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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Hematology-Oncology [J. T.], Biostatistics and Epidemiology [O. G., C. A. P.], Molecular Pharmacology [P. J. C., L. B. R., P. J. H.], and Pharmaceutical Sciences [M. M., C. F. S.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105-2794, and Department of Pediatrics, Princess Beatrix Hospital, Groningen, the Netherlands 9700RB [S. S. N. d. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.5. These results show that the therapeutic effect of combining topotecan with vincristine was greater than additive in most tumor models of childhood solid tumors, and toxicity data suggest that this can be administered to mice with only moderate reduction in the dose levels for each agent. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite this interest in combination therapy, relatively few in vivo preclinical studies evaluating such drug combinations have been performed. In part, this is a consequence of the difficulty and expense of undertaking such studies. However, the importance of animal testing is that synergism between drug combinations is only valuable if it is tumor selective and does not result in synergistic toxicity to limiting tissues of the host. Waud et al. (12) conducted preclinical studies using xenografts derived from small cell lung cancer or glioma to explore potential synergism of topotecan with other agents active against small cell lung tumors (e.g., cisplatin) and central nervous system tumors (e.g., temozolomide). Supra-additive activity was observed for both combinations of topotecan with cisplatin or temozolomide, but the former also demonstrated increased toxicity compared with the drugs as single agents. Topotecan and etoposide given simultaneously resulted in antagonism in agreement with results obtained when etoposide and CPT-11 were combined (13) . In contrast, initial treatment with topotecan was associated with increasing responsiveness of the xenografts to subsequent doses of etoposide (14) or doxorubicin (15) .
After exposure to camptothecin analogues, many cell types arrest in S phase or G2-M phase. We postulated that administration of topotecan followed by vincristine, an agent that causes depolymerization of microtubules leading to mitotic arrest and death, might have a synergistic antitumor effect. Topotecan also has different dose-limiting toxicities than the vincristine in vivo, and these agents may be combined without significant reduction in dose intensity of either agent. Administration of topotecan in combination with vincristine, therefore, might have an additive or synergistic antitumor effect and have tolerable toxicity. In addition, there are recent data to suggest that in yeast, TOP I (encoding DNA topoisomerase I) and TRF4 (16) participate in overlapping or dependent steps in mitotic chromosome condensation and serve to define a previously unrecognized biological function of topoisomerase I.
Vincristine has demonstrated activity in several xenograft models of pediatric tumors (17 , 18) and has an established role in the treatment of childhood cancers. We have shown also that topotecan is active in several childhood tumors when grown in mice, and data are emerging to support the clinical activity in childhood solid tumors (19, 20, 21) . In this study, we investigated the activity of the combination of topotecan and vincristine against a panel of childhood solid tumor xenografts and determined whether the response to the combination was more than would have been expected from the response to either drug alone. Moreover, we conducted pharmacokinetic studies of both topotecan and vincristine, given alone and in combination, to determine whether a pharmacokinetic interaction existed between the two drugs.
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MATERIALS AND METHODS |
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3 mm3 were implanted in the space of the dorsal lateral flanks of each mouse to initiate tumor growth. Tumor-bearing mice were randomized into groups of six or seven prior to initiating therapy. Animal care was in accord with institution guidelines.
Tumor Lines.
Briefly, 14 tumor lines were examined in this study. Each has been characterized and reported previously (1
, 3
, 23)
All six NB tumors were from young patients (1–3 years) with advanced disease. All are classified as Shimada poor risk. With the exception of xenograft NB-EB, each tumor demonstrated amplification of N-MYC. The RMSs (Rh prefix) were from previously untreated patients. Rh18/VCR was selected in mice for resistance to vincristine (24)
. Medulloblastoma lines (D283 and DAOY) were established from cell lines obtained from American Type Culture Collection, and the glioblastoma SJ-GBM2 was also established in vitro prior to growth in mice (3)
. Once established as xenografts, further transplantations were from mouse to mouse. For chemotherapy studies, all tumors were used within 30 passages of their engraftment in mice. Each tumor grew routinely in >95% of recipient mice, and all retained human origin as determined by karyotype.
Growth Inhibition Studies.
All mice bearing bilateral s.c. tumors received the chemotherapeutic agent when tumors were 0.2–1 cm in diameter. The procedures have been reported previously (1)
. Briefly, tumor diameters were measured every 7 days using Vernier calipers interfaced with a Macintosh computer. Tumor volumes were calculated, assuming tumors to be spherical, using the formula [(
/6) x d3], where d is the mean diameter. Tumor volumes were determined for at least 12 weeks after starting treatment.
Drug Formulation and Administration.
Topotecan was provided by SmithKline Beecham (King of Prussia, PA), and vincristine was commercially available (Eli Lilly and Company, Indianapolis, IN; 1 mg/ml). Topotecan was administered i.v. daily for 5 days on 2 consecutive weeks [(d x 5)2]. Cycles were repeated every 21 days over a period of 8 weeks. Vincristine was administered i.v. by a short infusion (<1 min) every 7 days for nine injections. Vincristine was administered on day 1 of each topotecan course, except in one experiment where vincristine was administered with the fifth dose of each topotecan course.
Evaluation of Pharmacokinetics.
For pharmacokinetic studies, vincristine was administered on day 1 at 1 mg/kg i.v. into a lateral tail vein, followed by topotecan at 1.25 mg/kg i.v. in a group of mice, and another group of mice received topotecan at 1.25 mg/kg alone. Serial blood samples were collected by cardiac puncture from three animals per time point for measurement of topotecan (pre, 0.25, 0.5, 1, 2, 4, and 6 h after topotecan) and vincristine (pre, 0.08, 0.25, 0.5, 1, 2, 4, 6, and 24 h after vincristine) plasma concentrations, respectively. Blood samples were processed and assayed for topotecan lactone by an HPLC assay described previously (25
, 26)
. Briefly, an isocratic HPLC assay with fluorescence detection was used to determine topotecan lactone plasma concentrations. The lower limit of assay sensitivity was 0.25 ng/ml. Vincristine samples were assayed by an isocratic HPLC assay, which used column-switching and on-line column extraction with a combination of UV and electrochemical detection. Sensitivity of the vincristine HPLC assay was 0.3 ng/ml (27
, 28)
.
A two-compartment linear model using maximum likelihood estimation was fit to topotecan lactone plasma concentration-time data after topotecan administration with or without vincristine (ADAPT II; Ref. 29
). A two-compartment linear model using maximum likelihood estimation was also used to fit the vincristine plasma concentration-time data after vincristine administration. Model parameters for the two-compartment pharmacokinetic model estimated included the volume of the central compartment (Vc), elimination rate constant (ke), and the intercompartment rate constants (kcp and kpc). Using standard equations, systemic clearance (Clsys) and volume of distribution at steady-state (Vdss) were calculated from parameter estimates (30)
. Area under the plasma versus concentration-time curve from zero to infinity (AUC0-
h) was calculated using log-linear trapezoidal method (31)
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Tumor Response and Tumor Failure Time.
For individual tumors, PR was defined as a volume regression >50%, but with measurable tumor at all times. CR was defined as disappearance of measurable tumor mass at some point within 12 weeks after initiation of therapy. Maintained CR is defined as no tumor regrowth within a 12-week study time frame. This time point was chosen because all studies lasted at least 12 weeks. Because some groups were studied up to week 27 to examine late regrowth, we also looked descriptively at maintained CRs at 20 weeks for those studies. A mouse was regarded as achieving a CR only if tumors on both flanks had CRs, and a PR only if the tumor of at least one flank had a PR and the tumor response on the other flank was not less than a PR. Mice that died before the end of the 12-week study time, and prior to achieving a response, were considered as failures for tumor response. If an initial tumor volume was <0.20 cm3 at the start of treatment, data on that tumor were excluded.
Tumor failure time was defined as the time (in weeks) required by individual tumors to quadruple their volume after the initiation of therapy. Tumor failure times were censored if a mouse died prior to week 12 and before a tumor grew to four times its initial volume. Because tumors were implanted in both lateral flanks, the tumor failure times from each mouse are clustered observations. Evidence of high correlation between such failure times has been reported previously (22) . To account for the clustering effect attributable to the mouse, without explicitly specifying the correlation structure, the time to failure was defined as the minimum of the failure times of the bilateral tumors.
Statistical Methods.
The different treatment regimens were analyzed using tumor failure time data and tumor response data. The primary objective of the analysis was to determine whether the outcomes obtained with the combination of topotecan and vincristine were significantly better than what would be expected when agents were administered alone. For each tumor line, a score test for similar action of two compounds, proposed by Giltinan et al. (32)
, was used to investigate whether the combination of topotecan and vincristine resulted in greater than expected responses compared with the activity of each agent when administered alone. A dose-response model with a logit link function was used in this analysis. Only CR was evaluated as response. It was assumed that vincristine and topotecan exhibit parallel linear regressions of the response (i.e., CR) on the log dose. This assumption could not be verified because vincristine was administered only at one dose level (1 mg/kg). However, plots of observed versus fitted CRs (illustrated in Fig. 1
) showed that the CR data sets were generally well fit by the four-parameter, dose-response model used. In tumor lines with sufficient design points (degrees of freedom), "goodness-of-fit" was tested using a Pearson 
2 statistic.
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is the relative potency of topotecan to vincristine, the relative potency and parameters 
, 
, 
are estimated by maximum likelihood method, with the last parameter representing indication of the agents’ interaction. F was chosen to be a logistic function. A score test was used to test for synergistic interaction, > 0. Additive and antagonism interactions correspond to = 0 and < 0, respectively.
In tumor lines for which the estimated value of the interaction parameter effect was positive, if the score test rejected the null hypothesis of additivity (P 
0.05), it was concluded that there was evidence in support of synergistic interaction of vincristine and topotecan. If the estimate of the interaction parameter was negative, then an antagonistic effect of the combination was suggested.
For comparisons of time to tumor failure for different treatment regimens, Kaplan-Meier estimates of survival distributions were obtained, and exact log-rank tests were used for pairwise comparisons of differences between treatment groups. The activities of the two-drug combinations of topotecan and vincristine were compared with those of the single agents administered at the same doses. In addition, tumor-failure distributions of each treatment group were compared with the failure distribution of the control group. Experiment-wise significance level was maintained at 0.05 by using the Bonferroni procedure (33) to adjust for the multiplicity of tests of significance within each tumor line. S-plus 4.0, SAS 6.12, and StatXact-3 were used for statistical analyses.
Evaluation of Toxicity.
Toxicity was assessed in tumor-bearing mice by measuring body weight changes in mice that received 75% (topotecan) and 80% (vincristine) of the MTD of each agent when administered alone on this schedule. During each of the 5-day courses of treatment with topotecan (1.5 mg/kg) and with vincristine (1 mg/kg), tumor-bearing mice were weighed daily. Weights were recorded every 7 days for the mice that received only vincristine. There were no toxicity-related deaths in any group. A separate study using non-tumor-bearing mice was conducted to investigate the myelotoxicity of vincristine and topotecan. Individual cohorts of mice (n = 5) were treated with vincristine (1 mg/kg) and topotecan (0.6 mg/kg) as single agents and in combination, in the same schedules that were used in the therapeutic study. The dose of topotecan used in this study gives clinically relevant plasma systemic exposure to topotecan lactone (22)
. Orbital blood samples (50 µl) were collected from anesthetized mice at 3–4-day intervals during one treatment course (20 days). Hemoglobin, platelets, and white cell count were determined for each sample (13)
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RESULTS |
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2 mg/kg) on an optimal schedule [daily x 5 for 2 consecutive weeks; (dx5)2; repeated every 21 days), topotecan caused regressions of most tumors. However, these dose levels are associated with topotecan lactone plasma systemic exposures that are in excess of that tolerated by children when drug is administered on the same schedule. Thus, for the present studies, we used topotecan dose levels that gave clinically relevant systemic exposures (approximately 20–90 ng·h/ml topotecan lactone). We chose to use a fixed dose of vincristine (i.e., 1 mg/kg every 7 days) that would achieve plasma systemic exposures consistent with those reported by Crom et al. (27)
in the range of 17–609 ng·h/ml. (For most antitumor studies, dose levels for topotecan were <30% of the MTD when given on the (dx5)2 schedule, and for vincristine were 80% of the MTD when drug was administered every 7 days for 9 consecutive weeks.) These studies, therefore, focus on synergistic antitumor activity, rather than on mouse toxicity.
Interaction of Topotecan and Vincristine.
For three NBs (NB-EB, NB-1382, and NB-1643), two RMSs (Rh12 and Rh18), and three brain tumors (D283, SJ-GBM2, and DAOY), the parameter estimates of interaction were positive, and this interaction was statistically significant (P < 0.001 in all cases). We were unable to fit two NB lines (NB-1691 and NB-SD) and one RMS line (Rh28) with the four-parameter model. A possible explanation for this is that the CR data for these tumor lines were clearly nonmonotonic, or as in the case of Rh28, 100% response rates were observed in all treatment groups, creating a very flat dose-response curve. For tumor lines Rh30 and NB-1771, the parameter estimates of interaction were negative, suggesting an antagonistic interaction of vincristine and topotecan. However, in the case of NB-1771, this interaction was not statistically significant (P = 0.38). As noted in "Materials and Methods," we could not directly verify the validity of the assumption that both vincristine and topotecan exhibited parallel linear regressions of the response. However, in the tumor lines with seven design points (i.e., treatment levels), the CR data were well fit by the four-parameter model, as the P values of goodness-of-fit tests show. Fig. 1
contains plots of observed CRs versus expected CRs (based on our model) for these tumor lines: Rh12, Rh18/VCR, NB-EB, and NB-1771. Notice that the expected responses are very close to the observed responses; the large P values (P > 0.30 for all four tests) indicate that there was no evidence of lack of fit of our model. In other tumor lines, we did not have adequate numbers of degrees of freedom for goodness-of-fit tests, although the plotted observed and fitted responses were close to each other (data not shown).
Control of Tumor Growth.
Results of the exact log-rank tests used to compare differences in the distributions of tumor failure times (i.e., time for tumors to grow to four times their volume at the start of treatment) among various treatment combinations are presented. Table 1
shows results of tumor lines for which topotecan and vincristine show improved activity over either individual agent. P values for these tests are adjusted by the Bonferroni procedure (33)
. Because this approach is rather conservative, we have included Table 2
to show tumor lines where the combination treatment showed marginal statistical differences over individual drugs. Tables 1
and 2
show the number of mice in each treatment group and the number of mice with censored failure times, that is, mice that died prior to week 12 and before a tumor grew to four times its initial volume. Overall, 10% of mice died prior to week 12 (64 of 640). Excluding two experiments where there was a higher rate of toxicity (30 deaths in 94 mice), the censored rate was 34 of 546 or 6.2%. Censored data occurred evenly in single-agent groups and combination groups. The exact log-rank test was used to compare each treatment group with the control group and also to compare the combination treatments with individual agents at the same dose levels. The actual P value of the test was multiplied by the number of tests. In all tumor lines, at least one of the treatment groups showed significant improvement in controlling tumor growth over the control groups.
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. The results in NB-1382 (Fig. 2)
and in NB-1691 show the dramatic effects of the combination treatments. The single NB-1382 tumor to regrow (week 25) was <0.2 cm3 at initiation of therapy and hence excluded from the statistical analysis. For example, in NB-1382, 100% of the tumors had complete responses to the combination treatments. The CR rate in the vincristine alone arm was 14%, whereas CR rates for the topotecan alone groups were 86 and 71% for 0.18 and 0.12 mg/kg doses, respectively. For the single-agent topotecan groups, 71 and 43% of the CRs were maintained at 12 weeks, but only 50 and 0% were maintained at 20 weeks. Except for one mouse, 100% of the CRs in the combination treatments were maintained at 20 weeks. One additional neuroblastoma line (NB-1643) showed slightly greater than anticipated activity of the combination. For example, NB-1643 xenografts were highly responsive to topotecan, but by week 17, tumors in four of five mice had regrown. Vincristine alone had marginal activity (zero of five CR and zero of five PR). In contrast, all but one mouse remained in CR at week 17 when treated with topotecan at 0.24 or 0.16 mg/kg in combination with fixed-dose vincristine.
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). Vincristine alone seemed to control tumor growth quite well. None of the mice in the combination treatment groups bearing Rh30 had tumors that quadrupled in volume, and every uncensored mouse had a CR to treatment. For all drug combination groups, these CRs were maintained at both 12 and 20 weeks. In contrast, only 29 and 43% of CRs were maintained at 12 weeks for the 0.24 and 0.36 mg/kg groups, respectively. Results for Rh12 tumors are shown in Fig. 3
. Although only marginally significant over single agents, the results indicate a considerable biological gain in combination treatment groups. For example, although palpable tumor could be detected, there was no tumor regrowth in any combination group over the 25 weeks of observation. In contrast, there were tumor regrowths in all topotecan-treated mice and in several animals treated with vincristine as a single agent. In Rh28, there were no differences in tumor failure times between the combination and single agents (P = 1 for all comparisons). All treatments seemed to have similar effects (i.e., maintained CR) on controlling tumor growth in the Rh28 line; none of the uncensored mice had tumors that quadrupled from their initial volumes.
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). For D283 medulloblastomas, 100% of CRs in the two combination treatments were maintained for up to 27 weeks. The maintained CR rates were 0% for the vincristine group and 43 and 0% with 0.24 and 0.16 mg/kg topotecan, respectively, at 20 weeks (Fig. 4
. The SJ-GMB2 glioblastoma line showed the most outstanding effect of the combination therapy (Table 2)
. All treatment groups in this line showed improvement over the control group, and in addition, the combination of topotecan and vincristine significantly delayed tumor growth over the drugs given individually (P = 0.009 for all comparisons). None of the mice receiving topotecan and vincristine together had tumors that grew to four times initial volume; in fact, all tumors in the combination treatment groups had CRs that were maintained at 12 weeks. Furthermore, 90% of the mice had tumors that maintained CRs for 22 weeks. Time to failure curves for the SJ-GBM2 line are shown in Fig. 5
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. Vincristine was administered every 7 days for 9 weeks, either on day 1 or day 5 of each topotecan course. Table 3
shows that both combination treatments significantly delayed tumor growth over the control groups (P = 0.045), but neither vincristine alone nor topotecan alone (0.36 mg/kg) showed improvement over the control group (P > 0.17). Furthermore, the combinations significantly delayed tumor growth over the individual agents (P = 0.009 for all tests). There was no evidence of a difference in giving vincristine on either day 1 or day 5 of each topotecan course with respect to comparisons of tumor failure times (P = 1).
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. The topotecan systemic clearance in mice receiving topotecan alone was similar to that in mice receiving topotecan along with vincristine. The topotecan lactone systemic exposure measured in the two groups of mice was similar, with or without vincristine. Plasma concentration plots for vincristine also showed a biphasic linear elimination. The estimated vincristine pharmacokinetic parameters are also summarized in Table 4
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. Clearly, the combination was more toxic than either agent individually; however, the total weight loss was <20% at doses that were 75 and 80% of the MTD for topotecan and vincristine, respectively. No drug-related deaths occurred in mice that received three cycles of therapy over a period of 8 weeks. As can be seen from Fig. 6, B and C
, a marked reduction in both hemoglobin and platelets resulted from combination treatment in non-tumor-bearing mice. Also, recovery of the blood count was more prolonged after the combination treatment. Of note is that vincristine was myelotoxic in this animal model, a toxicity not usually encountered in the clinical setting.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The model system using xenografts derived from childhood solid tumors grown in immune-deprived mice has been used extensively for identifying novel agents and for evaluating antitumor activity of drug combinations (1 , 3 , 13 , 17) . The model has been valuable in identifying novel agents that were subsequently demonstrated to have significant clinical activity against the respective tumor type (e.g., melphalan in RMS, topotecan in NB and RMS). Here, we have examined the interaction between vincristine and topotecan in a series of tumor models that represent some of the most frequently occurring tumors in children. Traditionally, therapeutic synergy has been defined as an antitumor effect that exceeds the optimal effect of the individual agents when used in combination. For these studies, drugs were used individually or in combination at dose levels up to those that were toxic to the host. However, whereas this would be appropriate for syngeneic rodent tumors, such an approach may not be suitable for assessing the activity of a combination against human tumor xenografts in mice. This may be particularly true for camptothecin analogues, where the systemic exposure (plasma AUC) at the MTD in the mouse may greatly exceed the AUC in humans at the MTD (22) . For the studies presented, we used a range of topotecan doses associated with systemic exposures that are clinically achievable (34) . Similarly, the dose of vincristine was fixed at a level consistent with achieving a plasma AUC achievable in children (27 , 28) . Therefore, we have examined the potential interaction of these agents against tumors separately from the potential synergistic toxicity to the rodent host.
For analysis of antitumor activity, a method proposed by Giltinan et al. (32) was used. This model assumes that both drugs exhibit parallel linear regressions of the response (CR) relative to the natural logarithm of the dose. Although we were unable to directly verify the model assumption, complete response data sets were generally well fit by the full parametric version of the model that we used. The combination of topotecan and vincristine was significantly synergistic in most tumor lines (9 of 14). Of the tumor lines we were able to fit, only two (NB 1771 and Rh30) suggested antagonistic interaction of topotecan and vincristine. Of interest is that whereas greater than additive activity was demonstrated against the Rh18 rhabdomyosarcoma, no interaction was shown against Rh18/VCR, a subline selected in situ for resistance to vincristine. This suggests that at least some mechanisms of acquired resistance can abrogate the interaction. This result suggests, also, that cellular mechanisms, rather than pharmacokinetic interactions, are responsible for the synergistic antitumor activity of this combination in vivo.
More CRs were observed in mice receiving the combination treatment compared with mice that received only vincristine or only topotecan, and these responses were maintained for longer duration in many lines. However, it is important to note that in most experiments, tumor responses were only followed for 4 weeks after the final cycle of therapy. Consequently, it is unlikely that treatment was curative in these mice. Overall, the combination of topotecan and vincristine showed improved or marginally improved activity over either individual agent in terms of time to tumor failure. Even in those tumors lines with only marginal statistical improvement for the combination therapy, impressive biological responses were observed. Examples are Rh12 and NB-1643 xenografts, where responses were far more durable in the combination therapy groups. Thus, conservatively, the combination of topotecan and vincristine had greater than anticipated antitumor activity in at least nine tumor lines.
Although in vitro studies of topotecan in combination with both vincristine and Taxol have been reported (10 , 11) , this is the first in vivo study to report the interaction of topotecan and vincristine. No clinical studies using this combination have been published. Studies in vitro established less-than-additive effects for combining topotecan with vincristine (11) ; in contrast, this in vivo preclinical study has demonstrated synergy in most tumor lines.
We evaluated toxicity by determining the fraction of the MTD for each agent that could be used in combination and determined hematological toxicity relative to each individual agent. Previous studies determined that for vincristine given i.v. every 7 days over a period of 9 weeks, immune-deprived mice tolerated 1.25 mg/kg per administration (data not presented). For topotecan given i.v. [(dx5)2] every 3 weeks for three cycles, the MTD was 2.0 mg/kg (3)
. We found that in combination, a dose of vincristine of 1 mg/kg and topotecan at 1.5 mg/kg was tolerated. Thus, approximately 80 and 75% of the MTD for each drug could be used in combination, suggesting less-than-additive toxicity. When a fixed dose of vincristine (1 mg/kg) was combined with topotecan administered at a dose that simulates clinically achievable AUC for topotecan lactone, there was increased myelotoxicity. Of note was prolonged thrombocytopenia and anemia in mice treated with the combination. However, how relevant these data will be to humans is unclear. In mice, vincristine caused significant myelosuppression, whereas for patients this is not observed. In an ongoing Phase I trial, where topotecan is given on the [(dx5)2] schedule with vincristine, 80% of the topotecan dose (targeting 80 ng·h/ml topotecan lactone) is tolerated with 1.5 mg/m2 vincristine given on the first day of each 5-day topotecan course. In this clinical study, vincristine does not appear to exacerbate myelotoxicity due to topotecan.4
Results of the pharmacokinetic studies of topotecan alone were similar to that we have published previously in non-tumor-bearing mice (2) . Although we noted a slight difference in topotecan pharmacokinetic parameters when it was coadministered with vincristine, the values for clearance and systemic exposure were within 17 and 9% of the topotecan alone group, respectively. Moreover, we have also shown that the presence of tumor does not alter the disposition of topotecan in mice bearing neuroblastoma xenografts. Thus, it is unlikely that the additional antitumor effect attributable to the combination of topotecan and vincristine has a pharmacokinetic basis.
In this study, we used a sensitive and specific HPLC assay for vincristine, whereas previous studies of vincristine pharmacokinetics in mice had used other nonspecific, less sensitive assays. Thus, no data exist with which to compare our vincristine-alone results. However, as with the topotecan data, the differences in vincristine disposition seen when administered in combination were within the range of expected variability.
In summary, our data suggest a significant interaction between topotecan and vincristine, resulting in an unanticipated level of antitumor activity. At this time, the mechanism that results in this synergy is unknown. However, our data suggest that it is not due to vincristine altering the pharmacokinetics of topotecan or vice versa. Thus, a cellular mechanism would appear to be important and requires further study. From practical considerations, this combination warrants evaluation against childhood solid tumors and potentially leukemias, particularly where both agents have demonstrated single-agent activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by USPHS Award CA23099, and Cancer Center Support Grant P30 CA21675 from the National Cancer Institute, and by grants from the American, Lebanese, Syrian Associated Charities. 
2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2974. Phone: (901) 495-3440; Fax: (901) 521-1668; E-mail: peter.houghton{at}stjude.org
3 The abbreviations used are: RMS, rhabdomyosarcoma; NB, neuroblastoma; HPLC, high-performance liquid chromatography; AUC, area under the concentration-time curve; MTD, maximum tolerated dose; CR, complete tumor regression; PR, partial tumor regression.
4 D. Reardon, personal communication.
Received 6/ 1/99; revised 8/12/99; accepted 8/16/99.
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B. H. Kushner, K. Kramer, S. Modak, and N.-K. V. Cheung Camptothecin Analogs (Irinotecan or Topotecan) plus High-Dose Cyclophosphamide as Preparative Regimens for Antibody-Based Immunotherapy in Resistant Neuroblastoma Clin. Cancer Res., January 1, 2004; 10(1): 84 - 87. [Abstract] [Full Text] [PDF]
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P. J. Houghton, P. C. Adamson, S. Blaney, H. A. Fine, R. Gorlick, M. Haber, L. Helman, S. Hirschfeld, M. G. Hollingshead, M. A. Israel, et al. Testing of New Agents in Childhood Cancer Preclinical Models: Meeting Summary Clin. Cancer Res., December 1, 2002; 8(12): 3646 - 3657. [Abstract] [Full Text] [PDF]
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M. Leggas, C. F. Stewart, M. H. Woo, M. Fouladi, P. J. Cheshire, J. K. Peterson, H. S. Friedman, C. Billups, and P. J. Houghton Relation between Irofulven (MGI-114) Systemic Exposure and Tumor Response in Human Solid Tumor Xenografts Clin. Cancer Res., September 1, 2002; 8(9): 3000 - 3007. [Abstract] [Full Text] [PDF]
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A. S. Pappo, E. Lyden, J. Breneman, E. Wiener, L. Teot, J. Meza, W. Crist, and T. Vietti Up-Front Window Trial of Topotecan in Previously Untreated Children and Adolescents With Metastatic Rhabdomyosarcoma: An Intergroup Rhabdomyosarcoma Study J. Clin. Oncol., January 1, 2001; 19(1): 213 - 219. [Abstract] [Full Text] [PDF]
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P. J. Houghton, C. F. Stewart, P. J. Cheshire, L. B. Richmond, M. N. Kirstein, C. A. Poquette, M. Tan, H. S. Friedman, and T. P. Brent Antitumor Activity of Temozolomide Combined with Irinotecan Is Partly Independent of O6-Methylguanine-DNA Methyltransferase and Mismatch Repair Phenotypes in Xenograft Models Clin. Cancer Res., October 1, 2000; 6(10): 4110 - 4118. [Abstract] [Full Text]
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) and expected (
) responses for two RMSs (Rh12 and Rh18/VCR) and two NBs (NB-EB and NB-1771). Observed complete responses to single-agent vincristine (1 mg/kg; V1), topotecan administered at different dose levels (T.24 and others), or the combination (T.24/VCR) were plotted against expected responses from the model described in "Materials and Methods."
), topotecan (
), or the combination (