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Journal of Clinical Oncology, Vol 25, No 13 (May 1), 2007: pp. 1779-1784 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.9672 Phase I Trial of Imexon in Patients With Advanced Malignancy
From the College of Medicine, Arizona Cancer Center, University of Arizona; Arizona Clinical Research Center; AmpliMed Corp, Tucson; Premiere Oncology, Scottsdale, AZ; Scott & White Clinic, Temple, TX; and The Angeles Clinic and Research Institute, Santa Monica, CA Address reprint requests to Robert Dorr, PhD, RPh, Arizona Cancer Center, University of Arizona, 1515 N Campbell Ave, Tucson, AZ 85724; e-mail: bdorr{at}azcc.arizona.edu
Purpose Imexon, a pro-oxidant small molecule, has antitumor activity in preclinical models. The drug induces apoptosis through accumulation of reactive oxygen species. The purpose of this trial was to define the maximum-tolerated dose (MTD), toxicities, pharmacokinetics, and pharmacodynamics of imexon in patients with advanced cancers. Patients and Methods Forty-nine patients with metastatic cancer received intravenous imexon over 30 to 45 minutes for 5 consecutive days (one course) every other week (days 1 through 5 and 15 through 19) monthly. Doses were initially escalated using an accelerated trial design and then a modified Fibonacci method. Plasma imexon levels and six different thiols were measured by high-performance liquid chromatography assays.
Results There were 13 dose levels evaluated, from 20 mg/m2/d to 1,000 mg/m2/d. The MTD recommended for phase II studies was 875 mg/m2/d for 5 days every 2 weeks (n = 9 patients). The two dose-limiting toxicities at 1,000 mg/m2/d involved grade 3 abdominal pain and fatigue and grade 4 neutropenia, which occurred in one patient each. Other common toxicities included nausea and vomiting (58%) and constipation (63%); both were managed well with prophylactic medications. One partial response was obtained in a heavily pretreated patient with non-Hodgkin's lymphoma. Pharmacokinetic studies showed dose-independent clearance, with a 95-minute mean half-life. Plasma thiol studies showed a dose- and area under the curve–dependent decrease in cystine levels 8 hours after dosing at Conclusion The phase II recommended dose of imexon is 875 mg/m2/d for 5 days every other week. A decrease in plasma thiols did correlate with imexon exposure.
Imexon is a small molecule (molecular weight = 111.1) that binds to reduced sulfhydryls,1 causing an accumulation of reactive oxygen species2 and loss of the mitochondrial membrane potential,3 leading to apoptosis.4,5 The chemistry of binding to thiols such as glutathione (GSH) and cysteine has been demonstrated to occur by two types of nucleophilic attack.2 Imexon induces mitochondrial swelling in 8226 myeloma cells, causing the release of cytochrome C and induction of apoptosis via activation of caspases 3, 9,4,6 and 8.5 Similar apoptotic changes have been documented in imexon-treated pancreatic cell lines.7 Imexon was previously evaluated as a putative immunomodulator in a single-institution phase I trial in cancer patients that did not establish a maximum-tolerated dose (MTD) or define the drug's pharmacokinetic disposition.8,9 Preclinical studies showed that imexon was broadly active against human tumor cells in colony-forming assays wherein myeloma cells were most sensitive.10 The drug is also active in the murine LPBM-5 AIDS model and prevents the development of lymphoma in a human murine lymphoma model.11 Studies in human lymphoma cell lines showed that imexon causes cell cycle arrest in S phase and blocks protein synthesis.12 On the basis of these unique mechanistic findings, imexon was synthesized and formulated for clinical use by National Cancer Institute contractors via a Rapid Access to Intervention Development grant (to R.D.). The phase I trial included several added translational end points, including plasma thiol levels (reported herein) and gene expression studies in peripheral-blood mononuclear cells (reported elsewhere). The trial design was influenced by mouse leukemia studies showing that imexon's antitumor activity in vivo favored repeated daily dosing.13 A daily for 9 days schedule, which was active in mice,13 was felt to be impractical for the clinic, and an alternate schedule was used comprising two 5-day courses in a 1-month cycle, with each 5-day course separated by 9 days off therapy.
Eligibility Criteria Adult patients (  18 years) with histologically confirmed metastatic cancer or advanced malignancy, measurable or assessable disease, Karnofsky scale performance status 70%, and projected life expectancy of 3 months were eligible. All patients must have experienced relapse or progression on one or more regimens of systemic drug therapy. They must also have had laboratory values within the following criteria: hemoglobin 9 g/dL, WBC 3,500/µL, absolute neutrophil count 1,500/µL, platelets 100,000/µL, creatinine less than 2 mg/dL, total bilirubin  2.0 mg/dL, and hepatic enzymes 3x the upper limit of normal. Patients were also required to have a glucose-6-phosphate dehydrogenase activity greater than or equal to the lower limit of normal. Patients with CNS metastases were ineligible.
Study Treatment
Patients received daily IV infusions of imexon in 250 to 500 mL of 0.9% sodium chloride, depending on the imexon dose. For imexon doses less than 750 mg/m2, infusions were completed in 30 minutes. For imexon doses Based on the toxicity seen in the prior trial,9 all patients were prophylactically treated with antiemetics 30 minutes before imexon infusion using 1 to 2 mg of granisetron or 8 mg of ondansetron IV or orally. If necessary, 1 mg of IV or oral lorazepam was added. Prophylactic medications to control constipation were routinely administered on days 1 through 8 of each cycle once constipation occurred. This regimen included dioctyl sodium succinate 200 mg daily and a senna laxative twice daily at step 1, and then, if needed, polyethylene glycol (Miralax; Braintree Laboratories, Braintree, MA) was added (1 tablespoonful in 8 oz of water); if this was unsuccessful, 60 mL of Fleet Phospho Soda (Fleet, Lynchburg, VA) up to every 4 hours was added. Other standard medications were allowed, but acetaminophen was forbidden to avoid potential hepatotoxicity based on potential binding of imexon to reduced sulfhydryls, such as GSH in the liver.
Toxicity and Treatment Assessment
Pharmacokinetic and Thiol Pharmacodynamic Assays Blood samples were obtained at 11 time points up to 8 hours after infusion ended to characterize the pharmacokinetics of imexon on days 1 and 5 of the first 5-day infusion. The following pharmacokinetic parameters were estimated using the WinNonlin program version 5.0 (Pharsight Corp, Mountain View, CA), with a noncompartmental approach: the maximum plasma concentration (Cmax), the dose-normalized Cmax (Cmax/dose), the time to reach the maximum plasma concentration, the area under the plasma concentration-time curve (AUC), the dose-normalized AUC (AUC/dose), systemic clearance (CLs), the volume of distribution at steady-state (Vss), and the half-life (t1/2). A 200-µL aliquot of the pharmacokinetic plasma samples was used to measure thiol levels up to 8 hours after imexon administration. Samples were frozen at –80°C until analyzed by the method of Jones et al16 with minor modification. Thiol concentrations were calculated from the ratio of the thiol peak to the internal standard gamma-glutamyl-glutamate, expressed in µM.
The effects of dose on the pharmacokinetics of imexon were assessed by comparing Cmax/dose, AUC/dose, CLs, Vss, and t1/2 among different dose levels using one-way analysis of variance. Effects of treatment day on the pharmacokinetics of imexon were assessed by comparing the pharmacokinetic parameters between days 1 and 5 of course 1 using a paired t test. A P
Patient Characteristics and Treatment Forty-nine patients with advanced malignancies were entered onto this trial. The median age was 64 years (range, 35 to 81 years), and 55% of patients were female. The diagnoses included patients with advanced melanoma (n = 10), pancreatic cancer (n = 9), ovarian cancer (n = 6), colon cancer (n = 5), non–small-cell lung cancer (n = 3), and other solid tumor types (n = 16). There were 13 dose levels evaluated in the trial, with daily doses ranging from 20 mg/m2 (n = 1) to 1,000 mg/m2 (n = 2). Three patients were treated at most dose levels until the higher dose levels of 750 mg/m2 (n = 6) and 875 mg/m2 (n = 9) were reached. The median number of cycles administered in the entire population was two (range, 0.5 to seven cycles). The median number of prior therapies was three, and 51% of patients had four or more prior therapies. Nine patients discontinued the study early (three patients because of patient preference, four patients because of adverse events, and one patient each by investigator decision or compliance failure).
MTD and Toxicity The major adverse events of any cause are listed in Table 1. The mild hyperglycemia seen in 34 patients was presumably a result of the collection of nonfasting blood, whereas the nausea, fatigue, anorexia, and constipation pain were clearly a result of the drug. Disease-related effects noted in the trial include anemia, hypoalbuminemia, hyponatremia, and elevations in bilirubin and alkaline phosphatase. The most common non-DLT was constipation, which occurred in 31 patients (63% of all patients treated). This adverse effect was not associated with any peripheral neuropathy or paralytic ileus. It was universally seen after several days at all dose levels greater than 430 mg/m2/d and was well managed with the prophylactic bowel regimen.
There were 25 grade 3 toxicities and one grade 4 toxicity that were probably or definitely drug related. These included 11 events related to blood and bone marrow in two patients (lymphopenia, n = 1; leukocytes, n = 4; hemoglobin, n = 1; neutrophils, n = 4; and platelets, n = 1), 13 events related to the GI tract in eight patients (constipation, n = 8; nausea, n = 1; and abdominal pain and cramping, n = 4), fatigue in one patient, and bruising in one patient. Grade 3 or 4 myelosuppression occurred in only two patients on the first course, including one of nine patients treated at 875 mg/m2 (grade 3 thrombocytopenia that resolved off drug) and one of two patients treated at 1,000 mg/m2 (grade 3 anemia and grade 4 thrombocytopenia and neutropenia). Seven patients died within 30 days of going off study as a result of progressive disease, and in all of these patients, there was a determination of no relationship to imexon.
Pharmacokinetics and Pharmacodynamics
Plasma thiol pharmacodynamics. Plasma thiol levels were measured on each day before imexon dosing and then for 8 hours afterwards on day 1. At baseline, the cysteine-cysteine dimer, cystine, was present at the highest concentrations, ranging from 45.7 to 81.1 µmol/L. This was followed by the cysteine-glycine dimer, ranging from 42.1 to 56.8 µmol/L, and then cysteine, ranging from 22.9 to 48.7 µmol/L. Baseline GSH levels were the lowest of all measured thiols, ranging from 3.9 to 5.2 µmol/L. A comparison of these baseline levels for the 5 days of imexon dosing at the MTD showed only one statistically significant change; the mean (± standard deviation) plasma cystine concentration on day 5 was 78.6% (± 7.47%) of the day 1 baseline concentration (P = .019 by paired-sample t test). At imexon doses less than 750 mg/m2, there were no changes in any of the plasma thiols measured. At higher doses, only one thiol, cystine, decreased as a function of time after the imexon infusion ended. The decline in cystine levels on day 1 was cumulative, peaking at the last 8-hour sampling point. At the dose level of 750 mg/m2/d, there was a mean 10% decrease in plasma cystine levels at 8 hours (P = .003), and this increased to 15% at the dose level of 875 mg/m2/d (P = .04) and to 33% at the dose level of 1,000 mg/m2/d (P = .04). The correlation between the decrease in plasma cystine and the AUC of imexon was also significant, as shown in Figure 2 (r2 = 0.82, P = .0001). The relationship between the decrease in cystine levels and overall survival was not significant (r2 = 0.25, P = .09). Similarly, the comparison of cystine decrease in patients with stable disease or partial response with patients with progressive disease was also not statistically significant (P = .08 by t test).
Clinical Activity Stabilization of disease was seen in 10 patients with a variety of tumor types (median, 3.7 months; range, 3.1 to 6.5 months). In addition, one patient with refractory follicular non-Hodgkin's lymphoma experienced a partial response lasting 6.5 months at the dose level of 875 mg/m2 based on complete disappearance of disease on computed tomography scan, with minimal residual disease (fluorodeoxyglucose uptake) on positron emission tomography scan (Fig 3). Bone marrow involvement was not assessed at entry, obviating any conclusions of response in the bone marrow. This patient had previously received cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab, high-dose chemotherapy, and autologous bone marrow transplantation, further rituximab single-agent chemotherapy, fludarabine, ibritumomab tiuxetan–based radioimmunotherapy, and a phase I topoisomerase I/II inhibitor, elsamitrucin. This patient came off study after 6.5 months because of the development of a relapse in the bone marrow associated with grade 4 thrombocytopenia followed in 1 month by pancytopenia. The patient requires transfusions of platelets weekly and RBCs monthly with growth factor support. Bone marrow biopsies before imexon in 1996 and 2005 showed marrow involvement, but an immediate prestudy marrow biopsy was not performed. Biopsies during the pancytopenia at 5.5 and 6.5 months showed marrow relapse; the later biopsy at 6.5 months showed 30% cellularity, a mass to charge ratio of 0.6:1, 0.2 megakaryocytes per high-power field, and decreased iron stores. There was a 1% B lymphoma cellularity by flow cytometry (CD10+ and CD19+, CD20–). The aspirate differential showed reduced myelocytic precursors, no band neutrophils, 43% erythroid precursors, 33 lymphoblasts, and no monocytic precursors. Thus, imexon contributed to the pancytopenia in this heavily pretreated patient.
Imexon is a novel pro-oxidant small molecule.1-5 Other anticancer agents with pro-oxidant properties include arsenic trioxide,17 motexafin gadolinium,18 and to some degree, bortezomib.19 The selectivity of such pro-oxidant agents is based on increased susceptibility of cancer cells to oxidative stress,20 possibly as a result of reduced expression of antioxidant defense enzymes such as superoxide dismutase-2.21 The myelosuppression was infrequent with imexon, suggesting that combinations of imexon with other myelosuppressive drugs should be explored. Agents shown to be synergistic with imexon in vitro include DNA-binding agents, such as melphalan and cisplatin, and pyrimidine-based antimetabolites, such as fluorouracil and gemcitabine.22 The following three combinations are currently in clinical trials: imexon with dacarbazine in a phase II trial in patients with metastatic malignant melanoma, a phase I trial of imexon with gemcitabine in patients with advanced pancreatic cancer, and a phase I trial of imexon with docetaxel in patients with refractory breast, lung, and prostate cancer. The pharmacokinetic studies of imexon showed that the drug is rapidly eliminated in a dose-independent, linear fashion. This was expected based on the prior mouse pharmacokinetic studies23 and on the physicochemical characteristics of imexon, notably its small size (molecular weight = 111.1) and water solubility (log P = –1.35). The pharmacodynamic effect of imexon on plasma cystine suggests that imexon may be binding to reduced sulfhydryls in cells, leading to a need to replenish intracellular cysteine stores. Cystine is the cysteine-cysteine dimer that comprises the primary cysteine storage form used to replenish intracellular GSH stores via the cystine/glutamate antiporter, which transports extracellular cystine inside the cell in exchange for glutamate.24 However, the degree of plasma cystine decrease (10% to 40%) is probably too small to comprise a significant biologic effect of imexon. This is based on the 50% or greater decrease in WBC cystine levels in cystinosis patients, who respond to cysteamine therapy.25 The 8-hour time at which cystine levels were maximally depressed represents almost five plasma t1/2 of imexon, showing that the decrease in plasma cystine is a cumulative effect of imexon exposure. In conclusion, imexon is well tolerated when administered at a dose of 875 mg/m2/d for 5 days every other week for 2 months. The partial response in one heavily pretreated lymphoma patient and the 10 patients with stable disease suggest that imexon should be evaluated in patients with refractory lymphomas and solid tumors.
Although all authors completed the disclosure declaration, the following authors or their 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. Employment: Kathryn Grenier, AmpliMed Corporation; Evan Hersh, AmpliMed Corporation; Robert Dorr, AmpliMed Corporation Leadership: Evan Hersh, Medical Director and Vice President/Medical Affairs; Robert Dorr, Vice President for Research, AmpliMed Corp Consultant: Michael Gordon, AmpliMed Corporation; H.-H. Sherry Chow, AmpliMed Corporation Stock: Kathryn Grenier, AmpliMed Corporation; Robert Dorr, AmpliMed Corporation Honoraria: H.-H. Sherry Chow, AmpliMed Corporation Research Funds: Tomislav Dragovich, AmpliMed Corporation AMP-001 Trial; Michael Gordon, AmpliMed Corporation; David Mendelson, AmpliMed Corporation; Lucas Wong, AmpliMed Corporation; Manuel Modiano, Arizona Clinical Research Center; Robert Dorr, AmpliMed Corporation Testimony: N/A Other: N/A
Conception and design: Tomislav Dragovich, Evan Hersh, Robert Dorr Financial support: Evan Hersh, Robert Dorr Provision of study materials or patients: Tomislav Dragovich, Michael Gordon, David Mendelson, Lucas Wong, Manuel Modiano, Steven O'Day Collection and assembly of data: Kathryn Grenier, Robert Dorr Data analysis and interpretation: Tomislav Dragovich, Michael Gordon, David Mendelson, Lucas Wong, H.-H. Sherry Chow, Betty Samulitis, Kathryn Grenier, Evan Hersh, Robert Dorr Manuscript writing: Manuel Modiano, Evan Hersh, Robert Dorr Final approval of manuscript: Tomislav Dragovich, Michael Gordon, David Mendelson, Lucas Wong, Manuel Modiano, Steven O'Day, Kathryn Grenier, Evan Hersh, Robert Dorr
Supported by Grant No. CA-17094 (R.D.) from the National Institutes of Health, National Cancer Center, Bethesda, MD; a Rapid Access to Intervention Development grant from the National Cancer Center; and a grant from the AmpliMed Corporation, Tucson, AZ. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Iyengar BS, Dorr RT, Remers WA: Chemical basis for the biological activity of imexon and related cyanoaziridines. J Med Chem 47:218-223, 2004[CrossRef][Medline] 2. Dvorakova K, Payne CM, Tome ME, et al: Induction of oxidative stress and apoptosis in myeloma cells by the aziridine-containing agent imexon. Biochem Pharmacol 60:749-758, 2000[CrossRef][Medline] 3. Dvorakova K, Waltmire CN, Payne CM, et al: Induction of mitochondrial changes in myeloma cells by imexon. Blood 97:3544-3551, 2001 4. Dvorakova K, Payne CM, Landowski TH, et al: Imexon activates an intrinsic apoptosis pathway in RPM18226 myeloma cells. Anticancer Drugs 13:1031-1042, 2002[CrossRef][Medline] 5. Evens AM, Prachand S, Shi B, et al: Imexon-induced apoptosis in multiple myeloma tumor cells is caspase-8 dependent. Clin Cancer Res 10:1481-1491, 2004 6. Samulitis BK, Landowski TH, Dorr RT: Correlates of imexon sensitivity in human multiple myeloma cell lines. Leuk Lymphoma 47:97-109, 2006[CrossRef][Medline] 7. Dorr RT, Raymond MA, Landowski TH, et al: Induction of apoptosis and cell cycle arrest by imexon in human pancreatic cancer cell lines. Int J Gastrointest Cancer 36:15-28, 2005[CrossRef][Medline] 8. Bicker U: BM 06 002: A new immunostimulating compound, in Chirigos MA (ed): Immune Modulation and Control of Neoplasia by Adjuvant Therapy. New York, NY, Raven Press, 1978, pp 389-401 9. Micksche M, Kokoschka EM, Sagaster P, et al: Phase I study for a new immunostimulating drug, BM 06 002, in man, in Chirigos MA (ed): Immune Modulation and Control of Neoplasia by Adjuvant Therapy. New York, NY, Raven Press, 1978, pp 403-443 10. Salmon SE, Hersh EM: Sensitivity of multiple myeloma to imexon in the human tumor cloning assay. J Natl Cancer Inst 86:228-230, 1994 11. Funk CY, Eisman J, Hersh EM: Treatment of the murine, retrovirus-induced lymphoproliferative immunodeficiency disease (LP-BM5) in C57BL/10 mice with the immunomodulator imexon. AIDS Res Hum Retroviruses 8:633-638, 1992[Medline] 12. Hersh EM, Gschwind CR, Taylor CW, et al: Antiproliferative and antitumor activity of the 2-cyanoaziridine compound imexon on tumor cell lines and fresh tumor cells in vitro. J Natl Cancer Inst 84:1238-1244, 1992 13. Dorr RT, Little JD, Klein MK, et al: Preclinical pharmacokinetics and antitumor activity of imexon. Invest New Drugs 13:113-116, 1995[CrossRef][Medline] 14. Simon R, Freidlin B, Rubinstein L, et al: Accelerated titration designs for phase I clinical trials in oncology. J Natl Cancer Inst 89:1138-1147, 1997 15. Therasse P, Arbuch SG, Eisenhauer EA, et al: New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst 92:205-216, 2000 16. Jones DP, Carlson JL, Samiec PS, et al: Glutathione measurement in human plasma evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC. Clin Chim Acta 275:175-184, 1998[CrossRef][Medline] 17. Woo SH, Park IC, Park MJ, et al: Arsenic trioxide sensitizes CD95/Fas-induced apoptosis through ROS-mediated upregulation of CD95/Fas by NF-kappaB activation. Int J Cancer 112:596-606, 2004[CrossRef][Medline] 18. Evens AM, Lecane P, Magda D, et al: Motexafin gadolinium generates reactive oxygen species and induces apoptosis in sensitive and highly resistant multiple myeloma cells. Blood 105:1265-1273, 2005 19. Fribley A, Zeng Q, Wang C-Y: Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells. Mol Cell Biol 24:9695-9704, 2004 20. Pelicano H, Carney D, Haung P: ROS stress in cancer cells and therapeutic implications. Drug Resist Updat 7:97-110, 2004[CrossRef][Medline] 21. Hodge DR, Peng B, Pompeia C, et al: Epigenetic silencing of manganese superoxide dismutase (SOD-2) in KAS 6/1 human multiple myeloma cells increases cell proliferation. Cancer Biol Ther 4:585-592, 2005[Medline] 22. Scott J, Dorr RT, Samulitis B, et al: Imexon-based combination chemotherapy in A375 human melanoma and RPMI 8226 human myeloma cell lines. Cancer Chemother Pharmacol PMID: 17333195 [epub ahead of print on February 28, 2007] 23. Pourpak A, Meyers RO, Samulitis BK, et al: Preclinical antitumor activity, pharmacokinetics and pharmacodynamics of imexon in mice. Anticancer Drugs 17:1179-1184, 2006[CrossRef][Medline] 24. Rimaniol AC, Mialocq P, Clayette P, et al: Role of glutamate transporters in the regulation of glutathione levels in human macrophages. Am J Physiol Cell Physiol 281:C1964-C1970, 2001 25. Thoene JG: Cystinosis. J Inherit Metab Dis 18:380-386, 1995[CrossRef][Medline] Submitted August 31, 2006; accepted January 30, 2007.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
