Rational Design of Irinotecan Administration Based on Preclinical Models
Most clinical drug regimens for irinotecan (CPT-11 [Camptosar]) have been empirically based on classic in vivo pharmacokinetic and pharmacodynamic considerations. We propose an alternative approach that attempts to
ABSTRACT: Most clinical drug regimens for irinotecan (CPT-11 [Camptosar]) have been empirically based on classic in vivo pharmacokinetic and pharmacodynamic considerations. We propose an alternative approach that attempts to provide a rationally designed schedule of irinotecan administration based on preclinical data. HL60 cells grown in suspension or as subcutaneously implanted solid xenografts in nude mice served as in vitro and in vivo models to test the activity of irinotecan or its active metabolite, SN-38. For SN-38, within an effective drug concentration range, scheduling drug administration based on duration of DNA synthesis inhibition significantly potentiated cell kill in vitro, and increasing drug concentrations at suboptimal scheduling did not result in additive cell kill. These data suggested that even though high drug doses may be attainable in vivo, they may not be required to achieve maximum antitumor activity. To test this hypothesis, a sensitive in vivo model to test the toxicity and antitumor activity of CPT-11 is required, which is provided in the human myeloid HL60 xenograft model grown in nude mice. In this model, CPT-11 at a dose 50 mg/kg, daily × 5 (MTD) achieved 100% complete tumor regression. This model should be useful to test the hypothesis that for irinotecan, administration of a minimum effective dose (MED) at an optimal schedule can achieve maximum antitumor activity and should therefore prevail over the classic approach of administering the MTD. [ONCOLOGY 12(Suppl 6):22-30, 1998]
Introduction
The antitumor activity of the plant alkaloid camptothecin has been known for more than 2 decades, but initial enthusiasm for this compound was tempered by the recognition of its severe side effects. (Hematologic toxicities are dose-limiting.)[1-5] The discovery that camptothecin perturbs the catalytic cycle of the topoisomerase I enzyme offered a novel target for chemotherapy and revitalized interest in this drug.[6-9]
Topoisomerase enzymes help regulate translation and transcription processes by controlling the topologic structure of DNA and relaxing the supercoiled DNA helix. The topoisomerase I catalytic cycle involves transient covalent binding of the enzyme to DNA (cleavable complex formation), which nicks one DNA strand. This, in turn, allows for DNA strand passage, religation of the DNA strand break, and subsequent release of the enzyme from the DNA. Consequently, the DNA helix is unwound, relieving torsional stress associated with, for instance, DNA replication. Camptothecin and most of its analogs act by mediating stabilization of the topoisomerase I/DNA cleavable complex,[8,9] initiating a series of events that ultimately lead to cell death.
The renewed interest in camptothecin resulted in the development of camptothecin derivatives, which display less severe side effects[10] and better water solubility than the parent compound. Topotecan (Hycamtin) and irinotecan (CPT-11 [Camptosar]) are two of the most prominent topoisomerase I-interactive agents for which antitumor activity has been evaluated in clinical trials.
The design of clinical drug regimens of irinotecan has been based on classic in vivo pharmacokinetic and pharmacodynamic considerations, which call for the administration of maximum tolerated doses (MTDs) for any given schedule. Preclinical in vivo data of the antitumor activity of irinotecan against human neuroblastoma xenografts, however, have indicated that antitumor activity depends more on the schedule of administration than on drug dose.[11]
Notwithstanding their empirical design, early phase II-III clinical trials evaluating the topoisomerase I-interactive agents topotecan and irinotecan have had encouraging results in patients with heavily pretreated secondary and refractory leukemias, showing indications of response in these otherwise poor responders.[12-16] Moreover, currently ongoing clinical trials in colorectal cancer combining irinotecan with fluorouracil (5-FU)/leucovorin have shown promising response rates of up to 60%.[17-19]
Many parameters relevant for studying the action of topoisomerase I-interactive agents have been identified. These include: catalytic activity, protein levels, and mutations of the topoisomerase I enzyme; topoisomerase I messenger RNA (mRNA) levels; and DNA-topoisomerase I protein cross-links.
Known resistance mechanisms against topoisomerase I-interactive agents include decreased topoisomerase I protein levels[20] due to posttranslational or posttranscriptional changes or gene rearrangements[21] and topoisomerase I mutations[22,23] affecting cleavable complex formation (decreased catalytic activity) or binding of topoisomerase I-interactive agent.[23,24] (Catalytic activity is preserved, but the topoisomerase I agent-induced stabilization of cleavable complex formation is reduced.) One mechanism specifically related to irinotecan resistance is a change in the activity of carboxylesterase,[25] the enzyme required for the metabolic conversion of irinotecan to its active metabolite, SN-38. Although many of the above resistance mechanisms have been described in different cell line models, their prognostic value for in vivo sensitivity to topoisomerase I-interactive agents has yet to be established.
The demonstration that (temporary) inhibition of DNA/RNA synthesis could protect from topoisomerase I agent-induced toxicity but not from drug-induced formation of protein/DNA cross-links has led to the "DNA-replication fork collision" model. This model assumes that the protein-DNA cross-links are prelethal lesions, which become lethal only when they encounter a moving DNA replication fork. This encounter results in irreparable double-strand DNA breaks[26-28] and chromosomal aberrations.[29-33]
Supporting evidence for this model comes from drug interaction studies of topoisomerase I- and topoisomerase II-interactive agents.[34,35] These studies have shown that simultaneous exposure of cells to camptothecin and etoposide results in antagonistic effects, which can be changed to additive effects if the drug exposures are separated by an interval that exceeds the time necessary to restore drug-induced inhibition of DNA and RNA synthesis.[35]
These data suggest that, to a certain extent, inhibition of DNA/RNA synthesis may provide a cell with an opportunity to restore the DNA/protein cross-link lesions before a fatal collision with DNA replication forks occurs. The fact that topoisomerase I- and topoisomerase II-interactive agents are effective as single agents despite concomitant drug-induced inhibition of DNA/RNA synthesis appears to be contradictory. We propose, therefore, that the extent and duration of DNA/RNA-synthesis must be taken into consideration.
Evidence that these parameters may play a role in ultimate drug-induced cytotoxicity may come from another study exploring the interaction between topoisomerase I- and topoisomerase II-interactive agents, which showed that simultaneous drug exposure results in drug synergism[36] rather than antagonism. In this study, very low concentrations of the drugs were used (< IC10, or the concentration inhibiting 10% of cell growth). One theory that may explain the two seemingly contradictory findings suggests that, at the low drug concentrations used in the latter study, the inhibition of DNA/RNA synthesis is insufficient to interfere with the moving replication forks and to affect drug efficacy.
This article describes a series of experiments designed to determine how exposure to topoisomerase I inhibitors (specifically, camptothecin and irinotecan) affect DNA synthesis, and how the extent and duration of this inhibition may be used to optimize scheduling of these drugs. In addition, a preclinical in vivo model is presented in which a 100% complete response (CR) rate can be achieved with irinotecan in a single-drug regimen. This model should prove to be useful to test the hypotheses developed from in vitro data in an in vivo setting.
Materials and Methods
Cell Lines and Xenografts
The human myeloid leukemia cell line HL60 was propagated in RPMI1640 media supplemented with 10% heat-inactivated fetal bovine serum. Cell cultures were maintained at exponential growth by maintaining cell densities below 1 × 106 cells/mL.
Xenografts were initially established by subcutaneous (SC) injection of 200 mL of a cell suspension (1× 107 cells/mL) in 8- to 12-week-old, female athymic nude mice (Sprague Dawley Inc., Indianapolis, Indiana). Subsequently, xenografts were maintained for several generations by SC transplantation of 50-mg nonnecrotic solid tumor tissue.
Drugs
Camptothecin and SN-38 for the in vitro studies were donated by BioNumerik Pharmaceuticals, Inc. (San Antonio, Texas). Stock solutions of each drug (5 mmol) were made in 100% dimethyl sulfoxide (DMSO) and stored at -20 °C until use. Drug dilutions were made from these frozen stocks with media to achieve the desired final concentrations. Irinotecan for the in vivo studies was donated by the Pharmacia & Upjohn Company (Kalamazoo, Michigan) as a sterile solution of 20 mg/mL in 0.9% saline.
Cell Kinetic Studies by Bivariate BrdU/DNA Labeling
Prior to SN-38 exposure, cells were labeled for 20 minutes with 10 µM of bromodeoxyuridine (BrdU). Following drug exposure, cell samples were obtained at specific time points: 30 min, 2h, 4h at 0h and 22h following drug exposure washed, fixed in aliquots of 1×106 cells in 70% ice-cold ethanol, and stored at -20 °C until analysis. Fixed samples for flow cytometric evaluation were prepared according to the instructions of the manufacturer of the anti-BrdU monoclonal antibody (Boehringer Mannheim, catalog no. 1202 693). Stained cell samples were analyzed with the FACScan flow cytometer (Becton Dickinson, San Jose, California) and WinList software (Verity Software House, Topsham, Maine).
3H-Thymidine Incorporation
3H-Thymidine incorporation, used as a parameter for DNA synthesis inhibition, was determined at specific time points specified above by incubating aliquots of 1 × 106 cells for 10 minutes in the presence of 1 mCi/mL of 3H-thymidine (Amersham). Cells were then washed twice with ice-cold Hanks Balanced Salt Solution, precipitated in 10% trichloric acid, and lysed in 0.4N sodium hydroxide. Radioactivity was determined using a Beckman LS1701 Scintillation Counter after resuspending 1-mL samples in 9-mL scintillation fluid (Polyfluor, Packard Chemical Co., Meriden, Connecticut).
In Vitro Assessment of Cytotoxicity
To perform an in vitro assessment of cytotoxicity as a function of exposure time and drug concentration, cells were resuspended in RPMI1640 + 10% fetal calf serum at a cell density of 0.5 ×106 cells/mL. Camptothecin and SN-38 were added from frozen stocks to achieve the final desired drug concentrations. Cell suspensions were then incubated at 37° C for up to 30 hours in a fully humidified atmosphere of 5% carbon dioxide. Aliquots were removed at 3-hour time intervals, washed twice with phosphate buffered saline, and plated in 96-well plates to assess cell survival. Cell survival was assessed following four cell doublings by the mitochondrial activity (MTT) assay, as described previously.[37]
Administration of Drugs and in Vivo Growth Inhibition Studies
Treatments were initiated at a tumor size of 150 to 200 mg. Irinotecan was administered via intravenous (IV) push at 50 mg/kg/d daily × 5. Tumor weight was measured with a Vernier caliper and calculated assuming unit density by the following formula: 1/2 (L × W²), where L is the length and W is the width in millimeters. Relative tumor volumes (RTV) were calculated using the following formula: RTV (%) = Vx/Vi ×100, where Vx represents the tumor volume on day x and VI the initial tumor volume.
Antitumor activity was assessed by the mean maximum inhibitory ratio (MIR) of the relative tumor volume of the treated mice over the untreated controls. Tumor doubling time (TD) was defined as the mean time for the tumor to reach twice its initial size. A partial response (PR) was defined as a > 50% reduction in tumor size and a complete response (CR) as the inability to detect tumor by palpation at the initial site of the tumor appearance for more than 60 days.
Results
In Vitro Cytotoxicity of Camptothecin and SN-38
In order to compare the potency of camptothecin and SN-38 against HL60 cells in vitro, the cytotoxicity of 24 hours exposure to each of the drugs was evaluated by the MTT assay. As shown in Figure 1, camptothecin and SN-38 were equipotent at equimolar concentrations in vitro. Shortening the exposure time to 2 hours decreased the potency of both drugs 10- to 20-fold (data not shown in figure).
DNA Synthesis Inhibition Following Exposure to Different Camptothecin Concentrations
Since camptothecin and SN-38 displayed similar behavior in terms of cytotoxicity against the HL60 cells in vitro, the initial studies regarding inhibition of DNA synthesis were performed using only the parent compound, camptothecin. As expected, following short-term exposure (30 minutes) to camptothecin, a concentration-dependent decrease in thymidine incorporation was observed. However, the dynamic evaluation revealed that thymidine incorporation resumed at the same time point (4 to 5 hours) following drug exposure regardless of the drug concentration and extent of initial inhibition. The ultimate level of restoration of thymidine incorporation, again, was dependent on drug concentration and extent of initial inhibition of thymidine incorporation (Figure 2).
Figure 3 shows the kinetic bivariate analysis of DNA content and BrdU label (cells were labeled before drug exposure) of HL60 cells following a 30-minute exposure to camptothecin. The first column (control) of Figure 3 demonstrates how the fate of the BrdU-labeled cells can be monitored over time. Up to the 4-hour time point, the cells are moving toward the G2/M compartment; at 8 hours, this trend continues, and the first cells that were in S phase during BrdU labeling are now reaching the G1 phase of the next cell cycle. Twenty-two hours after drug treatment, the cells have reached the S phase of the next cell cycle.
Data from the drug-treated cells (columns 2 through 5 of Figure 3) demonstrate that within 4 hours following drug exposure, cells that were in S phase during the camptothecin exposure (BrdU-labeled) undergo a concentration-dependent cell death. The appearance of the BrdU-labeled cells in the sub-G1 compartment suggests that the cell death occurs through an apoptotic pathway. For each drug concentration, even the highest one (10µM), a cell population is entering the S phase compartment at the 8-hour time point. The fact that the majority of these cells entering S phase are BrdU-negative (except for those exposed to the lowest drug concentration) indicates that these cells were in the G1 phase of the cell cycle during drug exposure.
Two important observations were made at the 22-hour time point:
The cells that entered S phase at the 8-hour time point were able to continue the cell cycle at least up to the G2 /M phase. (The G2/ M compartments that were emptied at the 8-hour time point are filled with BrdU-negative cells at 22 hours.)
The BrdU-negative population that entered the S phase at 8 hours seems to consist of a select population of "escapees." This is evidenced by the fact that entrance of the drug-treated cells into S phase is not a continuous process, as is observed in the control cells. (At 22 hours, there is a concentration-dependent appearance of a gap between the G1 and G2/ M compartments, indicating a blockade of S phase entry from G1.)
Effect of Dose Fractionation of Camptothecin on Cell Survival
The kinetic data on DNA synthesis inhibition and cell-cycle kinetics indicated the existence of a sanctuary compartment within the G1 cell-cycle phase that allowed the cells to escape short-term exposure to camptothecin. These data suggested that reexposure of cells at the right time interval following initial drug exposure (when the escapees enter S phase) could potentiate the toxicity of the drug treatment.
To test this hypothesis, HL60 cells were exposed to a single dose of 5 or 10 µM of camptothecin for 1 hour or to two exposures of 5 µM of camptothecin for 30 minutes spaced at several time intervals. Figure 4 demonstrates that the potency of a (10-µM/h) camptothecin treatment could be increased by fractionation into two 30-minute treatments of 5µM, and that this potentiation increased with increasing time intervals up to 8 hours.
Exposure Duration, Drug Concentration, and Cell Survival
The dose fractionation data demonstrated that, for short-term exposures, drug cytotoxicity could be significantly improved by timing subsequent exposures at optimal time intervals. Without proper timing, the drug could be present but was much less effective.
In order to determine the optimal exposure time for camptothecin and SN-38, a bivariate analysis was performed evaluating cell survival with various drug concentrations and exposure durations. Figure 5 shows the effect of various exposure durations on the IC50 (concentration inhibiting 50% of cell growth) of camptothecin and SN-38 against HL60 cells. With exposure durations of less than 6 hours, the IC50 concentrations are relatively stable, despite a greater than 100-fold increase in drug concentration. Furthermore, prolonging drug exposure time beyond 12 hours was no longer beneficial.
Thus, for both camptothecin and SN-38, a relatively narrow window is evident in which a more-or-less linear concentration × time relationship with cell survival exists. This window spans approximately 1 log of drug concentration and from 6 to 12 hours of exposure duration.
In Vivo Antitumor Activity and Toxicity of Irinotecan
The in vivo antitumor activity and toxicity of irinotecan against HL60 xenografts was next evaluated in the nude mouse. Maximum tolerated dose (MTD) of irinotecan for IV push administration daily× 3 was determined to be 100 mg/kg/d, while that of a daily × 5 schedule was 50 mg/kg/d. The antitumor activity of irinotecan was evaluated using the 50 mg/kg/d daily × 5 schedule.
Irinotecan was highly effective against the HL60 tumors, achieving 100% CRs in mice bearing both HL60 and HL60/ADR xenografts. Antitumor activity and toxicity are summarized in Figure 6 and Table 1. The data represent four independent experiments with five mice per treatment group.
For comparison, the in vivo response of the tumors to doxorubicin, administered at MTD concentrations for single IV push injection (10 mg/kg) and daily × 5 administration (2 mg/kg) is shown. Although the HL60 cells are sensitive to doxorubicin in vitro,[37] no response to the drug could be detected in vivo. In vitro, the HL60/ADR cells are highly (180-fold) resistant to doxorubicin because of the presence of the multidrug resistance-associated protein (MRP). In vivo, irinotecan was equally effective against the HL60 and HL60/ADR tumors (100% CRs), regardless of the presence of MRP. Expression of MRP in the tumor was confirmed by immunohistochemical staining (data not shown).
Discussion
Preclinical in vitro and in vivo studies (recently reviewed in reference 17) indicate that for most topoisomerase I inhibitors, continuous exposure significantly increases toxicity. However, in vivo long-term continuous infusions are less favored, generally because of toxicity and practical logistic considerations.
An alternative to long-term continuous infusions is dose fractionation and repetitive drug exposure. Our in vitro data confirm the findings of other studies showing that the topoisomerase I inhibitor camptothecin, serving as a representative of the camptothecin derivatives, is highly S phase-specific, and that the potency of a given dose can be increased by dose fractionation and appropriate scheduling.
The cell kinetic data obtained with the BrdU study reveal the existence of a sanctuary compartment within the G1 phase of the cell cycle, in which cells are not affected by the presence of a topoisomerase I inhibitor. The existence of such a sanctuary compartment is also suggested by the thymidine incorporation data, which showed that, despite the dose-dependent extent of inhibition, thymidine incorporation resumed at the same time point following drug exposure, regardless of the degree of inhibition. The entrance of the escapees into the more vulnerable S phase of the cell cycle may explain why continuous exposures and dose fractionation potentiate the cytotoxicity of these drugs.
The in vitro cytotoxicity data show that for both camptothecin and SN-38, there is a relatively narrow window in which an approximately linear relationship exists between cell survival and both drug concentration and exposure time. Outside of this window, extending drug exposure duration or increasing drug concentration has relatively little effect on cell survival. Mathematic modeling should be able to define the extent of this window more precisely.
These in vitro data support the in vivo findings of Thompson et al.[11] They demonstrated that the efficacy of irinotecan is highly schedule-dependent, and that, for a given schedule, concentrations lower than the MTD can still achieve the maximum effect. The notion that irinotecan, if administered according to an optimal schedule, does not have to be given at the MTD to achieve maximum antitumor activity requires the establishment of a minimum effective dose (MED). In this pursuit, an approach using the in vitro and in vivo models presented in this article should prove to be useful in the design of preclinical and clinical trials of irinotecan. With current knowledge, the drawback noted in an early review[38]--namely, that it is difficult for irinotecan to be used at full dose in combination chemotherapy--may not be relevant for drug efficacy.
Anticipated benefits of establishing MEDs for irinotecan are numerous and include decreases in side effects and cost. Our preclinical in vitro and in vivo data, combined with traditional pharmacokinetic data relating to effective drug plasma concentrations, should allow for the rational design of an irinotecan administration regimen.
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