etoposide-induced DNA-topoisomerase II complexes are more cytotoxic in cells which are in S-phase. EurJ Cancer, Vol. 28, No. 1, pp. 6671,1992. INTRODUCTION

IT HASbeen recently reported by this laboratory that pretreatment of the human histiocytic U937 cell line with non-toxic concentration of methotrexate that did not cause any detectable cytotoxicity, significantly enhanced the cytotoxic potential of etoposide. It was proposed that the methotrexate pretreatment was inducing synchronisation of U937 cells and that the higher sensitivity to etoposide was related to a increased proportion of S-phase cells [ 11. The studies with U937 cells also indicated that methotrexate pretreatment was able to enhance the ability of etoposide to induce DNA breaks, presumably due to a relative increase in intracellular DNA-topoisomerase II enzyme levels.

Correspondence to E. Erba. E. Erba, S. Sen and M. D’Incalci are at the Istituto di Ricerche Farmacologiche “Mario Negri” Via Eritrea 62, I-20157, Milano; and A. Lorico is at Oncologia Sperimentale, CR0 Aviano (PN), Italy. Revised 23 Sep. 1991; accepted 3 Oct. 1991.

However, the possibility that the enhanced cytotoxicity of etoposide was due to some unknown mechanism related to complex perturbations of biochemical process(es) induced by the antifolate could not be excluded. In order to evaluate whether the phenomenon seen could be generalised for the different inhibitors of DNA synthesis and also for cancer cells with different biological properties and sensitivities to etoposide, the present study was carried out. The aim was to ascertain whether etoposide cytotoxicity was enhanced by pretreatment with nontoxic concentrations of methotrexate or by aphidicolin, a drug causing specific and reversible inhibition of DNA polymerase a and 6 [2]. The study involved utilisation of an epithelial cancer cell line SW626, derived from a human ovarian carcinoma, to investigate the mechanism of potentiation of cytotoxicity. MATERIALS

AND METHODS

Chemicals

Methotrexate was obtained from the National Cancer Institute, Bethesda, Maryland. Aphidicolin-17.glycinate HCl was a