Z. Krebsforsch. 87, 301--309 (1976) ~ r i f t f i i r Krebsforschung und K]inische Onkologie �9 by Springer-Verlag 1976

The Effect of Fractionated Exposure to Thio-TEPA upon Chinese Hamster Cells in vitro

K.-R.Trott and E. B61tikbasi* Institut fiir Biologie der GSF, D-8042 Neuherberg und Strahlenbiologisches Institut der Universit~it MiJnchen, Federal Republic of Germany

Summary. The dependence of the surviving fraction of Chinese hamster cells in vitro upon the concentration of Thio-TEPA, upon the duration of drug exposure and upon fractionation of drug exposure has been studied. Given a constant exposure time, the surviving fraction decreased according to a "shoulder" curve with increasing concentration. Given a constant concentra- tion, the surviving fraction decreased exponentially with increasing exposure time. Splitting exposure of 60 min into two 30 minutes' exposures with drug- free intervals of several hours increased the cytostatic effect of Thio-TEPA.

Der Effekt der fraktionierten Einwirkung von Thio-TEPA auf Zellen des chinesischen Hamsters in vitro

Zusammenfassung. Die Abh~ingigkeit der Oberlebensrate von Zellen des chi- nesischen Hamsters in vitro von der Thio-TEPA-Konzentration, der Exposi- tionsdauer und dem Fraktionierungsschema wurde untersucht. Bei konstan- ter Expositionsdauer nahm die (]berlebensrate mit zunehmender Konzentra- tion nach einer ,,Schulterkurve" ab, bei konstanter Konzentration nahm die (]berlebensrate exponentiell mit zunehmender Expositionsdauer ab. Die Fraktionierung einer 60miniJtigen Exposition in 2 30minfitige Expositionen mit Intervallen von einigen Stunden erh6hte den zytostatischen Effekt von Thio-TEPA.

Introduction

Chemotherapy with cytostatic agents is a fast-growing field in modern cancer therapy, fertilized by a close collaboration between clinical and laboratory re- search. For the majority of the cytostatic drugs currently used, experiments on the mode of action have been performed, survival curves for different cell lines in vitro

* On leave from the Cekmece Nuclear Research and Training Center, Istanbul

302 K.-R. Trott and Esin B61iJkbasi

surviving fraction

1.0"

0.5-

0.3-

0.1

0.03"

not washed washed

o.oi

0 :20 /.'0 6'0 8'0 160 .uglml concentration

Fig. 1. The surviving fraction of Chinese hamster cells after treatment to various concentrations of Thio-TEPA for 30 min. See text for experimental details

and in vivo have been established and the changes of the sensitivity of the cells dur ing their cell cycle have been studied (Mauro and Madoc-Jones , 1970; Bhuyan

et al., 1972). However, in clinical practice, the various modes of drug appl icat ion and

t rea tment schedules lead to complicated pat terns of exposure of the t u m o u r cells to rapidly changing concentra t ions of the drugs. The r61e of this t ime factor for the effectiveness of the chemotherapy has not been given the necessary at tent ion, yet (Mellett, 1975). Therefore, the dependence of the act ion of a widely used mul t i funct ional alkylat ing agent, Thio-TEPA, on rapidly proliferating mam- mal ian cells in vitro upon the concent ra t ion of the drug, upon the dura t ion of exposure, and upon fract ionat ion of exposure has been studied.

Material and Methods

B-14-FAF 28 Chinese hamster cells (Ford and Yerganian, 1958) with a modal chromosome number of 22 were used throughout these experiments. The stock cultures were propagated in pyrex bottles with Eagle's minimal essential medium, supplemented with 10% calf serum, 100 mg/1 neomycine and 350 mg/l NaHCO 3. The cultures were kept in an incubator at 37 ~ C in a humid atmosphere with 3% CO2. The cells were subcultured from the stock culture with the aid of trypsin (0.25% for 5 rain). For the determinations of the fraction of cells surviving Thio-TEPA treatment, single cell suspensions were appropriately diluted with full medium and seeded into 4 replicate bottles for each point. 1 week later, the bottles were removed from the incubator, the medium was discarded and the cells were stained with methylene blue. The number of colonies having more than 50 cells was counted under a dissecting microscope and the fraction of surviving colony forming cells was calculated.

Effect of Fractionated Exposure to Thio-TEPA 303

surviving I f rac t ion

01

0.01

0.001

0.0001 . . . . . . . . . . . . 0 30 60 90 120 rain

exposure t ime

Fig. 2. The surviving fraction of Chinese hamster cells after treatment with 60 lag/ml Thio-TEPA for various exposure times. Data of one typical experiment. 3 repeats gave comparable results

15 mg of Thio-TEPA (Lederle) was dissolved in Hank's solution to give a final concentration of 300/.tg/ml. The required concentrations were prepared from this stock solution with Hank's salt solution and kept at 4 ~ C for up to 1 week.

Cells were treated with Thio-TEPA 18--24 h after seeding. The medium was decanted and 10 ml pre-warmed Hank's solution containing the desired concentration of Thio-TEPA was added. During exposure which lasted from 15 min to 4 h cells were put back into the incubator. After exposure, the drug solution was carefully decanted and full medium added. Since the traces of the drug remaining after this procedure showed a pronounced cytotoxic effect (Fig. 1) this medium was removed again and fresh medium added for a second time and the cells put back into the incubator. The same procedure was repeated in split exposure experiments after the cells had been kept in the incubator during the drug-free interval.

Each of the various protocols was repeated several times with 4 replicate dishes. The figures show the means of the results of the individual experiments and their standard error of the mean.

Results

Given a cons tan t exposure t ime of 30 min, the cy tos ta t ic effect of T h i o - T E P A increased with concent ra t ion . F igure 1 shows the combined results of 6 experi- ments.

The exper imenta l p rocedu re had a big influence on the shape and s lope of the survival curve. Smal l a m o u n t s of the drug might r ema in in the cul ture vessel if the cultures are not washed carefully after drug exposure and lead to m a r k e d l y en- hanced cell killing. The do t t ed response curve therefore is not representa t ive of the cell ki l l ing effect of the ind ica ted concen t ra t ion for 30 min but ra ther of the effect of the traces left after changing the med ium, ac t ing dur ing the fol lowing incuba t ion time.

If T h i o - T E P A is careful ly r emoved by repea ted washing with full m e d i u m the s lope of the survival curve is less steep. The full l ine was d rawn th rough the exper imenta l da t a by eye and can be descr ibed by the fol lowing pa r a me te r s (Alper et al., 1962): the s lope of the survival curve in its exponen t ia l pa r t co r re sponds to a D O of 23 lag/ml, the ex t r apo l a t i on n u m b e r is a b o u t 1.4, the shoulder width is equivalent to a concen t ra t ion of a b o u t 10 ~tg/ml Th io -TEPA.

304 K.-R. Trott and Esin B61iikbasi

Table 1. The surviving fraction of Chinese hamster cells exposed to various concentrations of Thio-TEPA for various times

Concentration (pg/ml) 200 100 50 25 12.5 Exposure time (min) 15 30 60 120 240

Experiment I 0.009 0.064 0.025 0.033 0.069 II 0.0025 0.016 0.035 0.075 0.075 III no data 0.008 0.04 0.062 0.08

surviving fraction

0.1

0.05

0.02

0.01

~' x"~,x \ \

\ X

X X

X \

\ \ \ \

\

6 2 ~h intervo[

Fig. 3. The surviving fraction of Chinese hamster cells after treatment with 40 tag/ml Thio-TEPA for 60 min or twice 30 rain with various drug-free intervals. Data from 4 experiments

The cytostatic effect of Thio-TEPA increased with duration of exposure. The surviving fraction of cells exposed to a concentration of 60 ~tg/ml for various time decreased exponentially with increasing exposure time (Fig. 2).

The dependence of the cytostatic effect on both concentration and duration of exposure suggested an experimental protocol in which the product of concentra- tion and time was kept constant but the individual values were varied by a factor of 16. The results in table I show that the increase in concentration more than outweights the shortening of the exposure times.

Effect of Fractionated Exposure to Thio-TEPA 305

surviving fraction

1.0

0.5

0.1

0.05

0.01�9

\ \ "**,%,

"-, ~ ".~,,

\ 6h %

\

O. i0 ~'0

2b ~b 6~ eb

interval

60 80 pg/ml second exposure

' 1(3'0 ' pg/ml concentration

Fig. 4. The surviving fraction of Chinese hamster cells after a second treatment with various concen- trations of Thio-TEPA for 30 rain following a first exposure to 30 ~tg/ml for 30 min either 6 h or 24 h earlier. Dotted curve from Figure 1. Data from 3 experiments

60 min exposure to a concentration of 40 pg/ml has been divided into two 30 rain exposures to the same concentration but with drug-free intervals of up to 4 h. This experimental protocol corresponds to the type of experiments com- monly performed in radiobiology to quantitate recovery phenomena. In contrast, however, to the usual dose splitting effect in radiobiology the combined data of 4 experiments as well as each individual experiment show that fewer cells survive the split exposure protocol than the same exposure given without interval. With 4 h interval between two 30 minutes' exposures to 40 ~tg/ml the surviving

fraction was smaller by a factor of 4 compared to a single exposure which would be equivalent to an exposure time of 90 rain to the same concentration without interval.

In a separate set of experiments, we studied whether the small "shoulder" apparent in the survival curves of Figure 1 was still present in cells which had survived a first exposure to Thio-TEPA. Cells surviving a 30 minutes' exposure to 30 pg/ml exhibit a "shoulder" 6 h later which is not significantly different from the "shoulder" of the original population (Fig. 4). The slope of the survival curve of the pre-treated population is the same as that of the original population. 24 h after pre-treatment the effect of a second exposure is generally less than the same exposure 6 h after pre-treatment (Fig.4).

The decrease of the sensitivity of the pre-treated cells to a second exposure of Thio-TEPA 24 h later, was even more pronounced in cells which were re-seeded 24 h after the first exposure and given a second exposure 48 h after the first one

306 K.-R.Trott and Esin B/Slfikbasi

surviving fraction

1.0

0.5"

0.1

0.05

0.01

\ \~ . \ \

%'\ %, \

\ \ \ . %\\

\

2~ ~'o 6'0 io 1~o ~/m, concentration

Fig. 5. The surviving fraction of Chinese hamster cells after treatment with various concentrations of Thio-TEPA for 30 min following a first exposure to 30 pg/ml for 30 rain 48 h earlier and reseeding of the cells 24 h earlier. Dotted line from Figure 1. Data from 3 experiments

(Fig. 5). The final slope of the survival curve was about the same as that of the original population but the "shoulder" size increased by a factor of 2.

Discussion

Thio-TEPA is a multifunctional alkylating agent which is mainly used in the treatment of cancerous effusions where it gives good clinical palliation (Schwandt and Ehrhard, 1966). It has also been given intravenously in the treatment of various malignant diseases (Shay et al., 1953). With both regimes, the malignant cells are exposed to high concentrations of the drug for only a short time since Thio-TEPA is rapidly metabolized and excreted with a serum half life time of about 1 h (Mellett, 1975). With each new dose, the concentration rises again to fall off to low levels within a few hours. Similar changes in drug concentrations apply to all cytostatic agents given either orally or parenterally. These undulating changes in drug concentration with short periods of high concentrations and long periods of low concentrations might have great influence on the effectiveness of a chemotherapeutic regime. This problem has been given little attention in the pharmacological literature on cytostatic agents, however (e.g. Skipper, 1965; Mel- lett, 1975).

In some ways, the repeated peaks of high drug concentrations are similar to the pattern of radiation doses given in clinical radiotherapy. Since alkylating

Effect of Fractionated Exposure to Thio-TEPA 307

agents are commonly called "radiomimetic" agents this analogy has been ex- plored further.

The shape of the survival curves after exposure to Thio-TEPA (Fig. 1) and to radiation (Trott, 1972) are very similar. The curve describing the decrease of the surviving fraction with increasing concentration at constant exposure times even shows a "shoulder". Such curves are commonly associated with split dose recov- ery phenomena in radiobiology (Elkind and Sutton, 1960). If a dose of radiation is split into two half doses with an interval of a few hours or days the surviving fraction increases by a factor of 2--10. This effect has been observed in nearly all types of mammalian cells with the type of radiations presently used in radiother- apy and is of great importance for the radiotherapeutic practice (Elkind, 1974).

However, if the same concentration of Thio-TEPA is given to the cells for the same exposure time but with an interval of a few hours no recovery takes place but an increase in the effect which becomes larger with increasing interval. The same effect has been observed in Hela cells treated with a split exposure to Thio- TEPA (Philipp, 1975). In other mammalian cells exposed in vitro to two fractions of various alkylating agents no increase in the surviving fraction of cells after split exposure has been observed (Walker and Thatcher, 1968; Fox and Nias, 1968; Sakamoto and Elkind, 1969; Fox et al., 1970; Barranco et al., 1975).

Although the survival curves after cytostatic drugs and radiations are similar these obvious similarities in shape may reflect different mechanisms of action. The "shoulder" curve after exposure to different concentrations of Thio-TEPA at constant exposure time should not be confused with the "shoulder" curve after different radiation doses. The correct analogy to the dose response curve after radiation doses given at constant dose rate is the curve describing the dependence of the surviving fraction after different exposure times at a constant concentration of the drug (Fig. 2). Since this is an exponential curve no recovery in split dose exposure was to be expected similar to the observations after split dose exposure to high LET radiations (Barendsen, 1968). The "shoulder" curve rather corres- ponds to conditions of irradiation with different dose rates but constant exposure time. Although the dose response curves for the various dose rates may be exponential, the dependence of the surviving fraction after different doses given at constant time with varying dose rates has been shown to follow a "shoulder" curve (Kellerer and Rossi, 1972).

The increased effectiveness of a fractionated regime compared to a single exposure was somewhat unexpected. If this effect occurs in vivo as well as in vitro it may play an important part in clinical chemotherapy. The most likely explana- tion for the effect is progression of surviving cells from resistant cell cycle phases to more sensitive phases. In DON Chinese hamster cells the surviving fraction after exposure to Thio-TEPA depends on the cell cycle phase of the cells the most resistant cells being those in early S-phase (Bhuyan et al., 1972). The sensitivity increases gradually during S-phase to reach a peak value around mitosis. Experi- ments are in progress to check whether the increased effectiveness of fractionated exposure to Thio-TEPA can be explained by cell cycle progression alone.

The cytostatic effect depends on the concentration of the drug as well as on the duration of drug exposure. In their extensive treatise on dose-effect-relationships in pharmacology, Druckrey and Kiipfmtiller (1949) deduced that the effect of all

308 K.-R.Trott and Esin BNiikbasi

cytotoxic agents was proportional to the product of concentration and exposure time, suggesting a general equation relating concentration and time for equal effect as

( C - C~)". ( t - t~) = constant

in which C ist the effective (intracellular) concentration, C s a threshold concentra- tion for infinite exposure time, t the actual exposure time and ts the minimal exposure time for infinitely high concentrations of the drug. The exponent n may be smaller or greater than 1. Druckrey and Ktipfmfiller suggested that this equation was valid for all drugs of which either the binding to the receptor or the effect was irreversible. Experimental evidence with carcinogens lead the authors to propose for practical application the simplified version of the equation as c. t= constant. This simplified equation has been adopted by Mellett (1975) for his discussion of dose-effect-relationships of cytostatic drugs. However, to our knowledge, no experimental data on any cytostatic drug has actually been published on the validity of this equation for mammalian cells. Skipper (1965) analyzed the relationship for the action of NSC 409962 on E. coli and found the equation

C 2/3 �9 t = c o n s t a n t .

Our data of table I are consistent with an equation like

C 1'2 �9 t= constant.

The surviving fraction of Chinese hamster cells decreases therefore after exposure to Thio-TEPA according to the following equation:

S~e-5, to 4.t-r

if S is the surviving fraction of the cells and t is given in minutes and c is given in tag/ml.

We agree with the conclusions of Druckrey and KiJpfmiJller that although the relevance of these concentration-time-effect-relationships might be great, the ac- tual equation, i.e. the exponent n derived from the experimental data does not give any indication of the nature of the underlying biochemical processes. Only the practical information can be drawn from the data that concentration has more influence on the cytostatic effect of Thio-TEPA on mammalian cells in vitro than exposure time.

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Effect of Fractionated Exposure to Thio-TEPA 309

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Received June 14, 1976; accepted July 7, 1976