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Extending Principles Learned in Model Systems to Clinical Trials Design

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 (AUC0)
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?

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