Improved Targeting of Brain Tumors Using Dexrazoxane Rescue of Topoisomerase II Combined with Supralethal Doses of Etoposide and Teniposide’

Vol. 4, 1367-1373, June 1998 Clinical Cancer Research 1367

Advances in Brief

Bente Hoim, Maxwell Sehested, and spread and infiltration of liver, spleen, and lymph nodes, Peter BuM Jensen2 whereas EHR2 cells acted as a solid tumor with no evidence of extracerebral disease. In all experiments, the combination Laboratory of Experimental Medical Oncology [B. H., P. B. J.], The Finsen Center, and Department of Pathology [B. H., M. S.], The of high-dose etoposide and ICRF-187 was significantly su- Laboratory Center, The National University Hospital, DK-2 100 perior to an equitoxic dose of etoposide alone. Such superi- Copenhagen 0, Denmark ority was also seen when treatment was given on days 4, 8, and 12 after tumor inoculation. Here etoposide alone re- suited in a mean increased life span of 12.3%, whereas the Abstract rescue regimen yielded an increase of 47% (P < 0.0001). In Dexrazoxane (ICRF-187) is a catalytic inhibitor of the conclusion, DNA topo II rescue by catalytic inhibitors is a nuclear enzyme DNA topoisomerase II (topo II). It protects new strategy enabling significant epipodophyllotoxin dose cells against topo II poisons, such as etoposide and tenipo- escalations; in this study, we have demonstrated the superi- side, by hindering the DNA cleavage reaction of the target ority of this strategy in two in vivo CNS tumor models. This enzyme. We have previously shown that this antagonism concept is now being tested in a clinical trial. also extends to an in vivo model. Thus, ICRF-187 protected mice against supralethal doses of etoposide and amsacrine, Introduction and the etoposide LD10 dose increased as much as 3.6-fold The essential nuclear enzyme DNA topo II is the target of when combined with ICRF-187 (B. Holm, Cancer Che- important antitumor agents such as etoposide (VP-l6) and doxo- mother. Pharmacol., 38: 203-209, 1996). We describe here rubicin (Adriamycin), which are used in leukemia, malignant how scheduling of this drug combination can be optimized lymphoma, breast and testicular cancer, and SCLC (1). Topo II and used. Interestingly, ICRF-187 can protect when it is mediates a cleavage/rejoining reaction of double-stranded DNA, given after etoposide. Although timing is very critical here, allowing the separation of intertwined DNA strands (2). During ICRF-187 was able to completely protect when given 10 mm the catalytic cycle of topo II, the enzyme binds covalently to after etoposide. This rescue principle resembles methotrex- DNA, creating a transient double-strand DNA break. Through ate rescue by folinic acid. We also found scheduling to be this break, the enzyme allows passage of another DNA double- crucial because ICRF-187 did not protect when etoposide strand helix. Finally, DNA is rejoined, and topo II dissociates was given once a day for five days, whereas effective pro- from DNA. tection was seen when etoposide was used three times, once topo Il-targeting anticancer agents can conveniently be every four days. Similar investigations were performed with classified into poisons [including etoposide and doxorubicin teniposide in combination with ICRF-187. The combination (I)], and catalytic inhibitors [including aclarubicin (3, 4), with ICRF-187 allowed a 3.4-fold teniposide dose escalation. chloroquine (5), and ICRF-187 (6-8)]. Both drug classes Such dose escalations could be advantageous in specific inhibit the enzyme, but only topo II poisons result in the situations. One such case is when the tumor is situated in a accumulation of topo II linked DNA breaks. This accumula- pharmacological sanctuary, e.g., in the brain. ICRF-187 is tion of DNA breaks is due to an inhibition of the rejoining hydrophilic and does not cross the blood-brain barrier, step of the enzyme. The breaks are called cleavable com- whereas the lipophilic etoposide and teniposide do. There- plexes, because their detection relies on SDS fixation and fore, ICRF-187 would protect normal tissues and allow a proteinase treatment to remove the covalently linked topo II cytotoxic dose of etoposide to reach the central nervous from the DNA break. Thus, a topo II poison acts at a stage of system (CNS). We therefore studied the combinations using the catalytic cycle of the enzyme when the gate DNA strand L1210 or EHR2 cells inoculated into the CNS of mice. L1210 is open or cleaved ( 1), whereas a catalytic inhibitor interacts presented a leukemic CNS model with leptomeningeal with the enzyme at a stage when there are no DNA breaks, i.e., the gate DNA strand is intact (9-13). This distinction may have some practical implications. Thus, a topo II poison converts the essential enzyme to a poisonous one, whereas Received 1 1/14/97; revised 3/26/98; accepted 3/27/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 The abbreviations used are: topo H, topoisomerase II; SCLC, small cell I This investigation was supported by the Danish Cancer Society. lung cancer; MTX, methotrexate; EHR2, Ehrlich ascites tumor cells; 2 To whom requests for reprints should be addressed, at The Finsen ICRF-l87, dexrazoxane I(+)-l,2-bis(3,5-dioxopiperazinyl-l-yl)pro- Center, The National University Hospital, 9 Blegdamsvej, DK-2l00 panel; BBB, blood-brain barrier; CNS, central nervous system; ABMT, Copenhagen, Denmark. Phone: 45-3545-4947; Fax: 45-3545-6966. autologous bone marrow transplantation.

Table 1 Dose escalation of teniposide in combination with ICRF-187 % The drugs were given once i.p. ICRF-187 was given 20 mm before teniposide, and the dose was fixed to 125 mg/kg. Ihe dose of S teniposide (VM-26) varied. As seen, ICRF-187 provided significant U protection of teniposide lethality. Calculation of LD10 values using the maximum-likelihood estimation (40) shows a 3.4-fold increase in V the LD10 of teniposide when combined with ICRF-187. The four V experiments listed are included in the computations. LD30 with 95% confidence interval when teniposide is given alone was 15.3 mg/kg n (9-25 mg/kg), and when given in combination with ICRF-187, it was 9 52.3 mg/kg (47-58 mg/kg).

ICRF-187 Experiment VM-26 Survivors/ + VM-26 Survivors! -500 -400 -300 -200 -W 0 130 no. (mg/kg) treated (mg/kg) treated C e time (rrin) I 10 5/5 20 4/5 Fig. 1 liming of ICRF-187 treatment in relation to etoposide admin- 30 4/5 istration. Shown are compiled data from four experiments, as also 40 2/5 shown in Table 2. Mice were treated at the indicated times with 50 0/5 ICRF-187. Etoposide was administered at time 0, and survival was registered. ICRF-187 conferred full protection when given 180 mm 2 20 3/5 before etoposide and up to 10 mm after etoposide. ICRF-l87 adminis- 40 0/5 125 + 40 5/5 tered 500 mm before etoposide showed diminished but not abolished 50 1/5 125 + 50 5/5 protection. 60 0/5 125+60 2/5 70 0/5 125 + 70 1/5

3 50 1/5 60 1/5 70 0/5 Materials and Methods 125 + 80 0/5 Drugs. ICRF-187 (Cardioxane) was purchased from Chi- 125 + 90 0/5 ron B.V. (Amsterdam, the Netherlands) and dissolved in 0.9% 4 50 2/5 125 + 50 5/5 saline to a concentration of 25 mg/ml. Etoposide (20 mg/ml) and 55 1/5 125 + 55 3/5 teniposide (10 mg/ml) were bought from Bristol-Myers Squibb 60 1/5 125 + 60 4/5 (Copenhagen, Denmark) in solution for clinical use and further diluted with saline. Mice. Female B6D2F1-hybrids were purchased from the catalytic inhibitor appears to exhibit toxicity by blocking BomholtgArd Breeding and Research Center Ltd. (Ry, Den- enzyme activity. A logical consequence of this distinction is mark). The animals were given free access to food and water. that a catalytic inhibitor should be able to inhibit a topo II They were acclimatized to the local animal facility for at least S poison by interfering with the catalytic cycle in such a way as days before entering the investigation and randomized to cages to reduce the amount of cleavable complex formation, in before experiments. Only mice weighing between 19 and 22 g at other words, decrease the available target of the poison. This the start of the experiment were included. has indeed been shown to be the case for aclarubicin (3, 4), In Vivo Experiments. Doses of etoposide and teniposide merbarone (14), fostriecin (15), novobiocin (16), suramin were adjusted according to weight, and the dose of ICRF-187 (17), chloroquine (5), distamycin (18), and the bisdiox- was given in a fixed volume of 0.2 ml. Etoposide, teniposide, opiperazines (6-8). The question naturally arises of whether and ICRF- 1 87 were all administered i.p. because we have pre- the knowledge gained on the different drug interactions of viously shown that separation of administration sites has no topo II catalytic inhibitors and poisons can be put to clinical influence on the drug action (20). Lethality was recorded, and use. surviving mice were sacrificed after 60 days. The BBB is considered a pharmacological sanctuary for In conventional therapy studies, mice were inoculated i.p. many drugs, and the hypothesis for the present study is that with 15 x 106 EHR2 cells on day 0 and treated with drugs i.p. etoposide/teniposide as lipophilic drugs cross the BBB, according to the schedule listed in “Results.” In the brain tumor whereas ICRF-187, being a more hydrophilic drug, does not model, cells were inoculated day 0 into the right temporal region in (19). Therefore, ICRF-l 87 would protect normal tissues and a volume of 30 pJ isotonic NaCI with 10% penicillin. We used allow a cytotoxic dose of etoposide to reach the CNS. Pre- 15 x l0 EFIR2 cells or 1 X l0 Ll210 leukemia cells because viously, we have shown that the LD10 of etoposide in mice these inoculates gave reproducible and comparable median survival increased 3.6-fold when used together with nontoxic ICRF- times. All mice were treated i.p. according to the schedules listed in 187 doses (20). Most promisingly, we found that this com- “Results.” Lethality was recorded, and surviving mice were sacri- bination was superior to an equitoxic dose of etoposide alone ficed after 60 days. The results were statistically analyzed by the in mice inoculated with L1210 cells into the CNS. We here Mann-Whitney test or by SAS software using the nonparametric describe our results with teniposide and our efforts to opti- one-way procedure (NPAR1 WAY). mize the timing and scheduling of etoposide administration. To evaluate the tumor pathology of EHR2 cells and L1210 Finally, we describe the effect of the combinations in vivo in cells inoculated in the CNS, we euthanized the mice on day 9 mice with a solid tumor CNS model. after inoculation and removed the brain, spleen, lymph nodes,

Table 2 Timing of drug administration Table 3 Scheduling of the drug combination ICRF-187 + etoposide liming of ICRF-187 treatment is shown in relation to etoposide ICRF-187 was given 20 mm before etoposide (VP-16). The ICRF- (VP-16) administration. Mice were treated at the stated times with 187 dose was fixed, and etoposide doses varied. ICRF-187 protected ICRF-187 and etoposide using the LD10 level obtained when adminis- against etoposide-induced lethality when drugs were administered tering ICRF-187 20 mm before etoposide, i.e., 125 mg/kg ICRF-l87 + q4dX3. ICRF-l87 did not protect in a schedule administered qldX5. 120 mg/kg VP-l6. Survival was registered, and as seen, ICRF-187 ICRF-l87 conferred full protection when given 180 mm before etoposide and up to Experiment VP-16 Survivors! + VP-16 Survivors! 10 mm after etoposide. ICRF-187 administered between 180 and 480 no. (mg/kg) treated (mg/kg) treated mm before etoposide attenuated protection. Results are data compiled from four experiments. Five or six mice were treated at each time point Administration i.p. on days 1-5 in each experiment. 1 0 100+0 Il/Il 20 8/11 100+20 9/11 ICRF-l87 + VP-l6 (mm) Survivors/treated 25 5/11 100+25 5/11 ICRF- 187 administered before VP-l6 30 2/11 100+30 3/11 480 4/5 Administration i.p. on days 0, 4, and 8 450 3/5 1 100+0 11/11 420 2/5 50 9/11 100 + 50 11/11 390 4/5 70 2/11 100+70 11/11 360 6/10 90 0/11 100 + 90 11/11 330 4/10 2 100+0 5/5 300 6/10 70 2/5 270 8/10 90 0/5 100+90 5/5 240 4/10 120 0/5 100 + 120 4/5 210 7/10 100+150 2/5 180 5/5 150 5/5 120 5/5 90 5/5 60 5/5 toxin and studied the timing of drug administration. In Fig. I 20 5/5 and Table 2, it is shown that ICRF-187 can be given up to 10 10 5/5 mm after etoposide and still prevent etoposide-induced lethality 5 4/5 in healthy animals. This is in line with folinic acid rescue used ICRF- 187 administered after VP-16 5 3/5 in combination with high-dose MTX (22). However, the timing 10 8/10 is critical: when ICRF- 1 87 was given 20 mm after etoposide 20 1/10 there was only minimal protection. In contrast, when ICRF-l87 30 5/21 was administered first, we found that it could be given up to 180 45 0/5 60 3/16 mm before etoposide without diminished protection. If etopo- 90 0/16 side is given between 180 and 480 mm after ICRF-187, the protection is diminished but not abrogated. The dose of etoposide chosen for these experiments is 120 mg/kg because this is the LD10 level in combination with 125 mg/kg ofICRF-187. As gut, lungs, liver, kidneys, and pancreas. The organs were im- controls, the experiments contained one group of mice treated mediately fixed in 4% buffered formalin and embedded in with 120 mg/kg etoposide alone, all of whom died. paraffin, and sections were stained with H&E for light micros- Repeated Administrations. In the clinical setting, eto- copy. poside is often administered at daily doses for 3-S days per treatment cycle. We therefore investigated possible schedules Results for repeated administration of the drug combination in mice. In ICRF-187 Antagonizes Supralethal Teniposide Doses. Table 3, the results of a schedule in which mice are treated with In Table 1, the results from the toxicological studies with etoposide alone or etoposide in combination with ICRF-l87 teniposide are presented. As expected from previous studies once a day for five days (qldXS) are shown. Curiously, this did (21), we found that teniposide was more toxic than etoposide; not convey any protection of etoposide-induced lethality in thus, the LD10 level of teniposide alone is 15 mg/kg, as corn- healthy mice. Neither did treatment three times, once every pared to 33 mg/kg of etoposide. Previously, we showed that second day (q2dX3) or three times, once every third day ICRF-187 was able to protect against etoposide-induced lethal- (q3dX3) (data not shown). Next we separated the injections to ity in normal mice (20). Similarly, we here demonstrate that the once a week for 3 weeks (qlwX3) and three times, once every dose of teniposide can be escalated significantly when mice are 4 days (q4dX3). With both of these schedules, ICRF- 187 co- pretreated with non toxic doses of ICRF-187. Thus, it is possible treatment enabled important dose escalations of etoposide. In to increase the dose of teniposide from 15 to 52 mg/kg when the Table 3, the results from treatment q4dX3 demonstrate that all drug is combined with 125 mg/kg ICRF-187. This corresponds protected mice (16 of 16) survived 90 mg etoposidelkg times to a 3.4-fold dose escalation at the LD10 toxicity level. This is three, whereas none of the unprotected mice did (0 of 16). remarkably similar to the 3.6-fold etoposide dose increment that Two Types of CNS Tumors. These significant dose we previously obtained with a similar ICRF-187 dose. escalations could be advantageous in specific situations. One Timing of Drug Administration. We next returned to such case is when the tumor is situated in a pharmacological etoposide because this is the most widely used epipodophyllo- sanctuary, e.g., in the brain. ICRF-187 is hydrophilic and does

Table 4 CNS tumor treated on day 4 after inoculation Results are shown from three experiments in which mice inoculated intracerebrally with ERH2 cells were treated on day 4 with either saline, 33 mg/kg etoposide (VP-l6), or the equitoxic dose of 125 mg/kg ICRF-187 + 120 mg/kg etoposide. The doses used have previously been shown to represent the LD10 level (20). Only experiment 3 exhibited significant benefit of the high-dose treatment (P < 0.005). However, when the compiled data were analyzed after normalizing saline treated controls to 100%, there was a significant increase in life span (P < 0.04) in the high-dose etoposide

group. n = 9 in each group, i.e. , 27 mice were studied in Experiment 1 and so forth.

Experiment 1 Experiment 2 Experiment 3 ICRF-187 + VP-l6 (mg/kg) MSV’ (days) ILS (%) MST (days) ILS (%) MSI (days) ILS (%) 0+0 10 9 9 0+33 10 0 12 33 12 33 125 + 120 11 10 12 33 15 56

a MST, median survival time; ILS, increase in life span.

Table 5 CNS tumor treated on days 4, 8, and 12 after inoculation of tumor cells

Experiment 1 Experiment 2 Experiment 3 ICRF-187 + VP-16 (mg/kg) MST’ (days) ILS (%) MST (days) ILS (%) MST (days) ILS (%)

0+ob 11 13 9.5 0+33” 12 9 13 0 10 5 125 + 120” 19 73 17 31 14 47 l25+0’ 12 9 13 0 10 5 0+0c 11 11 12 0+30’ 11 0 12 9 14 17

100 + 9#{216}C 15 36 17 55 18 50 l00+0c 12 9 12 5 12 0

a MST, median survival time; ILS, increase in life span. b Mice were inoculated ic. with ERH2 cells and treated on days 4, 8, and 12 using the indicated doses. Evaluation of compiled data shows a significant increase in life span in the group treated with high-dose etoposide (VP- 16) in combination with ICRF-187 as compared to groups treated with etoposide alone (P < 0.0001). When each experiment is analyzed individually, experiment I shows a significant difference (P < 0.001), whereas

the other two experiments are just above the 5% significance limit. n = 9 in each group, i.e. , 36 mice were studied in experiment 1 and so forth.

C- Mice were inoculated ic. with ERH2 cells and treated on days 4, 8, and 12 using the indicated doses. Statistical evaluation of pooled data shows a highly significant increase in life span in the high-dose etoposide treated group (P < 0.0001). Analysis of each experiment also reveals significance

(P < 0.05). The table also shows that ICRF- 187 itself does not have any antitumor activity. n = 9 in each group, i.e. , 36 mice were studied in experiment 1 and so forth.

not cross the BBB, whereas the lipophilic etoposide and teni- tumor and no evidence of extracranial disease was found. poside do to some extent. We have previously shown that in Thus, EHR2 cells and L12l0 cells serve as appropriate mod- mice inoculated with L12l0 leukemia cells in the CNS, there is els of malignant epithelial and hematological CNS disease, a significant increase in life span when treated with ICRF-187 respectively. and high-dose etoposide as compared to mice treated with an Antitumor Effect. We compared etoposide to high-dose equitoxic dose of etoposide alone (20). To further study this etoposide + ICRF-l87 in three experiments shown in Table 4. brain tumor model, we included EHR2 cells in our investiga- Mice were inoculated ic. with EHR2 cells and treated i.p. once, tions. This carcinoma cell line has the capacity to form solid on day 4, with the calculated LD10 doses of etoposide and tumors and, being 3-fold less sensititive than Ll2lO cells, it high-dose etoposide + ICRF-187. EHR2 cells are 3-fold less exhibits etoposide and teniposide sensitivities in a range similar sensitive than L1210 cells in vitro, and correspondingly, the to several human SCLC cell lines (21). survival data from mice with this tumor were not as impressive. When 15 X l0 EHR2 cells were inoculated in the right Thus, etoposide alone resulted in an increased median survival temporal region, we obtained reproducible mean and median time in only two out of three experiments. However, in agree- survival times of 10.4 and 10 days, respectively (95% confi- ment with our previous data from the L1210 tumor (20), we dence intervals of 9.96-10.84 days). These results were ob- found a significant benefit from the high-dose treatment tamed by pooling control data from 15 separate experiments. compared to etoposide alone (P 0.04 on the pooled data in To evaluate the biological features of the inoculated Table 4). CNS tumors, we sacrificed untreated mice inoculated with We then studied the q4dX3 schedule, i.e., treatment on Ll210 or EHR2 cells on day 9. Histological examination of days 4, 8, and 12 after inoculation of tumor cells. In Table 5, L12l0 cells showed a typical leukemic phenotype with an results from three experiments in which we used the doses intracranial leptomeningeal spread, as well as extracranial are presented. In this setting there is a more significant increase disease with a diffuse infiltration of the spleen, lymph nodes, in life span in the group treated with high-dose etoposide + and liver. In contrast, EHR2 cells grew as a single solid CNS ICRF-l87 as compared to the group of mice treated with an

Table 6 i.p. tumor treated on days 4, 8, and I 2 after inoculation ERH2 cells were inoculated i.p. Mice were treated on days 4, 8, and 12 after inoculation. At the same dose level as used in the CNS tumor S model, the mice were cured. Therefore, the etoposide (VP- 16) dose was lowered by 50%; this produced fewer survivors. It can be seen that U survival at an etoposide dose of 15 mg/kg is comparable to survival in the group treated with 45 mg/kg etoposide in combination with 100 V mg/kg ICRF-187. ICRF-187 + VP-16 (mg/kg) Survivors/treated V 0+15 12/18 0 + 30 27/27 n 100 + 45 14/18 100 + 90 25/27 g

with ICRF-l87, respectively. The dosage of ICRF-187 was C unchanged. The compiled data from two experiments are shown in Table 6. The survival levels of mice in the two e groups treated with 50% of the initial etoposide dose were very similar. Thus, it appears that there is no advantage of the high-dose etoposide + ICRF-l87 treatment of an i.p. tumor when both drugs are given i.p. 0 5 10 15 Discussion Day Previously, we have shown that LD10 of etoposide increased 3.6-fold when used together with nontoxic ICRF-l87 Fig. 2 Survival curve of experiment 2 from Table S consisting of groups of nine mice inoculated in the cerebrum with 15 X l0 EHR2 doses (20). Most promisingly, we found that this combination cells. The tumor-bearing mice were treated i.p. on days 4, 8, and 12 after was superior to an equitoxic dose of etoposide alone in mice inoculation. The group treated with high-dose etoposide (VP-16) in inoculated with L1210 cells into the CNS. Similarly, teniposide combination with ICRF- 187 showed a significant increase in survival doses can be increased 3.4-fold without a change in lethality compared to etoposide alone in an equitoxic dose, to ICRF- 187 alone, (Table 1). Such dose escalations may have an important clinical and to saline-treated controls (P < 0.05). , 100 mg/kg ICRF-l87 + impact and are comparable to the escalation possible with 90 mg/kg VP-16; - - - -, 100 mg/kg ICRF-l87; , 30 mg/kg

VP-16; - - - - -, saline-treated controls. ABMT (23). Thus, in humans, the conventional dose of etoposide is 360 mg/m2, the maximum dose without ABMT is 1200 mg/m2, and the maximum dose with ABMT is 2400 mg/m2 (23), i.e., a 2-fold dose escalation. equitoxic etoposide dose without pharmacological rescue (P < Etoposide is known to be a cell-cycle phase specific drug, 0.0001). Because the LD10 was expected to give toxic deaths, because its toxicity starts to increase in late S phase and peaks we lowered the dose of etoposide to 30 and 90 mg/kg, when just before the cells enter mitosis (24). This increase in etopo- etoposide was used in combination with ICRF-187. In Table 5, side sensitivity is paralleled by an increase in topo IIa content the survival results from three experiments are presented, and (24). Quiescent cells are much less susceptible to etoposide and the survival curve from experiment 2 is presented in Fig. 2. In teniposide and also show a lower content of topo 1hz. These this setting, we also obtained a highly significant increase in life results can explain the fact that in a clinical setting, prolonged span when data were pooled (P < 0.0001), and here the supe- drug exposure increases effectiveness (25). Therefore, we in- nority of high-dose etoposide + ICRF-l87 was also statistically vestigated the possibility of administering the drug combination significant when each experiment was tested by itself. more than once and still retaining protection by ICRF-187. To evaluate whether there was a therapeutic difference Curiously, ICRF-l87 did not protect in an etoposide schedule of between the two regimens when there was no transport bar- once a day for five days, whereas effective protection was seen rier to separate the drugs, we inoculated tumor cells into the when etoposide was used three times, once every four days. This peritoneal cavity and treated the tumor-bearing mice i.p. on time dependency of ICRF-l87 protection was unexpected and is days 4, 8, and 12 after inoculation with 30 mg/kg etoposide at present unexplained. The problem is of course very compli- (when etoposide was used alone) and 90 mg/kg etoposide cated and includes drug pharmacokinetics in plasma, drug phar- (when etoposide was used in combination with ICRF-l87). macokinetics in tumor, drug binding constants to enzyme, en- As can be seen in Table 6, all mice survived the tumor with zyme turnover, and so forth. However, we have shown in mice these treatments. This is not surprising because the drugs that although ICRF-l87 provides protection against etoposide- were administered directly into the tumor. We therefore induced weight loss and death, it does not protect against eto- reduced the dose level of etoposide to 50% of the initial level, poside-induced leucopenia (20). Leukocyte counts were, how- i.e., 15 and 45 mg/kg etoposide alone and in combination ever, normalized at day S after treatment, and this may explain

our problems with daily repeated administration and why the ICRF-187 protects tumor cells against the cytotoxicity of eto- combination can be administered on a q4dX3 schedule. Thus, as poside. Thus, the in vivo models used in the present study demonstrated, it is possible to increase the accumulated etopo- strongly indicate that the advantageous result in treating CNS side dose, and our results demonstrate a more feasible way of tumors with high-dose etoposide + ICRF-187 is indeed due to using this potent drug combination in a clinical setting. differences in their passage across pharmacological barriers, in Teniposide was included in our investigations because it is this case in the brain. It should also be pointed out that the more lipophilic and also is eliminated at a slower rate than normal cells located in the CNS are quiescent cells with a low etoposide. Therefore, treatment with teniposide in the clinic can content of topo 1hz and are therefore less susceptible to the still retain antitumor activity on a once a week schedule (26). actions of etoposide. This is in contrast to tumor cells, in which Hermann et a!. (27) demonstrated that ICRF-l87 can pro- all dividing cells have a high content of topo ha and are tect hamsters against lethal doses of daunorubicin also when sensitive to etoposide. Therefore, the higher dose of etoposide in administered after daunorubicin. Also, it is possible to postin- neural tissue is not considered to be a problem. cubate cells with ICRF-187 after initial etoposide treatment and In conclusion, we find that etoposide and teniposide doses obtain an antagonistic effect on etoposide-induced breaks in the can be escalated by pretreatment with ICRF-187. In this setting, DNA (28). Similarly our studies with scheduling in mice the optimal schedule is treatment three times, once every four showed that ICRF-187 can protect when it is given after etopo- days. Postinfusion of ICRF-l87 can be used, but timing of the side. Although timing is very critical here, ICRF- 187 was able infusion is critical. We have developed a solid CNS tumor to completely protect when given 10 mm after etoposide. On the model without metastasis outside the CNS. In this model, as other hand, ICRF-187 can be given 3 h before etoposide with well as in a leukemic model in which the tumor cells metasta- full protection, and protection was significant, although dimin- size, our data showed that high-dose epipodophyllotoxin in ished, for up to 8 h. Pharmacokinetic studies of these drugs in combination with ICRF-187 is superior to epipodophyllotoxin humans have revealed that the terminal half-life for ICRF- 187 is alone. It is likely that the favorable effect is due to differences about 2 h (29-3 1), and for etoposide, it is about 8 h (32). No in transport across membranes. significant protein binding has been determined for ICRF-187 (30), whereas etoposide is 95% protein bound (32). ICRF-187 is Acknowledgments therefore eliminated faster than etoposide. If this can be trans- We thank Annette Nielsen and Lis Str#{248}bechfor expert technical lated into the mouse model, this could explain why ICRF-187 assistance. S. Olesen-Larsen is thanked for performing the statistical shows diminished protection when administered more than 180 evaluations. mm before etoposide. Thus, ICRF-l87 is a true rescuing agent; its action resem- References bles MTX rescue by folinic acid. Folinic acid is an antidote that 1 . Chen, A. Y., and Liu, L. F. DNA topoisomerases: essential enzymes is able to completely abolish the toxic effects of MTX. This and lethal targets. Annu. Rev. Pharmacol. Toxicol., 34: 191-218, 1994. external salvage pathway has enabled manipulation of MTX 2. Wang, J. C. DNA topoisomerases. Annu. Rev. Biochem, 65: 635- administration. High-dose MTX with folinic acid rescue has 692, 1996. been studied in a number of settings. It has been proposed to 3. Jensen, P. B., Jensen, P. S., Demant, E. J. F., Friche, E., S#{248}rensen, overcome problems of drug resistance and poor drug penetra- B. S., Sehested, M., Wassermann, K., Vindel#{216}v,L., Westergaard, 0., tion. Thus, MTX penetrates poorly into the cerebrospinal fluid; and Hansen, H. H. Antagonistic effect of aclarubicin on daunorubicin- induced cytotoxicity in human small cell lung cancer cells: relationship however, after high-dose MTX treatment, cytotoxic dose levels to DNA integrity and topoisomerase II. Cancer Res., 51: 5093-5099, can be achieved. These regimens, which use otherwise lethal 1991. infusions of MTX, have now found application in the treatment 4. Jensen, P. B., S#{216}rensen, B. S., Demant, E. J. F., Sehested, M., Jensen, of lymphomas, osteogenic sarcoma, and acute leukemia (22). P. S., Vindel#{216}v,L., and Hansen, H. H. Antagonistic effect of aclarubicin Interestingly, similar options now appear with topo II rescue, as on the cytotoxicity of etoposide and 4’-(9-acridinylamino)methanesul- shown in this preclinical study. fon-m-anisidide in human small cell lung cancer cell lines and on topoisomerase Il-mediated DNA cleavage. Cancer Res., 50: 331 1-33 16, It has been shown in clinical trials that SCLC metastases to 1990. CNS and that primary tumors of the brain respond to etoposide 5. Jensen, P. B., S#{248}rensen, B. S., Sehested, M., Grue, P., Demant, treatment (33-37). This implies that cytotoxic doses of etopo- E. J. F., and Hansen, H. H. Targeting the cytotoxicity of topoisomerase side cross the BBB or at least the endothelial cells supplying the Il-directed epipodophyllotoxins to tumor cells in acidic environments. intracerebral tumor (36, 38). Conventional doses of teniposide Cancer Res., 54: 2959-2964, 1994. have also shown activity against SCLC brain metastases (39). 6. lanabe, K., Ikegami, Y., Ishida, R., and Andoh, I. Inhibition of topoisomerase II by antitumor agents bis(2,6-dioxopiperazine) deriva- The BBB is considered a pharmacological sanctuary for many tives. Cancer Res., 51: 4903-4908, 1991. drugs, and the hypothesis for the treatment studies was that 7. Ishida, R., Miki, I., Narita, I., Yui, R., Sato, M., Utsumi, K. R., etoposide/teniposide, being lipophilic drugs, cross the BBB, Tanabe, K., and Andoh, I. Inhibition of intracellular topoisomerase II whereas ICRF-l87, being a more hydrophilic drug, does not by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth (19). Therefore, ICRF-l87 would protect normal tissues and inhibition distinct from that of cleavable complex-forming type inhibi- allow a cytotoxic dose of etoposide to reach the CNS. In this tors. Cancer Res., 51: 4909-4916, 1991. context, it is important to note that the high-dose etoposide 8. Sehested, M., Jensen, P. B., S#{248}rensen, B. S., Holm, B., Friche, E., and Demant, E. J. F. Antagonistic effect of the cardioprotector (+)-l ,2,- ICRF-l 87 combination yielded no advantage when adminis- bis(3-5-dioxopiperazinyl-lyl)propane (ICRF-187) on DNA breaks and tered i.p. to i.p. EHR2 cells (Table 6). This is to be expected cytotoxicity induced by the topoisomerase II directed drugs daunorubi- from results of coadministration in clonogenic assay (8), where cm and etoposide (VP-16). Biochem. Pharmacol., 46: 389-393, 1993.

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Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 1998 American Association for Cancer Research. Improved targeting of brain tumors using dexrazoxane rescue of topoisomerase II combined with supralethal doses of etoposide and teniposide.

B Holm, M Sehested and P B Jensen

Clin Cancer Res 1998;4:1367-1373.

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