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Journal of Clinical Oncology, Vol 22, No 9 (May 1), 2004: pp. 1553-1563 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.07.158 Phase I and Pharmacokinetic Study of the Ribonucleotide Reductase Inhibitor, 3-Aminopyridine-2-Carboxaldehyde Thiosemicarbazone, Administered by 96-Hour Intravenous Continuous InfusionFrom the Albert Einstein College of Medicine and the Albert Einstein Comprehensive Cancer Center, Bronx, NY; and Vion Pharmaceuticals, Inc, New Haven, CT. Address reprint requests to Scott Wadler, MD, Division of Hematology and Oncology, C 606, Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10021
PURPOSE: 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP; Triapine; Vion Pharmaceuticals Inc, New Haven, CT) is a potent inhibitor of ribonucleotide reductase, with activity in preclinical tumor model systems. A phase I trial was initiated to determine the dose-limiting toxicities, maximum-tolerated dose, and pharmacokinetics of a 96-hour intravenous (IV) continuous infusion in patients with advanced cancer. PATIENTS AND METHODS: Initially, courses were administered every 3 weeks, using an accelerated titration design. Subsequently, courses were administered every 2 weeks, and the dose was escalated in cohorts of three to six patients. RESULTS: Twenty-one patients were enrolled, seven on the every-3-week schedule and 14 on the every-other-week schedule. Three of six patients at 160 mg/m2/d developed dose-limiting toxicities including neutropenia, hyperbilirubinemia, and nausea or vomiting. Based on these initial results, the dose for 3-AP was re-escalated beginning at 80 mg/m2/d but administered every 2 weeks. At 120 mg/m2/d, three of seven patients had dose-limiting but reversible asthenia, hyperbilirubinemia, and azotemia or acidosis; however, in the case of renal and hepatic adverse events, the events were related to pre-existing borderline abnormal organ function. Therefore, the recommended phase II dose for 3-AP administered by 96-hour IV infusion is 120 mg/m2/d every 2 weeks. Detailed pharmacokinetic studies demonstrated linear kinetics up to 160 mg/m2, with substantial inter-patient variability. There was no correlation between dose and clearance (R2 = 0.0137). There were no objective responses, but there was prolonged stabilization of disease or decreases in serum tumor markers associated with stable disease in four patients. CONCLUSION: The 96-hour infusion of 3-AP is safe and well tolerated at the recommended phase II doses. Phase II trials of Triapine are ongoing.
Ribonucleotide reductase (RR) plays a central role in cell growth and proliferation by synthesizing deoxyribonucleoside diphosphates from ribonucleoside diphosphates, thus ensuring a balanced supply of nucleotide precursors for DNA synthesis.1 RR is an  2ß2 heterodimer consisting of two subunits: M1, the larger, allosterically regulated subunit, and M2, the smaller subunit, which contains the nonheme iron, necessary for the reduction reaction.2 RR is an S phase enzyme and is rate-limiting for the synthesis of DNA.1,3 Levels of expression of the enzyme, and particularly for the inducible small M2 subunit, are closely related to cytokinetic progression through the cell cycle.4,5 In addition, RR can be induced by exposure to a variety of exogenous factors, including the transforming agent, 12-O-tetradecanoylphorbol-13-acetate, cytotoxic drugs that result in DNA repair, and signal transduction factors, including cyclic adenosine monophosphate and protein kinase C.6,7-11 Finally, overexpression of RR is related to tumorigenesis, metastasis, invasive potential and drug resistance, further supporting treatment approaches aimed at RR inhibition.12-19 The development of agents that inhibit RR activity is an established strategy in cancer therapy. Hydroxyurea, the first agent to be targeted to RR, inhibits the M2 subunit, by inactivating the nonheme iron.20 Hydroxyurea's efficacy appears to be limited to myeloproliferative disorders, although in early clinical trials, objective responses were reported in several solid tumors.21,22 Unlike hydroxyurea, nucleoside analogues, including gemcitabine and fludarabine, inhibit the M1 subunit.23,24 Gemcitabine has modest clinical activity against pancreatic cancer, non–small-cell lung cancer, and various other solid tumors, and fludarabine is a useful agent in the treatment of chronic leukemia and lymphoma. Nevertheless, more effective RR inhibitors are desirable. 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP; Triapine, Vion Pharmaceuticals, New Haven, CT) is a new and potent inhibitor of the M2 subunit.25 Similar to hydroxyurea, 3-AP quenches the tyrosyl free radical in the M2 subunit of the enzyme.20,26 Unlike hydroxyurea, 3-AP is also an iron chelator.27 Because the M2 subunit requires iron to regenerate the tyrosyl free radical, sequestration of iron by 3-AP may account for its enhanced potency against RR compared with hydroxyurea.28 Preclinical studies suggested anticancer activity for 3-AP, including delays in the growth of several murine and human tumors in mouse models.29 Of interest, 3-AP inhibited enzyme activity and cell growth in vitro at 100 to 1,000 fold lower concentrations than hydroxyurea, and inhibited growth of some tumor cell lines that are resistant to hydroxyurea.25,29 Therefore, it was believed that 3-AP would be an attractive agent to move into clinical development in patients with cancer. Early clinical trials of 3-AP employed short, 2-hour intravenous (IV) infusions, administered once or daily x 5 every 2 to 4 weeks.30,31 However, preclinical data suggested that such a schedule was suboptimal. Specifically, in vitro, 3-AP antitumor effects against several tumor cell lines were dependent on achieving both a threshold concentration and a minimum duration of exposure (Michael Belcourt, Vion Pharmaceuticals, personal communication). Similarly, in at least one animal tumor model, 3-AP demonstrated antitumor activity only if administered twice daily, indicating that antitumor effects are schedule-dependent and are optimized when the RR enzyme is inhibited throughout most or all of the day for several days consecutively. Because preclinical data indicated that 3-AP's antitumor effects were schedule-dependent and may be increased if RR can be inhibited continuously for several days, the current phase I trial was initiated in order to study the tolerability of a prolonged, 96-hour IV infusion in patients with solid tumors. Pharmacokinetic studies were performed to assist in choosing the appropriate dosing for phase II studies.
Administrative This was a prospective, dose-escalation, single institution study. The aims of the study were to determine the dose-limiting toxicities and maximum-tolerated dose for a 96-hour IV infusion of 3-AP administered every 2 or 3 weeks, and to determine the pharmacokinetic parameters associated with specific dosing levels. The study was approved by the Protocol Review Committee of the Albert Einstein Comprehensive Cancer Center and the Institutional Review Board of the Montefiore Medical Center (Bronx, NY).
Patients
Treatment Plan Dose escalation was executed according to the accelerated titration design described by Simon et al32, in which one to two patients were assigned to a cohort, and doses were escalated by 100% for each new cohort, until the first instance of drug-related grade 2 toxicity or dose-limiting toxicity (DLT). Intrapatient dose escalation was also permitted if drug-related toxicity in a previous course was less than grade 2. Initially, courses of 3-AP were repeated every 3 weeks. At a dose of 160 mg/m2/d, grade 2 to 4 adverse events were observed including DLT in three of six patients, which indicated that this dose was beyond the maximum-tolerated dose (MTD). DLT included both hematologic and nonhematologic adverse events. Only two patients had been treated at the next lower dose level of 80 mg/m2/d, and according to the study design, it would have been necessary to expand the 80 mg/m2/d dose level to three to six patients and then continue escalating the dose in smaller increments of 25% to 33% to determine the MTD at a dose intermediate between 80 and 160 mg/m2/d. The clinical data suggested that the MTD would be determined by rapidly reversible nonhematologic adverse events rather than hematologic toxicity, and therefore courses could be repeated every other week instead of every 3 weeks. Furthermore, the every-other-week schedule provided an opportunity to investigate a schedule with greater dose-intensity. The study was then amended to determine the MTD of the 96-hour IV infusion administered every other week, beginning at 80 mg/m2/d. Dose escalation was planned to 100 and 120 mg/m2/d in standard cohorts of 3 to 6 patients each. In this portion of the trial, intrapatient dose escalation was also permitted if drug-related toxicity in a previous course was less than grade 2. Patients developing reversible DLTs could receive additional courses at the next lower dose level. The National Cancer Institute Common Toxicity Criteria version 2.0 (http://ctep.cancer.gov/reporting/ctc.html) were employed to grade toxicity. DLT was defined as any grade 3 or greater nonhematologic toxicity; grade 4 neutropenia lasting more than 3 days or associated with infection; grade 4 thrombocytopenia lasting more than 3 days or associated with clinically significant bleeding; or persisting toxicities of any grade requiring delay of scheduled treatment by more than 2 weeks. MTD was defined as the highest dose level in which less than two of six patients develop first course DLT.
Drug Supply and Administration
Study Monitoring and Measurement of Effect Complete response was defined as disappearance of all clinical evidence of active tumor and symptoms, for a minimum of 4 weeks. Partial response was defined as a decrease (by greater than 50%) in the sum of the product of the perpendicular diameters of all measured lesions for at least four weeks. No simultaneous increase in the size of any lesion or the appearance of new lesions could occur. The disease was considered stable when the response was less than partial and did not meet evidence for progression for a minimum of 8 weeks. Progressive disease was defined as an unequivocal increase of at least 50% in the size of any measured lesion, or the appearance of significant new lesions.
Pharmacokinetic Studies High performance liquid chromatography with UV detection was used to analyze the serum and urine samples for 3-AP concentration, as previously described.30,31 The validated assay has a nominal curve range of 0.02 to 10 µg/mL for serum and 0.05 to 10 µg/mL for urine. Pharmacokinetic modeling and pharmacokinetic parameter calculations were conducted using WINNonlin (Scientific Consultant, Apex, NC) software (Pharsight Corp, Mountain View, CA) with compartmental as well as noncompartmental methods. The following pharmacokinetic parameters were computed: area under the serum concentration-time curve from time zero to the last data point, peak serum concentration (Cmax), elimination half-life (T1/2), volume of distribution at steady-state (Vss), and total body clearance (Cl). Descriptive statistics (mean and standard deviation [SD]) were calculated and used to characterize the pharmacokinetic parameters at each dose level. For urine samples, cumulative urinary recovery of unchanged drug (3-AP) was determined for the time period of 0 (start of the infusion) to 8 hours after the end of the infusion.
Patient Characteristics Twenty-one patients were entered onto the study between May 2000 and August 2001 (Table 1). The majority of patients had gastrointestinal or head and neck primary tumors. All patients had received prior chemotherapy; 13 (62%) had received at least two prior chemotherapy regimens.
Seven patients received 3-AP on the every-3-week schedule, and 14 were treated on the every-other-week schedule (Table 2). Intrapatient dose escalation or reduction was permitted; thus, some patients were treated at more than one dose level. Three dose levels (60, 132, 224 mg/m2/d) were the result of dose modification and did not contain new patients. The median number of courses delivered for all patients on both schedules was four, with a range of one to 26.
Adverse Events Table 3A lists the worst grade of toxicity for each patient for the first course received at the designated dose level in the every-3-week schedule. Patients treated at multiple dose levels are represented more than once in Table 3A. Table 3B lists the worst grade of toxicity for each patient for the first two courses received at the designated dose level in the every-2-week schedule.
In the initial dose escalation on the 3-week schedule, grade 2 to 4 adverse events were first observed at a dose of 160 mg/m2/d. Among the six patients who received this dose, four developed grade 3 to 4 adverse events, including neutropenia (three patients, all grade 4), anemia (one patient, grade 3), thrombocytopenia (one patient, grade 4), hyperbilirubinemia (two patients, both events grade 3), and nausea and vomiting (one patient, grade 3). Three of the four patients with grade 3 to 4 adverse event met the criteria for DLT. The most severe toxicity occurred in one patient who entered the study with a low serum albumin of 1.6 gm/dL, large ascites, and an indwelling biliary stent. The WBC, hemoglobin and platelets dropped precipitously in the 48 hours following the infusion, which was followed by fungal septicemia and death. Serum 3-AP levels were 24, 2.6, and 14.8 µmol/L at 4, 48, and 96 hours of the infusion, respectively, which were 10 to 20 times higher than predicted. Although a causal relationship between low albumin levels, high drug serum concentrations, and severe hematologic toxicity was not certain, for safety reasons, patients with serum albumin less than 3.0 gm/dL were subsequently excluded from the trial. Grade 4 neutropenia occurred in two other patients, and in one the duration met the criteria for DLT. Anemia of grade 2 to 3 severity developed in three patients over several days following the completion of the infusion. Other nonhematologic events are described under Changes in Renal Function. Dose-limiting toxicity was not observed during the first two courses at the 80 and 100 mg/m2/d dose levels administered every other week. One patient on the 80 mg/m2/d cohort with a carcinoma of the paranasal sinuses developed a left middle cerebral artery stroke on the last day of the 3-AP infusion, but in view of the extent of local disease and lung metastases, the event was not considered related to treatment. Of the seven new patients entered at the dose of 120 mg/m2/d, one discontinued treatment after the first course, two received four consecutive courses without dose reduction, and four required dose reduction following the first course. Dose reductions following the first course were made for poor tolerance to treatment or a DLT, and included grade 3 anemia; grade 3 asthenia; grade 3 azotemia and decreased bicarbonate (see below); and grade 3 hyperbilirubinemia. Two of the latter patients had existing conditions which predisposed to severe toxicity. In the patient developing azotemia and acidosis, indwelling ureteral stents were present before treatment for bilateral hydronephrosis, and baseline renal function was abnormal as demonstrated by a serum creatinine of 1.8 mg/dL and a serum bicarbonate of 17 mEq/dL. In the patient developing grade 3 hyperbilirubinemia, an indwelling common bile duct stent was present before treatment. These same two patients had recurrence of toxicities when their dose was reduced to 100 and 80 mg/m2/d. In all instances, the adverse events resolved to baseline or grade 1 within several days following the completion of the 3-AP infusion, although resolution of creatinine elevation to baseline in the patient with indwelling ureteral stents was slower. The only other grade 3 hyperbilirubinemia on the every-2-week schedule occurred after several courses in a patient entered at the 100 mg/m2/d dose level. The latter patient had multiple liver metastases from an islet cell tumor and was accepted to the protocol with a baseline alkaline phosphatase more than 20 times the upper limit of normal. Hematologic toxicity in patients receiving the 80 to 120 mg/m2/d dose levels administered every other week was less prominent than in patients treated on the every-3-week schedule at a dose of 160 mg/m2/d. Only one of 14 patients developed grade 4 neutropenia during the first two courses of treatment, and none of the patients had greater than grade 2 thrombocytopenia. However, anemia was common; it was grade 3 in four patients and grade 2 in five patients. For all courses administered at the 80 to 120 mg/m2/d dose levels, grade 4 neutropenia and grade 3 anemia were observed among three and seven patients, respectively, indicating some cumulative effect. Based on these results, including the reversibility of dose-limiting events, the recommended phase II dose for 3-AP administered by 96 hour IV infusion is 120 mg/m2/d every other week. Because the renal and hepatic toxicities were more severe in patients with pre-existing borderline abnormal organ function, initial phase II trials should exclude patients with creatinine greater than 1.5 mg/dL or patients with liver dysfunction, for example, bilirubin greater than 1.5 mg/dL, or alkaline phosphatase or transaminases greater than three times the upper limit of normal.
Changes in Renal Function At the 160 mg/m2/d dose level, increases in serum creatinine (to a maximum of 1.2 to 1.9 mg/dL in five of six patients) and decreases in serum bicarbonate (to values of 12 to 17 mEq/dL in four of six patients) were observed. The serum creatinine and bicarbonate returned to baseline by the time of the next scheduled evaluation, at 3 to 5 days following the completion of the infusion, in all patients. At the lower doses of 80 to 120 mg/m2/d administered every other week, 13 of 14 patients had decreases in serum bicarbonate during the infusion in one or more courses of treatment. The toxicity grade was 4 in one patient (5 mEq/dL), grade 2 in four patients (11-15 mEq/dL), and grade 1 in five others. Notably, four patients had baseline serum bicarbonate in the range of 17 to 19 mEq/dL, which accounted for the one grade 4 and three of the grade 2 events. The nadir occurred on days 3 to 5 of the infusion. Infusions were not held if a low serum bicarbonate was detected on day 3, and with the exception of the single patient with grade 4 toxicity, patients did not receive corrective therapy. During the infusions, serum potassium levels were in the range of 4 to 4.8 mmol/L in all but one patient, whose potassium level was 3.1. Additional studies including arterial blood gases and urine electrolytes were not obtained systematically. In two patients, arterial blood gases obtained concurrent with the nadir of the serum bicarbonate showed decreased pH (in the range of 7.3) and CO2 consistent with a metabolic acidosis and partial respiratory compensation. Urinalysis in four patients demonstrated normal pH (5-6.5) without glycosuria. While increases in serum creatinine more than 0.2 mg/dL were observed in 11 of the 14 patients, maximum concentrations exceeded 2.0 mg/dL in only 3 patients (2.8, 2.8, and 4.6 mg/dL). Two of the latter patients had baseline creatinine values of 1.8 mg/dL, and although the third patient had an on-study value of 1.3 mg/dL, previous creatinine levels were higher and a history of renal insufficiency was present. Except for the patient with indwelling bilateral ureteral stents who developed the grade 3 creatinine and grade 4 bicarbonate adverse events, all other patients had return of creatinine and bicarbonate levels to baseline or grade 1 by the next scheduled evaluation (within 48-96 hours) after the end of the infusion. Only one other patient entered the study with a creatinine level above normal (1.6 mg/dL).
Pharmacokinetic Studies 3-AP was quantifiable in serum for up to eight hours after dosing. Table 4 summarizes the mean pharmacokinetic parameters at each dose level. Clearance was independent of dose (R2 = 0.0137, as demonstrated in Figure 1A) and body-surface area (data not shown). The mean elimination T1/2 ranged from 1 to 3 hours with a median value of approximately 2 hours. The steady-state concentration (Css) in individual patients versus total dose per day is shown in Figure 1B, and the mean of serum concentration versus time for three representative dose levels is shown in Figure 2. An initial analysis of Css showed no correlation with dose (R2 = 0.23). However, reanalysis of the data, excluding a single patient who had relatively high Css during two consecutive courses at 80 mg/m2/d, indicated linear pharmacokinetic behavior for 3-AP (R2 = 0.65) although with high inter-patient variability.
In urine, 3-AP was detected during the eight hours following drug administration. Cumulative urinary recovery averaged 0.6 to 1.2% of the administered dose, suggesting that the elimination of 3-AP was primarily through metabolism. In a previous clinical trial, hydroxylated and acetylated metabolites of 3-AP were identified in urine. The pharmacokinetics of these metabolites were not investigated in this study, and their activity and toxicity are not known. 3-AP concentrations in ascites or other body fluids were not measured in this trial.
Serum Iron Studies
Response to Treatment No partial or complete responses based on standard criteria of tumor size reduction were observed. However, several patients showed evidence of antitumor effects. Patient 11 with a squamous cell carcinoma involving the tonsillar area, progressing following regimens of cisplatin (plus cetuximab or placebo), local radiotherapy, and paclitaxel plus gemcitabine, began 3-AP at a dose of 80 mg/m2/d on the every-other-week schedule. The patient received 26 courses of treatment over 13 months; 17 courses were administered at 120 mg/m2/d and all but three at doses of 100-132 mg/m2/d. Following the last course, a CT scan demonstrated minimal change in the area of tumor involvement, and a PET scan failed to show evidence of disease (a pretreatment PET scan was not available for comparison). The patient has remained progression-free for an additional 10+ months. Patient 15 with uterine papillary serous carcinoma metastatic to hilar and paratracheal lymph nodes, and prior treatment with doxil and paclitaxel, began 3-AP at a dose of 100 mg/m2/d on the every-other-week schedule. She received a second course at the same dose, but then required dose reduction to 80 and 60 mg/m2/d. Serum CA-125 levels decreased from 1,807 to 325 over four courses, and although the CA-125 subsequently began to rise, she remained progression-free on CT scans for a total of 6 months from initiation of treatment. Decreases in serum tumor markers associated with stable disease of 3 months' duration were observed in patient 22 with pancreatic cancer (CA19-9, 33% decrease) and patient 14 with an islet cell carcinoma metastatic to liver (circulating vasoactive intestinal peptide). Patient 2 with metastatic colon cancer and patient 18 with recurrent head and neck cancer also achieved stable disease lasting 3 to 4 months from initiation of treatment.
This study demonstrated that 3-AP can be administered safely as a 96 hour IV infusion every two weeks. The dose-limiting toxicities at the recommended phase II dose (RP2D) of 120 mg/m2/d are primarily renal and hepatic; at higher doses, hematologic DLT is also observed. Patients with serum creatinine greater than 1.5 mg/dL and liver dysfunction (for example, presence of a biliary stent, bilirubin greater than 1.5 mg/dL, transaminases or alkaline phosphatase greater than three times the upper limit of normal) are at risk for more severe renal toxicity and hyperbilirubinemia and therefore should be excluded from the initial phase II trials. The pharmacokinetic profile for the drug was linear to 160 mg/m2, and serum drug concentrations at RP2D were at the lower limit of the target range of 1 to 2 µmol/L. Furthermore, some encouraging evidence of antitumor activity was observed in this heavily pretreated population of patients with refractory tumors. The use of body-surface area to calculate the dose of many anticancer agents in standard use or investigational trials has been questioned.33 Other than to choose the initial starting dose, no preclinical data for 3-AP were available to indicate that dosing on the basis of surface area was necessary during the dose escalation portion of the trial. After completion of the trial, an analysis of drug clearance versus body-surface area showed no correlation. Therefore, correction of dose for body-surface area may not be necessary in future studies. The actual doses received by patients at the RP2D of 120 mg/m2/d ranged from 205 to 268 mg/d, with an average of 220 mg/d. Based on the limited data in this trial, other variables such as age, sex, and baseline renal or hepatic function did not appear to affect clearance, although additional dose-finding and pharmacokinetic studies in patients with abnormal organ function are required. We undertook an investigation of the 96-hour continuous infusion schedule of 3-AP because in vitro and in vivo studies have shown that optimal antitumor effects are achieved when RR is inhibited continuously for prolonged durations. The data are consistent with the observation that RR is expressed principally in S phase, and therefore prolonged exposure to 3-AP allows the drug access to more cells in S phase.1 In addition, even for cells in S phase, cytotoxicity may be dependent on the extent and duration of perturbation in deoxyribonucleotide pools.34 Drug-induced continuous inhibition of the RR M2 subunit, and therefore the extent and duration of perturbation in deoxyribonucleotide pools, can be counteracted by an apparent feedback mechanism, whereby inhibition of RR results in synthesis of new enzyme in order to maintain adequate pools of nucleotide precursors for DNA synthesis. Preclinical studies have shown that cells increase their content of the RR M2 subunit after brief exposures to hydroxyurea, an agent which inhibits the RR M2 subunit similar to 3-AP.34 Although no data are available for 3-AP, a similar effect on the RR M2 subunit could be expected. Cells that overexpress the M2 RR subunit become resistant to hydroxyurea,35 therefore, prolonged infusions of hydroxyurea are likely to be effective only if steady-state concentrations are sufficient to inhibit the increased levels of enzyme that are rapidly induced in cells. In contrast, 3-AP has been shown to retain activity in cells overexpressing the M2 subunit,29 suggesting that continuous infusions of 3-AP are more likely to result in prolonged suppression of enzyme activity and sustained perturbation of nucleotide pools with resultant cell death. In the National Cancer Institute panel of 60 tumor cell lines, the median 3-AP concentration required for 50% growth inhibition (GI50) in a 48-hour exposure assay was 1.6 µmol/L. These data, and additional in vitro data from experiments employing 72- to 96-hour exposures, were the basis for our objective to achieve serum concentrations in the range of 1 to 2 µmol/L for the 96-hour IV infusion. These serum concentrations were achieved with a dose of 160 mg/m2/d administered every 3 weeks, but patients developed moderate to severe hematologic toxicity and moderate nonhematologic toxicity. At the next lower dose of 80 mg/m2/d, serum concentrations were in the range of 0.8 to 1.2 µmol/L, and the infusion was associated with less hematologic and nonhematologic toxicity. Since the lower dose levels produced acceptable serum concentrations and could possibly be administered on a more intensive schedule, which may be important for achieving optimal antitumor effects with this class of agents, we then elected to explore the feasibility and MTD of an every-other-week schedule at doses of 80 to 120 mg/m2/d. Overall, 3-AP doses of 80 to 120 mg/m2/d administered on the every-other-week schedule were well-tolerated in most patients. The major hematologic toxicity was anemia. Considering all courses, grade 4 granulocytopenia occurred in only 21% and was rapidly reversible. Nonhematologic toxicities included rapidly reversible hyperbilirubinemia, azotemia, acidosis, and nausea and vomiting. Although four of the seven patients treated at a dose of 120 mg/m2/d required dose reduction after the first course, the adverse events provoking a dose reduction, including grade 3 anemia, asthenia, decreases in serum bicarbonate, increases in creatinine, and hyperbilirubinemia, were rapidly reversible or could easily be managed with supportive care. In two of the patients, 3-AP-related toxicity was exacerbated by underlying renal or liver dysfunction. Of the total of eight patients that received the 120 mg/m2/d dose level, three tolerated multiple courses without difficulty. Thus, the 120 mg/m2/d dose level can still be considered as a safe starting dose for most patients in phase II trials, although additional studies will be required to determine safe doses for patients with baseline abnormal renal or liver function or compromised performance status. Mild, reversible acidosis and azotemia was observed commonly in patients receiving 3-AP. The mechanisms responsible for 3-AP-induced azotemia and acidosis remain undetermined; however there was a slight rise in the anion gap, from 13.1 to 16.8, without evidence of hyperkalemia. The most severe creatinine and bicarbonate adverse events occurred in patients with baseline compromise in renal function and/or baseline low serum bicarbonate levels; nevertheless, the events resolved rapidly after completion of the infusion. Thus, these are unlikely to compromise administration of full-dose therapy in the phase II setting, if patients are chosen without baseline renal insufficiency. Of interest, in a separate phase I trial of the 96-hour infusion conducted in patients with advanced hematological malignancies, 3-AP doses up to 140 mg/m2/d were administered without nonhematologic DLT, although as in the current trial, hyperbilirubinemia, rapidly reversible decreases in serum bicarbonate, and mild increases in creatinine were observed.36 In the phase I trial of 3-AP administered by 2-hour IV infusion daily x 5, hyperbilirubinemia, azotemia and decreases in serum bicarbonate were also observed at the MTD of 96 mg/m2/d, but the incidence and severity of these events appeared to be lower.31 In contrast, the 96 mg/m2 daily x 5 schedule was associated with more consistent grade 4 neutropenia. Because 3-AP is a potent iron chelator and could theoretically affect iron serum levels and total body stores, we obtained studies of serum iron, iron binding capacity, and ferritin at baseline and at the end of the infusion for at least one course in most patients. Consistently, serum iron and ferritin levels were increased at the end of the infusion, and returned to baseline by the start of the next course. The data indicate that 3-AP does not cause a net loss of iron from the body. The mechanisms responsible for the transient increases in serum iron and ferritin are not clear. Perhaps iron is bound by 3-AP in the circulation, and recovered during metabolism of 3-AP in the liver. The apparent increased delivery of iron to liver could induce the production of ferritin, accounting for the elevated serum levels. Unexpectedly high 3-AP serum concentrations of 5 to 35 µmol/L were detected in four patients. Other samples were run concurrently with those containing high concentrations, therefore, a problem with the assay methodology does not appear to explain the aberrant results. In three patients treated with 40, 160, and 120 mg/m2/d, the high levels were found in multiple samples, and in one patient in which pharmacokinetic studies were performed in four separate courses, the high concentration was in the baseline sample before his eighth course. The patient treated with 40 mg/m2/d had received a previous course at 20 mg/m2/d that was associated with the predicted 3-AP serum concentrations. No unusual toxicity was observed in three patients, but the patient receiving 160 mg/m2/d was treated early in the trial and developed severe and rapid myelosuppression. She was enrolled to study with a serum albumin less than 2 gm/dL, ascites, and an indwelling biliary stent. Because of the immediate concern that the high 3-AP serum concentrations were related to the low albumin levels, for safety reasons, the eligibility criteria were amended to exclude patients with albumin less than 3 gm/dL. However, a relationship between low albumin and abnormal pharmacokinetics is unlikely for several reasons; the other patients with high 3-AP concentrations did not have low albumin levels; in vitro, 3-AP is only 60% bound to human serum protein; and low albumin levels have not been associated with abnormal 3-AP serum concentrations in other trials. Only up to 1.2% of the administered 3-AP dose was recovered in urine, suggesting metabolism of the drug occurred in the liver. 3-AP concentrations were within the expected range in the patients with liver metastases, including the patient who entered the study by waiver with an alkaline phosphatase greater than 20 times the upper limit of normal. 3-AP concentrations were also within the expected range in a patient without liver metastases but with a biliary stent in place and subsequent development of treatment-related grade 3 hyperbilirubinemia. The serum concentrations achieved at the RP2D are in the range sufficient to cause decreases in one or more of the deoxyribonucleotide pools of tumor cell lines in vitro. Evidence that deoxyribonucleotide pools were affected in patients' tumor or a surrogate tissue would provide important proof of concept that the enzyme target was inhibited at safe doses of 3-AP. Unfortunately, measurement of deoxyribonucleotide pools are difficult in patients with solid tumors because of the prohibitive quantities of tissue required from sequential biopsies, which are necessary for quantitating nucleotide pools, and because surrogate tissues, such as peripheral blood mononuclear cells, are an unreliable guide to drug activity.37 In the phase I trial of continuous infusion 3-AP conducted in patients with advanced leukemias, deoxyribonucleotide pools and DNA synthesis were measured in circulating blasts of three patients before and during treatment.36 Steady-state 3-AP concentrations in the leukemia patients were similar or slightly lower compared to those achieved in the current trial, and all three leukemia patients demonstrated 3-AP induced decreases of dATP and dGTP beginning at approximately 24 hours and maintained throughout the infusion. DNA synthesis in blasts was also inhibited by 30% to 70%. The limited biologic data obtained in the hematologic malignancy trial suggest that the 3-AP RP2D established for solid tumors does indeed inhibit RR, and also provide the rationale for future trials involving 3-AP and other agents that inhibit RR through alternate mechanisms, for example, M2 subunit inhibitors or antisense agents. Some evidence of antitumor effect in these heavily pretreated patients with refractory tumors was observed in this phase I trial. Phase II studies are warranted to further define 3-AP's activity in several tumor types. To facilitate continuous administration, an oral formulation of 3-AP is under development, as well as pro-drugs that increase the effective 3-AP serum half-life.38 Preclinical data also indicate that 3-AP may yield the greatest benefit when combined with other DNA damaging or cytotoxic agents. In vitro and/or in vivo, 3-AP enhances the activity of various DNA-damaging agents and nucleoside analogues.29,39 The safety profile demonstrated in this trial suggests that combinations of 3-AP with other anticancer agents may be well tolerated and merit clinical exploration.
The authors indicated no potential conflicts of interest.
Supported by Vion Pharmaceuticals, Inc, and by Cancer Center Support Grant CA 13330 from the National Cancer Institute of the National Institutes of Health. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2.0 mg/dL; total bilirubin 
