Abstract
Purpose: To define the toxicities, pharmacodynamics, and clinical activity of the proteasome inhibitor, PS-341 (bortezomib), in patients with advanced malignancies.
Patients and Methods: Twenty-eight patients (14 male and 14 female) received PS-341 twice weekly for 4 of 6 weeks (schedule I). Because toxicity necessitated dose omissions on this schedule, 16 additional patients (12 male and 4 female) received PS-341 twice weekly for 2 of every 3 weeks (schedule II). A total of 73 courses of treatment was given (median, 2; range, 1-4). Inhibition of 20S proteasome activity in peripheral blood mononuclear cells (PBMC) and accumulation of proteasome-targeted polypeptides in tumor tissue were evaluated as pharmacodynamic markers of PS-341 activity.
Results: The most common toxicity was thrombocytopenia, which was dose limiting at 1.7 mg/m2 (schedule I) and 1.6 mg/m2 (schedule II), respectively. Sensory neuropathy was dose-limiting in a patient in schedule I. Grade ≥3 toxicities for schedule I were constipation, fatigue, myalgia, and sensory neuropathy. Grade ≥3 toxicities for schedule II were dehydration resulting from diarrhea, nausea and vomiting, fatigue, hypoglycemia, and hypotension. The maximum tolerated dose was 1.5 mg/m2 for both schedules. Reversible dose-dependent decreases in 20S proteasome activity in PBMCs were observed, with 36% inhibition at 0.5 mg/m2, 52% at 0.9 mg/m2, and 75% at 1.25 mg/m2. Accumulation of proteasome-targeted polypeptides was detected in tumor samples after treatment with PS-341. A patient with multiple myeloma had a partial response.
Conclusion: PS-341 given 1.5 mg/m2 twice weekly for 2 of every 3 weeks is well tolerated and should be further studied.
- PS-341
- phase 1
- solid tumors
- biomarkers
The ubiquitin-proteasome pathway plays an important role in regulating the cell cycle, neoplastic growth, and metastasis (1). A number of key regulatory proteins are temporally degraded during the cell cycle by the ubiquitin-proteasome pathway; the ordered degradation of these proteins is required for the cell to progress through the cell cycle to mitosis. One of the targets of ubiquitin-proteasome–mediated degradation is the tumor suppressor p53 (2), which acts as a negative regulator of cell growth. In addition, the ubiquitin-proteasome pathway is required for transcriptional regulation. Nuclear factor-κB (NF-κB) is a key transcription factor, whose activation is regulated by proteasome-mediated degradation of the inhibitor protein IκB (3). Cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are regulated by NF-κB (4) and are involved in tumor metastasis and angiogenesis in vivo. In addition, NF-κB is required in a number of cell types to maintain cell viability by activating transcription of a number of antiapoptotic genes. Consistent with this view, inhibiting NF-κB activation by stabilizing the IκB protein makes cells more sensitive to environmental stress and cytotoxic agents, ultimately leading to apoptosis.
PS-341 (bortezomib, Velcade) is a small, cell permeable molecule that specifically and selectively inhibits the proteasome by binding tightly to the enzyme's chymotrypsin-like active sites. Notably, inhibition of 20S proteasome activity can be reliably and directly detected after treatment with PS-341 in vitro, in vivo, and ex vivo in cells and tissues. Chemically, PS-341 is a modified dipeptidyl boronic acid derived from leucine and phenylalanine. In the NCI 60 cell line screen, PS-341 showed growth inhibitory and cytotoxic activity for many human tumor cell types. The average GI50 of PS-341 across the 60 cell lines was 3.8 nmol/L. The pattern of growth inhibition and cytotoxicity was unique, suggesting that PS-341 represents a novel class of cytotoxic compound.
Biological activity of PS-341 was observed in the hollow fiber assay system as well as the murine Lewis lung carcinoma model and in two human xenograft models, HT-29 colon tumors and PC-3 prostate tumors (5, 6).
As is the case with other targeted agents, there has been considerable interest in knowing whether PS-341 successfully inhibits its target in situ. PS-341 has a short plasma half-life following i.v. administration; measurement of drug levels does not provide an appropriate method for evaluating drug activity. However, the activity of the biochemical target of PS-341, the proteasome, can be measured in isolated WBC from mice, rats, monkeys, and man (7). Following i.v. administration of PS-341, 20S proteasome activity in circulating WBC is inhibited in a dose-dependent and reversible manner. Based on striking phase II data (8, 9), PS-341 has recently been approved for the therapy of refractory multiple myeloma by the U.S. Food and Drug Administration.
We undertook a phase I and pharmacologic study among patients with advanced malignancies to define the maximum tolerated dose (MTD) of PS-341 given as an i.v. bolus twice weekly for 4 weeks of every 6 weeks (schedule I). As the MTD reached with schedule I in patients with refractory hematologic malignancies was lower (10, 11) in comparison with other schedules tested among patients with solid tumors in phase I trials (12, 13), an alternate schedule of PS341 given twice weekly 2 weeks of every 3 weeks (schedule II) was likewise evaluated. In addition, this study aimed to describe the toxicities of PS-341 given on the schedules described above, to measure the inhibition of the activity of the 20S proteasome by PS-341 in whole blood as a biological assay for the plasma disposition of this rapidly cleared agent, to investigate biological end points of drug activity by examining the effect of PS-341 on the levels of proteasome targets in tumor tissue and to seek preliminary evidence of therapeutic activity of PS-341.
Patients and Methods
Patient selection. Patients with histologic or cytologic evidence of metastatic or locally advanced cancer, including non-Hodgkin's lymphoma and multiple myeloma for which there was no established life-prolonging therapy available, or who were unresponsive to conventional therapy and had measurable or evaluable disease, were eligible for this study. Eligibility criteria included age of ≥18 years; Eastern Cooperative Oncology Group performance status of ≤2; adequate bone marrow (platelets, ≥100 × 109 cells/L; absolute neutrophil count, ≥1.5 × 109 cells/L), hepatic (total bilirubin, ≤1.5 × upper limit of normal), and renal (serum creatinine, ≤1.5 times the upper limit of normal) functions; no chemotherapy, radiotherapy, biological, hormonal, or investigational drug therapy within 28 days before study entry and no prior nitrosourea or mitomycin C chemotherapy. Excluded from this study were patients with a diagnosis of acute leukemia; radiation therapy to >30% of the bone marrow; brain metastasis, unless disease had been resected by surgery or radiosurgery and patient had been stable for at least 8 weeks; or presence of an active infection requiring therapy. Written informed consent was obtained according to federal and institutional guidelines.
Dosage and administration. PS-341 (N-pyrazinecarbonyl-l-phenylalanine-l-leucine boronic acid) was supplied by the Cancer Therapy and Evaluation Program of the National Cancer Institute (Bethesda, MD), as a 3.5-mg vial for injection. Each sterile single-use 10-mL vial contained 3.5 mg PS-341 as a lyophilized powder with 35 mg mannitol, USP. The intact vials were refrigerated (2°C to 8°C) and protected from light. When the 3.5-mg vial was reconstituted with 3.5 mL normal saline, USP, each milliliter of solution contained 1 mg of PS-341. The drug was given without further dilution as an i.v. bolus (over 3-5 seconds).
A modified version of the standard “cohorts of three” phase I design (S1) was used for dose escalation. This design allowed the accrual of three to four patients to a given dose level initially and an additional two to three patients whenever a cohort of six was required [i.e., one dose-limiting toxicity (DLT) observed in the first three to four patients enrolled at a given dose level]. Patients were enrolled to schedule II, starting at the MTD determined for schedule I.
Dose-limiting toxicities. All toxicities were graded according to the National Cancer Institute Common Toxicity Criteria (version 2.0). The MTD was defined as one dose level below the dose that induced dose-limiting toxicities in one third or more of patients (at least two of a maximum of six new patients). MTD was defined based on toxicities documented in the first cycle of treatment only. Severe or life-threatening nonhematologic toxicity (grade 3 or 4), with the exception of nausea, vomiting, or diarrhea, was considered dose limiting. Nausea was not considered dose limiting. Grade 4 vomiting or diarrhea that persisted despite maximal prophylaxis and treatment with antiemetic or antidiarrheal therapy was considered dose limiting. Grade 4 neutropenia associated with fever or lasting for ≥5 days and grade 4 or 3 thrombocytopenia with grade ≥2 hemorrhage were likewise deemed dose limiting.
Pretreatment and follow-up studies. Complete patient histories, physical examinations, complete blood counts, serum electrolytes, chemistries, and coagulation profiles were obtained at baseline and before each course of treatment. Full supportive care, such as antiemetics, antidiarrheals, blood products, use of recombinant erythropoietin to maintain adequate hemoglobin and avoid blood transfusions, etc. were rendered as necessary. Routine or prophylactic use of granulocyte colony-stimulating factor or granulocyte macrophage colony-stimulating factor was not allowed. Therapeutic use of granulocyte colony-stimulating factor or granulocyte macrophage colony-stimulating factor in patients with serious neutropenic complications was allowed at the physician's discretion. Laboratory studies were done weekly while patients were on study. Radiologic studies (roentgenograms, computed axial tomographic scans, and magnetic resonance imaging) were done at baseline and at the end of every second cycle to assess tumor response. A partial response required at least a 50% reduction in the sum of the products of bidimensional measurements, separated by at least 4 weeks. A complete response was defined as the disappearance of all evidence of tumor on two measurements separated by a minimum of 4 weeks. Progressive disease was the appearance of new lesion(s) or an increase in the sum of the bidimensional products of all known disease by at least 25%. Stable disease was documented when there was persistence of disease without meeting the criteria for progression, partial response, or complete response.
Pharmacodynamic analyses
20S proteasome inhibition. Preclinical studies in animal models showed that PS-341 was rapidly removed from the circulation and distributed widely to non–vascular tissues, making detection in serum difficult. Thus, a pharmacodynamic assay measuring the amount of inhibition of the 20S proteasome in whole blood, developed by Millennium Pharmaceuticals, Inc. (Cambridge, MA; ref. 7), was employed. Blood samples were collected in sodium heparin-containing tubes and inverted several times before freezing at −80°C. Samples were sent frozen to Millennium Pharmaceuticals for 20S proteasome inhibition determination. The blood cells were lysed with 5 mmol/L EDTA (pH 8.0) for 1 hour and centrifuged at 6,600 × g for 10 minutes at 4°C. The resultant whole blood lysate samples were used in the 20S proteasome assay as described previously (7). Briefly, samples (10 mL) were added to 2 mL of buffered substrate (20 mmol/L HEPES, 0.5 mmol/L EDTA, 0.05% SDS, and 60 mmol/L Ys substrate-Suc-Leu-Leu-Val-Tyr-AMC; Bachem, King of Prussia, PA). The reaction was carried out at 37°C for 5 minutes; the rate of substrate cleavage by the 20S proteasome was determined. The protein content of the samples was estimated using a Coomassie protein assay (Pierce Co., Rockford, IL). Data were represented as means ± SE with statistical significance of P < 0.05. Two samples were obtained before study to establish a baseline for each patient. During the first cycle, samples were drawn on the following schedule: (a) day 1: 0, 1, and 24 hours; (b) day 4: 0 and 1 hour; (c) day 8: 0, 1, and 24 hours; and (d) day 11: 0 and 1 hour.
Pharmacodynamic measurements
A number of key regulatory proteins are degraded during the cell cycle by the ubiquitin-proteasome pathway; the ordered degradation of these polypeptides is required for the cell to progress through the cell cycle to mitosis. These polypeptides include p53 (2), p27 (14), NF-κB (3), cyclins (2), and apoptotic proteins such as BAX (15). As has been shown in preclinical models, exposure to PS-341 is expected to increase accumulation of such proteins in certain normal tissues as well as cultured cancer cell lines and human tumors (16, 17). In this phase I trial, we sought to show inhibition of PS-341 in situ by evaluating the accumulation of proteins whose degradation is mediated by the ubiquitin protease pathway.
Immunoblotting for changes in proteasome target proteins in tumor specimens. Tumor biopsies obtained before and 6 hours after treatment with PS-341 were snap-frozen and stored in liquid nitrogen until analysis. In brief, the frozen samples were solubilized by sonication in alkylation buffer [6 mol/L guanidine hydrochloride, 250 mmol/L Tris-HCl (pH 8.5 at 21°C), 10 mmol/L EDTA, 1% [v/v] β-mercaptoethanol, and 1 mmol/L 2-phenylmethylsulfonyl fluoride (freshly added from a 100 mmol/L stock in anhydrous isopropanol)]. After the cell lysates were dialyzed and lyophilized as previously described, aliquots containing 50 μg of protein (assayed by the bicinchoninic acid method; ref. 18) were subjected to electrophoresis on SDS-polyacrylamide gradient gels containing 5% to 15% (w/v) acrylamide, transferred to nitrocellulose, and probed with antibodies against Actin and Bax (Santa Cruz Biotechnologies, Santa Cruz, CA), IκB (Oncogene, San Diego, CA), p27 (PharMingen, San Diego, CA), p53 (NeoMarkers, Fremont, CA), and Cyclin E (Robert T. Abraham, Burnham Institute, La Jolla, CA). Antigen-antibody complexes were detected using peroxidase-coupled secondary antibodies and enhanced chemiluminescence reagents.
Results
Patient demographics. A total of 44 patients (Table 1) from Mayo Clinic (Rochester, MN) and University of Wisconsin Comprehensive Cancer Center (Madison, WI) received 73 assessable courses of therapy through six dose levels (Table 2). Thirty-six patients were enrolled using the aforementioned modified version of the standard “cohorts of three” phase I design. As two patients assigned to the 1.7 mg/m2 dose level for schedule I encountered DLTs, a total of eight additional patients were enrolled at 1.5 mg/m2 dose level of schedule I. Although three patients of the additional eight patients had DLTs, there were only four DLTs overall of a cumulative total of 14 patients registered to that dose level. As the protocol-defined MTD was not exceeded, dose level 1.5 mg/m2 was thus chosen to be the MTD for schedule I. The median number of courses given was 2 for both schedules I and II. Patients were of good performance status, with all patients, except two (one each schedule), having an Eastern Cooperative Oncology Group performance status of ≤1. The most common tumor type was colorectal cancer followed by kidney, pancreatic and prostate cancer.
Patient demographics
Treatment data
Hematologic toxicity. The only hematologic treatment–related grade >2 toxicities observed during cycle 1 were anemia (one schedule I) and thrombocytopenia (five schedule I and three schedule II). Reversible thrombocytopenia was dose limiting for both schedules at 1.7 and 1.6 mg/m2, respectively. This thrombocytopenia did not result in bleeding complications or necessitate platelet transfusions. Two patients on schedule I had a grade 2 thrombocytopenia in cycle 2 and two patients on schedule II had thrombocytopenia (one grade 1 and one grade 3) in cycle 2. Only one patient (schedule II) had documented grade 1 leukopenia in cycle 2.
Nonhematologic toxicity. The nonhematologic side effects of PS-341 were mostly mild to moderate. The common treatment-related toxicities in all cycles for schedule I were fatigue, diarrhea, nausea, anorexia, sensory neurotoxicity, rash, and vomiting. These were similarly encountered in all cycles for schedule II except rash and sensory neuropathy. Although two patients in schedule II had grade >3 diarrhea that required protocol-defined dose reduction for subsequent cycles upon resolution of symptoms, diarrhea was generally easy to control using loperamide for both schedules. Constipation was also observed with some frequency in both groups. Figure 1A and B displays all cycles of treatment-related, nonhematologic toxicities for schedules I and II. Figure 2A and B displays treatment-related nonhematologic toxicities in cycle 1 for schedules I and II.
A, all cycles of treatment-related nonhematologic toxicities in schedule I. B, all cycles of treatment-related nonhematologic toxicities in schedule II.
A, cycle 1 treatment-related nonhematologic toxicities in schedule I. B, cycle 1 treatment-related nonhematologic toxicities in schedule II.
Comparison of toxicity profiles. Schedules I and II had similar toxicity profiles (Table 3). This similarity was seen when comparing cycle 1 toxicities only, or cumulative toxicities over all treatment cycles. However, it is important to note that 12 of the 28 patients in schedule I received a dose of PS-341 lower than the lowest dose of schedule II.
Comparison of toxicities by schedule
Antitumor activity. Forty-one patients [27 schedule I (one patient died before disease assessment), 14 schedule II (one patient received only two doses on cycle 1, one patient deteriorated before evaluation)] of the 44 were assessable for antitumor activity. Ten patients (five schedule I, five schedule II) did not have a tumor response evaluation. A partial regression (>50%) of a perinephric plasmacytoma was noted before cycle 2 of schedule I for a 50-year-old male with multiple myeloma receiving PS-341 at a dose of 1.25 mg/m2. This response was sustained for approximately 4 months before disease progression. Five patients (Table 2) had at least one evaluation of stable disease. No other patients had tumor regression.
Pharmacodynamic analyses
20S proteasome inhibition. As seen in Table 4, there was a dose-dependent increase in the degree of proteasome inhibition in peripheral blood mononuclear cells within an hour of exposure to PS-341. This effect is reversible, being maximal in the first hour. There was recovery of proteasome activity to at least 85% of pretreatment levels by 24 hours except in dose level 4 of schedule I (1.5 mg/m2), where 35% inhibition was still observed at 24 hours.
Inhibition of 20S proteasome in peripheral blood mononuclear cells
Inhibition of ubiquitinated proteins in tumor tissue.Figure 3 shows the effect of exposure to PS-341 over time on the expression of selected proteins such as Bax and p53 on two different patient samples. A549 human non–small cell lung cancer cells, which were used as a control, showed a marked increase in p53 levels after exposure to PS-341 for 24 hours. Levels of several other polypeptides that are degraded by the proteasome were altered much less. The sample obtained 6 hours after the administration of PS-341 from the first patient showed increased accumulation of Bax, Cyclin E, IκB, NF-κB, and p27, whereas the posttreatment sample from the second patient did not show any change.
Western blots of patient tumor samples. Core biopsy samples were taken from untreated (U) tumors before the first dose of PS-341 and again from the same tumor site 6 hours after treatment (T). Data from two patients. Lysates from A549 non–small cell lung cancer cells treated with diluent (U) or 10 nmol/L PS-341 for 24 hours (T) were used as controls.
Discussion
The ubiquitin-proteasome system is an attractive target for anticancer drug development. Several key proteins that regulate important cellular processes such as proliferation and apoptosis are regulated by proteasome-dependent proteolysis. PS-341 is a boronic acid dipeptide derivative that acts as a reversible and selective inhibitor of the proteasome.
A number of phase I trials have been done with varying schedules of PS-341. Phase I studies tested weekly and twice weekly schedules for 2 to 4 weeks (10–13) repeated every 3 to 6 weeks. In general, PS-341 given on these schedules was well tolerated. Nonhematologic toxicities such as fatigue, diarrhea, nausea, vomiting, and sensory neuropathy were likewise observed in our study. Similar to our findings, thrombocytopenia was dose limiting in the phase I trials that employed the twice-weekly schedule for 4 of 6 weeks, reported by Orlowski et al. (10) and Cortes et al. (11). These studies, conducted among patients with hematologic malignancies, established a lower MTD compared with our current study conducted in a mixed population comprised mainly of patients with solid tumors. Altered bone marrow function among patients with hematologic malignancies and reduced tolerability for dose-dependent thrombocytopenia are probable explanations. On the other hand, in contrast to our study, the phase I trials of PS341 using different schedules conducted among patients with solid tumors (12, 13), dose-limiting thrombocytopenia did not occur. Although this may in part be attributable to the subset of patients with hematologic malignancies in our patient population, a schedule-dependent effect on platelets is a tenable hypothesis.
Our trial initially had patients receiving PS-341 biweekly for 4 weeks followed by a 2-week break. This schedule was found difficult to administer, with several patients missing doses in the later weeks of the schedule, due to grade 2 to 3 thrombocytopenia. Protocol-defined guidelines required dose omission and subsequent dose reduction by one dose level upon retreatment. In fact, 14 of 28 (50%) and 24 of 28 (86%) patients missed one or more doses or had a dose modification, in cycle 1 and all cycles, respectively. For this reason, the protocol was amended to allow biweekly administration given on a 2-week on/1-week off schedule. This schedule enabled delivery of intended doses better than schedule I. As a comparison, 6 of 16 (38%) and 10 of 16 (63%) patients enrolled in schedule II missed one or more doses or had a dose modification, in cycle 1 and all cycles, respectively. The recommended phase II dose on this 2-week on/1-week off schedule was 1.5 mg/m2. This dose is similar to the MTD seen in other studies and is the dose used in recently initiated phase II trials. We observed one partial response in a patient with multiple myeloma in this trial and no objective responses in patients with solid tumors. There were, however, five patients with at least one evaluation of stable disease.
Because PS-341 is rapidly eliminated from the circulation, the plasma levels are thought not to be illustrative of the biological effect. In this trial, we have instead measured proteasome activity of whole blood as a measure of PS-341's effect at its target. This simple, robust assay has been shown in animal models to correlate with proteasome activity in tissue, including that seen in tumors. Dose-related inhibition of 20S proteasome activity in human WBC has been shown, with 68% to 74% inhibition reported at the higher (>1.32 mg/m2) dose levels (10–13). There is consistent evidence to show that the dose-response curve is sigmoidal, with proteasome inhibition plateauing at the higher doses. Complete recovery of proteasome activity to baseline is typically seen before each 72-hour dose. In congruence with the aforementioned observations from other phase I trials, our pharmacodynamic analyses reaffirm the dose-dependent proteasome inhibition seen in earlier studies, with ∼65% proteasome inhibition observed within the first hour after drug administration at the MTD of 1.5 mg/m2. To determine whether this proteasome inhibition resulted in inhibition of the degradation of ubiquinated proteins, additional studies evaluated the levels of individual polypeptides such as p53, which is degraded by the ubiquitin-proteasome system, in tumor tissue. Results were not clear-cut. It is important to emphasize, however, that paired tumor tissue biopsies were available from only four patients. Moreover, because of the invasive nature of the procedures required to obtain these samples, comparison was limited to only one point (i.e., pretreatment and 6 hours after PS-341 administration). PS-341–induced elevations of several polypeptides were observed in two of the four paired samples (Fig. 3; data not shown). In the case of the two patients from whom data are not shown, the amount of protein obtained from the biopsy was insufficient to draw any conclusions as to whether polypeptides were changed following PS341 treatment. These observations show inhibition of the target enzyme in some cancer specimens but also indicate that effects at the target might vary in different tumors. Whether this heterogeneity reflects differences in drug elimination, drug uptake, sensitivity of the proteasome to inhibition, or some other variable will need to be further evaluated in the context of phase II trials.
In summary, PS-341 represents a novel targeted therapy that has shown evidence of anticancer activity in multiple myeloma. In our trial, we have found that PS-341 given biweekly on a 2-week on/1-week off schedule was well tolerated. Pharmacodynamic studies showed that the proteasome was significantly inhibited and that accumulation of ubiquinated proteins occurred. This schedule should be further examined in phase II trials and may prove a useful schedule to adapt in future combination chemotherapy trials.
Footnotes
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Grant support: CA 69912, CA 62491, CA15083, and RR00585.
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Accepted January 19, 2005.
- Received October 8, 2004.
- Revision received January 17, 2005.
References
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