Phase I clinical trial of bortezomib in combination with gemcitabine in patients with advanced solid tumors†
Presented in part at the 38th Annual Meeting of the American Society of Clinical Oncology, May 18–21, 2002, Orlando, FL, at the 39th Annual Meeting of the American Society of Clinical Oncology, May 31 to June 3, 2003, Chicago, IL, and at the 94th Annual Meeting of the American Association for Cancer Research, July 11–14, 2003, Washington, DC.
Bortezomib is the first proteasome inhibitor to show preliminary evidence of activity against solid tumors. Findings from preclinical studies prompted a Phase I trial to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of bortezomib in combination with gemcitabine in patients with recurring/refractory advanced solid tumors. The effect of gemcitabine on proteasome inhibition by bortezomib in whole blood was also investigated.
Bortezomib was administered as an intravenous bolus injection on Days 1, 4, 8, and 11, with gemcitabine (30-minute infusion) on Days 1 and 8 of a 21-day cycle. Groups of ≥3 patients were evaluated at each dose level. Escalating doses of gemcitabine 500 mg/m2 to 1000 mg/m2 with bortezomib 1.0 mg/m2 to 1.5 mg/m2 were planned.
There were no DLTs in patients receiving bortezomib 1.0 mg/m2 and gemcitabine 500 mg/m2 to 1000 mg/m2 in the first 3 dose levels. Dose-limiting nausea, vomiting, gastrointestinal obstruction, and thrombocytopenia occurred in 4 of 5 evaluable patients in dose level 4 (bortezomib 1.3 mg/m2, gemcitabine 800 mg/m2), establishing bortezomib 1.0 mg/m2 and gemcitabine 1000 mg/m2 as the MTD. Most common Grade ≥3 toxicities were neutropenia (6 patients), thrombocytopenia (5 patients), gastrointestinal disorders (6 patients), and general disorders (4 patients) such as fatigue. One patient with nonsmall cell lung carcinoma achieved a partial response and 7 achieved stable disease. Inhibition of 20S proteasome activity by bortezomib was unaffected by gemcitabine coadministration.
Dosages of bortezomib and gemcitabine suitable for further evaluation of antitumor activity have been established. Cancer 2006. © 2006 American Cancer Society
The degradation of intracellular proteins by the multicatalytic proteasome is essential in cell cycle regulation, neoplastic growth, and metastasis.1–7 Inhibition of the proteasome results in the activation of proapoptotic caspases,8 reduced nuclear factor-κB (NF-κB) expression,9–12 apoptosis of cells expressing B-cell leukemia/lymphoma 2 (Bcl-2),13 tumor cell-cycle arrest at the G2/M boundary,14 and accumulation of the cell-cycle inhibitors p21 and p27.15 Bortezomib is a peptide boronic acid derivative that effectively inhibits the proteasome and exhibits a broad spectrum of antitumor activity in preclinical models.16–18 It has activity in multiple myeloma patients19, 20 and was recently approved for second-line and salvage treatment of multiple myeloma. Preliminary evidence indicates that the drug may have activity in a variety of solid and hematologic tumors.21–25
Gemcitabine is an antimetabolite that has clinical activity against various solid tumors, most notably advanced pancreatic cancer and nonsmall cell lung cancer (NSCLC).26 Resistance to gemcitabine is associated with decreased deoxycytidine kinase activity, resulting in decreased intracellular deoxycytidine triphosphate levels, Bcl-2 overexpression leading to decreased apoptosis,17, 27 and high levels of NF-κB.17, 27, 28 The administration of gemcitabine in combination with bortezomib resulted in sensitization, or even enhancement, of the effect of gemcitabine in preclinical models.17, 29–32 Moreover, proteasome inhibitors may overcome tumor resistance to gemcitabine by reducing NF-κB activity,17, 28 down-regulation of Bcl-2,15, 17 and stabilization of the cyclin kinase inhibitors p21 and p27.15
A Phase I clinical trial to determine the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of bortezomib in combination with gemcitabine in patients with incurable advanced solid tumors was conducted. Patients were evaluated to provide a preliminary assessment of the antitumor activity of the combination regimen. The effect of the concurrent administration of gemcitabine on 20S proteasome inhibition by bortezomib in whole blood lysate was also investigated.
MATERIALS AND METHODS
Patients age ≥18 years with advanced solid tumors that had progressed after receiving at least 2 prior regimens of chemotherapy were eligible for this study. Patients were required to have measurable or evaluable disease measured radiographically by computed tomography. A Karnofsky performance status ≥60%, and life expectancy >3 months were required. An absolute neutrophil count ≥1500/mm3, a platelet count ≥100,000/mm3, a total bilirubin level ≤1.5 times the upper limit of normal (ULN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels ≤2.5 times ULN (≤5 times ULN for patients with liver metastases), and a serum creatinine concentration ≤1.5 mg/dL were required within 14 days of starting treatment. Patients who had received chemotherapy within 4 weeks, nitrosoureas within 6 weeks, antibody therapy within 8 weeks, or radiation therapy to more than 35% of the bone marrow within 4 weeks of enrollment were ineligible. Other exclusion criteria included the presence of malignant lymphoma, leukemia, residual toxicity from previous treatment, uncontrolled brain metastases or nonneoplastic central nervous system disease, active hepatitis B or C infection, human immunodeficiency virus infection, electrocardiographic evidence of acute ischemia or new conduction system abnormalities, myocardial infarction within 6 months of enrollment, major surgery within 4 weeks of enrollment, or any other intercurrent illness that could potentially interfere with completion of the study protocol. Patients who had been previously treated with gemcitabine and required a dose reduction because of drug-related toxicity were excluded. Use of acceptable birth control for all fertile participants was required, and women who were pregnant or breast feeding were excluded.
The trial was conducted in compliance with the Declaration of Helsinki and the International Conference on Harmonization for Good Clinical Practice. The protocol was approved by the Institutional Review Board of each participating center. All patients provided written informed consent before participation in the study.
Drug Administration and Dose Escalation
Bortezomib (Velcade; Millennium Pharmaceuticals, Cambridge, MA, and Johnson & Johnson Pharmaceutical Research & Development, Raritan, NJ) was administered by bolus intravenous (iv) injection on Days 1, 4, 8, and 11 of each 21-day treatment cycle. Gemcitabine (Gemzar; Eli Lilly, Indianapolis, IN) was given as a 30-minute i.v. infusion 1 hour after bortezomib on Day 1 and immediately after bortezomib on Day 8. A maximum of 8 treatment cycles over a 24-week period was permitted. Starting doses were bortezomib 1.0 mg/m2 and gemcitabine 500 mg/m2. Initially, the gemcitabine dose was increased to 800 mg/m2 and then to a maximum of 1000 mg/m2. The gemcitabine dose was decreased to the level below the highest dose given with acceptable tolerability and the bortezomib dose was increased to 1.3 mg/m2 and a maximum of 1.5 mg/m2. The use of other antineoplastic therapy and routine use of colony-stimulating factors were prohibited; however, the use of erythropoietin was permitted.
Groups of at least 3 patients were evaluated at each dose level. Intrapatient dose escalation was not permitted. Patients were enrolled at the next higher dose if none of the 3 patients treated at a given dose level experienced a DLT. Three additional patients were enrolled at the same dose level if 1 patient experienced a DLT. When 2 or more patients in a dose level group experienced a DLT, the previous dose level was declared the MTD. An additional 10 patients were then enrolled to receive bortezomib and gemcitabine at the MTD. A patient who discontinued from the study or had interruption of treatment for reasons other than a treatment-related toxicity before completing Cycle 1 were inevaluable and replaced by another patient. Toxicities were categorized and graded according to the National Cancer Institute's Common Toxicity Criteria v. 2.0. DLT was defined as Grade 4 neutropenia or any other type of Grade ≥3 toxicity occurring in cycle 1.
Gemcitabine was administered at a 25% reduced dose on Day 8 in the case of Grade 3 neutropenia and was withheld for Grade 4 neutropenia. For all other Grade ≥3 toxicities, study drugs were withheld until the toxicity returned to Grade 1 or better, at which time the study drug was restarted at a dose reduction of 25%.
The protocol was amended to allow patients with no prior therapy to enroll in a new group, designated dose level 4A (bortezomib 1.3 mg/m2 and gemcitabine 800 mg/m2) to allow assessment of the potential effect of prior chemotherapy on DLTs.
A complete physical examination was performed before treatment, 10 days after the last study drug dose, and 6 weeks after the last day of the final treatment cycle (end of study). Routine laboratory tests for clinical chemistry were conducted at baseline, on Days 1 and 8 of each cycle, after the last study drug dose, and 6 weeks after the end of the study. Routine hematology and electrolyte studies were performed at baseline, before drug administration on Days 1, 4, 8, and 11 of each cycle, after the last study drug dose, and 6 weeks after the end of study. Coagulation studies were performed at baseline, on Day 1 of each cycle, and 6 weeks after the end of the study.
Adverse events were closely monitored throughout the study period and for 6 weeks after the final treatment cycle using symptom-directed histories, physical examinations, and laboratory assessments. Karnofsky performance status was determined on Days 1 and 11 of each treatment cycle, after the study drug dose, and 6 weeks after the last day of the final cycle.
Evaluation of Response
The largest measurable lesions (≥20 mm with conventional radiography or ≥10 mm with spiral computed tomography) were selected as target lesions. Patients with nonmeasurable tumors were required to have detectable tumor markers. Every 6 weeks during treatment, target lesions were measured, nontarget lesions were assessed, and overall disease response was determined according to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines, with minor modifications.34 A complete response (CR) was defined as disappearance of all target lesions. A partial response (PR) was defined as a ≥30% decrease from baseline in the sum of the longest diameter of target lesions. Stable disease (SD) was defined as neither sufficient reduction to qualify for PR nor sufficient increase to qualify for progressive disease (PD). Patients who experienced PD at any time, defined as a ≥20% increase in the sum of the longest diameter of target lesions or the appearance of 1 or more new lesions, were removed from the study. Patients were reassessed 6 weeks after observation of a CR, PR, or SD for confirmation.
Inhibition of 20S proteasome was determined using a spectrofluorometric assay to assess 20S activity in circulating blood cells, as previously described.35 Percentage inhibition of the proteasome was established by measuring the rate of substrate cleavage per 20S proteasome activity relative to protein content. Blood samples for the assay were obtained immediately before and 1 hour after administration of bortezomib on Days 1 and 4 of treatment cycles 1 and 2.
The characteristics of the 31 patients enrolled in the study are presented in Table 1. Almost one-third of the patients had primary tumors of the pancreas or biliary tract. Twenty-seven (87%) patients completed at least 1 treatment cycle, 12 (39%) completed 3 cycles, and 6 (19%) completed 8 cycles. The median number of cycles administered per patient was 2 (range, 0–9), and the total number of completed cycles was 97.
Table 1. Patient Characteristics
|No. of patients||31|
|Median age, y (range)||58 (39–82)|
|Sex, no. (%)|
| Male||18 (58)|
| Female||13 (42)|
|Karnofsky performance status|
| Median (range)||90 (70–100)|
|Tumor type, no. (%)|
| Pancreas||8 (26)|
| Sarcoma/gastrointestinal stromal tumor||6 (19)|
| Lung, nonsmall cell||5 (16)|
| Colon or rectum||3 (10)|
| Ampulla or bile duct||2 (6)|
| Other*||7 (23)|
|No. of prior chemotherapy regimens|
| Median (range)||1.0 (0–4)|
Determination of DLT and MTD
Five of the 31 patients were deemed inevaluable for DLT (1 withdrew consent, 3 PD during cycle 1, 1 allergic reaction). The tested dose levels were 1.0 mg/m2 bortezomib and 500 mg/m2 gemcitabine (dose level 1), 1.0 mg/m2 and 800 mg/m2 (dose level 2), 1.0 mg/m2 and 1000 mg/m2 (dose level 3), and 1.3 mg/m2 and 800 mg/m2 (dose level 4). There were no DLTs in patients receiving 1.0 mg/m2 bortezomib and 500 mg/m2 to 1000 mg/m2 gemcitabine in the first 3 dose levels (Table 2). Grade 3 or 4 DLTs were first observed in 4 of 4 evaluable patients in dose level 4 and included Grade 3 small gastrointestinal obstruction in a patient with prior surgery for rectal cancer, Grade 3 thrombocytopenia in 2 patients, and Grade 3 nausea and vomiting in 1 patient. Thus, the MTD was bortezomib 1.0 mg/m2 and gemcitabine 1000 mg/m2 and this dose level was expanded. Among the 10 evaluable patients enrolled in the expanded group, 2 patients experienced DLTs, with Grade 3 thrombocytopenia occurring in both patients and 1 patient with concomitant leukopenia.
Table 2. Dose Escalation and DLTs
Upon establishing the MTD for previously treated patients, the protocol was amended to determine whether patients who had not been previously treated with cytotoxic chemotherapeutic agents could tolerate bortezomib 1.3 mg/m2 in combination with gemcitabine 800 mg/m2. For treatment-naive patients, the only modification to the definition of a DLT was that Grade 4 neutropenia was considered a DLT only when the duration was >5 days and thrombocytopenia was a DLT only if the nadir platelet count was <20,000/μL. DLTs occurred in 2 of 4 evaluable patients (myocardial infarction in 1 patient and 1 Grade 3 elevation of ALT/AST in the other patient). Further evaluation of this dose level was discontinued.
All patients experienced at least 1 treatment-related adverse event during the study. Discontinuations due to toxicity occurred in 7 (23%) of the 31 patients. Four of the 7 patients who discontinued were in dose level 4 or 4A, and 3 had adverse events that were considered possibly or probably related to therapy: Grade 2 fatigue and Grade 3 peripheral sensory neuropathy (1 patient in dose level 4), Grade 3 nausea and vomiting (1 patient in dose level 4), and myocardial infarction (1 patient in dose level 4A). There were no patients in the first 3 dose levels who discontinued because of treatment-related toxicity.
No patient discontinued the study because of hematologic toxicities. Neutropenia was the most frequent hematologic adverse event (Tables 3, 4), attributed to bortezomib in 7 (23%) patients, and was bortezomib-related Grade ≥3 in 6 (19%) patients. Thrombocytopenia was the second most common hematologic toxicity, occurring in 9 of 31 patients (29%), and was bortezomib-related Grade ≥3 in 5 patients (16%). Two of these patients with Grade ≥3 thrombocytopenia received bortezomib 1.0 mg/m2 with gemcitabine 1000 mg/m2 (dose level 3), and 3 received bortezomib 1.3 mg/m2 with gemcitabine 800 mg/m2 (dose levels 4 and 4A). Anemia was observed in 7 of 31 (23%) patients and exceeded Grade 2 in only 1 patient, who experienced Grade 3 anemia. However, 4 of 31 (13%) patients received at least 1 red blood cell transfusion (1 patient each from dose level groups 1 and 4, and 2 patients from dose level 3) and 10 of 31 (32%) received erythropoietin (2 patients each from dose level groups 1, 2, and 4; 3 patients from dose level 3; and 1 patient from dose level 4A). No significant changes in coagulation parameters were observed.
Table 3. Number of Patients Experiencing Treatment-Emergent Grade 3/4 Hematologic Toxicities during the Study
Table 4. Number of Patients Experiencing the Most Commonly Reported (>20%) Adverse Events (All Grades)
|Abdominal pain NOS||0||1||3||3||3||10|
|Dizziness (excluding vertigo)||0||1||6||2||1||10|
|Edema, lower limb||1||0||4||2||1||8|
|Appetite decreased NOS||1||2||3||0||1||7|
Nausea, vomiting, and constipation were the most common gastrointestinal disturbances (Table 4). Nausea, vomiting, and diarrhea led to study discontinuation (DLT) in 1 patient in dose level 4. The frequency of nausea and vomiting or diarrhea did not appear to be associated with the dose of bortezomib. Nausea was the most frequent nonhematologic toxicity, occurring in 23 of 31 (74%) patients and was Grade ≥3 in 1 patient (dose level 4). Neuropathy was recorded as peripheral sensory, peripheral motor, hypoesthesia, paresthesia “neurotoxicity,” or peripheral neuropathy not otherwise specified. Hypoesthesia was the most common subtype, occurring in 6 patients. One patient had Grade 3 sensory neuropathy involving the hands and feet. Grade 3 possible treatment-related elevations in AST and ALT both occurred in 1 of 31 (3%) patients and Grade 3 hypercalcemia in 1 of 31 (3%) patients.
Response to Treatment
Of the 31 patients evaluable for response (defined as all patients treated with bortezomib or gemcitabine who had any postbaseline assessment), only 1 patient (3%) achieved an objective response and 7 (23%) of patients had SD. A PR was observed in 1 of the 5 patients with NSCLC, a 51-year-old woman with Stage IIIA (T2N2M0) disease treated with dose level 3 (bortezomib 1.0 mg/m2 and gemcitabine 1000 mg/m2). Prior treatment of this patient included single-agent paclitaxel followed by carboplatin/gemcitabine combination therapy. After a thoracotomy and radiation therapy, she underwent further chemotherapy with vinorelbine but again progressed. Computed tomography performed at screening for this clinical trial revealed measurable lesions with a total sum of 7.7 cm. The PR to bortezomib/gemcitabine was first observed after cycle 4, as a 35% decrease in tumor size that was maintained without progression or the development of new lesions through cycle 8. She was then enrolled in an extension protocol where she continued to receive treatment for an additional 6 months and received >10 cycles of the bortezomib/gemcitabine regimen at the same dose, which was well tolerated.
Among the 7 patients with SD (3 women and 4 men), 2 patients had tumors of the pancreas and 2 patients had NSCLC. None of the patients with SD had received prior gemcitabine treatment. No patients in the 2 lowest dose levels achieved SD; 5 of the 7 patients were treated at dose level 3 (1.0 mg/m2 and 1000 mg/m2), 1 patient was treated at dose level 4 (1.3 mg/m2 and 800 mg/m2), and 1 was treated at dose level 4A (1.3 mg/m2 and 800 mg/m2).
The mean percentage of inhibition of 20S proteasome activity in whole blood lysates after dosing, expressed relative to baseline, is shown in Table 5. Although the mean maximum percentage inhibition was similar between the 2 bortezomib dose levels, a trend toward greater inhibition was observed with increasing doses of both bortezomib and gemcitabine. The maximum proteasome inhibition observed 1 hour after dosing on Day 4 did not differ markedly from that determined on the first day of the same cycle. The mean proteasome inhibition before administering the second weekly dose of bortezomib in cycle 2 was approximately twice that in cycle 1 in patients treated at the MTD; however, this relation is not consistently seen at other dose levels. The percentage of patients returning to ≤10% of baseline proteasome activity 72 hours after dosing in cycle 2 was approximately half of that observed in cycle 1 (data not shown).
Table 5. Mean Percentage Inhibition (Standard Deviation) of the 20S Proteasome in the Dose Level Groups
|1||48.5 (6.70)||21.7 (12.0)||56.3 (8.65)||57.7 (1.57)||24.5 (9.63)||62.2 (3.25)|
|2||50.2 (2.03)||9.3 (2.78)||60.2 (4.66)||65.5 (7.40)||33.6 (5.35)||69.7 (2.23)|
|3||54.7 (7.44)||13.4 (19.4)||63.6 (11.92)||65.8 (7.13)||32.2 (17.4)||72.0 (10.2)|
|4||63.1 (6.07)||18.2 (7.80)||66.8 (9.32)||65.9 (2.45)||18.8 (3.72)||75.8 (8.44)|
|4A||63.5 (1.88)||19.8 (11.9)||69.8 (5.06)||82.7 (1.69)||25.9 (47.0)||74.1 (16.8)|
Bortezomib is active for the treatment of multiple myeloma, producing durable remissions and survival benefits in recurring patients.19 The potential antitumor activity of bortezomib is currently an area of active investigation in other types of malignancies. In this Phase I trial, the administration of bortezomib in combination with gemcitabine was evaluated in patients with advanced solid tumors. The MTD was bortezomib 1.0 mg/m2 given twice a week and gemcitabine 1000 mg/m2 given once a week for 2 weeks followed by a 1-week rest. This sequence was shown in preclinical models to increase sensitivity to gemcitabine.15 Bortezomib was administered based on doses previously used in Phase I and II clinical trials,19, 22, 33 and lower than the recommended dose of 1.3 mg/m2 for recurring myeloma. For gemcitabine, the dose is the same as that recommended for use in patients with NSCLC (4-week schedule) and pancreatic cancer, but with less frequent administration.26
The combination of bortezomib and gemcitabine was generally well tolerated and showed a predictable safety profile, with manageable toxicities. The DLTs were mainly gastrointestinal and hematologic in nature. Grade 3/4 neutropenia or thrombocytopenia was observed in fewer than one-third of patients and was rarely dose limiting. Notably, gemcitabine alone at a dose of 1000 mg/m2 once weekly for 3 weeks in a 4-week cycle has been associated with Grade 3/4 neutropenia in approximately one-third and thrombocytopenia in half of the patients,36 whereas this study evaluated a regimen of gemcitabine for 2 weeks of a 3-week cycle. The addition of twice-weekly bortezomib for 2 weeks of a 3-week cycle did not appear to significantly alter the hematologic profile of the gemcitabine when administered in a standard regimen. Nevertheless, we were unable to demonstrate tolerability for the standard single-agent dose of bortezomib 1.3 mg/m2. Peripheral neuropathy occurred in only 1 patient who discontinued treatment, but the neuropathy was not considered dose limiting. The myocardial infarction observed in dose level 4A was possibly related to treatment, but this has not been associated with either gemcitabine or bortezomib use in the past. In addition, elevation of hepatic transaminases is a frequent side effect of gemcitabine. Therefore, it is possible that a more liberal definition of dose-limiting toxicities in previously untreated patients could have allowed dose escalation.
The combination of bortezomib and gemcitabine exhibited preliminary evidence of antitumor activity as reflected by a PR in a patient with advanced NSCLC. Notably, this patient had previously experienced recurrence after combined modality therapy that had included gemcitabine; however, the patient was not known to be resistant to gemcitabine. Whether proteasome inhibitors such as bortezomib may help to overcome gemcitabine resistance is unclear. Studies on the intracellular kinetics of gemcitabine phosphorylation, accumulation, and disposition in tumors exposed to both gemcitabine and bortezomib could yield important information about potential synergy between these agents, and mechanisms of action, if any, by bortezomib to prevent or counteract gemcitabine resistance.30
The mean maximum percentage of proteasome inhibition was similar between the 2 bortezomib dose levels (1.0 mg/m2 and 1.3 mg/m2), consistent with the sigmoidal model of dose response with bortezomib in which proteasome inhibition reaches a plateau phase at doses between 1.0 mg/m2 to 1.3 mg/m2.34, 37 In groups of patients receiving the same dose of bortezomib, but escalating doses of gemcitabine, the mean proteasome inhibition showed a slight increase.
Preclinical studies demonstrate conflicting results regarding the effect of drug sequencing. In this study, we administered gemcitabine after bortezomib on the basis of drug sequencing studies demonstrating enhanced activity in pancreatic cancer cell lines.15 However, preclinical studies with the A549 NSCLC cell line suggest that the opposite drug sequencing may be important when combining bortezomib with gemcitabine.29 When the A549 cells were treated with bortezomib alone, the levels of cell cycle inhibitor p21 increased, inducing a G(2)/M arrest, resulting in a small amount of apoptosis. However, when bortezomib was administered before gemcitabine/carboplatin chemotherapy, the apoptotic effect of chemotherapy was eliminated. When bortezomib was given simultaneously with or after chemotherapy, apoptosis was increased. Further studies are necessary to determine if drug sequencing with bortezomib and gemcitabine is clinically important.
Overall, the results from this Phase I trial suggest that the combination of bortezomib and gemcitabine is generally safe with manageable toxicities. Coadministration with gemcitabine did not appear to interfere with the pharmacodynamics of bortezomib. However, given the limited number of treatment cycles administered, and the evidence that different dosing and sequencing can increase drug exposure, additional studies of this drug combination are warranted. Based on a Phase I analysis,38 an ongoing Phase II study by the Southwest Oncology Group is evaluating bortezomib in combination with gemcitabine/carboplatin as a treatment for NSCLC. Additionally, a Phase IB study of bortezomib and gemcitabine/cisplatin is currently ongoing.39
We thank Rosemary Washbrook, a medical editor with Gardiner-Caldwell London, for editorial contribution to the article.