Extending Principles Learned in Model Systems to Clinical Trials Design

ABSTRACT: Clinical results with irinotecan (CPT-11 [Camptosar]) and other camptothecin derivatives in various cancers, although encouraging, have fallen short of the expectations predicted by preclinical models. One proposed explanation for this is that preclinical xenograft models do not predict for the sensitivity of human cancer. In this article, we describe the results of several studies suggesting that this explanation is incorrect. Instead, our results indicate that the discrepancy between clinical response rates and findings in preclinical models may be due to a failure to incorporate the principles learned from preclinical studies into the design of clinical trials. Our analysis suggests that if differences in host tolerance are taken into account, the xenograft models are quite accurate predictors of clinical response. Moreover, application of the principles derived from preclinical models to the design of clinical trials may significantly enchance clinical response rates. Thus, the camptothecin analogs provide a paradigm for better integrated, pharmacokinetically driven, preclinical and clinical development of new drugs. [ONCOLOGY 12(Suppl 6):84-93, 1998]

Introduction

Irinotecan (CPT-11 [Camptosar]), [7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxy-camptothecin], formerly known as CPT-11, is one of approximately five camptothecin derivatives currently under clinical investigation. Of these, irinotecan and topotecan (Hycamtin) (9-dimethylamino methyl-10-hydroxy camptothecin) have demonstrated significant clinical activity.

Given as a single intravenous dose, irinotecan caused objective responses in previously treated patients with sarcoma of the stomach, melanoma, colon adenocarcinoma, and non-small-cell lung (NSCLC) cancer.[1] Irinotecan, administered weekly by short infusion, has demonstrated antitumor activity against refractory or relapsed small-cell lung cancer (SCLC),[2,3] and responses have been measured in non-Hodgkin’s lymphoma, acute lymphocytic leukemia,[4] NSCLC,[5,6] and colon adenocarcinoma[7-11] with different schedules. In most studies, dose-limiting toxicity has been neutropenia and severe diarrhea.[12]

Despite these encouraging clinical results, however, it is clear that irinotecan (and other camptothecin derivatives) has not fulfilled the expectations predicted by results in preclinical models.[13-22] One explanation (unfortunately offered with increasing frequency) is that the "preclinical xenograft models do not predict for sensitivity of human cancer." In this article, we will discuss whether this explanation is, in fact, correct, or whether the difference between the response rates predicted by preclinical models and current clinical experience may be due to a failure to incorporate some of the principles learned from the models into the design of clinical trials.

Our analysis suggests that if differences in host tolerance (mouse vs human) are taken into account, xenograft models predict clinical response rates quite accurately. Furthermore, if the principles learned in preclinical models are incorporated into the design of clinical trials, they may significantly enhance clinical response rates. Thus, rather than providing an example of how preclinical results have failed to translate to the clinical setting, the camptothecin agents appear to offer a paradigm for better integrated, pharmaco- kinetically driven, preclinical and clinical development of new agents.

Materials and Methods

Immune Deprivation of Mice--Female CBA/CaJ mice, 4 weeks of age, were immune-deprived by thymectomy, followed 3 weeks later by whole-body irradiation (1,200 cGy) using a cesium-137 source. The mice received 3 × 10⁶ nucleated bone marrow cells within 6 to 8 hours of irradiation.[20] Tumor pieces of approximately 3 mm³ were implanted in the space of the dorsal lateral flanks of the mice to initiate tumor growth. Tumor-bearing mice were randomized into groups of seven before therapy was initiated.

Tumor Lines--Each of the xenografts used has been described previously.[20,22]

Growth Inhibition Studies--Each mouse bearing bilateral subcutaneous tumors received the agent when tumors were approximately 0.20 to 1 cm in diameter. The procedures (tumor growth measurement) have been reported previously.[23] Briefly, two perpendicular diameters were determined at 7-day intervals using digital Vernier calipers interfaced with a microcomputer. Tumor volumes were calculated, based on the assumption that the tumors were spherical, using the formula [ (p/6) × d³], where d is the mean diameter. Mice were observed for at least 12 weeks after treatment was started.

Irinotecan Formulation and Administration--Irinotecan powder, provided by The Pharmacia & Upjohn Company (Kalamazoo, Michigan), was dissolved in sorbitol (70% with water) and lactic acid. The irinotecan was heated at 65° C for 20 minutes and cooled to room temperature. The pH of the solution was adjusted to 3.9 by adding hydrochloric acid. The solution was diluted in sterile water to 20 mg/mL, filter-sterilized, and stored in foil-wrapped tubes (-20 °C). The drug was further diluted prior to administration (0.1 mL/20 g body weight).

Irinotecan antitumor activity was evaluated on a schedule of intravenous daily administration for 5 days per week for 1 week, or for 2 consecutive weeks [days 1-5, 8-12; abbreviated (d ×5)2] repeated every 21 days for 3 cycles of therapy {[(d ×5)2]3} over 8 weeks. Control mice received vehicle [0.26 mL of sorbitol (70% with water), 0.9 mg of lactic acid per mL, pH 3.9], diluted appropriately.

Sample Collection and Drug Analysis--Following a single intravenous dose of irinotecan (10 mg/kg), blood samples were collected from non-tumor-bearing mice (four animals per time point) at 0.125, 0.25, 0.5, 1, 2, 4, 8, and 10 hours after the dose. All samples were immediately centrifuged at 12,500g for 2 minutes on a tabletop centrifuge. Plasma proteins were precipitated by the addition of 200 µL of plasma to 800 µL of cold methanol (-30 °C), followed by vigorous agitation on a vortex mixer and centrifuging again at 1,250g for 2 minutes. The supernatant was decanted and stored at -70 °C until analysis.

Concentrations of irinotecan and its more potent metabolite SN-38 (7-ethyl-10-hydroxy-camptothecin) in plasma were determined by an isocratic high-pressure liquid chromatography (HPLC) assay with fluorescence detection; this technique has been described in detail previously.[23] The lower level of quantitation was 2.5 ng/mL for both irinotecan and SN-38. All calibrators and controls were prepared in murine plasma.

Pharmacokinetic Analysis--A four-compartment model using maximum likelihood estimation (ADAPT II) was fit simultaneously to the irinotecan and SN-38 plasma concentration data after intravenous administration.[24] Model parameters included volume of the central compartment for irinotecan and SN-38 (Vc), irinotecan elimination rate (k₁₀), irinotecan intercompartment rate constants (k₁₂, k₂₁), SN-38 elimination rate constant (k₃₀), carboxylesterase conversion of irinotecan to SN-38 (k₁₃), and SN-38 intercompartment rate constants (k₃₄, k₄₃). Irinotecan and SN-38 undergo linear disposition at intravenous doses < 20 mg/kg in mice; thus a linear rate constant (k₁₃) was used to represent hydrolysis by carboxylesterase.[25] Systemic clearance of irinotecan was calculated by Vc and the sum of k₁₃ and k₁₀, and SN-38 systemic clearance was calculated from Vc and k₃₀. Area under the irinotecan and SN-38 plasma concentration-time curve from zero to infinity (AUC_0-¥) was calculated using the log-linear trapezoidal method.[26]

Tumor Response and Statistical Analysis--For comparison of different treatment regimens, tumor responses were analyzed for: (1) the time (days) individual tumors required to reach four times their volume at initiation of therapy, and (2) tumor regression. The proportion of tumors that failed to reach four times their volume at the start of treatment was estimated by the Kaplan-Meier method,[27] in which time to reach four times the original volume was censored in mice that died for any reason. The exact log-rank test[28,29] was used to assess differences among treatment groups with respect to time for tumors to increase fourfold in volume, as described previously.[23]

For individual tumors, partial response (PR) was defined as a volume regression > 50% but with measurable tumor at all times. Complete response (CR) was defined as disappearance of measurable tumor mass at some point after therapy was initiated. Maintained complete response (MCR) was CR with-out tumor regrowth within the study period (usually 12 weeks). Procedures for evaluating intracranial tumor responses have been reported previously.[30]

Results

Schedule-Dependent Antitumor Activity

In human xenograft models, different camptothecin analogs demonstrate both qualitative and quantitative differences in antitumor activity. However, certain principles have emerged that may be more generally applied to this class of topoisomerase I poisons.

Our group[19,20] and others[31-33] have clearly shown that the antitumor activity of camptothecin agents is highly schedule-dependent. The duration of treatment appears to be critical for optimal antitumor activity. Thus, as shown in Figure 1, irinotecan administered to mice bearing a human neuroblastoma on the (d ×5)2 schedule is more effective than the same total dose given over 5 days [(d × 5)1]. Similar results have been obtained with 9-aminocamptothecin and topotecan.

Data in Figure 2 further substantiate the schedule-dependent activity of irinotecan. In this experiment, mice bearing a colon tumor were treated on the (d × 5)2 schedule at 40 or 10 mg/kg/dose for one cycle {[(d ×5)2]1}, or on the same schedule at lower dose levels, where cycles were repeated every 21 days {[(d ×5)2]3}. Two points are noteworthy:

On the [(d ×5)2]1 schedule, the antitumor activity of 10 and 40 mg/kg/dose was identical, suggesting self-limiting antitumor activity.

Much better responses were obtained with the [(d ×5)2]3 schedule, despite a lower total dose administered (400 vs 150 mg/kg total dose).

Similar results have been obtained with other camptothecin analogs that, unlike irinotecan, do not require activation. Since the cytotoxicity of camptothecins depends on active DNA replication (at least at pharmacologically achievable levels), the most likely explanation is that increased antitumor activity is a consequence of killing more cells in S-phase by giving the drug over a longer period.

Table 1, Table 2, and Table 3 summarize results for rhabdomyosarcoma, colon carcinomas, and pediatric brain tumor xenografts treated with the optimal schedule {[(d × 5)2]3} of irinotecan. Figure 3 shows the responses to different cytotoxic drugs of a childhood glioblastoma xenograft (SJ-GBM2) established from a previously treated patient. Each agent was administered at the maximum tolerated dose (MTD) to mice bearing SJ-GBM2. The only agents that had significant activity were irinotecan and topotecan, whereas doxorubicin, vin- cristine, and melphalan (Alkeran) were inactive.

Additional data from adult and pediatric brain tumors, as subcutaneous grafts, are presented in Table 4. Against intracranial implanted D456 (glioblastoma) or D341 (medulloblastoma) tumors, irinotecan enhanced survival time by 114% and 73%, respectively. The response rates of these tumors as xenografts clearly exceed the response rates of their clinical counterparts, when these have been determined (eg, colon carcinoma). Why, then, are the models overpredicting the clinical activity of irinotecan?

Systemic Exposures to Irinotecan and SN-38 and Antitumor Responses

A likely explanation is that mice tolerate much higher systemic exposures of irinotecan and SN-38 (and other camptothecins tested) than do humans. We estimate that the systemic exposure of SN-38 at the MTD in humans represents ~ 6% of the MTD in mice. Using in vitro clonogenic assays, Erickson-Miller et al[34] have shown that murine hematopoietic progenitors of the myeloid lineage from humans, mice, and dogs exhibit the differential sensitivity to camptothecins, with mouse cells being intrinsically resistant. Consequently, most of the results presented in Table 1, Table 2, Table 3, and Table 4 may be highly artificial if the systemic exposures associated with these dose levels exceed those tolerated by patients.

To address this issue, we determined the relationship between systemic exposure and tumor response in a series of neuroblastoma models.[23] Using the [(d × 5)2]3 schedule, the minimum daily dose that produced a CR varied between 1.25 and 5.0 mg/kg. Complete regressions of all tumors in four independent lines were obtained at 2.5 or 1.25 mg/kg/dose, whereas partial remissions were obtained at a dose level of 1.25 mg/kg in all six neuroblastoma models (Table 5). This dose was associated with a daily systemic exposure (AUC) of 396 ng · h/mL (range, 351 to 440 ng · h/mL) and 114 ng · h/mL (range, 99 to 129 ng · h/mL) for lactone forms of irinotecan and SN-38, respectively.[23]

Comparison of SN-38 lactone exposures for a 21-day course of irinotecan administered at a dose of 100 mg/m² every 7 days shows that an equivalent exposure over 21 days in mice is achieved at a daily dose of ~ 0.61 mg/kg using the [(d × 5)2] schedule.[35] Therefore, from Table 5, the model would prediºct a PR rate of ~ 50% if similar exposures could be achieved in patients.

Thus, against childhood malignancies, the models predict that systemic exposures achieved in patients should be adequate to induce a significant frequency of regressions in rhabdomyosarcoma, neuroblastoma, and childhood brain tumors. Data derived from adult glioma xenografts also suggest sensitivity, although minimal dose levels inducing regressions have not been defined in these models.

Phase I Clinical Evaluation of Irinotecan in Children

To test whether adequate systemic exposure can be achieved in children, we completed a phase I evaluation of irinotecan in patients with relapsed solid tumors. The schedule used in this trial was based directly on the (d × 5)2 schedule found to be optimal in xenograft studies.[36] Eligible patients were £ 21 years old and had a minimum life expectancy of 8 weeks, ECOG performance status < 2, and adequate major organ function.

A total of 23 evaluable children received one of three dose levels of irinotecan (20, 24, or 29 mg/m²/d) by 60-minute intravenous infusion daily × 5 weekly × 2 [(d × 5) 2] repeated every 21 days. No intrapatient dose escalation was allowed.

The median age of patients was 14.5 years (range, 2.75 to 21.25 years). The predominant tumor types were neuroblastoma (N = 5), osteosarcoma (N = 5), and rhabdomyosarcoma (N = 4). Patients received a total of 84 courses of therapy (median, 3 courses; range, 2 to 10 courses).

The dose-limiting toxicity (DLT) was grade 3 or 4 diarrhea and/or abdominal cramps, which occurred despite aggressive use of loperamide at the first onset of loose stools. Other nonDLTs included leukopenia, neutropenia, nausea, vomiting, and abdominal pain.

Clinically significant responses (partial responses ³ 50% decrease in product of tumor diameters) were seen in this very heavily pretreated group of children. Extensive pharmacokinetic studies were conducted after the first and tenth irinotecan dose in 19 children. The average SN-38 lactone AUC_{0-7 h} was 103.9 (.SD 54.2) ng ·.h/mL.

Thus, our preliminary study indicates that systemic exposure of SN-38 lactone can be achieved in heavily pretreated children using the same schedule of administration shown to be effective in xenograft models. Interestingly, as shown in Figure 4, when irinotecan is administered on this schedule, plasma SN-38 lactone exposures are significantly higher than have been reported in adults when irinotecan was administered every 7 days.[37]

Phase II Clinical Evaluation of Irinotecan in Adults With Brain Tumors

 Irinotecan has been evaluated at Duke University on a more standard schedule of 125 mg/m²weekly for 4 weeks followed by a 2-week rest.[38] This study involved 60 patients with recurrent or progressive malignant gliomas, including 50 with glioblastoma multiforme, 8 with anaplastic astrocytoma, and 2 with anaplastic oligodendroglioma.

Partial responses were seen in 10 of 49 evaluable patients with glioblastoma and 1 of 8 evaluable patients with anaplastic astrocytoma. In addition, three patients with glioblastoma had minimal responses, and 13 patients (10 with glioblastoma and 3 with anaplastic astrocytoma) demonstrated stable disease beyond two cycles (range, 18 to 36+ weeks). Toxicity was limited to infrequent grade 3 myelosuppression and diarrhea.

These preliminary results suggest that irinotecan, when given on this schedule, has significant activity against recurrent brain tumors in adults.

Discussion

In these investigations, we have attempted to apply principles learned in preclinical models to the therapeutic use of the camptothecin analog irinotecan. Studies using mice bearing human tumor xenografts have shown that the antitumor activity of camptothecin drugs is highly schedule-dependent, and self-limiting at high dose levels. Furthermore, data not presented here indicate that there is a sharp decrease in antitumor activity as irinotecan dose is reduced. Thus, these results suggest that a minimum threshold exposure must be achieved for any antitumor activity to occur, and that, above some concentration, no further tumor cell killing is achieved.

This is consistent with the proposed mechanism of action; specifically, that poisoning of topoisomerase I is lethal only to cells in the S-phase of the cell cycle. Thus, protracted exposures of camptothecin drugs would appear to optimize the probability of killing S-phase cells. However, as the period of therapy is protracted, the daily dose must be reduced because of toxicity. Consequently, there is a balance between optimizing the duration of therapy without reducing the daily systemic exposure to a level below the threshold for antitumor activity.

Our studies using a variety of xenografts derived from both pediatric and adult malignancies have shown that daily treatment for 10 days with cycles repeated at 21-day intervals produces optimal antitumor activity of irinotecan. This schedule is significantly better than schedules in which irinotecan is administered at high dose levels at 21-day intervals or over 5 days out of every 21-day cycle.

Are Systemic Exposures That Cause Tumor Regression Achievable in Humans?

One remaining question that cannot be predicted from the xenograft studies is whether systemic exposures to camptothecins that cause tumor regression in mice can be achieved in patients. Our data show that mice tolerate far greater systemic exposures of irinotecan and SN-38 than do patients. Thus, tumor responses determined at the mouse MTD may significantly overpredict the clinical utility of this agent. For irinotecan, a prodrug that requires metabolic activation, potential species differences pose an additional complexity for accurate translation of preclinical data.

To overcome some of these problems, we have attempted to equate the responses of human tumors to systemic exposure of SN-38 lactone, rather than responses at the MTD in mice. Using the (d × 5)2 schedule, the dose giving similar exposures of SN-38 lactone in mice and patients is ~ 0.61 to 1.25 mg/kg/d. This gives an exposure of ~100 ng.h/mL for SN-38 lactone. From the antitumor activity of irinotecan in the xenograft models, we would anticipate a response rate of ~ 50% in childhood neuroblastoma, and from two of eight tumor xenografts that we have studied, we would expect a response rate of ~ 25% in colon cancer. The latter response rate appears to be consistent with emerging clinical results.

If we apply the same type of analysis to topotecan, a similar pattern emerges. We originally reported significant activity of topotecan in childhood rhabdomyosarcoma models,[39] and subsequently against medulloblastoma and neuroblastoma xenografts.[20,30,40] Clinically, these histiologic types are sensitive to topotecan when administered (d × 5)1 every 21 days, with response rates of ~40% to 50% reported.[References 39, 41, and unpublished results of the Intergroup Rhabdomyosarcoma Study, April, 1998] Of interest is the observation that xenografts of alveolar rhabdomyosarcoma are more sensitive than the embryonal subtype; a similar pattern of responses is seen clinically.

Furthermore, at systemic exposures of topotecan lactone reported in adult trials, human colon tumor xenografts are quite insensitive to this agent, again paralleling the clinical findings. At greater systemic exposures, some responses are observed in the models, which may predict some activity against these tumors if more intensive therapy is used in patients.

Thus, the preclinical models predict activity of irinotecan in rhabdomyosarcoma, neuroblastoma, and certain pediatric brain tumors. Models of adult glioblastoma also indicate sensitivity, although optimal scheduling and the relationship between systemic exposure and response have not been established.

Results of Clinical Trials

In this article, we have presented initial results from two clinical trials. We initiated a clinical phase I trial of irinotecan in children that simulated the optimal schedules in mice. Our data suggest that systemic exposure of SN-38 lactone associated with tumor responses in preclinical models can be achieved at dose levels that are tolerated by children. Our results indicate that the systemic exposure to SN-38 lactone, which given on the daily schedule at relatively low doses, is significantly greater than that reported from the adult studies in which a higher dose was administered every 7 days. Although the true efficacy of this schedule must be determined in a formal phase II trial, the incidence of responses in a population of heavily pretreated patients in our phase I trial is encouraging.

Similarly, a second phase II trial suggested that irinotecan given weekly appears to have some activity against recurrent glioblastoma in adults. Again, such results are encouraging, but must be confirmed in subsequent phase II trials.

Of importance will be clinical experiments that seek to validate or refute the value of the approach proposed here. Specifically, the models would predict that the (d × 5)2 schedule is more active than the (d × 5)1 schedule, which, in turn, is superior to a q7d or q21d schedule. This hypothesis could be tested clinically in randomized trials.

Conclusions

Our investigations suggest that, through better integration of preclinical and clinical studies, a new paradigm for drug development may be established. Specifically, prediction of clinical utility from studies of efficacy in human xenograft murine models will always be difficult because of differences in host tolerance, limiting organ toxicities, and metabolism. The approach we have taken, to equate the responses of human tumors in mice to drug systemic exposure, rather than to host toxicity, has applications beyond camptothecin analogs. Potentially, comparison of mouse-derived exposure-response data with the systemic exposure at the MTD defined in phase I clinical trials may be valuable in determining further development for many new anticancer agents.

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