Phase I Trial of the Cyclin-Dependent Kinase Inhibitor and Protein Kinase C Inhibitor 7-Hydroxystaurosporine in Combination With Fluorouraci
http://www.100md.com
▲還散笫雖悝◎
the Gastrointestinal Oncology Service, Division of Solid Tumor Oncology, Department of Medicine
Program of Molecular Pharmacology and Experimental Therapeutics
Departments of Pediatrics and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY
ABSTRACT
PURPOSE: Preclinical studies indicate that the cyclin-dependent kinase and protein kinase C inhibitor 7-hydroxystaurosporine (UCN-01) potentiates the cytotoxic effects of fluorouracil (FU). We designed a phase I clinical trial of FU in combination with UCN-01.
PATIENTS AND METHODS: FU was administered as a weekly 24-hour infusion. Doses were escalated in successive cohorts according to a modified Fibonacci design. UCN-01 was administered once every 4 weeks, immediately after disconnection from FU, at a dose of 135 mg/m2 over 72 hours in cycle 1 and 67.5 mg/m2 over 36 hours in subsequent cycles. FU and UCN-01 pharmacokinetics were obtained on all patients, and thymidylate synthetase (TS) activity was measured in peripheral-blood mononuclear cells by reverse-transcriptase polymerase chain reaction.
RESULTS: We escalated the weekly FU dose to 2,600 mg/m2 in combination with once a month infusions of UCN-01. Dose-limiting toxicity included arrhythmia and syncope. Other toxicities included hyperglycemia, headache, and nausea and vomiting. The mean maximal plasma concentration of UCN-01 was 33.5 米mol/L. There was significant interpatient variability, which correlated with plasma concentrations of alpha-1 acid glycoprotein. FU was rapidly cleared and the dose had no effect on the area under the curve of UCN-01. Changes in TS expression were detectable in peripheral-blood mononuclear cells after administration of UCN-01 but did not correlate with toxicity or activity. We observed no objective response, although seven patients had stable disease, six of whom had received prior fluoropyrimidines.
CONCLUSION: The combination of weekly infusions of FU and monthly UCN-01 can be administered safely and warrants further study in phase II trials. The recommended phase II dose of FU in combination with monthly UCN-01 is 2,600 mg/m2.
INTRODUCTION
7-Hydroxystaurosporine (UCN-01) is a staurosporine analog isolated from the culture broth of Streptomyces spp that has antitumor activity in several in vitro and in vivo preclinical models. Although UCN-01 is a potent inhibitor of Ca2+-dependent protein kinase C isoenzymes, its antiproliferative activity cannot be solely explained by this mechanism. Other proposed targets include cyclin-dependent kinases, Chk11每4 and AKT.5 In vitro, UCN-01 has growth-inhibitory effects on numerous human and murine tumor cell lines, with median inhibitory concentration values in the nanomolar range.6每8 UCN-01 has been shown to induce G1 arrest and cause hypophosphorylation of the retinoblastoma protein. In addition, there is an emerging body of literature that UCN-01 synergistically enhances radiation- and chemotherapy-induced apoptosis.9每18
UCN-01 has been shown to synergistically enhance fluorouracil (FU) -induced apoptosis in a sequence-dependent fashion.19 The enhancement of apoptosis induction was greatest when tumor cells were first exposed to FU for 24 hours followed by UCN-01. There was also enhancement with the reverse combination but to a lesser degree. Consistent with effects on inducing G1 arrest and hypophosphorylation of retinoblastoma protein, UCN-01 has been shown to decrease thymidylate synthetase (TS) mRNA and protein expression in a dose-dependent manner.19 Further studies showed that UCN-01 induces E2F-1-specific proteasome degradation, which subsequently leads to decreased transcription of TS mRNA.20
Results from two separate phase I clinical trials of UCN-01 have been reported.21每23 These studies revealed that the pharmacokinetics of UCN-01 administered as a 72- or 3-hour infusion have distinctive features that could not have been predicted from preclinical data. The distribution volume and the systemic clearance were extremely low. More importantly, the elimination half-life was unusually long (253 to 1,660 hours). In vitro protein-binding experiments demonstrated that UCN-01 is significantly bound to human alpha-1 acid glycoprotein (AGP) with high affinity. In fact, the association constant for UCN-01 and human AGP was found to be more than 60-fold the affinity between UCN-01 and dog AGP. In light of these observations, frequency of administration was extended from every 14 days to every 28 days. Also, after the first 72-hour infusion of UCN-01, subsequent doses were halved (36-hour infusion) to prevent cumulative drug buildup. With this schedule, the maximum-tolerated dose (MTD) was 42.5 mg/m2/d or 127.5 mg/m2 total over the 72-hour infusion. Dose-limiting toxicities (DLTs) were myalgias, hyperglycemia, nausea, vomiting, and hypoxemia.21每23 Side effects most commonly occurred during or shortly after drug infusion and resolved after cessation of drug administration. No clinical responders were noted in this dose-finding trial.
On the basis of our preclinical data, we initiated a phase I study combining FU and UCN-01. The UCN-01 dose and schedule were based on the single-agent phase I study of UCN-01 (42.5 mg/m2/d infused continuously over 72 hours in week 1 and the same dose infused over 36 hours repeated every 4 weeks). The initial FU dose was set at 250 mg/m2 was administered as a weekly 24-hour infusion during the entire course of therapy. The FU dose and schedule were designed to maximize the inhibition of TS. Infusional FU may act more specifically to inhibit TS24 when compared with bolus FU and has been associated with longer survival and less myelosuppression.25,26 In addition, our preclinical studies used a 24-hour exposure of FU to maximize the synergy observed with UCN-01. FU was administered before UCN-01 on the first week of each cycle to take advantage of the superior schedule.
PATIENTS AND METHODS
Patient Selection
Adult patients ( 18 years old) with histologically confirmed solid tumor that was refractory to standard therapy (or for which there was no standard therapy) were eligible for the study. The patient had to be off all previous chemotherapy, immunotherapy, or radiotherapy for 4 weeks before study entry (6 weeks for nitrosoureas and mitomycin). Prior FU therapy was allowed. Additional eligibility criteria included Karnofsky performance status of more than 60%, adequate hematopoietic function (WBC 3,500/米L, absolute neutrophil count 1,500/米L, and platelets 100,000/米L), adequate renal function (creatinine 1.5 mg/dL), adequate hepatic function (total bilirubin 1.5 mg/dL and AST and ALT < 2.5 x upper limit of normal), and adequate pulmonary function (lung diffusing capacity 60% predicted).
Patients were excluded from participation in the study for any of the following reasons: presence of any ongoing toxic effect from a prior treatment; presence of any serious or uncontrolled infection; known CNS metastasis or CNS primary tumor; prior mediastinal radiation; pregnant or lactating; history of cardiac arrhythmias, angina, or myocardial infarction in the preceding 6 months; HIV infection; or use of anticonvulsant medications. Patients with a distant history (> 6 months before study) of coronary artery disease required a cardiac stress examination and clearance by a cardiologist before enrollment. Patients using medications known to bind AGP were changed to an alternative medication, when possible. A central venous access device or peripherally inserted central catheter line was required.
The protocol was reviewed and approved by the National Cancer Institute (NCI) and by the Institutional Review Board of the Memorial Sloan-Kettering Cancer Center. Written informed consent was obtained from each patient.
Treatment Plan
This phase I trial was designed as an open-label, nonrandomized, dose-escalation study in which groups of three to six patients were to receive increasing doses of intravenous FU over 24 hours in combination with a fixed dose of intravenous UCN-01 until DLT was demonstrated in at least two of six patients or a FU dose of 2.6 g/m2 was achieved (the MTD of single-agent FU was administered as a weekly 24-hour infusion27每29). FU was administered weekly as a 24-hour continuous infusion on days 1, 8, 15, and 22 of each 4-week cycle. UCN-01 was administered on day 2 of each cycle immediately after disconnection from FU. UCN-01 was administered at a dose of 45 mg/m2/d as a 72-hour continuous infusion in cycle 1 (total dose, 135 mg/m2) and a 36-hour continuous infusion in each subsequent 4-week cycle (total dose, 67.5 mg/m2), in accordance with the NCI-recommended dosing schedule for UCN-01. Both FU and UCN-01 were administered through a central venous catheter. In the initial trial design, patients received UCN-01 alone (72-hour infusion) in the first cycle to determine whether FU affected the pharmacokinetics of UCN-01. Combination therapy with FU started with the second cycle on the schedule described earlier. Four of the first five patients progressed before the completion of three cycles of therapy, and the protocol was subsequently amended to begin weekly FU infusions with the first cycle of UCN-01 therapy. Prophylactic antiemetics were not routinely administered unless clinically indicated. To avoid exacerbation of UCN-01-induced hyperglycemia, corticosteroids were avoided unless other antiemetic choices were ineffective.
Because of potential hemodynamic effects, vital signs, including orthostatic blood pressure and oxygen saturation, were measured 15 and 55 minutes after initiation of UCN-01 and at disconnection from the UCN-01 infusion. Routine physical examination, including vital signs, performance status, and weight, was performed weekly before chemotherapy. Routine laboratory studies, including a CBC with differential and platelets, liver and renal function, and electrolytes with blood glucose, protein, albumin, lactate dehydrogenase, and uric acid, were conducted on the day of therapy and weekly thereafter. Treatment responses were evaluated after two cycles of combination therapy using Response Evaluation Criteria in Solid Tumors Group criteria.
The initial dose of FU was 250 mg/m2 over 24 hours, which represented approximately 10% of the standard dose for 24-hour infusions of FU. FU doses were to be escalated by approximately 50% between cohorts until a dose of 2,600 mg/m2 was reached or until any one patient developed grade 3 or greater toxicity of any type that was attributable to the combination of FU and UCN-01. Subsequent dose escalations of the FU were to be by 33% until the MTD was reached. Intrapatient escalation of FU to a higher dose level was permitted only if one cohort of three patients completed one cycle of therapy at the higher dose level without grade 3 or greater toxicity. If unexpected grade 3 or higher toxicity was observed in the first cohort of patients, the FU dose was to be reduced in 33% decrements to a minimum of 50 mg/m2. The NCI Common Toxicity Criteria (version 2.0) was used to evaluate toxicity and adverse events.
DLT was defined as the occurrence in cycle 1 of grade 4 neutropenia, grade 4 anemia, grade 3 or 4 thrombocytopenia, grade 4 hyperglycemia, or grade 3 or 4 nonhematologic toxicities not directly attributable to UCN-01 therapy as a single-agent. Toxicities that were described to be attributable to UCN-01 included headache, nausea, vomiting, hyperglycemia, muscle cramps, hypotension, and hypoxemia close to time of infusion. Grade 3 hyperglycemia refractory to insulin therapy was considered a DLT. Furthermore, a DLT was considered to have occurred if any nonhematologic toxicity failed to improve sufficiently within 7 days after causing a dose delay or if the platelet count failed to recover to 100,000/米L or the absolute neutrophil count failed to return to 1,500/米L within 7 days after causing a dose delay. A missed dose of FU was not considered a DLT, unless the patient received less than three doses of FU in a 4-week cycle. In the event that the MTD was reached because of dose-limiting hyperglycemia in patients with known diabetes (defined as fasting blood sugar 126 mg/dL, any blood sugar 200 mg/dL, or blood sugar 200 mg/dL after a glucose tolerance test), we allowed patients without a history of diabetes to be enrolled. Further dose escalation would then proceed to determine the MTD of the combination in nondiabetic patients.
A minimum of three patients was observed for at least one complete cycle of combination therapy (4 weeks after initiation of combination therapy) before escalation to the next dose level. If none of the three patients experienced a DLT, then three new patients were entered at the next higher FU dose level. All patients in the prior cohort must have completed one cycle of therapy before enrollment in the next cohort began. If one instance of DLT was observed among the initial three patients treated at any dose level, an additional three patients were to be treated at that dose level, with no further DLT, so that dose escalation could proceed. If two instances of DLT were observed at any dose level, the MTD was considered to have been surpassed, and a total of six patients were to be treated at the previous dose level to assure its tolerability. In the clinical setting of stable or responding disease, patients could continue treatment after experiencing a DLT, with the FU reduced to one lower level, once all unacceptable toxicity had completely resolved. Patients were continued on therapy unless one of the following criteria was met: objective disease progression, unacceptable toxicity not responsive to dose attenuation, investigator considered it unsafe to continue treatment, patient was unwilling or unable to continue (dropped out), patient died, or patient was lost to follow-up.
Drug Supply
UCN-01 (NSC 638850) was supplied by the NCI in sterile, single-use 10-mg and 50-mg vials. The 10-mg vial contained UCN-01 lyophilized powder, 11 mg of citric acid, United States Pharmacopeia (USP), 22.2 mg of anhydrous dibasic sodium phosphate, USP, and 200 mg of lactose, National Formulary (NF). The 50-mg vial contained 50 mg of UCN-01 lyophilized powder, 55 mg of citric acid, USP, 111 mg of anhydrous dibasic sodium phosphate, USP, and 1,000 mg of lactose, NF. The 10-mg vial was reconstituted with 10 mL of water for injection, USP, or 0.9% sodium chloride for injection, USP. The 50-mg vial was reconstituted with 50 mL of either diluent to produce a 1-mg/mL solution of UCN-01. Further dilution with normal saline to a final concentration as low as 10 米g/mL was permitted. Concentrations less than 10 米g/mL were not permitted. Normal saline was the diluent of choice. D5W or D51/2NS could not be used. Polyvinylchloride bags or glass bottles could be used for drug infusions. An in-line filter with a pore size of 1.2 米m was used.
FU (Adrucil; Pharmacia and Upjohn, Mississauga, Ontario, Canada) was supplied as a 50-mg/mL solution in vials of 50 mL and 100 mL. The appropriate volume was withdrawn into a syringe, which was then used for administration by 24-hour infusion. No dilution was required.
Pharmacokinetics
Assessment of plasma UCN-01 and FU concentrations for pharmacokinetic studies were performed on all patients according to published methods with the first two cycles of UCN-01.30 Blood samples to measure UCN-01 were drawn at the following time points during cycle 1: just before the start of UCN-01; 15 minutes, 55 minutes, and 24 hours after the initiation of the 72-hour UCN-01 infusion; and immediately after the completion of the UCN-01 infusion. During the second cycle, blood samples were drawn just before the start of the UCN-01 infusion and at the completion of the 36-hour infusion. For the five patients treated at 2,600 mg/m2, an additional blood draw was obtained 14 days after the completion of the UCN-01 infusion (day 16). Because UCN-01 is known to be heavily protein bound to AGP, plasma measurements of this protein were collected just before the initiation of the UCN-01 dose in the first two cycles. AGP was measured by radial immunodiffusion31 using commercially available kits (The Binding Site Ltd, Birmingham, United Kingdom). Blood samples to measure FU were drawn at the following time points during the first two cycles: just before the start of FU, at the completion of the FU infusion, and at 15 and 55 minutes after the initiation of the UCN-01 infusion. A limited sampling strategy was used for FU as validated by Moore et al.32
Biologic Monitoring
Ex vivo studies of the effects of treatment on TS activity in peripheral mononuclear cells were performed in the first two cycles. Whole-blood samples were collected according to the manufacturer's instructions in a Vacutainer CPT tube (Becton Dickinson, Franklin Lakes, NJ) with EDTA before the FU infusion, before the UCN-01 infusion, and at the completion of the UCN-01 infusion (72 hours in cycle 1 and 36 hours in cycle 2). In the five patients treated with FU 2,527 and 2,600 mg/m2, an additional sample was collected 14 days from the start of the UCN-01 infusion to measure midcycle effects. Centrifugation was performed with a Ficoll-Hypaque gradient to recover the mononuclear layer, as described by Wakamiya et al.33 TS mRNA expression was determined by quantitative reverse-transcriptase polymerase chain reaction (PCR; ABI Prism 7700 Sequence Detection System; Applied Biosystems, Foster City, CA) using TS sequence-specific complementary primers and beta-actin as the endogenous control.
RESULTS
Patient Characteristics
August 1999 through June 2002, 35 patients with advanced solid tumors were treated with the combination of FU and UCN-01. Table 1 lists the patient characteristics. The median age was 59 years (range, 38 to 79 years), and the median Karnofsky performance status was 90% (range, 70% to 90%). The majority of patients on study carried a diagnosis of metastatic colon cancer (n = 21). Thirty-four patients had received prior chemotherapy (mean number of regimens, three; range, zero to six regimens); of these patients, 26 had received prior FU or a FU derivative. Twenty-four of 26 patients discontinued their FU-based therapy because of progression of disease; one patient received FU as adjuvant therapy for colon cancer more than 2 years before enrolling onto our study; and one patient received FU as a radiosensitizing agent as adjuvant therapy for ampullary cancer. Eleven patients had received prior radiotherapy.
A total of 84 cycles of treatment were administered (median, two cycles; range, one to seven cycles) over eight cohorts at the following dose levels of FU: 250, 375, 560, 845, 1,265, 1,900, 2,527, and 2,600 mg/m2 (Table 2). All 35 patients enrolled onto the study were considered assessable for toxicity; two patients in cohort 2 and one patient in cohort 4 experienced rapid disease progression before completion of the first cycle and were replaced on study. One patient in cohort 6 did not complete the 72-hour UCN-01 infusion in cycle 1; to be certain of toxicity, an additional patient was added to this cohort. Thirty-three patients were assessable for response to combination therapy; two patients who experienced arrhythmia during the first week of treatment were removed from study.
DLT
We observed four DLTs in cycle 1 (Table 2). A patient receiving 560 mg/m2 of FU experienced a grade 3 syncopal episode 1 day after the second FU infusion in cycle 1. The patient reported observed loss of consciousness for several seconds on two separate occasions during the same night, after changing quickly from the supine to upright position. These symptoms were unreported until he presented to a physician for routine follow-up 6 days later. The described symptoms appeared orthostatic in nature, although no documented change in blood pressure or pulse was available at the time of the incident. A cardiac (orthostatic blood pressure assessment, ECG, and echocardiogram) and neurologic evaluation (head computed tomography) were both negative. We believed the event was likely related to the UCN-01 alone and not to the combination, and we elected to continue the patient on therapy without dose adjustment in FU. He completed two cycles without a second episode before discontinuing treatment because of progression.
A patient receiving 845 mg/m2 of FU experienced atrial fibrillation after the first dose of FU and UCN-01 and was removed from study. This cardiac event was observed in the setting of pneumonia and chronic obstructive pulmonary disease exacerbation. Normal sinus rhythm returned with conservative medical management. Because of the underlying medical issues, this arrhythmia was initially believed to be unrelated to therapy and was not considered a DLT. However, a patient receiving 1,900 mg/m2 of FU also developed atrial fibrillation during the first cycle of therapy. The arrhythmia was noted before receiving the second weekly dose of FU. The patient had no prior history of arrhythmia or coronary artery disease and reverted to a normal sinus rhythm with pharmacologic management. A cardiac evaluation, including cardiac enzymes and echocardiogram, was negative for a predisposing comorbidity. This patient was also removed from study because her performance status deteriorated over the ensuing weeks, and she was unfit to resume treatment.
In addition, one patient experienced grade 4 hyperglycemia, which was defined as a DLT on this protocol. The grade 4 toxicity occurred during the 72-hour infusion of UCN-01 in a patient treated at the 560 mg/m2 FU dose level. This patient had a known underlying diagnosis of diabetes, and hospitalization for intravenous fluids and insulin drip was required. The hyperglycemia proved to be refractory, requiring nearly 44 hours of insulin infusion before blood sugars resolved to less than 200 mg/dL. After this adverse event, we elected to amend the protocol to exclude patients with a known underlying diagnosis of diabetes mellitus. Diabetes mellitus was defined as fasting blood sugar 126 mg/dL, any blood sugar 200 mg/dL, or blood sugar 200 mg/dL after a glucose tolerance test.
Hematologic Toxicity
Therapy was well tolerated. No grade 4 hematologic toxicity was observed. One patient treated at a FU dose of 560 mg/m2 experienced grade 3 anemia.
Common Nonhematologic Toxicity
Common nonhematologic toxicities for all cycles of therapy that occurred in 10% of patients are listed in Table 3. As shown, diarrhea, constipation, fatigue, headache, nausea, and vomiting were observed with increased frequency. These toxicities have been reported in a previous phase I study of UCN-01.21 As was noted in other studies, hyperglycemia remains a significant problem with UCN-01. In addition to the patient who experienced grade 4 toxicity (DLT), five patients experienced grade 3 toxicity. Four of the five episodes of grade 3 hyperglycemia were observed after the protocol revision to exclude patients with underlying diabetes.
Additional observed grade 3 toxicities that were at least possibly attributable to the combination therapy include hyperbilirubinemia (n = 1), fatigue (n = 2), fever (n = 1), myalgia (n = 1), nausea (n = 5), and vomiting (n = 2). Nausea and vomiting were controlled with appropriate antiemetics in all instances. One patient experienced grade 4 fatigue, which occurred after the third FU infusion in cycle 1; however, this was attributed to rapid disease progression. In all patients, side effects of treatment resolved to grade 1 or lower before the next dose of UCN-01. We did not observe cumulative toxicities in patients who received more than two cycles of treatment.
Pharmacokinetics and Surrogate Studies
UCN-01 and AGP assays were available for 34 patients treated on this study. A total of 327 plasma samples were available for analysis of UCN-01 concentration (92% of planned samples). The mean maximal concentration (Cmax) of UCN-01 at the completion of the 72-hour infusion was 33.5 ㊣ 12.2 米mol/L (range, 3.7 to 53.5 米mol/L). The Cmax after completion of the 36-hour infusion of UCN-01 in the second cycle was 33.5 ㊣ 13.4 米mol/L (range, 15.0 to 65.5 米mol/L). There was no significant difference between the Cmax of UCN-01 achieved at the completion of the 72- and 36-hour infusions of UCN-01 in cycles 1 and 2, respectively (P = .64, signed-rank test). Starting with a FU dose of 1,900 mg/m2, there was a trend toward decreasing UCN-01 Cmax with increasing FU dose (Table 4); however, this difference was not statistically significant. We measured the area under the curve (AUC) of UCN-01, which was calculated using a noncompartmental model, from pre-UCN-01 infusion in cycle 1 to pre每UCN-01 infusion in cycle 2. Figure 1 demonstrates that, despite the increasing dose of FU in each cohort, there was no change in the AUC for UCN-01 (P = .77, Kruskal-Wallis test). We observed no correlation between UCN-01 Cmax or AUC and either toxicity or response.
We measured UCN-01 concentrations on day 16 in five patients treated in the FU 2,527 and 2,600 mg/m2 cohorts. The mean concentration of UCN-01 decreased to 17.6 ㊣ 6.2 米mol/L on day 16, which was approximately 50% of the Cmax at the completion of the 72-hour infusion. As predicted by its prolonged half-life in human models, UCN-01 was still measurable in plasma immediately before the infusion in the second cycle. At this time point, the mean concentration of UCN-01 was 13.0 ㊣ 2.7 米mol/L or approximately 40% of the Cmax.
Because of high binding affinity to UCN-01, plasma levels of AGP are postulated to correlate with peak UCN-01 concentrations. We measured AGP before the UCN-01 infusion in each cycle. Figure 2 demonstrates a linear correlation between UCN-01 concentration and AGP (P = .01).
We collected peripheral-blood mononuclear cells for ex vivo TS analysis before the FU infusion, before the UCN-01 infusion, and at the completion of the UCN-01 infusion (72 hours in cycle 1 and 36 hours in cycle 2). We compared TS expression by quantitative PCR before and after the UCN-01 infusion in each of the first two cycles. In this analysis, we had 13 matched pairs (seven in cycle 1 and six in cycle 2), representing nine different patients. Additionally, four matched pairs were available to compare TS mRNA levels at the completion of UCN-01 (cycle 1) with day 16 levels (midcycle). We observed no correlation between UCN-01 infusion and change in TS mRNA levels and no correlation between change in TS mRNA levels and response or toxicity to treatment.
Response to Therapy
Thirty-two patients were assessable for response. We observed no objective response, although eight patients (six colon, one small bowel, and one unknown primary) had stable disease. Seven of eight patients with stable disease had previously received and progressed on FU as part of a standard regimen for their disease. Patients with stable disease remained on study for a median of 4 months (3, 3, 4, 4, 4, 4, 5, and 7 months). This included one patient with colon cancer who had significant regression of lung metastases (Fig 3) after one cycle of treatment. This patient had received FU and levamisole in the adjuvant setting, but his disease recurred within 4 months of completion of treatment. Two months before enrolling onto this phase I study, the patient had progressed on FU, leucovorin, and oxaliplatin. Unfortunately, after the first cycle of treatment with FU and UCN-01, the patient developed an elevated bilirubin secondary to bile duct obstruction. A polyethylene stent was placed endoscopically, but treatment was delayed by 1 month until the bilirubin returned to normal. A computed tomography scan after one additional cycle of treatment revealed progression of disease in the lungs, and the patient was removed from study. His initial response was adjudicated as stable disease (minor response).
DISCUSSION
In this study, patients received escalating doses of FU administered once weekly as a 24-hour continuous infusion. UCN-01 was administered as a fixed dose of 45 mg/m2/d on the second day of week 1 of each 4-week cycle, immediately after disconnection from FU. As recommended by Sausville et al,21 UCN-01 was infused over 72 hours in the first cycle and then over 36 hours with each additional cycle. Our starting dose of FU was 250 mg/m2, and we successfully escalated the FU to our planned end point of 2,600 mg/m2 as a 24-hour infusion. This is our recommended phase II dose for future studies with FU in combination with fixed dose UCN-01 (45 mg/m2/d). This target dose of FU was based on a number of infusional FU regimens for gastrointestinal malignancies that have used 24-hour, 48-hour, 5-day, 28-day, and longer durations of therapy. Published studies using 24-hour infusions of FU have reported MTD of 2,600 mg/m2.27每29 We elected not to expand the final cohort to six patients because we observed no DLTs at FU doses of either 2,527 or 2,600 mg/m2.
We observed DLTs of syncope in the third cohort and arrhythmia in the sixth cohort with the combination. Hemodynamic complications have been previously reported with UCN-01. In the phase I study by Sausville et al,21 grade 1 and 2 hypotension was observed in patients treated at doses 24 mg/m2/d, with grade 3 toxicity in one patient treated with 53 mg/m2/d. Dees et al34 demonstrated dose-limiting hypotension and readily reversible respiratory failure in a patient receiving a 1-hour infusion of UCN-01 at a dose of 95 mg/m2/d. A Japanese study of UCN-01 administered as a 3-hour infusion did not observe dose-limiting hypotension.23 In our study, we evaluated patients for orthostatic hypotension at 15 and 55 minutes into the UCN-01 infusion and immediately after completion of the UCN-01 in the first two cycles. No patient experienced documented hemodynamic changes. For the one patient who experienced syncope, the event occurred 4 days after disconnection from the UCN-01. Although hypotension is probably attributable to UCN-01, it is unclear whether this effect is a consequence of rapid infusion or an idiosyncratic event.
Likewise, it is not clear whether the arrhythmia (atrial fibrillation) was truly a treatment-related side effect. In one patient, the event was observed in the setting of pneumonia and chronic obstructive pulmonary disease exacerbation and may have been coincidental. The second event occurred in an elderly woman and may represent an appropriate comorbidity. However, since completion of our study, grade 3 atrial fibrillation was observed in a patient enrolled onto a phase I trial of UCN-01 and cisplatin, suggesting that this toxicity may, in fact, be related to UCN-01.35
The majority of grade 3 and 4 toxicities observed on this study can be attributed to UCN-01 alone and include nausea, vomiting, headaches, and hyperglycemia. However, hyperglycemia remains a difficult problem in patients receiving UCN-01. On our study, one patient with hyperglycemia required hospitalization for continuous insulin infusion. Furthermore, after amending the eligibility criteria to exclude patients with diabetes mellitus, we observed four additional episodes of grade 3 hyperglycemia. Despite these toxicities, we remain encouraged by the observation of stable disease in eight patients who had previously experienced treatment failure with multiagent chemotherapy regimens, including seven patients who had previously progressed on FU-based therapy.
The etiology of the hyperglycemia is unclear, although it has been postulated that it is related to UCN-01-induced peripheral resistance to glucose. In their phase I study, Sausville et al21 noted hyperglycemia in association with hyperinsulinemia. An alternative hypothesis involves inhibition of AKT and downstream insulin-receptor pathways. Staurosporine inhibits phosphorylation on Thr-308 in the activation loop of AKT via inhibition of 3-phosphoinositide-dependent protein kinase-1.36 In mouse models, overexpression of AKT was associated with increased pancreatic beta-cell growth and increased overall insulin secretion.37 Interestingly, insulin secretion in response to glucose was impaired when corrected for beta-cell mass. There was no apparent effect on somatostatin or glucagon. Therefore, inhibition of AKT by UCN-01 may have the opposite effect (ie, decreased pancreatic beta-cell mass and decreased insulinemia). We plan to evaluate the effects of UCN-01 on AKT signaling pathways in future studies.
After the 72- and 36-hour infusions of UCN-01, the mean Cmax was 33.5 ㊣ 12.2 米mol/L (range, 3.7 to 53.4 米mol/L) and 33.5 ㊣ 13.4 米mol/L (range, 14.9 to 65.4 米mol/L), respectively. The mean Cmax is similar to published pharmacokinetics; at a dose of 42.5 mg/m2/d, Sausville et al21 reported a mean Cmax of 36.4 米mol/L. With increasing FU dose, there was a trend towards a decrease in UCN-01 Cmax starting at a FU dose of 1,900 mg/m2. However, these differences were not statistically significant because the ranges were overlapping. We suspect these changes are secondary to small patient numbers and wide interpatient variability. The wide interpatient variability in our study is likely related to differences in AGP, which is linearly correlated with the UCN-01 concentration. In contrast, intrapatient variability was relatively narrow, and the mean Cmax after the 72-hour infusion was similar to the mean Cmax after the 36-hour infusion. The pharmacokinetics of FU at the recommended phase II dose of 2,600 mg/m2 are being further tested in combination with the fixed doses of UCN-01 in ongoing phase II clinical trials.
In their phase I study of UCN-01 administration by 72-hour continuous infusion, Sausville et al21 demonstrated pharmacokinetic features of UCN-01 that were not predicted by preclinical studies in rodent and dog species. These unusual pharmacokinetics are attributed to strong binding to human AGP. There is a positive correlation between AGP concentration and plasma concentration for UCN-01. Unfortunately, this correlation likely has little clinical utility because it does not capture free UCN-01 levels, which are probably more predictive of toxicity and activity. The difficulty in measuring free drug is a major concern in the development of UCN-01. The current dosing schedule of UCN-01 is based on the prolonged half-life of the drug-AGP complex but may not reflect optimal tissue exposure to pharmacologically active free drug. Current methods to measure free drug, including ultracentrifugation, are imperfect.
Because of this difficulty in predicting free-UCN-01 using pharmacokinetic assays, we postulated that surrogate markers of drug activity may provide information regarding the appropriate dosing schedule of this agent and may maximize its antitumor effect. In this clinical trial, we used real-time PCR to assess changes in TS mRNA levels in peripheral-blood mononuclear cells with the hope that a correlation between mRNA levels and UCN-01 dosing might allow this ex vivo study to serve as a surrogate marker of drug activity. We were unable to detect a correlation between TS expression and UCN-01 dosing in peripheral-blood mononuclear cells. Furthermore, a decrease in TS mRNA levels failed to predict for response (stable disease) or toxicity to therapy. This lack of effect may be related to the fact that lymphocytes are noncycling cells and are unlikely to be affected by a cyclin-dependent kinase inhibitor without phytohaemagglutinin stimulation. Changes in TS expression within the tumor may be more representative of the true effects of UCN-01 and will be considered in further studies with this combination.
Despite a prolonged infusion, FU was rapidly cleared and was undetectable within the first hour of the UCN-01 infusion. The dose level of FU had no impact on UCN-01 pharmacokinetics, which remained constant throughout all the cohorts.
In the present study, we successfully escalated FU to a dose of 2,600 mg/m2 over 24 hours in combination with UCN-01 and recommend this dose for future disease-specific phase II studies. This represents the first clinical trial to combine chemotherapy with UCN-01. There was minimal toxicity attributed to the combination, although expected toxicities associated with UCN-01 were observed. The most troublesome side effect was hyperglycemia, which proved to be refractory to insulin infusion in one patient. After improved screening to exclude known diabetics, significant hyperglycemia was observed but was manageable. We plan to further study this combination in a phase II clinical trial of patients with metastatic, gemcitabine-refractory pancreatic cancer. This trial is based on Japanese data that indicate potentiation of FU cytotoxicity by UCN-01 through downregulation of TS mRNA in pancreatic cancer cell lines.16 TS is reported to be overexpressed in approximately 50% of pancreatic cancers.38 Although this population would be considered at high risk for hyperglycemia from UCN-01, we have reported that, with appropriate glucose monitoring, this toxicity can be well controlled.39 In the future study, we will attempt to demonstrate the effects on TSexpression in intratumoral specimens and to better clarify the etiology of hyperglycemia with this drug.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by National Cancer Institute grant No. U01-CA69856.
Presented in part at the 37th Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001 and the American Association for Cancer Research-National Cancer Institute-European Organisation for Research and Treatment of Cancer Molecular Targets and Cancer Therapeutics Meeting, Miami, FL, October 29-November 2, 2001.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Wang Q, Worland PJ, Clark JL, et al: Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2. Cell Growth Differ 6:927每936, 1995
Kawakami K, Futami H, Takahara J, et al: UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem Biophys Res Commun 219:778每783, 1996
Akiyama T, Yoshida T, Tsujita T, et al: G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 57:1495每1501, 1997
Graves PR, Yu L, Schwarz JK, et al: The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 275:5600每5605, 2000
Sato S, Fujita N, Tsuruo T: Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine). Oncogene 21:1727每1738, 2002
Takahashi I, Saitoh Y, Yoshida M, et al: UCN-01 and UCN-02, new selective inhibitors of protein kinase C: II. Purification, physico-chemical properties, structural determination and biological activities. J Antibiot (Tokyo) 42:571每576, 1989
Akinaga S, Gomi K, Morimoto M, et al: Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res 51:4888每4892, 1991
Seynaeve CM, Stetler-Stevenson M, Sebers S, et al: Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 53:2081每2086, 1993
Tsuchida E, Urano M: The effect of UCN-01 (7-hydroxystaurosporine), a potent inhibitor of protein kinase C, on fractionated radiotherapy or daily chemotherapy of a murine fibrosarcoma. Int J Radiat Oncol Biol Phys 39:1153每1161, 1997
Husain A, Yan XJ, Rosales N, et al: UCN-01 in ovary cancer cells: Effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of p53 status. Clin Cancer Res 3:2089每2097, 1997
Bunch RT, Eastman A: Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 2:791每797, 1996
Akinaga S, Nomura K, Gomi K, et al: Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C. Cancer Chemother Pharmacol 32:183每189, 1993
Nieves-Neira W, Pommier Y: Apoptotic response to camptothecin and 7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell lines of the NCI Anticancer Drug Screen: Multifactorial relationships with topoisomerase I, protein kinase C, Bcl-2, p53, MDM-2 and caspase pathways. Int J Cancer 82:396每404, 1999
Jones CB, Clements MK, Wasi S, et al: Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: Abrogation of S/G(2) arrest. Cancer Chemother Pharmacol 45:252每258, 2000
Tang L, Boise LH, Dent P, et al: Potentiation of 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial damage and apoptosis in human leukemia cells (U937) overexpressing bcl-2 by the kinase inhibitor 7-hydroxystaurosporine (UCN-01). Biochem Pharmacol 60:1445每1456, 2000
Abe S, Kubota T, Otani Y, et al: UCN-01 (7-hydroxystaurosporine) enhances 5-fluorouracil cytotoxicity through down-regulation of thymidylate synthetase messenger RNA. Jpn J Cancer Res 91:1192每1198, 2000
Shi Z, Azuma A, Sampath D, et al: S-phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine. Cancer Res 61:1065每1072, 2001
Hirose Y, Berger MS, Pieper RO: Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res 61:5843每5849, 2001
Hsueh CT, Kelsen D, Schwartz GK: UCN-01 suppresses thymidylate synthase gene expression and enhances 5-fluorouracil-induced apoptosis in a sequence-dependent manner. Clin Cancer Res 4:2201每2206, 1998
Hsueh CT, Wu YC, Schwartz GK: UCN-01 suppresses E2F-1 mediated by ubiquitin-proteasome-dependent degradation. Clin Cancer Res 7:669每674, 2001
Sausville EA, Arbuck SG, Messmann R, et al: Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 19:2319每2333, 2001
Fuse E, Tanii H, Kurata N, et al: Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein. Cancer Res 58:3248每3253, 1998
Tamura T, Sasaki Y, Minami H, et al: Phase I study of UCN-01 by 3-hour infusion. Proc Am Soc Clin Oncol 18:159a, 1999 (abstr 611)
Sobrero AF, Aschele C, Bertino JR: Fluorouracil in colorectal cancer: A tale of two drugs〞Implications for biochemical modulation. J Clin Oncol 15:368每381, 1997
Meta-Analysis Group in Cancer: Toxicity of fluorouracil in patients with advanced colorectal cancer: Effect of administration schedule and prognostic factors. J Clin Oncol 16:3537每3541, 1998
Meta-Analysis Group in Cancer: Efficacy of intravenous continuous infusion of fluorouracil compared with bolus administration in advanced colorectal cancer. J Clin Oncol 16:301每308, 1998
Ardalan B, Singh G, Silberman H: A randomized phase I and II study of short-term infusion of high-dose fluorouracil with or without N-(phosphonacetyl)-L-aspartic acid in patients with advanced pancreatic and colorectal cancers. J Clin Oncol 6:1053每1058, 1988
Haas NB, Hines JB, Hudes GR, et al: Phase I trial of 5-fluorouracil by 24-hour infusion weekly. Invest New Drugs 11:181每185, 1993
Weh HJ, Wilke HJ, Dierlamm J, et al: Weekly therapy with folinic acid (FA) and high-dose 5-fluorouracil (5- FU) 24-hour infusion in pretreated patients with metastatic colorectal carcinoma: A multicenter study by the Association of Medical Oncology of the German Cancer Society (AIO). Ann Oncol 5:233每237, 1994
Bauer KS, Lush RM, Rudek MA, et al: A high-performance liquid chromatography method using ultraviolet and fluorescence detection for the quantitation of UCN-01, 7- hydroxystaurosporine, from human plasma and saliva. Biomed Chromatogr 14:338每343, 2000
Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235每254, 1965
Moore MJ, Bunting P, Yuan S, et al: Development and validation of a limited sampling strategy for 5-fluorouracil given by bolus intravenous administration. Ther Drug Monit 15:394每399, 1993
Wakamiya N, Stone N, Takeyama H, et al: Detection of tumor necrosis factor gene expression as a cellular level in human acute myeloid leukemia. Leukemia 3:51每56, 1989
Dees C, O＊Reilly S, Figg WD, et al: A phase I and pharmacologic study of UCN-01, a protein kinase C inhibitor. Proc Am Soc Clin Oncol 19:205a, 2000 (abstr 797)
Gandara DR, Lara PN, Longmate J, et al: The cyclin-dependent kinase (CDK) inhibitor UCN-01 plus cisplatin in advanced solid tumors: A California Cancer Consortium phase I trial. Proc Am Soc Clin Oncol 22:246, 2004 (abstr 987)
Hill M, Andjelkovic M, Brazil D, et al: Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase. J Biol Chem 276:25643每25646, 2001
Tuttle R, Gill N, Pugh W, et al: Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKB-alpha. Nat Med 7:1133每1137, 2001
Takamura M, Nio Y, Yamasawa K, et al: Implication of thymidylate synthase in the outcome of patients with invasive ductal carcinoma of the pancreas and efficacy of adjuvant chemotherapy using 5-fluorouracil or its derivatives. Anticancer Drugs 13:75每85, 2002
Kortmansky J, Sauter N, O'Reilly EM, et al: Management of hyperglycemia in patients with metastatic pancreatic cancer receiving UCN-01 and fluorouracil. Proc Am Soc Clin Oncol 23:162a, 2004 (abstr 2140)(Jeremy Kortmansky, Manish)
Program of Molecular Pharmacology and Experimental Therapeutics
Departments of Pediatrics and Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY
ABSTRACT
PURPOSE: Preclinical studies indicate that the cyclin-dependent kinase and protein kinase C inhibitor 7-hydroxystaurosporine (UCN-01) potentiates the cytotoxic effects of fluorouracil (FU). We designed a phase I clinical trial of FU in combination with UCN-01.
PATIENTS AND METHODS: FU was administered as a weekly 24-hour infusion. Doses were escalated in successive cohorts according to a modified Fibonacci design. UCN-01 was administered once every 4 weeks, immediately after disconnection from FU, at a dose of 135 mg/m2 over 72 hours in cycle 1 and 67.5 mg/m2 over 36 hours in subsequent cycles. FU and UCN-01 pharmacokinetics were obtained on all patients, and thymidylate synthetase (TS) activity was measured in peripheral-blood mononuclear cells by reverse-transcriptase polymerase chain reaction.
RESULTS: We escalated the weekly FU dose to 2,600 mg/m2 in combination with once a month infusions of UCN-01. Dose-limiting toxicity included arrhythmia and syncope. Other toxicities included hyperglycemia, headache, and nausea and vomiting. The mean maximal plasma concentration of UCN-01 was 33.5 米mol/L. There was significant interpatient variability, which correlated with plasma concentrations of alpha-1 acid glycoprotein. FU was rapidly cleared and the dose had no effect on the area under the curve of UCN-01. Changes in TS expression were detectable in peripheral-blood mononuclear cells after administration of UCN-01 but did not correlate with toxicity or activity. We observed no objective response, although seven patients had stable disease, six of whom had received prior fluoropyrimidines.
CONCLUSION: The combination of weekly infusions of FU and monthly UCN-01 can be administered safely and warrants further study in phase II trials. The recommended phase II dose of FU in combination with monthly UCN-01 is 2,600 mg/m2.
INTRODUCTION
7-Hydroxystaurosporine (UCN-01) is a staurosporine analog isolated from the culture broth of Streptomyces spp that has antitumor activity in several in vitro and in vivo preclinical models. Although UCN-01 is a potent inhibitor of Ca2+-dependent protein kinase C isoenzymes, its antiproliferative activity cannot be solely explained by this mechanism. Other proposed targets include cyclin-dependent kinases, Chk11每4 and AKT.5 In vitro, UCN-01 has growth-inhibitory effects on numerous human and murine tumor cell lines, with median inhibitory concentration values in the nanomolar range.6每8 UCN-01 has been shown to induce G1 arrest and cause hypophosphorylation of the retinoblastoma protein. In addition, there is an emerging body of literature that UCN-01 synergistically enhances radiation- and chemotherapy-induced apoptosis.9每18
UCN-01 has been shown to synergistically enhance fluorouracil (FU) -induced apoptosis in a sequence-dependent fashion.19 The enhancement of apoptosis induction was greatest when tumor cells were first exposed to FU for 24 hours followed by UCN-01. There was also enhancement with the reverse combination but to a lesser degree. Consistent with effects on inducing G1 arrest and hypophosphorylation of retinoblastoma protein, UCN-01 has been shown to decrease thymidylate synthetase (TS) mRNA and protein expression in a dose-dependent manner.19 Further studies showed that UCN-01 induces E2F-1-specific proteasome degradation, which subsequently leads to decreased transcription of TS mRNA.20
Results from two separate phase I clinical trials of UCN-01 have been reported.21每23 These studies revealed that the pharmacokinetics of UCN-01 administered as a 72- or 3-hour infusion have distinctive features that could not have been predicted from preclinical data. The distribution volume and the systemic clearance were extremely low. More importantly, the elimination half-life was unusually long (253 to 1,660 hours). In vitro protein-binding experiments demonstrated that UCN-01 is significantly bound to human alpha-1 acid glycoprotein (AGP) with high affinity. In fact, the association constant for UCN-01 and human AGP was found to be more than 60-fold the affinity between UCN-01 and dog AGP. In light of these observations, frequency of administration was extended from every 14 days to every 28 days. Also, after the first 72-hour infusion of UCN-01, subsequent doses were halved (36-hour infusion) to prevent cumulative drug buildup. With this schedule, the maximum-tolerated dose (MTD) was 42.5 mg/m2/d or 127.5 mg/m2 total over the 72-hour infusion. Dose-limiting toxicities (DLTs) were myalgias, hyperglycemia, nausea, vomiting, and hypoxemia.21每23 Side effects most commonly occurred during or shortly after drug infusion and resolved after cessation of drug administration. No clinical responders were noted in this dose-finding trial.
On the basis of our preclinical data, we initiated a phase I study combining FU and UCN-01. The UCN-01 dose and schedule were based on the single-agent phase I study of UCN-01 (42.5 mg/m2/d infused continuously over 72 hours in week 1 and the same dose infused over 36 hours repeated every 4 weeks). The initial FU dose was set at 250 mg/m2 was administered as a weekly 24-hour infusion during the entire course of therapy. The FU dose and schedule were designed to maximize the inhibition of TS. Infusional FU may act more specifically to inhibit TS24 when compared with bolus FU and has been associated with longer survival and less myelosuppression.25,26 In addition, our preclinical studies used a 24-hour exposure of FU to maximize the synergy observed with UCN-01. FU was administered before UCN-01 on the first week of each cycle to take advantage of the superior schedule.
PATIENTS AND METHODS
Patient Selection
Adult patients ( 18 years old) with histologically confirmed solid tumor that was refractory to standard therapy (or for which there was no standard therapy) were eligible for the study. The patient had to be off all previous chemotherapy, immunotherapy, or radiotherapy for 4 weeks before study entry (6 weeks for nitrosoureas and mitomycin). Prior FU therapy was allowed. Additional eligibility criteria included Karnofsky performance status of more than 60%, adequate hematopoietic function (WBC 3,500/米L, absolute neutrophil count 1,500/米L, and platelets 100,000/米L), adequate renal function (creatinine 1.5 mg/dL), adequate hepatic function (total bilirubin 1.5 mg/dL and AST and ALT < 2.5 x upper limit of normal), and adequate pulmonary function (lung diffusing capacity 60% predicted).
Patients were excluded from participation in the study for any of the following reasons: presence of any ongoing toxic effect from a prior treatment; presence of any serious or uncontrolled infection; known CNS metastasis or CNS primary tumor; prior mediastinal radiation; pregnant or lactating; history of cardiac arrhythmias, angina, or myocardial infarction in the preceding 6 months; HIV infection; or use of anticonvulsant medications. Patients with a distant history (> 6 months before study) of coronary artery disease required a cardiac stress examination and clearance by a cardiologist before enrollment. Patients using medications known to bind AGP were changed to an alternative medication, when possible. A central venous access device or peripherally inserted central catheter line was required.
The protocol was reviewed and approved by the National Cancer Institute (NCI) and by the Institutional Review Board of the Memorial Sloan-Kettering Cancer Center. Written informed consent was obtained from each patient.
Treatment Plan
This phase I trial was designed as an open-label, nonrandomized, dose-escalation study in which groups of three to six patients were to receive increasing doses of intravenous FU over 24 hours in combination with a fixed dose of intravenous UCN-01 until DLT was demonstrated in at least two of six patients or a FU dose of 2.6 g/m2 was achieved (the MTD of single-agent FU was administered as a weekly 24-hour infusion27每29). FU was administered weekly as a 24-hour continuous infusion on days 1, 8, 15, and 22 of each 4-week cycle. UCN-01 was administered on day 2 of each cycle immediately after disconnection from FU. UCN-01 was administered at a dose of 45 mg/m2/d as a 72-hour continuous infusion in cycle 1 (total dose, 135 mg/m2) and a 36-hour continuous infusion in each subsequent 4-week cycle (total dose, 67.5 mg/m2), in accordance with the NCI-recommended dosing schedule for UCN-01. Both FU and UCN-01 were administered through a central venous catheter. In the initial trial design, patients received UCN-01 alone (72-hour infusion) in the first cycle to determine whether FU affected the pharmacokinetics of UCN-01. Combination therapy with FU started with the second cycle on the schedule described earlier. Four of the first five patients progressed before the completion of three cycles of therapy, and the protocol was subsequently amended to begin weekly FU infusions with the first cycle of UCN-01 therapy. Prophylactic antiemetics were not routinely administered unless clinically indicated. To avoid exacerbation of UCN-01-induced hyperglycemia, corticosteroids were avoided unless other antiemetic choices were ineffective.
Because of potential hemodynamic effects, vital signs, including orthostatic blood pressure and oxygen saturation, were measured 15 and 55 minutes after initiation of UCN-01 and at disconnection from the UCN-01 infusion. Routine physical examination, including vital signs, performance status, and weight, was performed weekly before chemotherapy. Routine laboratory studies, including a CBC with differential and platelets, liver and renal function, and electrolytes with blood glucose, protein, albumin, lactate dehydrogenase, and uric acid, were conducted on the day of therapy and weekly thereafter. Treatment responses were evaluated after two cycles of combination therapy using Response Evaluation Criteria in Solid Tumors Group criteria.
The initial dose of FU was 250 mg/m2 over 24 hours, which represented approximately 10% of the standard dose for 24-hour infusions of FU. FU doses were to be escalated by approximately 50% between cohorts until a dose of 2,600 mg/m2 was reached or until any one patient developed grade 3 or greater toxicity of any type that was attributable to the combination of FU and UCN-01. Subsequent dose escalations of the FU were to be by 33% until the MTD was reached. Intrapatient escalation of FU to a higher dose level was permitted only if one cohort of three patients completed one cycle of therapy at the higher dose level without grade 3 or greater toxicity. If unexpected grade 3 or higher toxicity was observed in the first cohort of patients, the FU dose was to be reduced in 33% decrements to a minimum of 50 mg/m2. The NCI Common Toxicity Criteria (version 2.0) was used to evaluate toxicity and adverse events.
DLT was defined as the occurrence in cycle 1 of grade 4 neutropenia, grade 4 anemia, grade 3 or 4 thrombocytopenia, grade 4 hyperglycemia, or grade 3 or 4 nonhematologic toxicities not directly attributable to UCN-01 therapy as a single-agent. Toxicities that were described to be attributable to UCN-01 included headache, nausea, vomiting, hyperglycemia, muscle cramps, hypotension, and hypoxemia close to time of infusion. Grade 3 hyperglycemia refractory to insulin therapy was considered a DLT. Furthermore, a DLT was considered to have occurred if any nonhematologic toxicity failed to improve sufficiently within 7 days after causing a dose delay or if the platelet count failed to recover to 100,000/米L or the absolute neutrophil count failed to return to 1,500/米L within 7 days after causing a dose delay. A missed dose of FU was not considered a DLT, unless the patient received less than three doses of FU in a 4-week cycle. In the event that the MTD was reached because of dose-limiting hyperglycemia in patients with known diabetes (defined as fasting blood sugar 126 mg/dL, any blood sugar 200 mg/dL, or blood sugar 200 mg/dL after a glucose tolerance test), we allowed patients without a history of diabetes to be enrolled. Further dose escalation would then proceed to determine the MTD of the combination in nondiabetic patients.
A minimum of three patients was observed for at least one complete cycle of combination therapy (4 weeks after initiation of combination therapy) before escalation to the next dose level. If none of the three patients experienced a DLT, then three new patients were entered at the next higher FU dose level. All patients in the prior cohort must have completed one cycle of therapy before enrollment in the next cohort began. If one instance of DLT was observed among the initial three patients treated at any dose level, an additional three patients were to be treated at that dose level, with no further DLT, so that dose escalation could proceed. If two instances of DLT were observed at any dose level, the MTD was considered to have been surpassed, and a total of six patients were to be treated at the previous dose level to assure its tolerability. In the clinical setting of stable or responding disease, patients could continue treatment after experiencing a DLT, with the FU reduced to one lower level, once all unacceptable toxicity had completely resolved. Patients were continued on therapy unless one of the following criteria was met: objective disease progression, unacceptable toxicity not responsive to dose attenuation, investigator considered it unsafe to continue treatment, patient was unwilling or unable to continue (dropped out), patient died, or patient was lost to follow-up.
Drug Supply
UCN-01 (NSC 638850) was supplied by the NCI in sterile, single-use 10-mg and 50-mg vials. The 10-mg vial contained UCN-01 lyophilized powder, 11 mg of citric acid, United States Pharmacopeia (USP), 22.2 mg of anhydrous dibasic sodium phosphate, USP, and 200 mg of lactose, National Formulary (NF). The 50-mg vial contained 50 mg of UCN-01 lyophilized powder, 55 mg of citric acid, USP, 111 mg of anhydrous dibasic sodium phosphate, USP, and 1,000 mg of lactose, NF. The 10-mg vial was reconstituted with 10 mL of water for injection, USP, or 0.9% sodium chloride for injection, USP. The 50-mg vial was reconstituted with 50 mL of either diluent to produce a 1-mg/mL solution of UCN-01. Further dilution with normal saline to a final concentration as low as 10 米g/mL was permitted. Concentrations less than 10 米g/mL were not permitted. Normal saline was the diluent of choice. D5W or D51/2NS could not be used. Polyvinylchloride bags or glass bottles could be used for drug infusions. An in-line filter with a pore size of 1.2 米m was used.
FU (Adrucil; Pharmacia and Upjohn, Mississauga, Ontario, Canada) was supplied as a 50-mg/mL solution in vials of 50 mL and 100 mL. The appropriate volume was withdrawn into a syringe, which was then used for administration by 24-hour infusion. No dilution was required.
Pharmacokinetics
Assessment of plasma UCN-01 and FU concentrations for pharmacokinetic studies were performed on all patients according to published methods with the first two cycles of UCN-01.30 Blood samples to measure UCN-01 were drawn at the following time points during cycle 1: just before the start of UCN-01; 15 minutes, 55 minutes, and 24 hours after the initiation of the 72-hour UCN-01 infusion; and immediately after the completion of the UCN-01 infusion. During the second cycle, blood samples were drawn just before the start of the UCN-01 infusion and at the completion of the 36-hour infusion. For the five patients treated at 2,600 mg/m2, an additional blood draw was obtained 14 days after the completion of the UCN-01 infusion (day 16). Because UCN-01 is known to be heavily protein bound to AGP, plasma measurements of this protein were collected just before the initiation of the UCN-01 dose in the first two cycles. AGP was measured by radial immunodiffusion31 using commercially available kits (The Binding Site Ltd, Birmingham, United Kingdom). Blood samples to measure FU were drawn at the following time points during the first two cycles: just before the start of FU, at the completion of the FU infusion, and at 15 and 55 minutes after the initiation of the UCN-01 infusion. A limited sampling strategy was used for FU as validated by Moore et al.32
Biologic Monitoring
Ex vivo studies of the effects of treatment on TS activity in peripheral mononuclear cells were performed in the first two cycles. Whole-blood samples were collected according to the manufacturer's instructions in a Vacutainer CPT tube (Becton Dickinson, Franklin Lakes, NJ) with EDTA before the FU infusion, before the UCN-01 infusion, and at the completion of the UCN-01 infusion (72 hours in cycle 1 and 36 hours in cycle 2). In the five patients treated with FU 2,527 and 2,600 mg/m2, an additional sample was collected 14 days from the start of the UCN-01 infusion to measure midcycle effects. Centrifugation was performed with a Ficoll-Hypaque gradient to recover the mononuclear layer, as described by Wakamiya et al.33 TS mRNA expression was determined by quantitative reverse-transcriptase polymerase chain reaction (PCR; ABI Prism 7700 Sequence Detection System; Applied Biosystems, Foster City, CA) using TS sequence-specific complementary primers and beta-actin as the endogenous control.
RESULTS
Patient Characteristics
August 1999 through June 2002, 35 patients with advanced solid tumors were treated with the combination of FU and UCN-01. Table 1 lists the patient characteristics. The median age was 59 years (range, 38 to 79 years), and the median Karnofsky performance status was 90% (range, 70% to 90%). The majority of patients on study carried a diagnosis of metastatic colon cancer (n = 21). Thirty-four patients had received prior chemotherapy (mean number of regimens, three; range, zero to six regimens); of these patients, 26 had received prior FU or a FU derivative. Twenty-four of 26 patients discontinued their FU-based therapy because of progression of disease; one patient received FU as adjuvant therapy for colon cancer more than 2 years before enrolling onto our study; and one patient received FU as a radiosensitizing agent as adjuvant therapy for ampullary cancer. Eleven patients had received prior radiotherapy.
A total of 84 cycles of treatment were administered (median, two cycles; range, one to seven cycles) over eight cohorts at the following dose levels of FU: 250, 375, 560, 845, 1,265, 1,900, 2,527, and 2,600 mg/m2 (Table 2). All 35 patients enrolled onto the study were considered assessable for toxicity; two patients in cohort 2 and one patient in cohort 4 experienced rapid disease progression before completion of the first cycle and were replaced on study. One patient in cohort 6 did not complete the 72-hour UCN-01 infusion in cycle 1; to be certain of toxicity, an additional patient was added to this cohort. Thirty-three patients were assessable for response to combination therapy; two patients who experienced arrhythmia during the first week of treatment were removed from study.
DLT
We observed four DLTs in cycle 1 (Table 2). A patient receiving 560 mg/m2 of FU experienced a grade 3 syncopal episode 1 day after the second FU infusion in cycle 1. The patient reported observed loss of consciousness for several seconds on two separate occasions during the same night, after changing quickly from the supine to upright position. These symptoms were unreported until he presented to a physician for routine follow-up 6 days later. The described symptoms appeared orthostatic in nature, although no documented change in blood pressure or pulse was available at the time of the incident. A cardiac (orthostatic blood pressure assessment, ECG, and echocardiogram) and neurologic evaluation (head computed tomography) were both negative. We believed the event was likely related to the UCN-01 alone and not to the combination, and we elected to continue the patient on therapy without dose adjustment in FU. He completed two cycles without a second episode before discontinuing treatment because of progression.
A patient receiving 845 mg/m2 of FU experienced atrial fibrillation after the first dose of FU and UCN-01 and was removed from study. This cardiac event was observed in the setting of pneumonia and chronic obstructive pulmonary disease exacerbation. Normal sinus rhythm returned with conservative medical management. Because of the underlying medical issues, this arrhythmia was initially believed to be unrelated to therapy and was not considered a DLT. However, a patient receiving 1,900 mg/m2 of FU also developed atrial fibrillation during the first cycle of therapy. The arrhythmia was noted before receiving the second weekly dose of FU. The patient had no prior history of arrhythmia or coronary artery disease and reverted to a normal sinus rhythm with pharmacologic management. A cardiac evaluation, including cardiac enzymes and echocardiogram, was negative for a predisposing comorbidity. This patient was also removed from study because her performance status deteriorated over the ensuing weeks, and she was unfit to resume treatment.
In addition, one patient experienced grade 4 hyperglycemia, which was defined as a DLT on this protocol. The grade 4 toxicity occurred during the 72-hour infusion of UCN-01 in a patient treated at the 560 mg/m2 FU dose level. This patient had a known underlying diagnosis of diabetes, and hospitalization for intravenous fluids and insulin drip was required. The hyperglycemia proved to be refractory, requiring nearly 44 hours of insulin infusion before blood sugars resolved to less than 200 mg/dL. After this adverse event, we elected to amend the protocol to exclude patients with a known underlying diagnosis of diabetes mellitus. Diabetes mellitus was defined as fasting blood sugar 126 mg/dL, any blood sugar 200 mg/dL, or blood sugar 200 mg/dL after a glucose tolerance test.
Hematologic Toxicity
Therapy was well tolerated. No grade 4 hematologic toxicity was observed. One patient treated at a FU dose of 560 mg/m2 experienced grade 3 anemia.
Common Nonhematologic Toxicity
Common nonhematologic toxicities for all cycles of therapy that occurred in 10% of patients are listed in Table 3. As shown, diarrhea, constipation, fatigue, headache, nausea, and vomiting were observed with increased frequency. These toxicities have been reported in a previous phase I study of UCN-01.21 As was noted in other studies, hyperglycemia remains a significant problem with UCN-01. In addition to the patient who experienced grade 4 toxicity (DLT), five patients experienced grade 3 toxicity. Four of the five episodes of grade 3 hyperglycemia were observed after the protocol revision to exclude patients with underlying diabetes.
Additional observed grade 3 toxicities that were at least possibly attributable to the combination therapy include hyperbilirubinemia (n = 1), fatigue (n = 2), fever (n = 1), myalgia (n = 1), nausea (n = 5), and vomiting (n = 2). Nausea and vomiting were controlled with appropriate antiemetics in all instances. One patient experienced grade 4 fatigue, which occurred after the third FU infusion in cycle 1; however, this was attributed to rapid disease progression. In all patients, side effects of treatment resolved to grade 1 or lower before the next dose of UCN-01. We did not observe cumulative toxicities in patients who received more than two cycles of treatment.
Pharmacokinetics and Surrogate Studies
UCN-01 and AGP assays were available for 34 patients treated on this study. A total of 327 plasma samples were available for analysis of UCN-01 concentration (92% of planned samples). The mean maximal concentration (Cmax) of UCN-01 at the completion of the 72-hour infusion was 33.5 ㊣ 12.2 米mol/L (range, 3.7 to 53.5 米mol/L). The Cmax after completion of the 36-hour infusion of UCN-01 in the second cycle was 33.5 ㊣ 13.4 米mol/L (range, 15.0 to 65.5 米mol/L). There was no significant difference between the Cmax of UCN-01 achieved at the completion of the 72- and 36-hour infusions of UCN-01 in cycles 1 and 2, respectively (P = .64, signed-rank test). Starting with a FU dose of 1,900 mg/m2, there was a trend toward decreasing UCN-01 Cmax with increasing FU dose (Table 4); however, this difference was not statistically significant. We measured the area under the curve (AUC) of UCN-01, which was calculated using a noncompartmental model, from pre-UCN-01 infusion in cycle 1 to pre每UCN-01 infusion in cycle 2. Figure 1 demonstrates that, despite the increasing dose of FU in each cohort, there was no change in the AUC for UCN-01 (P = .77, Kruskal-Wallis test). We observed no correlation between UCN-01 Cmax or AUC and either toxicity or response.
We measured UCN-01 concentrations on day 16 in five patients treated in the FU 2,527 and 2,600 mg/m2 cohorts. The mean concentration of UCN-01 decreased to 17.6 ㊣ 6.2 米mol/L on day 16, which was approximately 50% of the Cmax at the completion of the 72-hour infusion. As predicted by its prolonged half-life in human models, UCN-01 was still measurable in plasma immediately before the infusion in the second cycle. At this time point, the mean concentration of UCN-01 was 13.0 ㊣ 2.7 米mol/L or approximately 40% of the Cmax.
Because of high binding affinity to UCN-01, plasma levels of AGP are postulated to correlate with peak UCN-01 concentrations. We measured AGP before the UCN-01 infusion in each cycle. Figure 2 demonstrates a linear correlation between UCN-01 concentration and AGP (P = .01).
We collected peripheral-blood mononuclear cells for ex vivo TS analysis before the FU infusion, before the UCN-01 infusion, and at the completion of the UCN-01 infusion (72 hours in cycle 1 and 36 hours in cycle 2). We compared TS expression by quantitative PCR before and after the UCN-01 infusion in each of the first two cycles. In this analysis, we had 13 matched pairs (seven in cycle 1 and six in cycle 2), representing nine different patients. Additionally, four matched pairs were available to compare TS mRNA levels at the completion of UCN-01 (cycle 1) with day 16 levels (midcycle). We observed no correlation between UCN-01 infusion and change in TS mRNA levels and no correlation between change in TS mRNA levels and response or toxicity to treatment.
Response to Therapy
Thirty-two patients were assessable for response. We observed no objective response, although eight patients (six colon, one small bowel, and one unknown primary) had stable disease. Seven of eight patients with stable disease had previously received and progressed on FU as part of a standard regimen for their disease. Patients with stable disease remained on study for a median of 4 months (3, 3, 4, 4, 4, 4, 5, and 7 months). This included one patient with colon cancer who had significant regression of lung metastases (Fig 3) after one cycle of treatment. This patient had received FU and levamisole in the adjuvant setting, but his disease recurred within 4 months of completion of treatment. Two months before enrolling onto this phase I study, the patient had progressed on FU, leucovorin, and oxaliplatin. Unfortunately, after the first cycle of treatment with FU and UCN-01, the patient developed an elevated bilirubin secondary to bile duct obstruction. A polyethylene stent was placed endoscopically, but treatment was delayed by 1 month until the bilirubin returned to normal. A computed tomography scan after one additional cycle of treatment revealed progression of disease in the lungs, and the patient was removed from study. His initial response was adjudicated as stable disease (minor response).
DISCUSSION
In this study, patients received escalating doses of FU administered once weekly as a 24-hour continuous infusion. UCN-01 was administered as a fixed dose of 45 mg/m2/d on the second day of week 1 of each 4-week cycle, immediately after disconnection from FU. As recommended by Sausville et al,21 UCN-01 was infused over 72 hours in the first cycle and then over 36 hours with each additional cycle. Our starting dose of FU was 250 mg/m2, and we successfully escalated the FU to our planned end point of 2,600 mg/m2 as a 24-hour infusion. This is our recommended phase II dose for future studies with FU in combination with fixed dose UCN-01 (45 mg/m2/d). This target dose of FU was based on a number of infusional FU regimens for gastrointestinal malignancies that have used 24-hour, 48-hour, 5-day, 28-day, and longer durations of therapy. Published studies using 24-hour infusions of FU have reported MTD of 2,600 mg/m2.27每29 We elected not to expand the final cohort to six patients because we observed no DLTs at FU doses of either 2,527 or 2,600 mg/m2.
We observed DLTs of syncope in the third cohort and arrhythmia in the sixth cohort with the combination. Hemodynamic complications have been previously reported with UCN-01. In the phase I study by Sausville et al,21 grade 1 and 2 hypotension was observed in patients treated at doses 24 mg/m2/d, with grade 3 toxicity in one patient treated with 53 mg/m2/d. Dees et al34 demonstrated dose-limiting hypotension and readily reversible respiratory failure in a patient receiving a 1-hour infusion of UCN-01 at a dose of 95 mg/m2/d. A Japanese study of UCN-01 administered as a 3-hour infusion did not observe dose-limiting hypotension.23 In our study, we evaluated patients for orthostatic hypotension at 15 and 55 minutes into the UCN-01 infusion and immediately after completion of the UCN-01 in the first two cycles. No patient experienced documented hemodynamic changes. For the one patient who experienced syncope, the event occurred 4 days after disconnection from the UCN-01. Although hypotension is probably attributable to UCN-01, it is unclear whether this effect is a consequence of rapid infusion or an idiosyncratic event.
Likewise, it is not clear whether the arrhythmia (atrial fibrillation) was truly a treatment-related side effect. In one patient, the event was observed in the setting of pneumonia and chronic obstructive pulmonary disease exacerbation and may have been coincidental. The second event occurred in an elderly woman and may represent an appropriate comorbidity. However, since completion of our study, grade 3 atrial fibrillation was observed in a patient enrolled onto a phase I trial of UCN-01 and cisplatin, suggesting that this toxicity may, in fact, be related to UCN-01.35
The majority of grade 3 and 4 toxicities observed on this study can be attributed to UCN-01 alone and include nausea, vomiting, headaches, and hyperglycemia. However, hyperglycemia remains a difficult problem in patients receiving UCN-01. On our study, one patient with hyperglycemia required hospitalization for continuous insulin infusion. Furthermore, after amending the eligibility criteria to exclude patients with diabetes mellitus, we observed four additional episodes of grade 3 hyperglycemia. Despite these toxicities, we remain encouraged by the observation of stable disease in eight patients who had previously experienced treatment failure with multiagent chemotherapy regimens, including seven patients who had previously progressed on FU-based therapy.
The etiology of the hyperglycemia is unclear, although it has been postulated that it is related to UCN-01-induced peripheral resistance to glucose. In their phase I study, Sausville et al21 noted hyperglycemia in association with hyperinsulinemia. An alternative hypothesis involves inhibition of AKT and downstream insulin-receptor pathways. Staurosporine inhibits phosphorylation on Thr-308 in the activation loop of AKT via inhibition of 3-phosphoinositide-dependent protein kinase-1.36 In mouse models, overexpression of AKT was associated with increased pancreatic beta-cell growth and increased overall insulin secretion.37 Interestingly, insulin secretion in response to glucose was impaired when corrected for beta-cell mass. There was no apparent effect on somatostatin or glucagon. Therefore, inhibition of AKT by UCN-01 may have the opposite effect (ie, decreased pancreatic beta-cell mass and decreased insulinemia). We plan to evaluate the effects of UCN-01 on AKT signaling pathways in future studies.
After the 72- and 36-hour infusions of UCN-01, the mean Cmax was 33.5 ㊣ 12.2 米mol/L (range, 3.7 to 53.4 米mol/L) and 33.5 ㊣ 13.4 米mol/L (range, 14.9 to 65.4 米mol/L), respectively. The mean Cmax is similar to published pharmacokinetics; at a dose of 42.5 mg/m2/d, Sausville et al21 reported a mean Cmax of 36.4 米mol/L. With increasing FU dose, there was a trend towards a decrease in UCN-01 Cmax starting at a FU dose of 1,900 mg/m2. However, these differences were not statistically significant because the ranges were overlapping. We suspect these changes are secondary to small patient numbers and wide interpatient variability. The wide interpatient variability in our study is likely related to differences in AGP, which is linearly correlated with the UCN-01 concentration. In contrast, intrapatient variability was relatively narrow, and the mean Cmax after the 72-hour infusion was similar to the mean Cmax after the 36-hour infusion. The pharmacokinetics of FU at the recommended phase II dose of 2,600 mg/m2 are being further tested in combination with the fixed doses of UCN-01 in ongoing phase II clinical trials.
In their phase I study of UCN-01 administration by 72-hour continuous infusion, Sausville et al21 demonstrated pharmacokinetic features of UCN-01 that were not predicted by preclinical studies in rodent and dog species. These unusual pharmacokinetics are attributed to strong binding to human AGP. There is a positive correlation between AGP concentration and plasma concentration for UCN-01. Unfortunately, this correlation likely has little clinical utility because it does not capture free UCN-01 levels, which are probably more predictive of toxicity and activity. The difficulty in measuring free drug is a major concern in the development of UCN-01. The current dosing schedule of UCN-01 is based on the prolonged half-life of the drug-AGP complex but may not reflect optimal tissue exposure to pharmacologically active free drug. Current methods to measure free drug, including ultracentrifugation, are imperfect.
Because of this difficulty in predicting free-UCN-01 using pharmacokinetic assays, we postulated that surrogate markers of drug activity may provide information regarding the appropriate dosing schedule of this agent and may maximize its antitumor effect. In this clinical trial, we used real-time PCR to assess changes in TS mRNA levels in peripheral-blood mononuclear cells with the hope that a correlation between mRNA levels and UCN-01 dosing might allow this ex vivo study to serve as a surrogate marker of drug activity. We were unable to detect a correlation between TS expression and UCN-01 dosing in peripheral-blood mononuclear cells. Furthermore, a decrease in TS mRNA levels failed to predict for response (stable disease) or toxicity to therapy. This lack of effect may be related to the fact that lymphocytes are noncycling cells and are unlikely to be affected by a cyclin-dependent kinase inhibitor without phytohaemagglutinin stimulation. Changes in TS expression within the tumor may be more representative of the true effects of UCN-01 and will be considered in further studies with this combination.
Despite a prolonged infusion, FU was rapidly cleared and was undetectable within the first hour of the UCN-01 infusion. The dose level of FU had no impact on UCN-01 pharmacokinetics, which remained constant throughout all the cohorts.
In the present study, we successfully escalated FU to a dose of 2,600 mg/m2 over 24 hours in combination with UCN-01 and recommend this dose for future disease-specific phase II studies. This represents the first clinical trial to combine chemotherapy with UCN-01. There was minimal toxicity attributed to the combination, although expected toxicities associated with UCN-01 were observed. The most troublesome side effect was hyperglycemia, which proved to be refractory to insulin infusion in one patient. After improved screening to exclude known diabetics, significant hyperglycemia was observed but was manageable. We plan to further study this combination in a phase II clinical trial of patients with metastatic, gemcitabine-refractory pancreatic cancer. This trial is based on Japanese data that indicate potentiation of FU cytotoxicity by UCN-01 through downregulation of TS mRNA in pancreatic cancer cell lines.16 TS is reported to be overexpressed in approximately 50% of pancreatic cancers.38 Although this population would be considered at high risk for hyperglycemia from UCN-01, we have reported that, with appropriate glucose monitoring, this toxicity can be well controlled.39 In the future study, we will attempt to demonstrate the effects on TSexpression in intratumoral specimens and to better clarify the etiology of hyperglycemia with this drug.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by National Cancer Institute grant No. U01-CA69856.
Presented in part at the 37th Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12-15, 2001 and the American Association for Cancer Research-National Cancer Institute-European Organisation for Research and Treatment of Cancer Molecular Targets and Cancer Therapeutics Meeting, Miami, FL, October 29-November 2, 2001.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Wang Q, Worland PJ, Clark JL, et al: Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2. Cell Growth Differ 6:927每936, 1995
Kawakami K, Futami H, Takahara J, et al: UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem Biophys Res Commun 219:778每783, 1996
Akiyama T, Yoshida T, Tsujita T, et al: G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 57:1495每1501, 1997
Graves PR, Yu L, Schwarz JK, et al: The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 275:5600每5605, 2000
Sato S, Fujita N, Tsuruo T: Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-hydroxystaurosporine). Oncogene 21:1727每1738, 2002
Takahashi I, Saitoh Y, Yoshida M, et al: UCN-01 and UCN-02, new selective inhibitors of protein kinase C: II. Purification, physico-chemical properties, structural determination and biological activities. J Antibiot (Tokyo) 42:571每576, 1989
Akinaga S, Gomi K, Morimoto M, et al: Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res 51:4888每4892, 1991
Seynaeve CM, Stetler-Stevenson M, Sebers S, et al: Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 53:2081每2086, 1993
Tsuchida E, Urano M: The effect of UCN-01 (7-hydroxystaurosporine), a potent inhibitor of protein kinase C, on fractionated radiotherapy or daily chemotherapy of a murine fibrosarcoma. Int J Radiat Oncol Biol Phys 39:1153每1161, 1997
Husain A, Yan XJ, Rosales N, et al: UCN-01 in ovary cancer cells: Effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of p53 status. Clin Cancer Res 3:2089每2097, 1997
Bunch RT, Eastman A: Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 2:791每797, 1996
Akinaga S, Nomura K, Gomi K, et al: Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C. Cancer Chemother Pharmacol 32:183每189, 1993
Nieves-Neira W, Pommier Y: Apoptotic response to camptothecin and 7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell lines of the NCI Anticancer Drug Screen: Multifactorial relationships with topoisomerase I, protein kinase C, Bcl-2, p53, MDM-2 and caspase pathways. Int J Cancer 82:396每404, 1999
Jones CB, Clements MK, Wasi S, et al: Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: Abrogation of S/G(2) arrest. Cancer Chemother Pharmacol 45:252每258, 2000
Tang L, Boise LH, Dent P, et al: Potentiation of 1-beta-D-arabinofuranosylcytosine-mediated mitochondrial damage and apoptosis in human leukemia cells (U937) overexpressing bcl-2 by the kinase inhibitor 7-hydroxystaurosporine (UCN-01). Biochem Pharmacol 60:1445每1456, 2000
Abe S, Kubota T, Otani Y, et al: UCN-01 (7-hydroxystaurosporine) enhances 5-fluorouracil cytotoxicity through down-regulation of thymidylate synthetase messenger RNA. Jpn J Cancer Res 91:1192每1198, 2000
Shi Z, Azuma A, Sampath D, et al: S-phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine. Cancer Res 61:1065每1072, 2001
Hirose Y, Berger MS, Pieper RO: Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res 61:5843每5849, 2001
Hsueh CT, Kelsen D, Schwartz GK: UCN-01 suppresses thymidylate synthase gene expression and enhances 5-fluorouracil-induced apoptosis in a sequence-dependent manner. Clin Cancer Res 4:2201每2206, 1998
Hsueh CT, Wu YC, Schwartz GK: UCN-01 suppresses E2F-1 mediated by ubiquitin-proteasome-dependent degradation. Clin Cancer Res 7:669每674, 2001
Sausville EA, Arbuck SG, Messmann R, et al: Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 19:2319每2333, 2001
Fuse E, Tanii H, Kurata N, et al: Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein. Cancer Res 58:3248每3253, 1998
Tamura T, Sasaki Y, Minami H, et al: Phase I study of UCN-01 by 3-hour infusion. Proc Am Soc Clin Oncol 18:159a, 1999 (abstr 611)
Sobrero AF, Aschele C, Bertino JR: Fluorouracil in colorectal cancer: A tale of two drugs〞Implications for biochemical modulation. J Clin Oncol 15:368每381, 1997
Meta-Analysis Group in Cancer: Toxicity of fluorouracil in patients with advanced colorectal cancer: Effect of administration schedule and prognostic factors. J Clin Oncol 16:3537每3541, 1998
Meta-Analysis Group in Cancer: Efficacy of intravenous continuous infusion of fluorouracil compared with bolus administration in advanced colorectal cancer. J Clin Oncol 16:301每308, 1998
Ardalan B, Singh G, Silberman H: A randomized phase I and II study of short-term infusion of high-dose fluorouracil with or without N-(phosphonacetyl)-L-aspartic acid in patients with advanced pancreatic and colorectal cancers. J Clin Oncol 6:1053每1058, 1988
Haas NB, Hines JB, Hudes GR, et al: Phase I trial of 5-fluorouracil by 24-hour infusion weekly. Invest New Drugs 11:181每185, 1993
Weh HJ, Wilke HJ, Dierlamm J, et al: Weekly therapy with folinic acid (FA) and high-dose 5-fluorouracil (5- FU) 24-hour infusion in pretreated patients with metastatic colorectal carcinoma: A multicenter study by the Association of Medical Oncology of the German Cancer Society (AIO). Ann Oncol 5:233每237, 1994
Bauer KS, Lush RM, Rudek MA, et al: A high-performance liquid chromatography method using ultraviolet and fluorescence detection for the quantitation of UCN-01, 7- hydroxystaurosporine, from human plasma and saliva. Biomed Chromatogr 14:338每343, 2000
Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235每254, 1965
Moore MJ, Bunting P, Yuan S, et al: Development and validation of a limited sampling strategy for 5-fluorouracil given by bolus intravenous administration. Ther Drug Monit 15:394每399, 1993
Wakamiya N, Stone N, Takeyama H, et al: Detection of tumor necrosis factor gene expression as a cellular level in human acute myeloid leukemia. Leukemia 3:51每56, 1989
Dees C, O＊Reilly S, Figg WD, et al: A phase I and pharmacologic study of UCN-01, a protein kinase C inhibitor. Proc Am Soc Clin Oncol 19:205a, 2000 (abstr 797)
Gandara DR, Lara PN, Longmate J, et al: The cyclin-dependent kinase (CDK) inhibitor UCN-01 plus cisplatin in advanced solid tumors: A California Cancer Consortium phase I trial. Proc Am Soc Clin Oncol 22:246, 2004 (abstr 987)
Hill M, Andjelkovic M, Brazil D, et al: Insulin-stimulated protein kinase B phosphorylation on Ser-473 is independent of its activity and occurs through a staurosporine-insensitive kinase. J Biol Chem 276:25643每25646, 2001
Tuttle R, Gill N, Pugh W, et al: Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKB-alpha. Nat Med 7:1133每1137, 2001
Takamura M, Nio Y, Yamasawa K, et al: Implication of thymidylate synthase in the outcome of patients with invasive ductal carcinoma of the pancreas and efficacy of adjuvant chemotherapy using 5-fluorouracil or its derivatives. Anticancer Drugs 13:75每85, 2002
Kortmansky J, Sauter N, O'Reilly EM, et al: Management of hyperglycemia in patients with metastatic pancreatic cancer receiving UCN-01 and fluorouracil. Proc Am Soc Clin Oncol 23:162a, 2004 (abstr 2140)(Jeremy Kortmansky, Manish)