Phase I Trial of Bortezomib and Carboplatin in Recurrent Ovarian or Primary Peritoneal Cancer
http://www.100md.com
《临床肿瘤学》
the Developmental Chemotherapy Service, Memorial Sloan-Kettering Cancer Center, New York, NY
The Program in Women's Oncology, Women & Infants' Hospital, Brown Medical School, Providence, RI
Department of Neurology, Northwestern University, Feinberg School of Medicine, Chicago, IL
ABSTRACT
PURPOSE: To determine the maximum-tolerated dose, pharmacodynamics, and safety of the combination of bortezomib and carboplatin in recurrent ovarian cancer.
PATIENTS AND METHODS: Fifteen patients were treated with a fixed dose of carboplatin (area under the curve [AUC] 5) and increasing doses of bortezomib (0.75, 1, 1.3, and 1.5 mg/m2/dose). Patients must have received upfront chemotherapy and up to two prior chemotherapy regimens for recurrent disease. Neurologic evaluation was performed at baseline and after every two cycles by the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire and examination by an attending neurologist. All patients received carboplatin alone in cycle 1 to establish baseline pharmacodynamics for nuclear factor-kappa B (NF-kB). Starting with cycle 2, patients were treated with carboplatin on day 1 and bortezomib on days 1, 4, 8, and 11.
RESULTS: Diarrhea, rash, neuropathy, and constipation (with colonic wall thickening on computed tomography) were dose-limiting toxicities, occurring in the two patients treated at the 1.5 mg/m2/dose level. The Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire was helpful in guiding the need for dose reductions. Neurotoxicity was manageable through six cycles, with appropriate dose reductions. Carboplatin had no effect on bortezomib pharmacodynamics as measured by percent inhibition of the 20S proteasome. Bortezomib decreased carboplatin-induced NF-kB. The overall response rate to this combination was 47%, with two complete responses (CR) and five partial responses, including one CR in a patient with platinum-resistant disease.
CONCLUSION: The recommended phase II dose of bortezomib administered in combination with carboplatin (AUC 5) is 1.3 mg/m2/dose.
INTRODUCTION
The ubiquitin proteasome pathway is a highly conserved intracellular pathway for the degradation of proteins. Many of the short-lived regulatory proteins that govern cell division, growth, activation, signaling, and transcription are substrates that are temporally degraded by the proteasome. Therefore, the proteasome represents a novel target for chemotherapy. Bortezomib is a dipeptidyl boronic acid inhibitor with high specificity for the proteasome.
The ubiquitin-proteasome pathway plays a significant role in neoplastic growth and metastasis. The ordered and temporal degradation of numerous key proteins, such as cyclins, cyclin-dependent kinase inhibitors, and tumor suppressors, is required for cell cycle progression and mitosis.1 The proteasome is also required for activation of nuclear factor-kappa B (NF-kB) by degradation of its inhibitory protein, I-kB.2 NF-kB is a transcription factor that upregulates a number of proteins involved in cancer progression, including several proangiogenic factors and antiapoptotic factors. NF-kB may be present as a homodimer or heterodimer; the p65/p50 heterodimer is the most common complex. In quiescent cells, NF-kB is sequestered in the cytoplasm by its inhibitory partner, I-kB, which renders NF-kB inactive. However, cellular stress signals, such as those induced by chemotherapy, radiation, viruses, growth factors, or antigens, trigger phosphorylation of I-kB, which targets it for proteasome-mediated degradation. Release of NF-kB by I-kB degradation allows NF-kB to translocate to the nucleus and effect its transcriptional activation functions. Inhibition of the proteasome via bortezomib prevents degradation of I-kB and, thus, downregulates NF-kB–mediated transcriptional activation. Thus, NF-kB is required, in part, to maintain cell viability through the transcription of inhibitors of apoptosis in response to environmental stress or cytotoxic agents.3-6 Stabilization of the Ik-B protein and blockade of NF-kB activity has been demonstrated to make cells more susceptible to apoptosis.3-5 Furthermore, NF-kB has been implicated in controlling the cell-surface expression of adhesion molecules such as E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1.7,8 Proteasome inhibitors have also been shown to overcome Bcl-2–mediated protection from apoptosis.9-11
Ovarian cancer was diagnosed in 25,280 women in the United States in 2004 and resulted in an estimated 16,090 deaths.12 In women treated with platinum combinations as primary therapy, the response rates are 60% to 80%, with complete responses (CRs) being most common in women who have had adequate surgical therapy.13 Unfortunately, however, the majority of patients eventually die of platinum-resistant disease persistence or recurrence, with the abdominal cavity being the most common site or recurrence. Long-term survival remains approximately 15% to 30%. Therefore, new approaches are needed to provide effective therapy with minimal induction of chemotherapy resistance. Certain types of chemoresistance observed in solid tumors are associated with NF-kB activation.14 Platinum agents have been shown to rapidly induce chemoresistance by activating NF-kB.15,16 Therefore, blocking NF-kB activation with bortezomib represents a potential target for overcoming the apoptosis failure that develops during ovarian cancer treatment.
In animal models, bortezomib is rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. It is difficult to correlate plasma concentration of the drug with the degree of proteasome inhibition in blood samples. Therefore, a pharmacodynamic assay has been developed that provides a reliable measure of 20S proteasome activity in peripheral leukocytes, whole blood, and tissue biopsy and is sensitive, accurate, and reproducible.17 This assay allows for measurement of bortezomib pharmacodynamics and reflects its target activity. The principal objectives of this phase I and pharmacodynamic study were as follows: (1) to determine the maximum-tolerated dose of bortezomib that can be administered with a standard dose of carboplatin in the treatment of recurrent or progressive epithelial ovarian or primary peritoneal cancer; (2) to evaluate the toxicity of bortezomib when administered with carboplatin in this patient population; (3) to characterize the pharmacodynamics of 20S proteasome activity in patients treated with bortezomib and carboplatin; and (4) to determine the effect of bortezomib on carboplatin-induced NF-kB activity.
PATIENTS AND METHODS
Patient Selection
Patients with recurrent or progressive epithelial ovarian or primary peritoneal carcinoma were eligible for this trial. Patients must have received a platinum-based chemotherapeutic regimen for the management of primary disease, which may have included consolidation or extended therapy after surgical or nonsurgical assessment. Patients may have received up to two prior regimens for recurrent disease, including one nonplatinum-containing regimen. Patients were considered platinum resistant if they had progressed on or within 6 months of their last platinum-based regimen. Prior chemotherapy must have been completed 4 weeks before beginning investigational therapy, and all treatment-related toxicities must have resolved to baseline. Other eligibility criteria were as follows: age 18 years; Karnofsky performance score 70; adequate bone marrow function (absolute neutrophil count 1,000/μL, platelets 100,000/μL, and hemoglobin 8 gm/dL), hepatic function (AST and ALT levels 2.5 x the upper limit of institutional normal and total bilirubin 1.8 mg/dL), and renal function (serum creatinine 1.5 mg/dL); normal ejection fraction (> 50%) by multiple-gated acquisition scan or echocardiogram; no grade 2 peripheral neuropathy; no major surgery within 2 weeks of study entry; no significant atherosclerotic disease (defined as peripheral vascular disease requiring surgical management, history of myocardial infarction or congestive heart failure, or history of cerebrovascular event); no ECG evidence of acute ischemia or significant conduction abnormality (bifascicular block, defined as left anterior hemiblock in the presence of right bundle branch block, or second- or third-degree atrioventricular blocks); no orthostatic hypotension; no CNS disease (brain metastases or leptomeningeal involvement); and no serious medical or psychiatric illness that would limit full compliance with the study. Informed consent was obtained according to federal and institutional guidelines.
Drug Administration
Six cycles of therapy administered every 21 days were planned. For the first cycle, all patients received only carboplatin on day 1 to establish baseline NF-kB activation. Beginning with cycle 2, patients received bortezomib at doses of 0.75, 1, 1.3, or 1.5 mg/m2 on day 1, followed 1 hour later by carboplatin; bortezomib was administered alone on days 4, 8, and 11.
Bortezomib was administered as a rapid intravenous bolus injection into the side arm of a running intravenous infusion of normal saline at 100 mL/h. Carboplatin was administered as an intravenous infusion over 30 minutes. The dose of carboplatin was calculated using the Calvert formula. The Jellife formula was used to estimate the glomerular filtration rate.
Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Dose-limiting toxicity was defined as grade 4 hematologic toxicity and any grade 3 nonhematologic toxicity occurring during the second cycle (first cycle of combination bortezomib/carboplatin) of therapy. Three to six patients were entered at each dose level. No intrapatient dose escalation was allowed. Dose escalation was not allowed until all patients treated at the prior dose level completed the second cycle of therapy. If one patient experienced dose-limiting toxicity, three additional patients were added to the dose level. If two of six patients experienced dose-limiting toxicity, the previous dose level was declared the maximum-tolerated dose. If only one of six patients experienced dose-limiting toxicity, dose escalation was permitted to continue.
Pretreatment and Follow-Up Studies
Pretreatment evaluation consisted of history and physical examination, assessment of Karnofsky performance status, completion of the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire, baseline neurologic evaluation by attending neurologist, ECG, measurement of left ventricular ejection fraction (by multiple-gated acquisition scan or echocardiogram), chest x-ray, urinalysis, CBC count, serum chemistries (electrolytes, glucose, blood urea nitrogen, creatinine, magnesium, calcium, phosphate, albumin, AST, ALT, alkaline phosphatase, and total bilirubin), CA-125 level, and documentation of measurable or assessable disease by computed tomography (CT) scan. During the study, interval history, physical examination, toxicity assessment, Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire (Table 1), CBC count, serum chemistries, and CA-125 level were obtained at the start of each cycle. Starting with cycle 2, a CBC count was obtained before each bortezomib dose. Follow-up neurologic evaluation by the attending neurologist was performed after every two cycles. CT scan to evaluate response was performed after every two cycles. Evaluation of response was performed according to Response Evaluation Criteria in Solid Tumors.
Pharmacodynamics
20S proteasome assay. In animal model studies, bortezomib was rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. Therefore, a pharmacodynamic assay that results in a calculation of percentage of inhibition of the proteasome was used as an alternative to pharmacokinetic measurements. The method for determination of 20S proteasome activity was developed by Millennium Pharmaceuticals, Inc (Cambridge, MA).17 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 then centrifuged at 6,600 x g for 10 minutes at 4°C. The resultant whole-blood lysate samples were then used in the 20S proteasome assay as previously described.17 Briefly, samples (10 mL) were added to 2 mL of substrate buffer (20 mmol/L HEPES, 0.5 mmol/L EDTA, 0.05% sodium dodecyl sulfate, 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, and the rate of substrate cleavage per 20S proteasome activity was determined. The protein content of the samples was determined using a Coomassie protein assay (Pierce, Rockford, IL). Data are presented as means ± SEM, with statistical significance at P < .05. Two samples were obtained before study to establish a baseline for each patient. During the second cycle of therapy (first cycle of combined bortezomib and carboplatin treatment), samples were drawn on the following schedule: (1) day 1 at 0 and 1 hour (1 hour after bortezomib, and before carboplatin), at 2 hours (30 minutes after end of carboplatin infusion), and at 24 hours; (2) day 4 at 0 and 1 hour; (3) day 8 at 0 and 1 hour; and (4) day 11 at 0 and 1 hour.
NF-kB determinations. To assess the inhibition of NF-kB by bortezomib when administered in combination with carboplatin, an aliquot of blood was collected in cell preparation tubes at baseline (time 0) and 30 minutes after the completion of carboplatin administration (time 1 hour) on day 1 of cycle 1. On day 1 of cycle 2, when both bortezomib and carboplatin were administered, specimens were collected at time 0 (before bortezomib administration), 1 hour (before carboplatin administration), and 2 hours (30 minutes after completion of carboplatin administration). Samples were separated by Ficoll-Hypaque density centrifugation with WBC pellet suspended in cold Buffer A (10 mmol/L HEPES-KOH at pH 7.9 at 4°C, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, and 0.2 mmol/L PMSF). Cells were allowed to swell on ice and were then vortexed, pelleted, resuspended in cold Buffer C (20 mmol/L HEPES-KOH at pH 7.9, 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, and 0.2 mmol/L PMSF), and incubated on ice for 20 minutes for high-salt extraction. Protein concentrations were determined by the Bradford method using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Electromobility shift assay was then performed. Consensus oligonucleotides (Promega, Madison, WI) were end labeled with [-32P]adenosine triphosphate using T4 polynucleotide kinase. Binding reactions were performed in a 10-μL volume containing 10 μg of nuclear extract, 0.1 μg of poly (dI-dC), 4% glycerol, 1 mmol/L of MgCl2, 0.5 mmol/L of EDTA, 5 mmol/L of dithiothreitol, 50 mmol/L of NaCl, and 10 mmol/L of Tris-HCl at pH 7.5 in the presence of labeled probe (20,000 cpm). The samples were incubated at room temperature for 20 minutes, loaded on a 4% polyacrylamide gel, and electrophoresed at 350 V for 1 hour in 1 x Tris-glycine buffer.
RESULTS
Patient Characteristics
Fifteen patients were enrolled onto the study, and all patients were assessable for toxicity. One patient in the 1.3 mg/m2/dose level did not complete the full dose of carboplatin in cycle 2 because of a hypersensitivity reaction and was not assessable for response. An additional patient was accrued to this level to ensure accurate assessment of toxicities for that dose level. Patient characteristics are listed in Table 2. The median age was 54 years (range, 45 to 68 years), and the median Karnofsky performance status was 90% (range, 80% to 90%). Four of the 15 patients were platinum resistant (had progression of disease on or within 6 months of receiving their last platinum therapy). The median number of prior chemotherapy regimens was one (range, one to three regimens). Two patients had prior pelvic radiation therapy.
Drug Delivery
The dose-escalation schema and the number of patients and courses administered as a function of dose level are listed in Table 3. At the 1.3 mg/m2/dose level of bortezomib, one patient was unable to receive the last two doses of bortezomib in cycle 6 because of grade 3 diarrhea, and one patient was unable to receive the last two doses of bortezomib in cycle 5 because off asthenia, anorexia, orthostatic dehydration (grade 2), and constipation. All other doses were able to be administered.
Toxicity
Both hematologic and nonhematologic toxicity were minimal at the first two dose levels (0.75 and 1 mg/m2/dose). Toxic effects that are reported were scored as being possibly, probably, or definitely drug related. These toxicities are reported as the worst toxicity per category exhibited by a patient during their treatment course (Table 4). Toxicities that were scored as not being drug related have been reviewed and have not been included if they were clearly not associated with the experimental treatment.
Hematologic Toxicity
There was no dose-limiting hematologic toxicity seen in the trial.
Nonhematologic Toxicity
The nonhematologic dose-limiting toxicities observed were diarrhea, constipation (in association with colonic wall thickening on CT), rash, and sensory neuropathy. The first two patients treated at the highest dose level of 1.5 mg/m2/dose of bortezomib developed dose-limiting toxicity. The first patient developed grade 3 diarrhea, grade 3 rash, and grade 3 sensory peripheral neuropathy. The second patient developed grade 3 constipation in association with colonic wall thickening on CT.
Neurotoxicity was accessed by both examination and assessment by an attending neurologist at baseline and then after every two cycles and by using the Gynecologic Oncology Group neurotoxicity questionnaire. At the first dose level (bortezomib 0.75 mg/m2/dose and carboplatin area under the curve [AUC] 5), all three patients entered the study with no baseline symptoms of neuropathy (all answers 0 on the neurotoxicity questionnaire). All three patients had diminished vibration noted on baseline examination by the neurologist, and all three patients had stable examination and stable assessment on the neurotoxicity questionnaire throughout the study. At the second dose level (bortezomib 1 mg/m2/dose and carboplatin AUC 5), two of the three patients entered the study with a baseline grade 1 neuropathy. All three patients had diminished vibration noted on baseline examination by the neurologist, and all three patients had stable examination and stable assessment on the neurotoxicity questionnaire throughout the study. Of the seven patients entered onto the third dose level (bortezomib 1.3 mg/m2/dose and carboplatin AUC 5), two had a baseline grade 1 neuropathy. These two patients remained stable on treatment. Two patients who entered the study without neuropathy developed a grade 1 neuropathy on treatment. An additional patient who entered the study without neuropathy developed a grade 2 neuropathy on treatment. Her neuropathy improved to grade 1 with a 1-week delay, and she resumed treatment with a one-level dose reduction (to bortezomib 1 mg/m2/dose and carboplatin AUC 5). After two additional doses of bortezomib at the reduced level, the neuropathy increased back to grade 2 and persisted for 4 weeks, when it again decreased to grade 1. The patient was removed from study. The examination by the attending neurologist did not find any change on any of these patients. The changes in neuropathy were well described by the patients on the neurotoxicity questionnaire. The first patient at the 1.5 mg/m2/dose level of bortezomib entered without neuropathy. She developed a grade 3 neuropathy after day 4 of cycle 3. She was removed from study. The neuropathy was painful, requiring narcotic analgesia and gabapentin, and was evident on examination by the attending neurologist in bilateral lower extremities by new diminished pinprick to the shins, diminished vibratory sense to the ankles, and lack of temperature sense. Her neurotoxicity questionnaire showed complaints beginning 4 weeks before the symptoms were evident at the standard office assessment and examination. Her neuropathy improved but continued to require narcotic analgesia and gabapentin to control pain at the last follow-up 9 months from the completion of therapy. The second patient at the 1.5 mg/m2/dose level entered with a grade 1 neuropathy and remained stable. She was taken off study after cycle 2 because of GI toxicity. Therefore, the maximum-tolerated dose of this combination was determined to be carboplatin AUC 5 and bortezomib 1.3 mg/m2/dose.
Pharmacodynamics
Fourteen patients were assessable for pharmacodynamics. The results of the 20S proteasome inhibition determinations for bortezomib alone and for the combination of bortezomib and carboplatin are shown in Figure 1. The bortezomib alone points represent an average of the 1-hour draws on days 1, 4, 8, and 11. The bortezomib/carboplatin points represent the 2-hour draw on day 1. The 1-hour time point was chosen because this was shown to be the time of maximum percent 20S proteasome inhibition in phase I trials of bortezomib administered as a single agent.18-20 The dose-response relationship between bortezomib and 20S proteasome inhibition is know to be steep up until to the range used in this study, followed by a plateau.18-20 Therefore, it is expected that the percent 20S proteasome inhibition seen across dose levels in this study is similar. There was no difference seen in percent inhibition of the 20S proteasome with and without carboplatin (0.75 mg/m2/dose level, P = .2; 1 mg/m2/dose level, P = .5; 1.3 mg/m2/dose level, P = .2; and 1.5 mg/m2/dose level, P = .9). As predicted by single-agent pharmacodynamics of bortezomib, the level of proteasome inhibition declined at 24 hours (average, 21 hours; range, 11 to 44 hours) and returned to baseline by 72 hours.18-20
NF-kB Determinations
Fourteen patients were assessable for NF-kB. One patient did not receive the full dose of carboplatin with cycle 2 because of a hypersensitivity reaction. Electromobility shift assays were successfully completed on 11 of the 14 patients (the remaining three patients had inadequate or missing samples). Blood samples were taken at baseline (time 0) and 30 minutes after the completion of carboplatin administration (time 1 hour) on day 1 of cycle 1. On day 1 of cycle 2, when both bortezomib and carboplatin were administered, specimens were collected at time 0 (before bortezomib administration), 1 hour (before carboplatin administration), and 2 hours (30 minutes after completion of carboplatin administration). A representative electromobility shift assay is shown in Figure 2. Eight of the 11 patients showed increased NF-kB activity after carboplatin treatment on day 1 of cycle 1. This increase in NF-kB activity persisted at the baseline sample on day 1 of cycle 2. Of the eight patients with increased NF-kB activity, seven showed decline after treatment with bortezomib and failure to completely reactivate on re-exposure to carboplatin.
Response
Fourteen of 15 patients were assessable for response. Because response evaluations were performed after every two cycles, the first response evaluation performed reflected one cycle of single-agent carboplatin and one cycle of the combination of bortezomib and carboplatin. There were two CRs and five partial responses, for an overall response rate of 47%. One CR occurred in a platinum-resistant patient. All partial responses were in platinum-sensitive patients. An additional four patients experienced stable disease as their best response. Both patients with CR normalized their CA-125. Four of the five patients with a partial response had measurable CA-125 levels; of these patients, three normalized their marker, and the remaining patient had a greater than 50% decline in CA-125. Of the four patients with stable disease, two normalized CA-125, one had a more than 50% decline in CA-125, and one had stable CA-125. All three patients with progression of disease had an increase in CA-125 on treatment.
DISCUSSION
The ubiquitin-proteasome pathway plays an essential role in the degradation of most short- and long-lived intracellular proteins in eukaryotic cells. At the heart of this degradative pathway is the 26S proteasome, an adenosine triphosphate–dependent, multicatalytic protease. Proteolytic degradation of damaged, oxidized, or misfolded proteins is part of the housekeeping role for the 26S proteasome. In addition, the 26S proteasome also plays a vital role in degrading regulatory proteins that govern the cell cycle, transcription factor activation, apoptosis, and cell trafficking. Inhibitors of the proteasome represent a novel approach to treating human malignancies. Bortezomib is a reversible, highly selective inhibitor of the proteasome with promising in vitro and in vivo activity. This phase I and pharmacodynamic trial was designed to study the feasibility of administering bortezomib as a rapid intravenous infusion twice weekly for 2 weeks, followed by a 1-week recovery period, in combination with a fixed dose of carboplatin in women with recurrent ovarian cancer.
There was no significant toxicity seen in the first two dose levels tested (0.75 and 1 mg/m2/dose). There was no dose-limiting hematologic toxicity seen. The nonhematologic toxicity seen was largely predicted by the single-agent experience with bortezomib. The dose-limiting toxicities observed were diarrhea, constipation (in association with colonic wall thickening on CT), rash, and sensory peripheral neuropathy. The recommended phase II dose of bortezomib administered in combination with carboplatin (AUC 5) is 1.3 mg/m2/dose. This is the same dose of bortezomib that is currently being tested in a single-agent study by the Gynecologic Oncology Group in recurrent ovarian cancer.
The preclinical activity of bortezomib makes it a promising agent for combination therapy in overcoming chemotherapy resistance. The CR seen in this study in a platinum-resistant patient is also encouraging. Preclinical testing has shown synergy between bortezomib and the topoisomerase I inhibitor irinotecan.21 Activation of the transcription factor NF-kB by ionizing radiation,5 irinotecan,22 cisplatin,15 and other chemotherapeutic agents has been found to protect from cell killing. Proteasome inhibitors have been shown to inhibit drug-induced NF-kB and, thus, sensitize the treated cells to drug-induced apoptosis. Proteasome inhibition has also been shown to enhance the radiosensitivity of cell lines in vitro.23
The results of this phase I trial demonstrate that carboplatin in combination with bortezomib administered in a twice weekly for 2 weeks cycle every 21 days results in predictable toxicity and proteasome inhibition associated with preclinical activity. In lieu of a sensitive pharmacokinetic assay, the 20S proteasome pharmacodynamic assay proved to be both useful and predictive. It confirms inhibition of the biochemical target, the proteasome, and provides insight into how long the target is affected by bortezomib. In this study, carboplatin did not alter the pharmacodynamics of bortezomib. In the future, it is hoped that this assay will be used to determine proteasome inhibition in tumor biopsies, thereby providing data in biologically relevant tissues. The demonstration that bortezomib administration resulted in a decrease in carboplatin-induced upregulation of NF-kB and the occurrence of one CR in a patient with platinum-resistant disease suggest that further investigation of this agent as a means to overcoming drug resistance is warranted. A Gynecologic Oncology Group phase II trial of single-agent bortezomib in recurrent ovarian cancer is ongoing. If single-agent activity of bortezomib is seen, further exploration of platinum-based combinations via randomized phase II or III studies is feasible and warranted.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
NOTES
Supported by grant No. 5RO1 CA84009 from the National Cancer Institute (Bethesda, MD).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Goldberg AL, Stein R, Adams J: New insights into proteasome function: From Archaebacteria to drug development. Chem Biol 2:503-508, 1995
Palombella VJ, Rando OJ, Goldberg AL, et al: The ubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein and the activation of NF-kB. Cell 78:773-785, 1994
Beg AA, Baltimore D: An essential role for NF-kB in preventing TNF-alpha-induced cell death. Science 274:782-784, 1996
Van Antwerp DJ, Martin SJ, Kafri T, et al: Suppression of TNF-alpha-induced apoptosis by NF-kB. Science 274:787-789, 1996
Wang CY, Mayo MW, Baldwin AS: TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kB. Science 274:784-787, 1996
Chu ZL, Mckinsey TA, Liu L, et al: Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kB control. Proc Natl Acad Sci U S A 94:10057-10062, 1997
Zetter BR: Adhesion molecules in tumor metastasis. Semin Cancer Biol 4:219-229, 1993
Read MA, Neish AS, Luscinskas FW, et al: The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression. Immunity 2:493-506, 1995
Benyi L, Ping Dou Q: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression. Proc Natl Acad Sci U S A 97:3850-3855, 2000
An B, Goldfarb RH, Siman R: Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts. Cell Death Differ 5:1062-1075, 1998
Herrmann JL, Briones F, Brisbay S, et al: Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53. Oncogene 17:2889-2899, 1998
Jemal A, Tiwari RC, Murray T, et al: Cancer statistics, 2004. CA Cancer J Clin 54:8-29, 2004
Ozols RF, Schwartz PE, Eifel PJ: Ovarian cancer, fallopian tube carcinoma, and peritoneal carcinoma, in DeVita VT, Hellman S, Rosenberg RA (eds). Cancer: Principles and Practice of Oncology (ed 6). Baltimore, MD, Lippincott Williams and Wilkins, 2001, pp 1597-1632
Cusack JC: Rationale for the treatment of solid tumors with the proteasome inhibitor bortezomib. Cancer Treat Rev 29:21-31, 2003
Yan XJ, Rosales N, Aghajanian C, et al: Cisplatin induces NF-kB activity through IkB kinase dependent pathway. Proc Am Assoc Cancer Res 40:195, 1999 (abstr 1299)
Aghajanian C: Clinical update: Novel targets in gynecologic malignancies. Semin Oncol 31:22-26, 2004
Lightcap ES, McCormack TA, Pien CS, et al: Proteasome inhibition measurements: Clinical application. Clin Chem 46:673-683, 2000
Aghajanian C, Soignet S, Dizon DS, et al: A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 8:2505-2511, 2002
Orlowski RZ, Stinchcombe TE, Mitchell BS, et al: Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol 20:4420-4427, 2002
Papandreou CN, Daliani DD, Nix D, et al: Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 22:2108-2121, 2004
Cusack JC Jr, Liu R, Houston M, et al: Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: Implication for systemic nuclear factor-vB inhibitor. Cancer Res 61:3535-3540, 2001
Huang TT, Wuerzberger-Davis SM, Seufzer BJ, et al: NF-kB activation by a camptothecin. J Biol Chem 275:9501-9509, 2000
Houston MA, Liu R, Abendroth K, et al: Proteasome inhibition enhances radiosensitivity of human pancreatic cancer cells in vitro. Proc Am Assoc Cancer Res 41:709, 2000 (abstr 4505)(C. Aghajanian, D.S. Dizon)
The Program in Women's Oncology, Women & Infants' Hospital, Brown Medical School, Providence, RI
Department of Neurology, Northwestern University, Feinberg School of Medicine, Chicago, IL
ABSTRACT
PURPOSE: To determine the maximum-tolerated dose, pharmacodynamics, and safety of the combination of bortezomib and carboplatin in recurrent ovarian cancer.
PATIENTS AND METHODS: Fifteen patients were treated with a fixed dose of carboplatin (area under the curve [AUC] 5) and increasing doses of bortezomib (0.75, 1, 1.3, and 1.5 mg/m2/dose). Patients must have received upfront chemotherapy and up to two prior chemotherapy regimens for recurrent disease. Neurologic evaluation was performed at baseline and after every two cycles by the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire and examination by an attending neurologist. All patients received carboplatin alone in cycle 1 to establish baseline pharmacodynamics for nuclear factor-kappa B (NF-kB). Starting with cycle 2, patients were treated with carboplatin on day 1 and bortezomib on days 1, 4, 8, and 11.
RESULTS: Diarrhea, rash, neuropathy, and constipation (with colonic wall thickening on computed tomography) were dose-limiting toxicities, occurring in the two patients treated at the 1.5 mg/m2/dose level. The Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire was helpful in guiding the need for dose reductions. Neurotoxicity was manageable through six cycles, with appropriate dose reductions. Carboplatin had no effect on bortezomib pharmacodynamics as measured by percent inhibition of the 20S proteasome. Bortezomib decreased carboplatin-induced NF-kB. The overall response rate to this combination was 47%, with two complete responses (CR) and five partial responses, including one CR in a patient with platinum-resistant disease.
CONCLUSION: The recommended phase II dose of bortezomib administered in combination with carboplatin (AUC 5) is 1.3 mg/m2/dose.
INTRODUCTION
The ubiquitin proteasome pathway is a highly conserved intracellular pathway for the degradation of proteins. Many of the short-lived regulatory proteins that govern cell division, growth, activation, signaling, and transcription are substrates that are temporally degraded by the proteasome. Therefore, the proteasome represents a novel target for chemotherapy. Bortezomib is a dipeptidyl boronic acid inhibitor with high specificity for the proteasome.
The ubiquitin-proteasome pathway plays a significant role in neoplastic growth and metastasis. The ordered and temporal degradation of numerous key proteins, such as cyclins, cyclin-dependent kinase inhibitors, and tumor suppressors, is required for cell cycle progression and mitosis.1 The proteasome is also required for activation of nuclear factor-kappa B (NF-kB) by degradation of its inhibitory protein, I-kB.2 NF-kB is a transcription factor that upregulates a number of proteins involved in cancer progression, including several proangiogenic factors and antiapoptotic factors. NF-kB may be present as a homodimer or heterodimer; the p65/p50 heterodimer is the most common complex. In quiescent cells, NF-kB is sequestered in the cytoplasm by its inhibitory partner, I-kB, which renders NF-kB inactive. However, cellular stress signals, such as those induced by chemotherapy, radiation, viruses, growth factors, or antigens, trigger phosphorylation of I-kB, which targets it for proteasome-mediated degradation. Release of NF-kB by I-kB degradation allows NF-kB to translocate to the nucleus and effect its transcriptional activation functions. Inhibition of the proteasome via bortezomib prevents degradation of I-kB and, thus, downregulates NF-kB–mediated transcriptional activation. Thus, NF-kB is required, in part, to maintain cell viability through the transcription of inhibitors of apoptosis in response to environmental stress or cytotoxic agents.3-6 Stabilization of the Ik-B protein and blockade of NF-kB activity has been demonstrated to make cells more susceptible to apoptosis.3-5 Furthermore, NF-kB has been implicated in controlling the cell-surface expression of adhesion molecules such as E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1.7,8 Proteasome inhibitors have also been shown to overcome Bcl-2–mediated protection from apoptosis.9-11
Ovarian cancer was diagnosed in 25,280 women in the United States in 2004 and resulted in an estimated 16,090 deaths.12 In women treated with platinum combinations as primary therapy, the response rates are 60% to 80%, with complete responses (CRs) being most common in women who have had adequate surgical therapy.13 Unfortunately, however, the majority of patients eventually die of platinum-resistant disease persistence or recurrence, with the abdominal cavity being the most common site or recurrence. Long-term survival remains approximately 15% to 30%. Therefore, new approaches are needed to provide effective therapy with minimal induction of chemotherapy resistance. Certain types of chemoresistance observed in solid tumors are associated with NF-kB activation.14 Platinum agents have been shown to rapidly induce chemoresistance by activating NF-kB.15,16 Therefore, blocking NF-kB activation with bortezomib represents a potential target for overcoming the apoptosis failure that develops during ovarian cancer treatment.
In animal models, bortezomib is rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. It is difficult to correlate plasma concentration of the drug with the degree of proteasome inhibition in blood samples. Therefore, a pharmacodynamic assay has been developed that provides a reliable measure of 20S proteasome activity in peripheral leukocytes, whole blood, and tissue biopsy and is sensitive, accurate, and reproducible.17 This assay allows for measurement of bortezomib pharmacodynamics and reflects its target activity. The principal objectives of this phase I and pharmacodynamic study were as follows: (1) to determine the maximum-tolerated dose of bortezomib that can be administered with a standard dose of carboplatin in the treatment of recurrent or progressive epithelial ovarian or primary peritoneal cancer; (2) to evaluate the toxicity of bortezomib when administered with carboplatin in this patient population; (3) to characterize the pharmacodynamics of 20S proteasome activity in patients treated with bortezomib and carboplatin; and (4) to determine the effect of bortezomib on carboplatin-induced NF-kB activity.
PATIENTS AND METHODS
Patient Selection
Patients with recurrent or progressive epithelial ovarian or primary peritoneal carcinoma were eligible for this trial. Patients must have received a platinum-based chemotherapeutic regimen for the management of primary disease, which may have included consolidation or extended therapy after surgical or nonsurgical assessment. Patients may have received up to two prior regimens for recurrent disease, including one nonplatinum-containing regimen. Patients were considered platinum resistant if they had progressed on or within 6 months of their last platinum-based regimen. Prior chemotherapy must have been completed 4 weeks before beginning investigational therapy, and all treatment-related toxicities must have resolved to baseline. Other eligibility criteria were as follows: age 18 years; Karnofsky performance score 70; adequate bone marrow function (absolute neutrophil count 1,000/μL, platelets 100,000/μL, and hemoglobin 8 gm/dL), hepatic function (AST and ALT levels 2.5 x the upper limit of institutional normal and total bilirubin 1.8 mg/dL), and renal function (serum creatinine 1.5 mg/dL); normal ejection fraction (> 50%) by multiple-gated acquisition scan or echocardiogram; no grade 2 peripheral neuropathy; no major surgery within 2 weeks of study entry; no significant atherosclerotic disease (defined as peripheral vascular disease requiring surgical management, history of myocardial infarction or congestive heart failure, or history of cerebrovascular event); no ECG evidence of acute ischemia or significant conduction abnormality (bifascicular block, defined as left anterior hemiblock in the presence of right bundle branch block, or second- or third-degree atrioventricular blocks); no orthostatic hypotension; no CNS disease (brain metastases or leptomeningeal involvement); and no serious medical or psychiatric illness that would limit full compliance with the study. Informed consent was obtained according to federal and institutional guidelines.
Drug Administration
Six cycles of therapy administered every 21 days were planned. For the first cycle, all patients received only carboplatin on day 1 to establish baseline NF-kB activation. Beginning with cycle 2, patients received bortezomib at doses of 0.75, 1, 1.3, or 1.5 mg/m2 on day 1, followed 1 hour later by carboplatin; bortezomib was administered alone on days 4, 8, and 11.
Bortezomib was administered as a rapid intravenous bolus injection into the side arm of a running intravenous infusion of normal saline at 100 mL/h. Carboplatin was administered as an intravenous infusion over 30 minutes. The dose of carboplatin was calculated using the Calvert formula. The Jellife formula was used to estimate the glomerular filtration rate.
Toxicities were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Dose-limiting toxicity was defined as grade 4 hematologic toxicity and any grade 3 nonhematologic toxicity occurring during the second cycle (first cycle of combination bortezomib/carboplatin) of therapy. Three to six patients were entered at each dose level. No intrapatient dose escalation was allowed. Dose escalation was not allowed until all patients treated at the prior dose level completed the second cycle of therapy. If one patient experienced dose-limiting toxicity, three additional patients were added to the dose level. If two of six patients experienced dose-limiting toxicity, the previous dose level was declared the maximum-tolerated dose. If only one of six patients experienced dose-limiting toxicity, dose escalation was permitted to continue.
Pretreatment and Follow-Up Studies
Pretreatment evaluation consisted of history and physical examination, assessment of Karnofsky performance status, completion of the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire, baseline neurologic evaluation by attending neurologist, ECG, measurement of left ventricular ejection fraction (by multiple-gated acquisition scan or echocardiogram), chest x-ray, urinalysis, CBC count, serum chemistries (electrolytes, glucose, blood urea nitrogen, creatinine, magnesium, calcium, phosphate, albumin, AST, ALT, alkaline phosphatase, and total bilirubin), CA-125 level, and documentation of measurable or assessable disease by computed tomography (CT) scan. During the study, interval history, physical examination, toxicity assessment, Functional Assessment of Cancer Therapy/Gynecologic Oncology Group neurotoxicity questionnaire (Table 1), CBC count, serum chemistries, and CA-125 level were obtained at the start of each cycle. Starting with cycle 2, a CBC count was obtained before each bortezomib dose. Follow-up neurologic evaluation by the attending neurologist was performed after every two cycles. CT scan to evaluate response was performed after every two cycles. Evaluation of response was performed according to Response Evaluation Criteria in Solid Tumors.
Pharmacodynamics
20S proteasome assay. In animal model studies, bortezomib was rapidly removed from the vascular compartment and distributed widely, quickly approaching the limits of detection. Therefore, a pharmacodynamic assay that results in a calculation of percentage of inhibition of the proteasome was used as an alternative to pharmacokinetic measurements. The method for determination of 20S proteasome activity was developed by Millennium Pharmaceuticals, Inc (Cambridge, MA).17 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 then centrifuged at 6,600 x g for 10 minutes at 4°C. The resultant whole-blood lysate samples were then used in the 20S proteasome assay as previously described.17 Briefly, samples (10 mL) were added to 2 mL of substrate buffer (20 mmol/L HEPES, 0.5 mmol/L EDTA, 0.05% sodium dodecyl sulfate, 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, and the rate of substrate cleavage per 20S proteasome activity was determined. The protein content of the samples was determined using a Coomassie protein assay (Pierce, Rockford, IL). Data are presented as means ± SEM, with statistical significance at P < .05. Two samples were obtained before study to establish a baseline for each patient. During the second cycle of therapy (first cycle of combined bortezomib and carboplatin treatment), samples were drawn on the following schedule: (1) day 1 at 0 and 1 hour (1 hour after bortezomib, and before carboplatin), at 2 hours (30 minutes after end of carboplatin infusion), and at 24 hours; (2) day 4 at 0 and 1 hour; (3) day 8 at 0 and 1 hour; and (4) day 11 at 0 and 1 hour.
NF-kB determinations. To assess the inhibition of NF-kB by bortezomib when administered in combination with carboplatin, an aliquot of blood was collected in cell preparation tubes at baseline (time 0) and 30 minutes after the completion of carboplatin administration (time 1 hour) on day 1 of cycle 1. On day 1 of cycle 2, when both bortezomib and carboplatin were administered, specimens were collected at time 0 (before bortezomib administration), 1 hour (before carboplatin administration), and 2 hours (30 minutes after completion of carboplatin administration). Samples were separated by Ficoll-Hypaque density centrifugation with WBC pellet suspended in cold Buffer A (10 mmol/L HEPES-KOH at pH 7.9 at 4°C, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, and 0.2 mmol/L PMSF). Cells were allowed to swell on ice and were then vortexed, pelleted, resuspended in cold Buffer C (20 mmol/L HEPES-KOH at pH 7.9, 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, and 0.2 mmol/L PMSF), and incubated on ice for 20 minutes for high-salt extraction. Protein concentrations were determined by the Bradford method using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Electromobility shift assay was then performed. Consensus oligonucleotides (Promega, Madison, WI) were end labeled with [-32P]adenosine triphosphate using T4 polynucleotide kinase. Binding reactions were performed in a 10-μL volume containing 10 μg of nuclear extract, 0.1 μg of poly (dI-dC), 4% glycerol, 1 mmol/L of MgCl2, 0.5 mmol/L of EDTA, 5 mmol/L of dithiothreitol, 50 mmol/L of NaCl, and 10 mmol/L of Tris-HCl at pH 7.5 in the presence of labeled probe (20,000 cpm). The samples were incubated at room temperature for 20 minutes, loaded on a 4% polyacrylamide gel, and electrophoresed at 350 V for 1 hour in 1 x Tris-glycine buffer.
RESULTS
Patient Characteristics
Fifteen patients were enrolled onto the study, and all patients were assessable for toxicity. One patient in the 1.3 mg/m2/dose level did not complete the full dose of carboplatin in cycle 2 because of a hypersensitivity reaction and was not assessable for response. An additional patient was accrued to this level to ensure accurate assessment of toxicities for that dose level. Patient characteristics are listed in Table 2. The median age was 54 years (range, 45 to 68 years), and the median Karnofsky performance status was 90% (range, 80% to 90%). Four of the 15 patients were platinum resistant (had progression of disease on or within 6 months of receiving their last platinum therapy). The median number of prior chemotherapy regimens was one (range, one to three regimens). Two patients had prior pelvic radiation therapy.
Drug Delivery
The dose-escalation schema and the number of patients and courses administered as a function of dose level are listed in Table 3. At the 1.3 mg/m2/dose level of bortezomib, one patient was unable to receive the last two doses of bortezomib in cycle 6 because of grade 3 diarrhea, and one patient was unable to receive the last two doses of bortezomib in cycle 5 because off asthenia, anorexia, orthostatic dehydration (grade 2), and constipation. All other doses were able to be administered.
Toxicity
Both hematologic and nonhematologic toxicity were minimal at the first two dose levels (0.75 and 1 mg/m2/dose). Toxic effects that are reported were scored as being possibly, probably, or definitely drug related. These toxicities are reported as the worst toxicity per category exhibited by a patient during their treatment course (Table 4). Toxicities that were scored as not being drug related have been reviewed and have not been included if they were clearly not associated with the experimental treatment.
Hematologic Toxicity
There was no dose-limiting hematologic toxicity seen in the trial.
Nonhematologic Toxicity
The nonhematologic dose-limiting toxicities observed were diarrhea, constipation (in association with colonic wall thickening on CT), rash, and sensory neuropathy. The first two patients treated at the highest dose level of 1.5 mg/m2/dose of bortezomib developed dose-limiting toxicity. The first patient developed grade 3 diarrhea, grade 3 rash, and grade 3 sensory peripheral neuropathy. The second patient developed grade 3 constipation in association with colonic wall thickening on CT.
Neurotoxicity was accessed by both examination and assessment by an attending neurologist at baseline and then after every two cycles and by using the Gynecologic Oncology Group neurotoxicity questionnaire. At the first dose level (bortezomib 0.75 mg/m2/dose and carboplatin area under the curve [AUC] 5), all three patients entered the study with no baseline symptoms of neuropathy (all answers 0 on the neurotoxicity questionnaire). All three patients had diminished vibration noted on baseline examination by the neurologist, and all three patients had stable examination and stable assessment on the neurotoxicity questionnaire throughout the study. At the second dose level (bortezomib 1 mg/m2/dose and carboplatin AUC 5), two of the three patients entered the study with a baseline grade 1 neuropathy. All three patients had diminished vibration noted on baseline examination by the neurologist, and all three patients had stable examination and stable assessment on the neurotoxicity questionnaire throughout the study. Of the seven patients entered onto the third dose level (bortezomib 1.3 mg/m2/dose and carboplatin AUC 5), two had a baseline grade 1 neuropathy. These two patients remained stable on treatment. Two patients who entered the study without neuropathy developed a grade 1 neuropathy on treatment. An additional patient who entered the study without neuropathy developed a grade 2 neuropathy on treatment. Her neuropathy improved to grade 1 with a 1-week delay, and she resumed treatment with a one-level dose reduction (to bortezomib 1 mg/m2/dose and carboplatin AUC 5). After two additional doses of bortezomib at the reduced level, the neuropathy increased back to grade 2 and persisted for 4 weeks, when it again decreased to grade 1. The patient was removed from study. The examination by the attending neurologist did not find any change on any of these patients. The changes in neuropathy were well described by the patients on the neurotoxicity questionnaire. The first patient at the 1.5 mg/m2/dose level of bortezomib entered without neuropathy. She developed a grade 3 neuropathy after day 4 of cycle 3. She was removed from study. The neuropathy was painful, requiring narcotic analgesia and gabapentin, and was evident on examination by the attending neurologist in bilateral lower extremities by new diminished pinprick to the shins, diminished vibratory sense to the ankles, and lack of temperature sense. Her neurotoxicity questionnaire showed complaints beginning 4 weeks before the symptoms were evident at the standard office assessment and examination. Her neuropathy improved but continued to require narcotic analgesia and gabapentin to control pain at the last follow-up 9 months from the completion of therapy. The second patient at the 1.5 mg/m2/dose level entered with a grade 1 neuropathy and remained stable. She was taken off study after cycle 2 because of GI toxicity. Therefore, the maximum-tolerated dose of this combination was determined to be carboplatin AUC 5 and bortezomib 1.3 mg/m2/dose.
Pharmacodynamics
Fourteen patients were assessable for pharmacodynamics. The results of the 20S proteasome inhibition determinations for bortezomib alone and for the combination of bortezomib and carboplatin are shown in Figure 1. The bortezomib alone points represent an average of the 1-hour draws on days 1, 4, 8, and 11. The bortezomib/carboplatin points represent the 2-hour draw on day 1. The 1-hour time point was chosen because this was shown to be the time of maximum percent 20S proteasome inhibition in phase I trials of bortezomib administered as a single agent.18-20 The dose-response relationship between bortezomib and 20S proteasome inhibition is know to be steep up until to the range used in this study, followed by a plateau.18-20 Therefore, it is expected that the percent 20S proteasome inhibition seen across dose levels in this study is similar. There was no difference seen in percent inhibition of the 20S proteasome with and without carboplatin (0.75 mg/m2/dose level, P = .2; 1 mg/m2/dose level, P = .5; 1.3 mg/m2/dose level, P = .2; and 1.5 mg/m2/dose level, P = .9). As predicted by single-agent pharmacodynamics of bortezomib, the level of proteasome inhibition declined at 24 hours (average, 21 hours; range, 11 to 44 hours) and returned to baseline by 72 hours.18-20
NF-kB Determinations
Fourteen patients were assessable for NF-kB. One patient did not receive the full dose of carboplatin with cycle 2 because of a hypersensitivity reaction. Electromobility shift assays were successfully completed on 11 of the 14 patients (the remaining three patients had inadequate or missing samples). Blood samples were taken at baseline (time 0) and 30 minutes after the completion of carboplatin administration (time 1 hour) on day 1 of cycle 1. On day 1 of cycle 2, when both bortezomib and carboplatin were administered, specimens were collected at time 0 (before bortezomib administration), 1 hour (before carboplatin administration), and 2 hours (30 minutes after completion of carboplatin administration). A representative electromobility shift assay is shown in Figure 2. Eight of the 11 patients showed increased NF-kB activity after carboplatin treatment on day 1 of cycle 1. This increase in NF-kB activity persisted at the baseline sample on day 1 of cycle 2. Of the eight patients with increased NF-kB activity, seven showed decline after treatment with bortezomib and failure to completely reactivate on re-exposure to carboplatin.
Response
Fourteen of 15 patients were assessable for response. Because response evaluations were performed after every two cycles, the first response evaluation performed reflected one cycle of single-agent carboplatin and one cycle of the combination of bortezomib and carboplatin. There were two CRs and five partial responses, for an overall response rate of 47%. One CR occurred in a platinum-resistant patient. All partial responses were in platinum-sensitive patients. An additional four patients experienced stable disease as their best response. Both patients with CR normalized their CA-125. Four of the five patients with a partial response had measurable CA-125 levels; of these patients, three normalized their marker, and the remaining patient had a greater than 50% decline in CA-125. Of the four patients with stable disease, two normalized CA-125, one had a more than 50% decline in CA-125, and one had stable CA-125. All three patients with progression of disease had an increase in CA-125 on treatment.
DISCUSSION
The ubiquitin-proteasome pathway plays an essential role in the degradation of most short- and long-lived intracellular proteins in eukaryotic cells. At the heart of this degradative pathway is the 26S proteasome, an adenosine triphosphate–dependent, multicatalytic protease. Proteolytic degradation of damaged, oxidized, or misfolded proteins is part of the housekeeping role for the 26S proteasome. In addition, the 26S proteasome also plays a vital role in degrading regulatory proteins that govern the cell cycle, transcription factor activation, apoptosis, and cell trafficking. Inhibitors of the proteasome represent a novel approach to treating human malignancies. Bortezomib is a reversible, highly selective inhibitor of the proteasome with promising in vitro and in vivo activity. This phase I and pharmacodynamic trial was designed to study the feasibility of administering bortezomib as a rapid intravenous infusion twice weekly for 2 weeks, followed by a 1-week recovery period, in combination with a fixed dose of carboplatin in women with recurrent ovarian cancer.
There was no significant toxicity seen in the first two dose levels tested (0.75 and 1 mg/m2/dose). There was no dose-limiting hematologic toxicity seen. The nonhematologic toxicity seen was largely predicted by the single-agent experience with bortezomib. The dose-limiting toxicities observed were diarrhea, constipation (in association with colonic wall thickening on CT), rash, and sensory peripheral neuropathy. The recommended phase II dose of bortezomib administered in combination with carboplatin (AUC 5) is 1.3 mg/m2/dose. This is the same dose of bortezomib that is currently being tested in a single-agent study by the Gynecologic Oncology Group in recurrent ovarian cancer.
The preclinical activity of bortezomib makes it a promising agent for combination therapy in overcoming chemotherapy resistance. The CR seen in this study in a platinum-resistant patient is also encouraging. Preclinical testing has shown synergy between bortezomib and the topoisomerase I inhibitor irinotecan.21 Activation of the transcription factor NF-kB by ionizing radiation,5 irinotecan,22 cisplatin,15 and other chemotherapeutic agents has been found to protect from cell killing. Proteasome inhibitors have been shown to inhibit drug-induced NF-kB and, thus, sensitize the treated cells to drug-induced apoptosis. Proteasome inhibition has also been shown to enhance the radiosensitivity of cell lines in vitro.23
The results of this phase I trial demonstrate that carboplatin in combination with bortezomib administered in a twice weekly for 2 weeks cycle every 21 days results in predictable toxicity and proteasome inhibition associated with preclinical activity. In lieu of a sensitive pharmacokinetic assay, the 20S proteasome pharmacodynamic assay proved to be both useful and predictive. It confirms inhibition of the biochemical target, the proteasome, and provides insight into how long the target is affected by bortezomib. In this study, carboplatin did not alter the pharmacodynamics of bortezomib. In the future, it is hoped that this assay will be used to determine proteasome inhibition in tumor biopsies, thereby providing data in biologically relevant tissues. The demonstration that bortezomib administration resulted in a decrease in carboplatin-induced upregulation of NF-kB and the occurrence of one CR in a patient with platinum-resistant disease suggest that further investigation of this agent as a means to overcoming drug resistance is warranted. A Gynecologic Oncology Group phase II trial of single-agent bortezomib in recurrent ovarian cancer is ongoing. If single-agent activity of bortezomib is seen, further exploration of platinum-based combinations via randomized phase II or III studies is feasible and warranted.
Authors' Disclosures of Potential Conflicts of Interest
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
NOTES
Supported by grant No. 5RO1 CA84009 from the National Cancer Institute (Bethesda, MD).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Goldberg AL, Stein R, Adams J: New insights into proteasome function: From Archaebacteria to drug development. Chem Biol 2:503-508, 1995
Palombella VJ, Rando OJ, Goldberg AL, et al: The ubiquitin-proteasome pathway is required for processing the NF-kB1 precursor protein and the activation of NF-kB. Cell 78:773-785, 1994
Beg AA, Baltimore D: An essential role for NF-kB in preventing TNF-alpha-induced cell death. Science 274:782-784, 1996
Van Antwerp DJ, Martin SJ, Kafri T, et al: Suppression of TNF-alpha-induced apoptosis by NF-kB. Science 274:787-789, 1996
Wang CY, Mayo MW, Baldwin AS: TNF- and cancer therapy-induced apoptosis: Potentiation by inhibition of NF-kB. Science 274:784-787, 1996
Chu ZL, Mckinsey TA, Liu L, et al: Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kB control. Proc Natl Acad Sci U S A 94:10057-10062, 1997
Zetter BR: Adhesion molecules in tumor metastasis. Semin Cancer Biol 4:219-229, 1993
Read MA, Neish AS, Luscinskas FW, et al: The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression. Immunity 2:493-506, 1995
Benyi L, Ping Dou Q: Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression. Proc Natl Acad Sci U S A 97:3850-3855, 2000
An B, Goldfarb RH, Siman R: Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts. Cell Death Differ 5:1062-1075, 1998
Herrmann JL, Briones F, Brisbay S, et al: Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53. Oncogene 17:2889-2899, 1998
Jemal A, Tiwari RC, Murray T, et al: Cancer statistics, 2004. CA Cancer J Clin 54:8-29, 2004
Ozols RF, Schwartz PE, Eifel PJ: Ovarian cancer, fallopian tube carcinoma, and peritoneal carcinoma, in DeVita VT, Hellman S, Rosenberg RA (eds). Cancer: Principles and Practice of Oncology (ed 6). Baltimore, MD, Lippincott Williams and Wilkins, 2001, pp 1597-1632
Cusack JC: Rationale for the treatment of solid tumors with the proteasome inhibitor bortezomib. Cancer Treat Rev 29:21-31, 2003
Yan XJ, Rosales N, Aghajanian C, et al: Cisplatin induces NF-kB activity through IkB kinase dependent pathway. Proc Am Assoc Cancer Res 40:195, 1999 (abstr 1299)
Aghajanian C: Clinical update: Novel targets in gynecologic malignancies. Semin Oncol 31:22-26, 2004
Lightcap ES, McCormack TA, Pien CS, et al: Proteasome inhibition measurements: Clinical application. Clin Chem 46:673-683, 2000
Aghajanian C, Soignet S, Dizon DS, et al: A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 8:2505-2511, 2002
Orlowski RZ, Stinchcombe TE, Mitchell BS, et al: Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol 20:4420-4427, 2002
Papandreou CN, Daliani DD, Nix D, et al: Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol 22:2108-2121, 2004
Cusack JC Jr, Liu R, Houston M, et al: Enhanced chemosensitivity to CPT-11 with proteasome inhibitor PS-341: Implication for systemic nuclear factor-vB inhibitor. Cancer Res 61:3535-3540, 2001
Huang TT, Wuerzberger-Davis SM, Seufzer BJ, et al: NF-kB activation by a camptothecin. J Biol Chem 275:9501-9509, 2000
Houston MA, Liu R, Abendroth K, et al: Proteasome inhibition enhances radiosensitivity of human pancreatic cancer cells in vitro. Proc Am Assoc Cancer Res 41:709, 2000 (abstr 4505)(C. Aghajanian, D.S. Dizon)