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A Phase I and Pharmacokinetic Study of ILX-295501,¹ an Oral Diarylsulfonylurea, on a Weekly for 3 Weeks Every 4-Week Schedule in Patients with Advanced Solid Malignancies

² Cancer Therapy and Research Center, Institute for Drug Development, San Antonio, Texas;
³ Brooke Army Medical Center, San Antonio, Texas; and
⁴ Eli Lilly and Company, Indianapolis, Indiana

	ABSTRACT

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Purpose: This study was conducted to assess the feasibility of administering the oral diarylsulfonylurea (DSU) ILX-295501 on a weekly for 3 weeks every 4-week schedule. The study also sought to determine the maximum tolerated dose (MTD) of ILX-295501 on this schedule, characterize its pharmacokinetic behavior, and seek preliminary evidence of anticancer activity.

Experimental Design: The initial starting dose of ILX-295501 was 100 mg/m², which was equivalent to one-sixth of the highest dose that did not induce irreversible toxicity in dogs, and, using a modified Fibonnaci search scheme to guide dose level selection, the following dose levels were evaluated: 100, 200, 400, 600, 900, 1350, and 1800 mg/m². Because severe toxicities were being reported in other trials at doses that encompassed this range and a cumulative toxicity profile was emerging, the study was suspended and then reinitiated to further reevaluate the lower dosing range. In the second part of the study, the following dose levels were selected a priori for evaluation: 400, 800, 1000, 1250, and 1500 mg/m²; and a modified continual reassessment model was used for dose assignment to determine the MTD, which was defined a priori as the highest dose in which the incidence of dose-limiting toxicity in the first course did not exceed 20%.

Results: Forty-nine patients were treated with 142 courses of ILX-295501 at doses ranging from 100 to 1800 mg/m². The incidences of dose-limiting toxicity, principally neutropenia and thrombocytopenia, were unacceptably high at ILX-295501 doses exceeding 1000 mg/m², which was determined to be the MTD for both minimally pretreated and heavily pretreated (HP) patients. In contrast to the first generation of DSUs, particularly sulofenur, clinically relevant levels of oxidized hemoglobin (methemoglobin) and secondary hemolytic anemia, were not noted. One HP patient with non-small cell lung carcinoma experienced a partial response. Pharmacokinetic studies revealed that ILX-295501 was absorbed slowly, with peak plasma concentrations (C_max) achieving 6.02 h, on average, after oral administration. The pharmacokinetic behavior of ILX-295501 was characterized by dose proportionality, a relatively small apparent volume of distribution at steady state (V_ss/F), averaging 8.02 ± 14.08 liters, and low apparent total body clearance (CL_t/F) rate (mean, 0.036 ± 0.116 liters/h). The initial drug distribution phase was rapid [harmonic mean half-life (t_1/2), 2.1 ± 7.0 min], whereas the terminal elimination phase was slow (harmonic mean t_1/2ß, 150.6 ± 80.2 h).

Conclusions: The recommended dose for Phase II studies of the oral DSU ILX-295501 administered weekly for 3 weeks every 4 weeks is 1000 mg/m²/day for both minimally pretreated and HP patients. The characteristics of the myelosuppressive effects of ILX-295501, the paucity of severe nonhematological toxicities, and preliminary antitumor activity warrant disease-directed evaluations of ILX-295501.

	INTRODUCTION

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The precise mechanisms that account for the potent cytotoxic effects of the DSUs,⁵ which differ structurally from the sulfonylureas by the substitution of aryl functionalities, are not known; however, the DSUs do not inhibit the synthesis of nucleic acids or proteins (1, 2, 3) . In early clinical evaluations of the first-generation DSU sulofenur (LY186641; Eli Lilly and Company, Indianapolis, IN), methemoglobinemia and secondary hemolytic anemia, most likely caused by relevant concentrations of a principal methemoglobinemia-inducing aniline metabolite, precluded further developmental efforts (4, 5, 6, 7) . Instead, subsequent developmental efforts were directed toward the evaluation of other DSU analogues such as ILX-295501 [N-(5-(2,3-dihydrobenzofuranyl)-sulfonyl)-N'-(3,4-dichlorophenyl)-urea; Ilex Oncology Inc., San Antonio, TX; formerly known as LY295501 (Eli Lilly and Company); see Fig. 1 ], which, in contrast to sulofenur, is principally metabolized by hydroxylation with negligible formation of aniline metabolites at relevant doses in rodents, dogs, and subhuman primates (2) .

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Structure of ILX-295501 and sulofenur.

In preclinical toxicology studies involving several animal species, rapidly proliferative tissues, including hematopoietic, gastrointestinal, and lymph nodal tissues, were most prone to the toxic effects of ILX-295501. In monkeys, which are most susceptible to the clinical effects associated with the oxidation of hemoglobin, neither hemolysis, methemoglobinemia, nor anemia were observed after treatment with ILX-295501.

The decision to develop ILX-295501 was based on its structural uniqueness, seemingly novel mechanism of action based on its unique activity spectrum, and its broad spectrum of antitumor activity, particularly against malignancies with the multidrug-resistant phenotype. In most early studies, the feasibility of administering oral ILX-295501 daily for 5–14 days every 2–3 weeks was evaluated, but these schedules were associated with a high incidence of intolerable, unpredictable, and apparently cumulative hematological and gastrointestinal effects (12, 13, 14, 15, 16) . All studies, including the present trial that evaluated a weekly for 3 weeks every 4-week oral dosing schedule, were suspended until an extensive toxicokinetic analysis indicated that several frequent dosing schedules and accelerated dose-escalation schemes, particularly those in which a single patient was treated at each dose level that was not associated with toxicity in course 1, similar to that used in the first part of this study, were not congruent with the unexpectedly slow clearance of ILX-295501, resulting in cumulative and unpredictable myelosuppression. After the institution of a more conservative dose-escalation schedule, which reflected preliminary knowledge about the pharmacological and toxicological profiles of ILX-295501 in humans, this Phase I and pharmacokinetic study recommenced. The principal objectives of the study were to: (a) determine the MTD of ILX-295501 administered as a single oral dose weekly for 3 weeks every 4 weeks; (b) determine the toxicities of ILX-295501 on this schedule; (c) characterize the pharmacokinetic behavior of ILX-295501; and (d) seek preliminarily evidence of anticancer activity in patients with advanced solid malignancies.

	PATIENTS AND METHODS

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Patient Selection.
Patients with histologically confirmed advanced solid malignancies that failed to respond to standard therapy or for whom adequate therapy was not available were eligible for this study. Eligibility criteria also included: age 18 years; a Zubrod performance status 2; life expectancy of at least 3 months; no prior chemotherapy, investigational medication, or wide-field radiation therapy (>25% of the bone marrow) within 3 weeks (6 weeks for nitrosoureas and mitomycin C); adequate hematopoietic (WBC count, 3000/µl; ANC, 1500/µl; hemoglobin, 9.0 g/dl; platelets, 100,000/µl; no RBC transfusions in the previous 3 weeks), hepatic [total bilirubin, 2.0 mg/dl; transaminase, 3.0 times institutional normal upper limit (<5 times if attributable to liver metastasis); coagulation times, 1.5 times institutional normal upper limit], and renal (creatinine, 2.0 mg/dl) functions; serum calcium, 11 mg/dl; no active brain metastases (defined as evidence of recent growth, peritumoral edema, requirement for corticosteroids), and no coexisting medical problem of sufficient severity to limit compliance with the study. Because of concerns about the potential for drug-induced hemolysis and methemoglobinemia, both direct and indirect Coombs’ tests were required to be negative, and patients with known glucose-6-phosphate dehydrogenase deficiency, prior history of hypersensitivity to sulfur-based drugs, or >2% methemoglobinemia were ineligible. Patients gave written informed consent according to federal and institutional guidelines before treatment.

Dosage and Drug Administration.
ILX-295501 was supplied by Eli Lilly and Company as an off-white colored tablet in 25-, 100-, and 400-mg strengths. The tablets were administered orally with 6–8 ounces of water. Patients were required to fast for 1 h before and 30 min after drug administration. The total dose was rounded to the nearest multiple of 25 mg.

Study Design.
The study was designed as an open-label, Phase I, and pharmacokinetic study of ILX-295501 administered weekly for 3 weeks every 4 weeks. The initial starting dose was 100 mg/m², which was equivalent to one-sixth of the highest dose that did not induce irreversible toxicity in dogs, and, using a modified Fibonnaci scheme to guide dose level selection in the first part of the study, the following dose levels were evaluated: 100, 200, 400, 600, 900, 1350, and 1800 mg/m². A minimum of three patients was to be treated at each dose level in the study. Before enrollment of the first patient at each successively higher dose level, all three patients at the preceding dose level must have completed at least one course of therapy and at least one patient must have completed a second course. Because severe adverse events were reported in other trials at dose levels that encompassed these dose levels and a cumulative toxicity profile was emerging, the study was temporarily suspended and then reinitiated to further evaluate the lower dosing range. In the second part of the study, the following dose levels were selected a priori for evaluation: 400, 800, 1000, 1250, and 1500 mg/m²; a modified continual reassessment model was used for dose level assignment to determine the MTD, which was defined a priori as the highest dose in which the incidence of DLT did not exceed 20% in the first treatment course. The MTD was to be defined separately for MP patients and HP patients if it appeared that HP patients were more susceptible to DLT. HP patients were defined as those who had been treated previously with more than six courses of alkylating agent-containing chemotherapy [or more than four courses of carboplatin; two or more courses of mitomycin C or a nitrosourea; or radiation therapy to >25% of hematopoietic reserves (with whole pelvic radiation equivalent to radiation to 30%)].

After determination of the MTD, at least six additional patients were to be treated at the next lower level to provide additional information about the tolerance of repetitive treatment at that dose level, which was used to recommend a dose for Phase II studies. DLT was defined as: (a) grade 3 nonhematological toxicity, except for nausea, vomiting, or diarrhea without optimal premedication and/or supportive measures and/or tolerable toxicity of brief duration; (b) any grade 4 nonhematological toxicity; (c) platelets <25,000/µl; (d) ANC <500/µl lasting longer than 5 days and/or associated with fever (38.5°); and (e) unresolved toxicity resulting in delay of retreatment >2 weeks. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria (version 1.0).

Patients could be retreated as long as hematological parameters recovered to grade 1 or less and all nonhematological toxicities, except alopecia, had completely resolved. If criteria required for retreatment could not be met after a delay of more than 3 weeks, the patient was taken off study. If a scheduled dose could not be administered, it was omitted. Intrapatient dose escalation was permitted as long as there was no disease progression and the patient completed at least three courses at the initial dose level without drug-related toxicity at grade 2 or higher, provided that the next highest dose level was demonstrated to be tolerable after treatment of at least three patients. Intraindividual dose reduction by one level was permitted for individuals who experienced DLT.

Pretreatment and Follow-up Studies.
Histories that included recording of performance status and concurrent medications, physical examinations, and routine laboratory evaluations were performed before treatment and weekly. Routine laboratory evaluations included complete blood counts, differential WBCs, reticulocyte counts, electrolytes, chemistries, urinalysis, blood clotting studies, and methemoglobin levels. Before treatment, direct and indirect Coombs’ testing and an electrocardiogram, as well as pregnancy testing in relevant females, were obtained. Complete blood counts and differential white blood counts were assessed every other day if the ANC and platelet counts decreased to <750/µl and <25,000/µl, respectively, and pertinent chemistry and/or electrolyte studies were obtained at least twice weekly in patients who developed abnormalities of at least one grade above pretreatment values. Pertinent tumor markers were assessed after every course, and radiological studies of all known sites of disease were obtained before treatment and after every other course. Patients were able to continue treatment in the absence of progressive disease, which was defined as 25% increase in the size (the sum of the product of the bidimensional measurements of the target lesions) or appearance of any new lesion. A complete response was scored if there was disappearance of all disease on two measurements separated by at least 4 weeks, and a partial response required at least 50% reduction in the sum of the product of the bidimensional measurements of all documented lesions separated by at least 4 weeks.

Pharmacokinetic Sampling and Assay.
Five-milliliter blood samples in heparanized tubes were collected before treatment, and 1, 3, 6, 8, 10, 24, 48, 72, and 96 h after the first dose and immediately before the second and third doses on days 8 and 15 in the first course. Plasma concentrations were also obtained during course 2 on days 1, 8, and 15 (predose and 6 h postdose) and also before course 3 day 1 dosing. The samples were centrifuged immediately and frozen at -70°C until analysis.

Briefly, after plasma samples were thawed at room temperature and vortexed, 250-µl aliquots of the patients’ samples, human plasma for preparation of standard curve (5–400 µg/ml), blank samples, and validation samples were transferred to polypropylene tubes, and known amounts of LY18664 (sulofenur; Fig. 1 ), which is structurally similar to ILX-295501, were added to each sample. The samples were then vortexed, 2 µl of acetonitrile were added to each sample, and the resultant mixtures were vortexed and centrifuged at 4°C. Next, the supernatants were decanted into glass tubes and evaporated to dryness under nitrogen and then reconstituted with 250 µl of acetonitrile and 500 µl of phosphate buffer. The reconstituted solution was then vortexed for 10 s, centrifuged through an Ultrafree-MC Centrifugal Filter Unit (Millipore, Bedford, MA), and a 10-µl sample was injected into the high-performance liquid chromatography system. The high-performance liquid chromatography system was equipped with a Varian model 5000 pump (Varian Inc., Walnut Creek, CA), a Shimadzu SIL-10A autosampler (Shimadzu Inc., Columbia, MD), a 3.9 x 150 x 0.004 mm octadecyl silane column (Waters Novapack Inc., Milford, MA), and a Kratos Spectroflow model 783 programmable absorbance detector (Kratos Inc., Chestnut Ridge, NY). Samples were isocratically eluted with 30% acetonitrile/70% phosphate buffer (pH 7.0, 20 mM) at a rate of 1.0 ml/min. The column effluent was monitored using a Kratos Spectroflow model 783 programmable absorbance detector with UV detection set at 260 nm. A Perkin-Elmer Access Chrom data system (Perkin-Elmer Inc., Shelton, CT) was used for data acquisition and peak area integration, and drug concentrations were determined from linear regression equations derived from calibration curves prepared with known standard samples. The lower limit of reliable detection for ILX-295501 was set at the concentration of the lowest non-zero standard, 0.200 ng/ml. The intra-assay CV percentage ranged from 1.6 to 8.9%, whereas the intra-assay accuracy was between 93.6 and 98.5%. Interassay CV values ranged from 1.4 to 14.7%, and the interassay precision was between 95.5 and 108.6%.

Two blank samples were assayed with each set of study samples. The first blank was prepared by spiking 50 µl of internal standard stock solution (sulofenur) into 250 µl of control human plasma. The second blank was prepared by processing 250 µl of control human plasma without the addition of the internal standard. Neither blank contained ILX-295501. To validate the analytical method, pools were prepared by spiking known quantities of ILX-295501 into human plasma. Five replicates of each validation pool were assayed every 3 days to determine the intraday and interday accuracy and precision. Validation pools were prepared at 5, 50, and 400 µg/ml. Two replicates of each QC level (5, 50, and 400 µg/ml) were assayed each time a set of study sample was analyzed. Control sample pools were prepared along with study samples and were stored with the study samples until analysis. The intraday and interday accuracy and precision were always ±15%, except at the limit of quantitation, in which values of ±20% were considered acceptable. Six QCs were analyzed with each analytical run (duplicate QCs at 5, 50, and 400 µg/ml). At least four of the six QCs, and one at each level, must have been within ±20% of their nominal concentration. The overall accuracy of the method during the validation, expressed as percentage of relative error, ranged from -4.9 to 1.4%; the overall precision, expressed as percentage of CV, ranged from 2.4 to 4.7%. Extraction efficiency was determined by comparing the peak areas of extracted samples at 5 and 400 µg/ml to equivalent concentrations of standards. The mean extraction efficiency of ILX-295501 (5 and 400 µg/ml) was 83%. The mean extraction efficiency of sulofenur was 81%. The lower and upper LOQ for ILX-295501 were established at 5 and 400 µg/ml, respectively. These concentrations represent the lowest and highest points on the standard curve. Data below the standard curve were reported as below the LOQ, and data above the curve were diluted with control human plasma and reanalyzed. If there was insufficient sample to reanalyze, the sample was reported as above the LOQ.

Pharmacokinetic Analysis.
The AUC for the first week of therapy from 0 to 168 h (AUC_{0–168 h}) was calculated using the partial area function of WinNonLin Standard (version 3.1; Pharsight Corporation, Mountain View, CA). Actual sampling times and the linear trapezoidal method were used to calculate the AUC values for each individual (17) . Peak plasma concentration (C_max) and time of maximal plasma concentration (T_max) values were determined by inspection. The dose proportionality of AUC_{0–168 h} or C_max was examined by application of the power model (17) . Noncompartmental analyses were performed initially using single-dose data from the first week of therapy, but because of the prolonged plasma half-life (t_1/2), the extrapolated area extending beyond the last data point was too large to accurately estimate the total AUC out to infinity. Instead, pharmacokinetic parameter estimation was performed using compartmental analytical methods as implemented by ADAPT II, release 4 (18) . Plasma concentration data from the first 4 weeks of therapy (course 1) were fit to one-, two-, and three-compartment extravascular input models with and without an absorption lag time. Nonlinear regression was performed using maximum likelihood estimation with variance modeled as a linear function of the plasma concentration data. Model discrimination was performed by examining graphs of the fitted and observed data, standardized residual plots, and by calculation of the Akaike’s information criteria (19 , 20) . The model found to most closely follow the data was a two-compartmental model with a lag time and parameterized for clearance. The following primary pharmacokinetic parameters were calculated based on that model: apparent volumes of distribution for the central and peripheral compartments and the apparent total volume of distribution (V_ss/F), apparent clearance for both kinetic phases and apparent total clearance (CL_t/F), and the absorption rate constant of the drug from the gut compartment to the central compartment (K_a). In addition, both and ß phase half-lives of distribution (t_1/2 and t_1/2ß, respectively) were calculated as secondary parameters from this model. Pharmacodynamic analyses were performed using linear and maximal effect (E_max) models to relate the AUC and C_max to observed drug effects using WinNonLin.

	RESULTS

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General.
Forty-nine patients, whose pertinent demographic characteristics are listed in Table 1 , were treated with 142 total courses of ILX-295501 at 11 dose levels ranging from 100 to 1800 mg/m². The dose escalation scheme and total numbers of new patients, fully assessable courses, dose reductions, and rates of DLT at each dose level are depicted in Table 2 . In the first part of the study, in which 20 patients received 63 total courses spanning seven dose levels (100, 200, 400, 600, 900, 1350, and 1800 mg/m²), the MTD was not determined because of the occurrence of severe adverse events in other concurrent Phase I studies that led to the premature suspension of all trials. In this part of the study, DLT, characterized by both grade 4 neutropenia and thrombocytopenia during the first course of ILX-295501 at the 1800 mg/m² dose level, was experienced by a single individual who was the last subject treated before study suspension. This 71-year-old HP male with colorectal cancer, who had received prior pelvic radiation and several chemotherapy regimens, experienced grade 4 neutropenia, grade 4 thrombocytopenia, grade 2 stomatitis, dehydration, nausea, and vomiting, and died of sepsis with clostridium butyricum and massive gastrointestinal hemorrhage on day 8 of his first course.

On the basis of the development of unacceptable toxicity in two of six patients at the 1250 mg/m² dose level, despite the fact that one of the patients experienced DLT in course 2, and DLT in the single patient treated at the 1500 mg/m² dose level, 13 total patients (10 MP and 3 HP), received 38 total courses at the 1000 mg/m² dose level. At this dose, a single HP patient experienced DLT, which consisted of grade 4 neutropenia for longer than 5 days in course 1. Although the MTD was not strictly determined based on the precise criteria established a priori, repetitive administration of ILX-295501 at 1000 mg/m² resulted in an acceptable rate of DLTs, and, therefore, this dose was recommended for subsequent disease-directed studies.

Hematological Toxicity.
Myelosuppression, particularly neutropenia and thrombocytopenia, was the principal toxicity of ILX-295501 on this schedule. The distributions of pertinent National Cancer Institute Common Toxicity Criteria grades of neutropenia and thrombocytopenia, as well as hematological DLTs, as a function of dose are listed in Table 3 . ANC nadirs occurred, on average, on day 24 of the first course and day 17 of subsequent courses. In patients who experienced either grade 3 or 4 neutropenia, ANC nadirs typically occurred in the week after the three consecutive weekly treatments, but ANC recovery was not sufficient to permit retreatment on day 29. At the 1000 mg/m² dose level, neutropenia of grade 1–2 severity occurred in 9 (24%) of 38 courses. Of 31 courses administered to MP patients, 8 (26%) courses were associated with grade 1–2 neutropenia, whereas grade 1–2 neutropenia and grade 4 neutropenia for longer than 5 days without fever occurred during one course each of seven courses administered to HP patients. Clinically relevant effects on platelets were uncommon.

Methemoglobin levels were mildly elevated (<2.5 times the upper limit of normal) in blood samples collected during 16 (11%) courses, but only one subject experienced a greater, albeit transient, magnitude of methemoglobinemia during one course. These elevations were isolated and not associated with elevations in reticulocyte counts, plasma concentrations of lactic dehydrogenase, or anemia.

Nonhematological Toxicity.
Metabolic abnormalities, including hyperglycemia, hypoglycemia, and hypocalcemia, were the most common nonhematological effects of ILX-295501. In many cases, metabolic abnormalities predated treatment. For example, hyperglycemia was noted in several patients before treatment, and its emergence during treatment was principally in subjects who had pretreatment abnormalities; therefore, it was not felt to be drug related. Before beginning treatment, 14 (28%), 3 (6%), and 1 (<1%) patients had grades 1, 2, and 3 hyperglycemia, respectively, and 20 (40%), 15 (30%), and 7 (14%) patients developed hyperglycemia of these respective grades at some time during treatment. With regard to hypoglycemia, grade 1–2 events, which were clinically asymptomatic, were experienced by five (10%) patients in five (3%) courses. In addition, mild to moderate (grade 1–2) hypocalcemia was evident in 17 (35%) patients before beginning treatment, whereas it was experienced by 27 (55%) patients, predominately those individuals who had pretreatment abnormalities, during 60 (42%) courses. Although most episodes were mild to moderate (grade 1–2) in severity, two individuals experienced more severe (grade 3), albeit asymptomatic, hypocalcemia concurrent with the development of dose-limiting hematological toxicity at the 1000 mg/m² (course 6) and 1500 mg/m² (course 1) dose levels. The incidences of these metabolic abnormalities were not related to dose.

Other nonhematological effects, which were possibly related to ILX-295501 and uncommon (<10% of courses), included elevations in hepatic function tests, nausea, vomiting, diarrhea, stomatitis, fatigue, and skin rash. Although elevations in hepatic transaminases and bilirubin were also observed, these events were uncommon, mild to moderate in severity, and typically noted in conjunction with the progressive growth of hepatic metastases. Except for one sporadic episode of grade 3 nausea and vomiting, along with diarrhea, in a patient who had not yet received optimal antiemetic premedication with ILX-295501 at the 1800 mg/m² dose level, nausea and vomiting were rare and mild to moderate in severity. Nausea and vomiting were generally managed successfully, and recurrences were prevented with prochlorperazine treatment alone, however, routine premedication was not necessary because most events consisted of nausea only. Delayed emesis was not observed. Additionally, alopecia did not occur. Two patients developed severe (grade 3) stomatitis, which occurred concomitant with dose-limiting myelosuppression at the 1250 and 1500 mg/m² dose levels.

Three patients developed manifestations suggestive of an acute respiratory distress syndrome, characterized by progressive pulmonary infiltrates and hypoxia. These manifestations occurred concomitant with the development of progressive pulmonary metastases and culminated in death in one patient after treatment with two courses of ILX-295501 at the 1000 mg/m² dose level. Two other individuals experienced progressive respiratory dysfunction associated with severe myelosuppression, mucositis, sepsis, and multiorgan dysfunction presumptively because of sepsis. One patient experienced these manifestations during his first course of ILX-295501 at the 1800 mg/m² dose level. The other individual experienced progressive respiratory failure and pulmonary infiltrates after an emergent surgical decompression of a vertebral mass during a third course of ILX-295501 at the 800 mg/m² dose level. This patient also developed grade 3 thrombocytopenia, grade 2 mucositis, and grade 1 neutropenia, massive gastrointestinal hemorrhage, acute renal failure, and sepsis, which culminated in death. A postmortem examination revealed interstitial pulmonary fibrosis and hyaline membrane formation consistent with acute respiratory distress syndrome, but the precise etiology of these manifestations could not be established. After these events, all subsequent patients enrolled onto study and treated with ILX-295501 at the 1000, 1250, and 1500 mg/m² dose levels underwent serial pulmonary function testing and chest radiography before treatment and after each course, which did not demonstrate pulmonary toxicity.

Antineoplastic Activity.
A 73-year-old male with advanced NSCLC and a history of coronary artery disease and diabetes mellitus, whose disease progressed on prior docetaxel-based treatment, experienced a PR. He was treated with a single course of ILX-295501 at the 1250 mg/m² dose level that was associated with DLT and seven additional courses of ILX-295501 at 1000 mg/m² without severe toxic effects. An 85% reduction in the size of his measurable lesions was noted after two courses, confirmed after four courses, and maintained until his death from an acute myocardial infarction. Two additional patients with NSCLC and prior histories of radiation therapy and progressive disease during treatment with platinum and taxane-based chemotherapy regimens experienced stable disease of note. Stable disease persisted for 10 and 5 months during treatment with ILX-295501 at the 400 and 1000 mg/m², respectively.

Pharmacokinetics.
Plasma sampling for pharmacological studies was performed in all 49 patients treated with ILX-295501 at doses ranging from 100 to 1800 mg/m². The agent was absorbed slowly with C_max values achieved 7.88 h, on average, after oral administration. A plasma concentration-time curve reflecting mean concentration values for patients treated with ILX-295501 at the 1000 mg/m² dose level is shown in Fig. 2 . Because of the low clearance rate of the agent, a terminal elimination phase could not be estimated accurately from plasma sampled over 7 days after a single dose because the mean percentage of AUC extrapolated beyond the last time point to infinity was unacceptably large (43.5 ± 18.6%; n = 49; Ref. 21 ). To examine the dose proportionality of ILX-295501 pharmacokinetics, both C_max and AUC_{0–168 h} parameter values were calculated for all 49 patients. An inspection of the scatterplots of dose versus either C_max or AUC_{0–168 h} revealed significant overlap in C_max and AUC_{0–168 h} values within the dosing range evaluated in the study (Fig. 3) . Although both parameter values increased with increasing doses (r² = 0.78 and 0.80, respectively), the 95% CI for the slope of the log(AUC_{0–168 h}) versus log(dose level) plot did not encompass a value of 1 (95% CI, 1.090–1.342), suggesting some degree of disproportionality in the relationship between dose and exposure. Similarly, the 95% CI for the slope of the log(C_max) versus log(dose level) relationship (range, 0.626–0.908) did not include a value of 1.0. However, if the two lowest dose levels (100 and 200 mg/m²; n = 8) are excluded from these analyses, the criteria for dose proportionality over the dose range of 400-1800 mg/m² are met.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Mean plasma ILX-295501 concentration-time profile (day 1) of patients treated with ILX-295501 at the 1000 mg/m2 dose level.

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Scatterplots showing the distributions of total ILX-295501 Cmax values (left) and AUC0–168 values (right) as a function of the ILX-295501 dose.

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View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Representative patient’s plasma ILX-295501 concentration data () over a 3-week course fit to the compartmental model. The patient was treated with ILX-295501 at the 1000 mg/m2 dose level.

View larger version (12K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Scatterplot depicting the relationships between apparent clearance (CLt/F) values and ILX-295501 dose.

	DISCUSSION

Top ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The development of new cytotoxic compounds, such as the DSUs, which are structurally and different than contemporary agents, have often resulted in incremental progress in treating a variety of malignancies. The DSUs are structurally similar to oral hypoglycemic agents such as glibenclamide, although the series was originally synthesized as potential herbicides (22) . The prototypic DSU, sulofenur, exhibited impressive antitumor activity against a broad spectrum of human tumor xenografts, including those with intrinsic resistance to a wide range of chemotherapeutic agents (2, 3, 4) . From a clinical development standpoint, however, sulofenur proved to be disappointing. Although antitumor activity was observed consistently in Phase I evaluations, the development of sulofenur was precluded by several toxicities, including methemoglobinemia and hemolytic anemia, which limited drug concentrations that could be achieved to levels that were substantially lower than those shown to elicit therapeutic responses in human tumor xenograft models (23, 24, 25) . These preclusive effects were considered to be consequences of two factors. First, the high protein binding of sulofenur in human plasma relative to that in other animal species necessitated the administration of much higher drug doses to induce relevant biological effects. Second, there are substantial differences in sulofenur metabolism between rodents and humans (23) . In humans, a p-chloroaniline metabolite, which is capable of oxidizing hemoglobin, may be formed by metabolic conversion of sulfofenur directly, or after initial metabolism (26) . Consequently, the DSU analogue ILX-295501 was synthesized to block the initial metabolic reactions observed with sulofenur, specifically hydroxylation at the 1-position of the indane ring that leads to the formation of the methemoglobinemia-inducing p-chloroaniline metabolite. Preclinical toxicology in primates and other animal species indicated the toxicity profile ILX-295501 was different from sulofenur, with toxicity principally noted in tissues with high rates of turnover. However, oxidation of hemoglobin was still detected, albeit to a minor degree (27) .

As predicted from preclinical studies, neutropenia was the principal DLT of ILX-295501. Severe thrombocytopenia and anemia were occasionally noted in patients who developed concomitant severe neutropenia. Although neutropenia was relatively common at the ILX-295501 dose level of 1000 mg/m², severe and/or protracted neutropenia occurred infrequently, and other dose-limiting hematological effects were uncommon. Furthermore, both MP and HP patients alike were able to repeatedly tolerate ILX-295501 on a schedule consisting of three weekly treatments every 4 weeks. At 1000 mg/m², the incidence of hematological DLT was acceptable, occurring in only 1 (7.7%) of 13 patients in the first course and in 1 (2.6%) of 38 total courses. In contrast, higher doses of ILX-295501 were associated with unacceptably high rates of dose-limiting hematological events. Although neutropenia and thrombocytopenia were generally more severe in HP patients at any given dose and pharmacological index of drug exposure, these differences were not clinically significant at ILX-295501 doses up to 1000 mg/m². The low rate of dose-liming myelosuppressive events, treatment delay, and cumulative hematological toxicity indicate that 1000 mg/m² ILX-295501 is an appropriate starting dose for both MP and HP patients with good performance status and organ function.

Nonhematological toxicities occurred less frequently than hematological effects and were rarely severe. There was heightened attention paid to the development of methemoglominemia and associated sequalae from the outset of this study because of the previous experience with sulofenur. However, in contrast to sulofenur, which was associated with unacceptably high rates of severe dose-related methemoglobinemia, hemolysis, and anemia, the tolerance of which varied from individual to individual, elevated methomoglobin levels of clinical relevance were uncommon in the present study. Mild methemoglobinemia was noted in 16 (11%) courses, but only one patient experienced methemoglobinemia of greater magnitude. Furthermore, the incidence of methemoglobinemia was not dose related or associated with the development of anemia, reticulocytosis, or elevations in lactic dehydrogenase.

The timing of the administration of ILX-295501 on the weekly for 3 weeks every 4-week schedule evaluated in the present study is congruent with the pharmacokinetic behavior of the agent. Similar to sulofenur, the clearance rate of ILX-295501 is low, with t_1/2ß values averaging 150.6 ± 80.2 h in the present study. Also consistent with sulofenur, many relevant aspects of the pharmacological behavior of ILX-295501 are explained by its high (>99%) plasma protein binding. The low clearance rate of ILX-295501 was, in part, responsible for the severe, cumulative hematological toxicities observed in clinical evaluations of more frequent dosing schedules, particularly in Phase I studies that used accelerated titration schemes with single patient cohorts at early, seemingly nontoxic, dose levels as well as inappropriately short observation periods before dose escalation (16) . In these studies, dose escalation was performed after a traditional observation period, consisting of a single course, at which time toxicity at the lower doses was still in evolution because of the slow clearance of ILX-295501. This exemplifies a principal shortcoming of accelerated titration dose-escalation schemes that consist of single-patient cohorts and short observation periods. Accelerated titration schemes with such features may not be appropriate for use in initial evaluations of anticancer agents with low clearance rates and/or the propensity to induce cumulative or irreversible toxicity.

The anticancer activity observed in a HP patient with NSCLC and other manifestations of potential clinical benefit are encouraging and provide impetus for the development of ILX-295501 and the DSUs, in general, as a new therapeutic class. The tolerability of repetitive treatment at the MTD and recommended Phase II dose, 1000 mg/m², is also encouraging, but the seemingly narrow therapeutic window of ILX-295501, with unacceptably high rates of DLTs at slightly higher doses, is, nonetheless, worrisome. Furthermore, ILX-295501 concentrations that were achieved at the recommended Phase II dose were substantially greater than those associated with prominent anticancer activity in preclinical studies of ILX-295501. For example, impressive inhibition of tumor growth was observed in 72% of a broad range of fresh human tumors treated with 100 mg/liter ILX-295501 for protracted periods, conditions that are readily achieved at the 1000 mg/m² dose level (10) . Although the ultimate therapeutic index for ILX-295501 will be defined only in appropriate Phase II/III trials, its specific pattern of myelotoxicity and relative paucity of nonhematological toxicity on the schedule evaluated in the present study, as well as the documentation of antineoplastic activity, warrant broad disease-directed evaluations of the agent on this administration schedule and developmental efforts directed at additional optimization of the DSUs as anticancer therapeutics.

	FOOTNOTES

 
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.

Requests for reprints: Dr. Eric K. Rowinsky, Institute for Drug Development, Cancer Therapy and Research Center, 7979 Wurzbach Road, 4th Floor Zeller Building, San Antonio, TX 78229. Phone: (210) 616-5945; Fax: (210) 692-7502; E-mail: erowinsk{at}idd.org

¹ Formerly known as LY295501.

⁵ The abbreviations used are: DSU, diarylsulfonylurea; ANC, absolute neutrophil count; AUC, area under the plasma concentration-time curve; CI, confidence interval; CV, coefficient(s) of variation; DLT, dose-limiting toxicity; HP, heavily pretreated; LOQ, lower limit(s) of quantitation; MP, minimally pretreated; MTD, maximum tolerated dose; NSCLC, non-small cell lung carcinoma; QC, quality control.

Received 6/16/03; revised 8/ 5/03; accepted 8/ 5/03.

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