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Cancer Therapy: Clinical |
Authors' Affiliations: Departments of 1 Medicine, 2 Epidemiology, 3 Pharmacology, and 4 Biostatistics, Roswell Park Cancer Institute; and 5 Department of Medicine, University at Buffalo School of Medicine and Biomedical Sciences; 6 Department of Pharmacy Practice, University at Buffalo School of Pharmacy and Pharmaceutical Sciences, Buffalo, New York; 7 Sabinsa Pharmaceutical, Inc., Piscataway, New Jersey
Requests for reprints: Marwan Fakih, Department of Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: 716-845-3362; Fax: 716-845-8008; E-mail: marwan.fakih{at}roswellpark.org.
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Abstract |
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Experimental Design: Selenomethionine was given orally as a single daily dose containing 2,200 µg of elemental selenium (Se) starting 1 week before the first dose of irinotecan. Irinotecan was given i.v. once weekly x 4 every 6 weeks (one cycle). The starting dose of irinotecan was 125 mg/m2/wk. Escalation occurred in cohorts of three patients until the MTD was defined. Pharmacokinetic studies were done for selenium and irinotecan and its metabolites.
Results: Three of four evaluable patients at dose level 2 of irinotecan (160 mg/m2/wk) had a dose-limiting diarrhea. None of the six evaluable patients at dose level 1 (125 mg/m2/wk irinotecan) had a dose-limiting toxicity. One patient with history of irinotecan-refractory colon cancer achieved a partial response. The long half-life of selenium resulted in a prolonged accumulation towards steady-state concentrations. No significant changes in the pharmacokinetics of CPT-11, SN-38, or SN-38G were identified; however, the coadministration of selenomethionine significantly reduced the irinotecan biliary index, which has been associated with gastrointestinal toxicity.
Conclusions: Selenomethionine at 2,200 µg/d did not allow the safe escalation of irinotecan beyond the previously defined MTD of 125 mg/m2. None of the patients receiving 125 mg/m2 of irinotecan had grade >2 diarrhea. Unexpected responses and disease stabilizations were noted in a highly refractory population. Further escalation of selenomethionine is recommended in future trials to achieve defined protective serum concentrations of selenium.
Given the favorable epidemiologic data and the decreased chemotherapy-related toxicity associated with selenium-replete patients, our group has investigated the utility of high-dose selenium supplementation with chemotherapy in preclinical models. We have shown that the organic selenium compounds selenomethionine and methylselenocysteine selectively modulate the therapeutic efficacy of anticancer drugs in human xenograft models (16). Both selenium compounds reduced the toxicity of irinotecan as evidenced by the ability to administer 2- to 3-fold the maximum tolerated dose (MTD) of irinotecan when mice were treated with daily selenomethionine or methylselenocysteine starting 1 week before chemotherapy (16). Although a dose of irinotecan of 200 mg/mouse was associated with 60% lethality, the addition of selenomethionine at 0.2 mg/mouse/d (starting 1 week before chemotherapy) was associated with 0% lethality (16). To ensure that chemotoxicity attenuation is not associated with a negative effect on antitumor activity, we evaluated the activity of equal doses of irinotecan with or without methylselenocysteine (duplicated for selenomethionine) in several xenograft models (16). The addition of methylselenocysteine (or selenomethionine) was associated with an increased cure rate across all tested tumors (16).
We, hereby, report the results of a phase I study of high-dose selenomethionine (2,200 µg Se) given orally once daily in combination with escalating doses of irinotecan. The dose of selenomethionine was extrapolated from the Watchful Waiting study, a study of a high dose of selenized yeast in patients with biopsy-proven prostate cancer (17). On this study, patients were treated with 3,200 µg/d Se in the form of selenized yeast daily for periods exceeding 1 year without the development of any serious adverse events. The speciation of the yeast used on this study showed that 70% of Se was present as a selenomethionine component. Extrapolating for the above, we considered that a 2,200 µg dose of Se in the form of selenomethionine daily would be considered as a relatively safe dose that could be assessed as a toxicity modulator of irinotecan.
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Materials and Methods |
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Patient criteria
Patients with a histologically or cytologically confirmed solid tumor that was metastatic or unresectable and for which standard curative or palliative measures did not exist or for whom single-agent irinotecan constituted a reasonable treatment option were eligible for the trial. The last chemotherapeutic or radiation treatment was at least 4 weeks (6 weeks for nitrosureas or mitomycin C) before trial enrollment. Other criteria included age 
18 years of age, Eastern Cooperative Oncology Group performance status of 
1, estimated life expectancy of >12 weeks, no central nervous system involvement, adequate bone marrow function (neutrophils 1,500/µL, hemoglobin 8.0 g/dL, platelets 100,000/µL), adequate hepatic function (serum bilirubin less than or equal to the upper limit of reference range, serum aspartate aminotransferase and alanine aminotransferase 2.5 x upper limit of reference range), and adequate renal function (creatinine 1.5 upper limit of reference range or creatinine clearance 60 mL/min). The study excluded patients unable to receive oral medications, patients with brain metastases, patients with a history of Gilbert's syndrome, and patients with active inflammatory bowel disease or chronic diarrhea. HIV-positive patients were not eligible because of possible pharmacokinetic interaction with antiretroviral drugs. Patients with grade 2 neuropathy were not eligible, as no safety data were available regarding selenomethionine effects in this patient population and because of concern about possible exacerbation of neurotoxicity. Patients with reproductive potential had to agree to use adequate contraception before study entry and for the duration of study participation. The study and consent form were approved by the Institutional Scientific and Review Committee and the Institutional Review Board before its activation. All patients provided signed informed consent before study entry. The study was conducted in accordance with the Good Clinical Practice Guidelines as issued by the International Conference on Harmonization and the Declaration of Helsinki.
Study design and treatment plan
Three patients were entered at each dose level. In the absence of dose-limiting toxicity (DLT), the next dose level was explored. If DLT was seen in one patient, three further patients were added at that dose level, and if no additional DLT was seen, escalation to the next dose level occurred. If at least two patients had DLT at a given dose level, accrual to that dose level was stopped; this was the maximally administered dose. Further patients were then added, as required, to the previous dose level (and if necessary, to lower dose levels) to establish the highest dose at which less than two of six patients had DLT. This was the MTD.
Patients received irinotecan in 500 mL of normal saline over 90 minutes once weekly x 4 every 6 weeks. Patients were medicated with 10 mg dexamethasone and 8 mg ondansetron i.v. before irinotecan.
The first dose level of irinotecan was 125 mg/m2, which is the recommended dose of single-agent weekly irinotecan (18). The second dose level of irinotecan was 160 mg/m2, and subsequent dose levels were to be escalated by 30% until the MTD was defined. No intrapatient escalation was allowed. Selenomethionine (manufactured by Sabinsa Pharmaceutical, Inc., Piscataway, NJ) was given at a fixed dose of 2,200 µg (Se) orally daily starting 1 week before the first dose of irinotecan at all investigated dose levels.
A DLT was any of the following attributable to study treatment on cycle 1: any nonhematologic grade 3 or 4 toxicity, with the exception of grade 3 diarrhea lasting <24 hours; any grade 4 thrombocytopenia or any grade 3 thrombocytopenia lasting >6 days; any grade 4 neutropenia lasting >6 days or any grade 4 neutropenia associated with fever; any dose delay secondary to toxicity that lasts 2 weeks or results in giving less than three of the four scheduled weekly irinotecan treatments on the first cycle.
Dose modifications for irinotecan were required for grade 2 toxicities (Table 1). Treatment was interrupted for any grade 3 toxicity; missed treatments were not made up. A cycle was not to be started, unless the absolute neutrophil count recovered to 1,500/mL and the platelets to 100,000/mL and nonhematologic treatment-related toxicities to grade 1. Patients were instructed to take 4 mg Imodium orally at the onset of diarrhea and 2 mg every 2 hours, until diarrhea resolved. No growth factors were allowed on the study with the exception of recombinant erythropoietin.
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Pharmacokinetics: sample collection, preparation, and analysis
Sample collection. Blood, for Se pharmacokinetic determinations, was collected in trace element free tubes and drawn on days 1, 2, 8, 15, 29, and 50 (also subsequently when possible). Multiple samples were drawn on day 8 (week 1) and again on day 29 (week 4; where possible) for complete pharmacokinetic studies of irinotecan and Se. Blood, for irinotecan pharmacokinetic determinations, was collected in a separate heparinized collection tube.
Selenium measurements. Selenium in plasma was measured by atomic absorption spectrophotometry using a PC-based ZL4100 Atomic Absorption Spectrophotometer equipped with autosampler. Plasma was diluted 1:5 in a diluent consisting of 0.4% nitric acid, 0.2% Triton X-100, 1% Pd(NO3)2, and 0.1% Mg(NO3)2 before a 20-µL injection. The matrix modifiers Pd(NO3)2 and Mg(NO3)2 decrease the volatility of Se, prevent its loss during thermal pretreatment, increase the volatility of matrix components, and promote their removal before atomization. Matrix-matched analytic standards were prepared and identically treated with the diluent consisting of the matrix modifiers. Standard curve range was 40 to 800 ng/mL. The graphite furnace program consisted of a two-step drying at 110°C and 130°C, pyrolysis at 1,300°C, atomization at 2,100°C, and clean out at 2,400°C. The method was validated. Quality control samples consisting of plasma added to selenium at two different concentrations [one at the high end of the standard curve (500 ng/mL) and one at the low end of the standard curve (150 ng/mL)] were used to ensure quality assurance throughout the study. The quality control samples were prepared in bulk at the time of assay validation, aliquoted, stored at –20°C, and assayed with the unknowns at time of patient sample analysis. If the observed quality control sample values deviated by >15% of the expected for the high quality control sample and >20% for the quality control sample near the limit of quantification, the assays were typically rerun.
Measurement of irinotecan (CPT-11), SN-38, and SN-38G. A validated high-performance liquid chromatography method with fluorescence detection was used to measure CPT-11. The method was a modification of that described by Warner and Burke (20). Campothecin was the internal standard. The ratio of the peak areas for the irinotecan (CPT-11) and internal standard and that for SN-38 and internal standard were used for quantification. The limit of quantification for both was 2.5 ng/mL. Quality assurance was maintained by simultaneously assaying the quality control samples prepared in bulk, before assay validation. CPT-11 and SN-38 from plasma were extracted with acidified methanol. The residue after evaporation of methanol was dissolved in 3% triethylamine acetate (pH 5.5) and acidified methanol (50:50) and injected on to high-performance liquid chromatography. Separation was carried out on a Waters Nova-Pak C18 column equipped with µBondapak C18 guard column, with mobile phase consisting of 20% acetonitrile and 80% of a 3% triethylamine acetate solution (pH 5.5). The detection was by fluorescence, with excitation at 370 and emission at 510 nm. Our adaptation of the method measures total irinotecan and SN-38 in the lactone form. For measurement of SN-38G, plasma was preincubated with 1,000 units ß-glucuronidase for 2 hours at 37°C, before extracting the plasma, and subjecting it to high-performance liquid chromatography. The ß-glucuronidase incubation of plasma resulted in the deconjugation of the glucuronide moiety from SN-38, thus giving the total SN-38 (SN-38 + SN-38G). The difference between the total and the SN-38 was a measure of SN-38G.
Pharmacokinetic data analysis. Pharmacokinetic analysis of the concentration-time data was conducted with standard noncompartmental methods as implemented in WinNonlin (Professional Version 5.0, Pharsight Corp., Lexington, KY). Area under the concentration-time curve (AUC) was determined by the trapezoidal rule. The terminal elimination rate constant (ke) was estimated by weighed least squares linear regression of the concentrations in the terminal elimination phase. Half-life was computed as the quotient of the natural log of 2 and the elimination rate constant. The maximum observed concentration (Cmax), and time of Cmax (Tmax) was determined by visual inspection of the raw data. Any samples that fell below the lower limit of quantification (BLQ) of the bioanalytic assay that occurred before the achievement of the first quantifiable concentration (>BLQ) was assigned a concentration of 0. Input values for dose, time of dose, plasma concentrations, and corresponding real-time values based on drug dosing times were used whenever possible. Summary statistics of pharmacokinetic variables and comparisons between irinotecan pharmacokinetic variables by study period (with or without selenomethionine) were accomplished using SAS statistical software (PROC MIXED, SAS version 8.02, Cary, NC).
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Results |
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Toxicity
All patients were evaluated for toxicity. Only grade 2 toxicity data were collected and reported. Treatment-related grade 2 to 4 toxicities are summarized in Tables 3 and 4.
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Nonhematologic toxicity. The most common grade 2 nonhematologic adverse event was diarrhea (five patients). One patient on each dose level developed grade 2 diarrhea, whereas three of four patients on dose level 2 developed grade 3 diarrhea. Nausea and vomiting (grade 3) was seen in one patient at dose level 2. Dehydration was present only in the setting of moderate to severe diarrhea. Nonhematologic toxicities are detailed in Table 4.
Selenomethionine toxicity. Selenomethionine was well tolerated in all patients. The only toxicity attributed to selenomethionine was garlic-like odor (breath and urine). This was relatively mild and noted in six patients, usually after receiving the morning dose of selenomethionine. The odor tended to ameliorate or disappear with prolonged treatment. None of the patients or their families voiced a complaint from this side effect.
DLTs, MTD, and recommended dose
No DLTs were noted in the first three patients at dose level 1. Three patients of four on dose level 2 experienced DLTs. These consisted of grade 3 diarrhea in two patients and grade 3 diarrhea, nausea, vomiting, dehydration, and fatigue in one patient. Dose level 2 was declared intolerable, and dose level one was expanded to a total of six evaluable patients, none of whom developed a DLT. Dose level 1 was declared the MTD.
Antitumor activity
Eight patients were assessable for response. One patient with metastatic colon cancer had a confirmed partial response but eventually progressed after 10 cycles of treatment (Fig. 1). This patient had previously progressed with enlarging hepatic metastases within 2 weeks from completing four cycles of FOLFIRI (5-fluorouracil, leucovorin, and irinotecan). She subsequently had liver metastases resection and hepatic intra-arterial chemotherapy with floxuridine. She had disease recurrence, which was treated with FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin) with progression after 2 months of initiation of this therapy. She then received palliative radiation therapy to enlarging porta hepatic lymph nodes. Her disease progressed after 3 months following radiation therapy, at which point she enrolled on this study. A patient with gemcitabine-refractory pancreatic cancer with liver metastases had confirmed stable disease that is ongoing (received eight cycles thus far). Stable disease was also confirmed in a colon cancer patient (three cycles followed by resection; patient free of disease for >1 year), a metastatic gastric cancer (progressed after seven cycles), and a non–small cell lung cancer (progressed after four cycles).
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Selenium pharmacokinetics. The overall accumulation of selenium in the plasma of patients receiving selenomethionine is shown in Fig. 2. As evident in the figure, selenium accumulation was significant in all patients, starting from a median (range) baseline value of 169.8 ng/mL (138-206 ng/mL). The rate of selenium accumulation was slow, with most patients not reaching steady-state concentrations for at least 4 to 6 weeks. The median steady-state plasma selenium concentration was 932 ng/mL and ranged from 540 to 1,609 ng/mL. Due to the long half-life and slow rate of accumulation, patients did not achieve the 14 µmol/L (1,100 ng/mL) target plasma concentrations before treatment with irinotecan.
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, where tau is the dosing interval, and A is the ratio between the trough concentration following the first dose of selenomethionine and the last measured trough concentration in each patient. The estimated mean (SD) half-life of selenium was 183 (94) hours. Thus, the time required to reach steady-state plasma selenium concentrations, on average, is 
1 month. Irinotecan pharmacokinetics. In addition to studying selenium pharmacokinetics, we also evaluated the pharmacokinetics of irinotecan and its SN-38 and SN-38G metabolites. A summary of the pharmacokinetics by week is shown in Table 5, and variables for each individual patient are shown in Table 6.
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26% lower at week 4 compared with week 1 (Table 5), but this did not reach statistical significance. Although we could not confirm an interaction between selenium and SN-38G, the trend towards a lower clearance and hence a larger AUC should be evaluated in larger studies.
Increasing biliary index values have been associated with higher rates irinotecan gastrointestinal toxicity (21–23) and is computed as (AUCCPT11 · AUCSN38) / AUCSN38G. The arithmetic mean irinotecan biliary index was reduced by 46% following administration of selenomethionine (Table 5). By repeated measures mixed effects modeling, the least squares mean biliary index at weeks 1 and 4 were 4,401 versus 3,416, respectively (P = 0.04). One patient studied only at week 1 had a very high biliary index (patient 4; Table 6). The statistical analysis was repeated with this patient excluded from the data set, and the results remained statistically significant. Thus, the conclusion of statistical significance for the observed changes in biliary index is not dependent upon this one individual.
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Discussion |
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We were unable to escalate irinotecan beyond the previously defined MTD of 125 mg/m2. Weekly doses of 160 mg/m2 were deemed nontolerable with three of four patients developing dose-limiting diarrhea, consistent with the previously defined DLT of irinotecan. The MTD of the combination of irinotecan and selenomethionine on this study was defined at 125 mg/m2 weekly x 4 every 6 weeks in combination with selenomethionine at 2,200 µg/d Se. It is interesting to note that minimal toxicities were noted at dose level 1 of irinotecan of 125 mg/m2. Of significance is the minimal gastrointestinal toxicity noted at this dose level with no grade 3 diarrhea in all six patients. This contrasts with the known historic grade 3 diarrhea of 30% described with single-agent irinotecan (25–28). However, we cannot rule out that the low rate of grade 3 toxicities at the MTD are secondary to chance given the small sample size of six patients. No significant alterations in CPT-11, SN-38, or SN-38G pharmacokinetics were identified following the administration of selenomethionine. A reduction in irinotecan biliary index was noted with concurrent selenomethionine administration. However, this was of borderline statistical significance and should be interpreted with caution given the small sample size. The biliary index has been reported to correlate with irinotecan gastrointestinal toxicity, with increasing values resulting in an increased risk of toxicity (20, 23). Our finding of a reduced biliary index with selenomethionine administration is consistent with the lower than expected incidence of grade 3/4 diarrhea at the 125 mg/m2 dose.
To our knowledge, this is the first intensive pharmacokinetic study of selenium following administration of selenomethionine. Selenium exhibited a longer-than-expected half-life, resulting in prolonged accumulation, taking 4 to 6 weeks to approach steady-state concentrations. Given the available data and study design, which included two intensively sampled study periods, and the unexpectedly long half-life, a precise estimate of half-life could not be determined. Future studies that incorporate sampling during a washout period after discontinuation of selenium would be useful to improve the accuracy of the half-life estimate. Based on the accumulation of trough concentrations, the half-life of selenium in humans is >7 days. Clearance of selenium was similarly slow, and the pharmacokinetic variability of selenium in this study was modest. As mentioned above, the slow accumulation prevented us from achieving the preclinical target concentrations, and larger doses, including a loading dose, would likely be useful to achieve the target concentrations more rapidly.
Selenomethionine was well tolerated with minimal garlic-like breath and urine odor in six patients. Garlic odor is attributed to the breakdown of selenomethionine into dimethylselenol (breath) and trimethyselenonium (urine; ref. 29). These toxicities were mild, non–dose limiting, and resolved promptly upon discontinuation of study treatment.
This is the first study that established the safety of administering high doses of selenomethionine in humans. To our knowledge, doses exceeding 800 µg (of pure selenomethionine) had not been tested previously. We have shown that a dose of 2,200 µg Se in the form of selenomethionine can be given safely in cancer patients for periods up to 13 months. This supports the feasibility of investigating high doses of selenomethionine in combination with chemotherapy in patients with advanced malignancies. These results cannot be extrapolated to the prevention setting, as the longer-term effects (years of supplementation) of high-dose selenomethionine supplementation have not been adequately evaluated.
We have noted one partial response on this study in an irinotecan-refractory patient with metastatic colon cancer. This patient achieved the highest plasma selenium concentrations (steady-state level 30 µmol/L) among the enrolled patients, likely secondary to her low body weight of only 40 kg. Her response was durable, lasting >1 year from initiation of treatment. Furthermore, unexpected prolonged disease stabilizations in patients who previously failed combination chemotherapy were noted in lung, gastric, and pancreatic adenocarcinomas. Although these findings are anecdotal, the ability to reverse irinotecan resistance for >1 year is encouraging and consistent with the preclinical efficacy data of selenomethionine and methylselenocysteine described by our group (16). We have recently determined that concentrations of selenium >14 µmol/L (1,100 ng/mL) are needed to achieve maximum protection against chemotherapy-induced toxicity, whereas concentrations >23 µmol/L (1,800 ng/mL) may be needed to achieve potentiation of irinotecan's antitumor activity in xenograft models (30). This may explain the ability to overcome irinotecan resistance in the patient achieving the highest selenium concentration on our study (30 µmol/L).
The dose of selenomethionine investigated on this study (2,200 µg Se) resulted in suboptimal concentrations of selenium on day 8 of treatment (<10 µmol/L in all patients on the first day of irinotecan), which may explain the lack of protective effects and the inability to escalate irinotecan beyond the previously defined MTD. To adequately test the ability of high-dose selenomethionine to ameliorate irinotecan toxicity or allow irinotecan dose escalation, the dose of selenomethionine that is recommended for further investigation should result in concentrations >14 µmol/L before initiation of irinotecan. We are currently conducting a phase I escalation study of selenomethionine with fixed-dose irinotecan to identify doses of Se that results in plasma selenium concentrations >14 and >23 µmol/L. Although selenomethionine doses resulting in concentrations >14 µmol/L may be necessary to ameliorate toxicity, selenomethionine doses resulting in concentrations >23 µmol/L will be required for the optimal study of tumor-potentiating effects and would be our dose of choice for future clinical studies.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9/14/05; revised 11/ 8/05; accepted 11/30/05.
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