Optimum Time Sequence for the Administration of Vincristine and Cyclophosphamide in Vivo1

[CANCER RESEARCH 34, 1857-1861, August 1974)

Aly Razek, Teresa Vietti, and Fred Valeriote Division of Hemalology and Oncology, St. Louis Children's Hospital, Mallinckrodt Department of Pediatrics [A. R., T. V.], and Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine [F. V.}, St. Louis, Missouri 63110

SUMMARY normal hematopoietic stem cells. The drug sequence- and interval-dependent inhibition and synergism of cell killing is The cytotoxic effect of the combination of vincristine interpreted in terms of cell kinetics for both the leukemic sulfate and cyclophosphamide was quantitated by the spleen and normal cell populations. colony assay for both AKR- and L1210-transplantable leukemias as well as for normal hematopoietic stem cells. The lethal effect of this combination on the leukemic cell MATERIALS AND METHODS population was found to be schedule dependent. Subadditive Drugs. VCR (Oncovin) (Lot 5JH82A) was supplied by Eli cell killing occurred when the drugs were administered Lilly and Co., Indianapolis, Ind. in 1-mg vials. CY (Cy- together or within a few hr of each other. A marked toxan) (Lot MHM45A), in 100-mg vials, was obtained enhancement of the cytotoxicity was noted when cyclophos phamide was given after vincristine, with a nadir obtained from Mead Johnson Laboratories, Evansville, Ind. when 12 hr had elapsed between the administration of the Both drugs were dissolved in sterile 0.15 M sodium chloride. The same diluent was used to prepare the required two drugs. The observed killing of normal hematopoietic stem cells by this combination was additive independent of drug doses that were injected in a volume of 0.5 ml via the tail vein of the mouse. the sequence or schedule of administration. Mice. AKR and BALB/c x DBA/2 (hereafter called CD2FO mice were obtained from National Animal Labora INTRODUCTION tories, CrevÃ©Coeur,Mo., and through the National Cancer Institute, Bethesda, Md., respectively. DBA/2J mice were VCR2 and CY are antineoplastic drugs with significant purchased from The Jackson Laboratory, Bar Harbor, biological activity against a variety of experimental tumors Maine. Mice of either sex were used in these studies when and human cancers (1, 10, 16). VCR causes metaphase they were 6 to 8 weeks old and weighed 18 to 25 g each. arrest, inhibits the synthesis of both DNA and RNA (5), Leukemic Cells. Two leukemic cell lines were utilized in and has been shown to kill cells selectively when the drug is these experiments: a transplanted AKR line, derived origi added during the DNA-synthetic (S) phase of the cell cycle nally from a mouse with spontaneous AKR lymphoma and (11). CY is a cycle-specific drug because it preferentially transplanted weekly into syngeneic mice as previously kills proliferating cells (3, 20). It is thought to alkylate many described (19); and an L1210 leukemia, obtained originally cellular constituents but probably exerts its lethality by in the ascites formed in DBA/2J mice from the National reacting with DNA (13). Cancer Institue and passaged weekly into groups of Clinical experience (7, 8, 14, 15, 17) suggests that the DBA/2J mice by the i.v. injection of IO5cells obtained from combined use of these 2 drugs is superior to the use of either the spleens of leukemic mice. The leukemic mice used in the drug alone in terms of antitumor effect and prolonged experiments described below received IO6 cells prepared duration of survival. However, the schedule for administra from the spleen of a mouse with advanced leukemia in a tion of both drugs is empirical and, in many instances, is volume of 0.5 ml i.v. 4 days prior to drug treatment. based on convenience of administration. Assay for LCFU. These assays were performed on groups In this report we examine the effect of sequence and time of 4 to 5 leukemic mice at specified times following drug interval for administration of VCR and CY on transplanta- administration. The femurs of control and treated mice ble AKR and L1210 leukemias. A quantitative cellular were removed and a monodispersed cell suspension of assay was used for these leukemic cell lines as well as for the femoral marrow was prepared. After appropriate dilutions were made in a-minimum essential media (Flow Laborato 'Supported by USPHS Grants 1P02CA13053 from the National ries, Rockville, Md.), 0.5 ml/mouse, was given via the tail Cancer Institute and GM02016 from NIH. ' The abbreviations used are: VCR, vincristine sulfate; CY, cyclophos vein to groups of 8 to 10 recipients (AKR mice for the AKR phamide; LCFU, leukemic colony-forming units; NCFU, normal hemato leukemia and CD2P! mice for the L1210 leukemia). Eight poietic colony-forming units. days later, the spleens of the recipient mice were removed, Received February 28, 1974; accepted April 26, 1974. and placed in Bouin's solution, and the number of macro-

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. A. Razek et al. scopic colonies were counted. The number of LCFU from the original donor femur could then be determined (4). The fractional survival of LCFU was determined by normalizing all results to an untreated control group assayed at either 4 or 5 days, depending on the experimental study. Assay for NCFU. This assay is similar to that for LCFU except that 5 normal donor and 15 recipient mice were used. After the recipients received 900 rads total-body irradiation they were given injections of the femoral marrow suspen sion. These mice were killed 9 to 10 days later, and the number of macroscopic colonies were'determined (18). The fractional survival of NCFU following treatment was determined by normalizing the results to an untreated control group.

RESULTS 0 05 10 IS 20 DOSE CYCLOPHOSPHAMIDEImg/mousei Dose-Survival Curves for VCR and CY on LCFU. Dose- Chart 2. Fractional survival of LCFU as a function of dose of CY. survival curves for each drug alone were constructed to Different symbols represent different experiments; vertical bars, 1 S.E. determine an appropriate dose of each to be used in the obtain a surviving fraction of LCFU between 10 ' and 10~2. combination experiments. Single, increasing doses of each drug were given i.v. to groups of leukemic mice and the Time-Survival Curvesfor VCR and CY on LCFU. Before fractional survival of LCFU was assayed 24 hr later. The the effect of the combination of the 2 drugs could be studied results were normalized to the 5-day control. it was necessary to determine the kinetics of the lethal effect The dose-survival curves obtained for VCR are shown in of the selected doses of the 2 agents. This provided a Chart I. The AKR leukemic cells were more sensitive than correction factor for proliferation that occurred during the the L1210 cells. The curve for AKR leukemia was exponen interval between drug combination. The kinetics also indi tial throughout 6 decades of survival. The curve for L1210 cated the time at which the fractional survival reached its leukemia was also exponential, but doses greater than 0.05 minimum so that the assays would not be done earlier than mg/mouse were lethal to the mice within 24 hr. A dose of the optimum time. The assay of LCFU was done at 0.025 mg VCR per mouse was chosen for the combination specified times after the administration of 0.025 mg VCR experiments to obtain a fraction survival of LCFU between per mouse. The results were normalized to the 4-day 10-' and IO-3. control. The fractional survival of LCFU with respect to The dose-survival curves for CY are shown in Chart 2 and time for both AKR and L1210 leukemia is shown in Chart 3. There was a gradual decrease in the surviving fraction of are exponential for both leukemic lines. A small shoulder LCFU, reaching minimum levels of 10~3and 10"', respec region was observed for AKR leukemia. A dose of 0.5 mg CY per mouse was chosen for the combination studies to tively, at about 12hr, following which repopulation predom inated. The kinetics of the reduction in the surviving fraction of LCFU after 0.5 mg CY per mouse is shown in Chart 4. There was a rapid decrease in survival for both cell lines, reaching a minimum within 2 hr after drug administration, followed by an increase of the LCFU with a doubling time of about 9 hr. The maximum reduction in fractional survival of LCFU was 10 ' for AKR leukemia and 10 2 for L1210 leukemia. Combined Effect of VCR and CY on LCFU. Either VCR or CY was given at the dose levels described above, and the alternate drug was administered at time intervals varying from 1 min to 24 hr. One group was untreated and served as the control, another group received VCR only, and a 3rd group received CY only. Each group was assayed for LCFU/femur 24 hr after administration of the last drug. The results were normalized to the 5-day control. Prolifera

10 tion of LCFU between the drug administrations was 0 0025 005 0075 corrected by using the times in Charts 3 and 4 at which DOSE VINCRISTINE (mg/mouse) repopulation commenced. If the 2nd drug was administered Chart I. Fractional survival of LCFU as a function of dose of VCR. during the time interval following the 1st drug when no net Different symbols represent different experiments; vertical bars, 1 S.E. increase in LCFU occurred, no correction was made. For

1858 CANCER RESEARCH VOL. 34

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. VCR and C Y at this level up to the 24-hr interval. However, when CY was given after VCR, the decrease in LCFU survival continued with the increase in the time interval between the 2 drugs, and a synergistic effect was found between the 6-hr and 24-hr intervals. The survival fraction returned to the range of the additive level after 24 hr. The results obtained with L1210 leukemia are shown in Chart 6. The pattern was similar to that found with the

4 8 12 16 20 24 TIME AFTER VINCRISTINE

ZO IO IO 20 CY BEFORE AFTER VCR (M VCR Ihr) VCR Chart 5. Fractional survival of LCFU for AKR leukemia as a function of sequence and interval between the administration of VCR (0.025 mg/mouse) and CY (0.5 mg/mouse). Q, geometric mean of 5 separate experiments; limits, range of individual values obtained; â€”¿.expected additive level; 0 geometric mean from 5 separate experiments; limits, range obtained. Different symbols represent different experiments; vertical bars, 1 S.E.

t 8 12 16 20 TIME AFTER CYCLOPHOSPHAMlDEthr) Chart 4. Fractional survival of LCFU as a function of time following the administration of 0.5 mg of CY per mouse. Different symbols represent different experiments; vertical bars, 1 S.E. longer intervals, it was assumed that the residual cell population increased in number, with a doubling time of 9 hr, and an appropriate correction in LCFU surviving fraction was made. The results for the AKR leukemia are shown in Chart 5. VCR given alone yielded a mean surviving fraction of 2.6 x 10~3 LCFU/femur and CY alone resulted in a mean survival level of 8.1 x 10~2 LCFU/femur. The predicted additive fractional survival for the 2 drugs when used in IO 20 combination is the product of their cytocidal effect when CY AFTER administered independently (2.1 x 10~4). When VCR was VCR (hr) VCR given simultaneously with CY, the fractional survival was 3 X 10 3, greater than the additive level and nearly equal to Chart 6. Fractional survival of LCFU for LI210 leukemia as a function of sequence and interval between the administration of VCR the level of VCR alone. As the interval between the (0.025 mg/mouse) and CY (0.5 mg/mouse). Q, geometric mean of 5 administration of VCR and CY was increased, a gradual separate experiments; limits, range of individual values obtained; â€”¿. decrease in the surviving fraction occurred. When CY was expected additive level; 0, geometric mean from 5 separate experiments; given before VCR, the cytocidal effect increased and limits, range obtained. Different symbols represent different experiments; reached the additive level in approximately 12hr, remaining vertical bars. 1 S.E.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. A. Razek et al. AKR leukemia. When VCR and CY were given at the same the agents were given together, the fractional survival of time or within a short interval, the cytotoxic effect was less leukemic cells was less than additive and equal to the level than additive. When the 2 drugs were separated by a time achieved by VCR alone. This is possibly because both interval ranging between 2 and 24 hr, the effect was agents kill cells in the S phase of the cell cycle preferentially, synergistic. The minimum fraction survival level was and thus are not independent in their mode of action. This reached when CY was given 8 hr after VCR and remained at does not totally explain the results, because CY presumably this level up to the 24-hr interval. However, the effect was is effective in other phases of the cell cycle and thus the not as great as that observed for the AKR leukemia. killing should have been greater than that observed with Combined Effect of VCR and CY on NCFU. VCR and VCR alone. CY, both singly and in combination, at the same dose levels Second, as the time interval was increased for either described above and for the same intervals as in the previous sequence of drug, the fractional survival decreased to the experiments, were given to normal mice. After an appropri additive effect. This possibly demonstrates that, if the drugs ate interval of time, analogous to the LCFU studies, NCFU are not given at the same time, one might not interfere with were assayed in the femoral marrow. The results are shown the other's independent production of its full cytotoxic in Chart 7. VCR given alone resulted in a fractional survival effect. of NCFU of 6 x 10~', and CY alone resulted in a survival Third, when CY was given about 8 hr after VCR, a level of 8 x 10 '. The predicted additive level is thus 4.8 x synergistic cell killing occurred. This could result from a 10 '. All combinations used were found to be additive. partial synchronization of the leukemic cells so that the maximum number passes through the CY-sensitive phase at that time. Such a synchronizing effect has been demon DISCUSSION strated in vivo with Ehrlich ascites cells with combined VCR-CY treatment (9). Another possibility considered is The results that we obtained can be divided into 2 that VCR may increase the permeability of the malignant categories, the effects observed with the drugs when used cells, leading to increased incorporation of CY and a greater alone, or combined. The dose-survival curves for the lethal effect. transplanted AKR leukemia for both drugs were similar to Our results on the effect of the combination of VCR and those that have been obtained previously (2, 3). The CY on the normal hematopoietic stem cells did not show differences found between the AKR and L1210 leukemias any schedule dependency. There was no potentiation of the might be reasonably attributed to some biochemical differ cytocidal effect of the 2 drugs when CY was administered ences between the cell lines. The time-survival curves before or-after VCR, contrary to that noticed for the indicate that the cell killing following CY seemed complete leukemic cell population. within 2 to 4 hr following drug administration, while the cell Clinically, these results may be useful in terms of killing following VCR continued for 12 hr. This probably scheduling the use of the 2 drugs to obtain an optimum reflects a difference in the pharmacokinetics of the 2 drugs, effect through an additional killing of the malignant cell in that the rate of disappearance of drug activity from the population without an increased toxic effect on the normal serum is rapid for the former agent (12) and slow for the cells. At the present time, we cannot be sure of the time .latter agent (6). interval with regard to human tumors because we do not For the combined drug studies, the results can be understand the basis for the synergy in the experimental considered in terms of 3 distinct time intervals. First, when system. However, regardless of the cell-cycle-generation time parameter, it would seem advantageous to separate the suCItfiIgI 1, administration of these 2 drugs by a time interval rather than giving them simultaneously.

-1 I i CY ALOW I â€”¿; V .[1 . l.i...Â«Â«*71Â«-,T j....Â£.i|LLJ ACKNOWLEDGMENTS ^- i 1 'Ã® ALONP- The authors appreciate the technical assistance of Sandra Tolen and I1,1.1,301 I Kathy Dale.

X'3 /0"l'I1-:' !O IO 0 IO K CYAFTERVCR BEFORE ' CY REFERENCES Ihrl VCR (nilLTÃ’:veÂ« VCR 1. Armstrong, J. G. Vincristine (NSC-67574) Symposiumâ€”Summary Chart 7. Fractional survival of NCFU as a function of sequence and and Prospectives. Cancer Chemotherapy Rept., 52: 527-535, 1968. interval between the administration of VCR (0.025 mg/mouse) and CY 2. Bruce, W. R., Meeker, B. E., Powers, W. E., and Valeriote, F. A. (0.5 mg/mouse). Q, geometric mean of 5 separate experiments; limits, Comparison of the Dose- and Time-Survival Curves for Normal range of individual values obtained; â€”¿-,additive fractional survival Hematopoietic and Lymphoma Colony-Forming Cells Exposed to expected; Q. geometric mean from 5 separate experiments; limits, range Vinblastine. Vincristine, Arabinosylcytosine. and Amethopterin. J. obtained. Different symbols represent different experiments; vertical bars, Nati. Cancer Inst., 42: 1015 1025, 1969. I S.E. 3. Bruce, W. R., Meeker, B. E., and Valeriote, F. A. Comparison of the

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Optimum Time Sequence for the Administration of Vincristine and Cyclophosphamide in Vivo

Aly Razek, Teresa Vietti and Fred Valeriote

Cancer Res 1974;34:1857-1861.

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