Camptothecin Radiation Sensitization: Mechanisms, Schedules, and Timing

ABSTRACT: Based on high tumoricidal activity of the camptothecin analogs topotecan (Hycamtin), irinotecan (CPT-11[Camptosar]), and 9-aminocamptothecin (9-AC) in preclinical studies, clinical trials began testing these agents against human cancers. The cytotoxic activity of camptothecins in the clinic has been lower than predicted from the laboratory, however, and new approaches are needed. One method that holds promise is the use of the camptothecins as radiation sensitizers. The camptothecin dose, schedule, method of administration, and timing of administration, when given with irradiation, are likely to be important factors for these new S-phase radiation sensitizers. Phase I trials of the camptothecins as radiation sensitizers have begun, and multicenter phase II studies are planned by the Radiation Therapy Oncology Group (RTOG). One new approach based on preclinical observations that deserves clinical evaluation is chronomodulated camptothecin delivery with irradiation in order to widen the therapeutic window. [ONCOLOGY 12(Suppl 6):114-120, 1998]

Introduction

Recent Food and Drug Administration approval of the camptothecin analog irinotecan (CPT-11 [Camptosar]) for the treatment of colorectal cancer resistant to fluorouracil (5-FU)[1] has opened a new chapter in chemotherapeutic radiation sensitization. High interest in using this and other camptothecins (eg, topotecan [Hycamtin], 9-aminocamptothecin) in combination with irradiation is based in part on past successes with 5-FU, an antimetabolite, as a radiation sensitizer.[2] Both camptothecin analogs and antimetabolites have cytotoxic activity against S-phase cells, and both have a defined role in the treatment of colorectal cancer, a disease in which radiation sensitization has improved locoregional control and overall survival.[3]

Radiation sensitization with either class of agents is dose- and schedule-dependent,[4] and the importance of the timing of administration of these drugs when given with fractionated irradiation is a new factor that is gaining attention. This knowledge combined with new laboratory data will be important in the design of new camptothecin radiosensitizer trials.

Cytotoxic Mechanisms of Camptothecins and Irradiation

The molecular basis for the lethal effects of ionizing radiation alone include the production of single- and double-strand breaks in DNA.[5] Another basic observation regarding repair of x-ray- or chemotherapeutic-induced genomic damage is the requirement for topoisomerase I, which is widely used in DNA metabolism.[6]

In the presence of camptothecin, the camptothecin-topoisomerase I-DNA complex becomes stabilized because the 5¢-phosphoryl terminus of an enzyme-catalyzed DNA single-strand break is bound covalently to a tyrosine of topoisomerase I. Irradiation can create thousands of single-strand breaks per cell per gray, leaving these sites to be attacked by topoisomerase I in the presence of camptothecin. The rate of topoisomerase I binding to nicked DNA is also more rapid (increased by a factor of 800 to 1,000) than its binding rate to undamaged supercoiled DNA.[7,8] These stabilized complexes interact with the advancing replication fork during S-phase or during unscheduled DNA replication after genomic stress and cause the conversion of single-strand breaks into irreversible DNA double-strand breaks, resulting in cell death (Figure 1).[7,9]

Topoisomerase I may also compete with DNA repair complexes (DNA ligases, poly-(adenosine diphosphate)-ribosyl-transferase) for the single-strand breaks. In the presence of camptothecins, this can result in unrepaired DNA damage that can be recognized by the p53 damage-sensing pathway, initiating and possibly amplifying the apoptotic pathway of cell death.[7,10-12] The camptothecins have also been found to modulate apoptosis independently of DNA synthesis in postmitotic neurons[13] and confluent cell cultures,[10] as well as in actively proliferating cell cultures[14] and in murine tumors in vivo.[15]

High levels of topoisomerase I are associated with a high frequency of cleavable complex formation.[16] High topoisomerase I levels have been detected in surgical specimens from malignant melanoma, colonic, ovarian, esophageal, breast, stomach, and lung cancers, and in cultures from non-Hodgkin’s lymphoma and leukemia cells.[17] One basis for selective camptothecin toxicity in malignant cells compared to normal tissues may relate to these enzyme levels. An additional biological basis for selective camptothecin activity is the low pH found in cancers that can stabilize the closed (active) lactone ring form.[17,18]

In addition, DNA-topoisomerase I-camptothecin cleavable complexes affect the repair of potentially lethal damage in plateau phase cells (Figure 1).[7] In contrast, in log phase cells, lethality caused by camptothecin and irradiation also appears to be determined by effects on the cell cycle; ie, there appears to be differential phase specificity of cell killing by drug and irradiation. The camptothecins are considered to be S-phase agents since selective cytotoxicity is observed in S-phase,[14,16,18,19] while G₁-, G₂-, and M-phase cells are relatively spared following pulse exposure to camptothecin.[19] Elimination by aphidocolin of camptothecin-induced cytotoxicity and radiation sensitization is consistent with these S-phase-selective effects.[20]

An additional contribution to radiation sensitization by combination treatment may be the synchronizing effect of irradiation itself that preferentially kills G₂- through M-cells, thus leaving S-phase cells intact and subject to attack by camptothecin.[14,19]

Clinical Studies

The clinical basis for chemoradiation is cytotoxic cooperation between systemic chemotherapy and irradiation when chemotherapeutic drugs are given concurrently with fractionated irradiation. Clinical research has established the superiority of fractionated irradiation, which spares late toxic effects. When S-phase-specific agents are administered with conventional irradiation (eg, 2 Gy/d), this regimen becomes a form of accelerated treatment.[21] With such regimens, the dose-limiting toxicity may consist not only of the late morbidity of irradiation (eg, fibrosis and necrosis) but also enhanced acute toxicity expressed in the rapidly proliferating cell compartments. Thus, the pattern of dose application used in camptothecin radiation sensitization studies will likely play a role in their success or failure.

Topotecan

Topotecan is a water-soluble topoisomerase I inhibitor with cytotoxic activity in a variety of preclinical models. Topotecan exhibits schedule-dependency in vivo and has high cytotoxic activity with frequently repeated (daily) dosing schedules.[22,23]

In murine systems, there is evidence that reducing the dose intensity (by prolonging the drug administration schedule using a small amount with each treatment) provides a therapeutic advantage because of reduced host toxicity and equal or superior tumor responses. In clinical studies, the short plasma half-life of topotecan also suggests that prolonged drug exposure by infusion could be effective.[24] In a phase I trial, an escalating low-dose topotecan infusion was found to have an increased therapeutic ratio when compared to an intermittent dosing schedule.[24] Neutropenia is usually the dose-limiting toxicity of topotecan.

Phase II studies have shown that topotecan alone has cytotoxic activity in lung cancer with intermittent (daily × 5 every 21 days) dosing schedules,[25] as well as in lung cancer patients with topoisomerase II-refractory disease.[26] In advanced head and neck cancer patients, topotecan is well-tolerated and has single-agent activity similar to that of cisplatin (Platinol), 5-FU, and methotrexate.[27]

Decreased production or mutation of topoisomerase I can cause resistance to the cytotoxic effects of topotecan and other camptothecins. Active efflux of the camptothecins by P-glycoprotein-mediated transport may also contribute to resistance.[28]

Radiation Sensitization--Topotecan has demonstrated radiation-sensitizing properties in log and plateau phase cell cultures[29,30] and in murine fibrosarcomas in vivo.[31,32] Clinical trials have begun in patients with non-small-cell lung cancer and in patients with central nervous system tumors.

Clinical evidence of radiation sensitization with topotecan has been demonstrated in a dose-escalation trial in patients with locally advanced, inoperable non-small-cell lung cancer.[33]. In this trial, 12 patients received 60 Gy (2 Gy/d) of radiation plus topotecan delivered by bolus injection on days 1 through 5 and on days 22 through 26, beginning on the same day as irradiation. The initial dose level of topotecan was 0.5 mg/m²; dose levels of 0.75 and 1.0 mg/m² were also tested. Doses higher than 0.5 mg/m² were associated with relatively high acute hematologic and gastrointestinal toxicity.

Of the 12 patients, 5 survived (2 without evident disease) and 7 died of their cancer. Severe late pulmonary toxicity was not reported, but pneumonitis was noted.

The Radiation Therapy Oncology Group (RTOG) is evaluating topotecan plus cranial irradiation in patients with glioblastoma multiforme. The Children’s Cancer Group (CCG) is also evaluating this combination in children with pontine gliomas. In both of these trials, topotecan is given daily as a 30-minute infusion 30 to 120 minutes before irradiation.

Irinotecan

Irinotecan is a semisynthetic derivative of camptothecin that has shown wide antineoplastic activity in vitro and in vivo. Irinotecan treatment schedules have differed by country: 125 to 150 mg/m² once a week for 4 weeks, followed by a 2-week drug-free interval, has been used in the United States; 350 mg/m² once every 3 weeks has been employed in Europe; and 100 mg/m²/wk or 150 mg/m² every 2 weeks has been used in Japan. Differing treatment schedules using cytokine support for neutropenia or intensive loperamide therapy to counteract diarrhea have also been reported.[34]

These tolerable irinotecan regimens have produced median durations of response ranging from 5.6 to 10.6 months in colorectal cancer patients; disease stabilization occurs in 30% to 71%.[34] Response rates of 26% and 32% have been reported in previously untreated colorectal cancer patients[35,36]; these rates are higher than those reported for 5-FU-refractory patients (only 7% to 21%).[1]

Diarrhea, nausea, and vomiting are common toxicities of irinotecan therapy; other side effects include asthenia, abdominal pain, leukopenia, and neutropenia. In the US trials, at least one of these adverse events occurred in > 50% of patients.[34] Grade 3 or 4 toxicity develops in about one third of patients, and the most common grade 3 or 4 event is severe late diarrhea.

The mechanism underlying gastrointestinal toxicity after single or repeated daily doses of camptothecin is determined by the high S-phase fraction in rapidly proliferating cell populations, such as the intestinal mucosa. In addition, biliary excretion of the camptothecin conjugated with glucuronic acid is an important route of drug elimination.[18] Glucuronidase of intestinal microflora can cleave the camptothecin-glucuronide conjugate, releasing the drug in the intestinal lumen.[37,38] This correlates with the occurrence of late diarrhea.[39]

Irinotecan has significant activity in non-small-cell lung cancer,[40] and it has been combined with irradiation in phase I/II trials in Japan. Irinotecan is a prodrug that must be converted to an active form, SN-38, by carboxylesterase, which is found in liver, blood, and lung cancer biopsies.[41] Carboxylesterase (detected by immunostaining with an antihuman carboxylesterase polyclonal antibody and by indirect immunostaining) occurs in significantly higher levels in squamous cell carcinomas compared to adenocarcinoma cells (P < .05). Other studies in 10 human lung cancer cell lines have shown that SN-38 levels increase significantly over 24 hours, suggesting that human lung cancer cells efficiently convert irinotecan to SN-38.[41]

Radiation sensitization with irinotecan has been reported in two human lung cancer xenografts.[42] In these experiments, irinotecan was administered in nontoxic doses 1 hour prior to a single dose of irradiation. In other reports, radiation sensitization with the camptothecin occurred when the drug was given either during or after irradiation.[43]

In a clinical phase I/II trial using irinotecan with concurrent irradiation (60 Gy in 30 fractions over 6 weeks) in patients with non-small-cell lung cancer, the maximum tolerated dose of irinotecan was 60 mg/m² (by 90-minute intravenous infusion) when given weekly for 6 weeks (Table 1).[44] A follow-up phase II trial was conducted in 24 previously untreated patients with unresectable stage IIIA/IIIB non-small-cell lung cancer and high performance status who had varied histologic diagnoses and a median age of 60 years (range, 44 to 72 years).[45] Six planned courses of irinotecan were delivered in 71% of patients, and another 21% of patients received five courses. External-beam irradiation to the thorax (60 Gy) was completed in 88% of patients, and treatment was delayed in three patients because of fever or fatigue.

The overall objective response rate was 79%. Acute toxicities were limited to one grade 3 leukocytopenia and two grade 3 neutropenias. Other toxicities included three cases of grade 3 hypoxemia due to pneumonitis, two cases of temporary grade 3 esophagitis, and one case of grade 3 fever. Grade 3 or 4 diarrhea was not observed.

These authors concluded that weekly irinotecan and concurrent radiation therapy has activity in the treatment of locally advanced non-small-cell lung cancer. The high incidence of pneumonitis with this treatment combination needs further investigation.

Phase I trials are now under way using irinotecan plus irradiation in patients with advanced or recurrent colorectal cancer. In one study, a single weekly dose of 30 mg/kg of irinotecan is given intravenously in combination with infusional 5-FU (300 mg/m²/d) and concurrent pelvic radiotherapy (50 Gy).[E. Mitchell, personal communication, February, 1998] This approach builds on an existing standard practice of infusional chemoradiation and modifies the drug dosing scheme for irinotecan now commonly used in the United States.

A different chemoradiation approach based on phase I data with daily low-dose irinotecan is also being investigated by Saltz et al.[46] The rationale for this approach is that camptothecin-stabilized DNA-topoisomerase I-cleavable complexes are reversible, and, thus, frequent irinotecan dosing may favor production of these complexes. This theory is supported by the facts that the half-life of SN-38 is relatively long and that daily bolus injections can result in a concentration ´ time product similar to that of a continuous infusion.

Saltz et al administered irinotecan in a phase I fashion for 5 consecutive days for 2 weeks followed by a 1-week rest. In 20 previously treated patients with advanced tumors (16 with colorectal cancer), acute toxicities over the first two cycles (total duration, 6 weeks) were mild diarrhea and neutropenia. Two patients with colorectal cancer achieved a partial response, and six others had stable disease.

Grade 3 diarrhea and neutropenic fever were seen at the highest dose level (22 mg/m²/d), and dose escalation was stopped. This treatment schema delivers 88% of the amount of drug given in a more standard weekly × 4 schedule.

The investigators feel that less heavily pretreated patients may tolerate higher doses. A phase I chemoradiation study using this schedule with concurrent pelvic radiation therapy for locally advanced rectal cancer is under way.[L. Saltz, personal communication, February, 1998}

9-Aminocamptothecin

The rationale for combining 9-AC with irradiation is based on in vitro research with human colon and pancreatic cancer cell lines showing dose- dependency for cytotoxicity and radiation sensitization.[T. Rich, unpublished observations, 1995] Other reports have also indicated that 9-AC is a potent radiation sensitizer in vitro.[47]

The optimal dose and timing of either irradiation or 9-AC have been investigated in vivo using the MCa-4 mouse mammary carcinoma.[48] These studies clearly indicated that radiation enhancement is dependent on the dose and schedule of drug administration, as well as radiation fraction size (Figure 2).

For example, 9-AC given for 2 weeks with daily fractionated irradiation (28 Gy in 14 fractions) resulted in dose modification factors of 2.4 and 3.7 in animals treated with 0.5 and 2 mg/kg twice weekly for 2 weeks, respectively. Evidence of schedule-dependency came from an identical treatment schedule in which the total dose was 4 mg/kg; in this case, the treatment was more effective when 9-AC was given as a divided dose either twice or four times a week compared to only once a week (dose modification factors of 2.8, 2.6, and 1.7, respectively).[48] In contrast, the dose modification obtained with a single dose of 9-AC plus single-fraction irradiation was markedly reduced (dose modification factor, 1.11; Table 2).

These results indicate that, in this murine tumor, fractionation of both the radiosensitizer dose and irradiation itself produces a greater effect than large single doses. Optimal camptothecin radiation sensitization in the clinic would thus favor a schedule of frequently repeated (daily) injections or a continuous infusion.

Chronomodulated Chemotherapy

In clinical trials with either 9-AC or irinotecan, response rates have been lower than expected based on laboratory reports. Acute toxicities partly explain the relatively limited doses that can be used. These toxicities, in turn, are greater than predicted by murine studies, and these differences are partly due to the high tolerance of mice to camptothecin, as compared with that of humans.

An important chronobiologic difference exists between humans and murine models. Camptothecin is generally administered to humans and rodents during the daylight hours. The human gastrointestinal tract actively proliferates during this portion of the day, whereas proliferation is relatively low in mice.[49] Several phase I and II trials using chronomodulated chemotherapy have shown that drug toxicity depends on the time of day of administration, which provides strong support for a relationship between trough values of gastrointestinal epithelial cell DNA synthesis rates and reduced toxicity to anti-S-phase chemotherapeutic agents.

Direct evidence for the importance of chronoregulated chemotherapy comes from a multicenter, phase III trial conducted by Levi et al. In this trial, patients with metastatic colorectal cancer who were treated with chronoregulated infusional 5-FU plus folinic acid (peak doses at 2200 hours) and oxaliplatin (peak dose at 1800 hours) had a fivefold decrease in acute toxicity compared to those given identical chemotherapy over 24 hours with nonchronoregulated infusions (89% vs 18%; P = .05).[50] Despite the decreased toxicity in patients treated with chronoregulated therapy, these patients received significantly higher dose intensity during each treatment course, resulting in an improved objective response rate (53% vs 32%; P < .05), better carcinoembryonic antigen (CEA) tumor marker regression, and superior median survival (19 vs 14.9 months; P = .03. These data strongly support the need for continued research in the area of chronoregulated drug administration.

A phase I trial of chronoregulated infusional chemoradiation has been reported by researchers at the University of Florida.[51] In this trial, circadian infusional 5-FU was combined with pelvic irradiation (~50 Gy in 5 weeks). The dose of 5-FU varied from 250 to 325 mg/m², with daily peak doses administered at midnight with the use of a portable programmable pump. In 18 patients, the maximum tolerated dose was 275 mg/m²; this represented a 22% increase in 5-FU dose, compared to the standard chemoradiation dose of 225 mg/m² determined in a large randomized trial.

Also of interest in the University of Florida study were the results of histologic examination of the resected rectal cancer specimens. This evaluation showed a 29% pathologic complete response rate (no identifiable tumor), which is five to six times higher than that achieved with nonchronoregulated 5-FU infusional chemoradiation in similarly staged patients.[52] These data support the value of further examination of chronomodulated radiosensitization with camptothecins in preclinical and clinical models.

In the laboratory, we have examined the use of irradiation combined with camptothecin delivered at different times of the day, based on the hypothesis that chronomodulated therapy could be delivered at a time when it would be better tolerated. There is evidence from both the literature and our own observations that irinotecan is more toxic to the mouse at night when the animals are active and there is higher proliferation in the gastrointestinal tract.[53-55] We have also observed a striking circadian dependence of 9-AC toxicity, similar to the results obtained with irinotecan (Figure 3). These data provide a rationale for the examination of chronomodulated administration of irinotecan as a radiosensitizer in order to ameliorate acute toxicity. This approach is also feasible through the use of portable, programmable pumps that have been employed in the infusional 5-FU chemoradiation trials.

Summary

The radiation-sensitizing properties of irinotecan and other camptothecin analogs are beginning to be evaluated clinically. There is evidence from murine solid tumor models that a daily dosing schedule or a short infusion may be optimal for this class of chemotherapeutic agents. Schedules using small repeated doses of the camptothecins show high efficacy in murine models and are supported by cytokinetic and pharmacologic data. The repeated dose schedule, in turn, fits well with the radiobiological principles of fractionated irradiation. Preclinical evidence also suggests that daily chronomodulated camptothecin administration could be combined with daily-fractionated irradiation, which could lead to further gains in the clinic by reducing normal tissue toxicity. These ideas await confirmation in prospective clinical trials.

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