Feasibility and Correlates of Arsenic Trioxide Combined with Ascorbic

3658 Vol. 8, 3658–3668, December 2002 Clinical Cancer Research

Advances in Brief Feasibility and Correlates of Arsenic Trioxide Combined with Ascorbic Acid-mediated Depletion of Intracellular Glutathione for the Treatment of Relapsed/Refractory Multiple Myeloma1

Nizar J. Bahlis,2 Jennifer McCafferty-Grad, development of arsenic resistance. Six patients with stage Ileana Jordan-McMurry, Jim Neil, Isildinha Reis, IIIA relapsed/refractory myeloma were studied. We found that 0.25 mg/kg/day As O ؉ 1000 mg/day AA could be Mohamed Kharfan-Dabaja,3 James Eckman, 2 3 given for 25 days (over a 35-day period) without dose- Mark Goodman, Hugo F. Fernandez, limiting toxicity. One episode of grade 3 hematological tox- 4 Lawrence H. Boise, and Kelvin P. Lee icity (leukopenia) and no grade 3 nonhematological toxicities Sylvester Comprehensive Cancer Center [N. J. B., J. M-G., I. J-M., (in particular, cardiac) were observed. The coadministration I. R., M. K-D., M. G., H. F. F., L. H. B., K. P. L.], Division of of AA did not alter the pharmacokinetics of As203, and Hematology and Oncology, Department of Medicine [N. J. B., elevated AA levels were associated with decreased intracel- M. K-D., M. G., H. F. F., K. P. L.], and Department of Microbiology and Immunology [J. M-G., L. H. B., K. P. L.], University of Miami, lular GSH. Serial in vitro studies demonstrated continued ؉ Florida, and Division of Hematology and Oncology, Department of sensitivity of patient myeloma cells to As203 AA. Two Medicine, Grady Memorial Hospital, Emory University, Atlanta, patients (both with thalidomide-refractory disease) had par- Georgia [J. N., J. E.] tial responses; four patients had stable disease. In conclu- ؉ sion, we have found that As203 AA has acceptable toxicity Abstract and that there is promising evidence of activity in refractory/relapsed myeloma. Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new agents that bypass these resistance mecha- Introduction 5 nisms. We have reported that ascorbic acid (AA) enhances MM remains an incurable disease with an overall 5-year survival of less than 30% (1). Even with intensive up-front the activity of arsenic trioxide (As203) against drug-resistant MM in vitro by depleting intracellular glutathione (GSH). treatment strategies with stem cell rescue, the majority of pa- These data led us to open a National Cancer Institute/ tients with MM eventually relapse with chemotherapy-resistant Cancer Therapy Evaluation Program-sponsored Phase I/II disease (2, 3). A major mechanism of multidrug resistance is ؉ overexpression of the mdr1 and mrp genes (4, 5). These drug trial of As203 AA for relapsed/refractory MM. We now present the completed Phase I component of this trial. The efflux pumps protect the MM cell by preventing the intracellular primary objective of the trial’s Phase I component was to accumulation of a wide range of chemotherapeutic agents. Pro- assess whether the addition of AA affected the well- survival cytokines (interleukin 6, tumor necrosis factor ␣, vas- cular endothelial growth factor), stromal interactions, enhanced described toxicity profile of As203 alone. Correlative studies drug metabolism via the GSH redox pathway, alterations in drug were undertaken of As203 and AA pharmacokinetics, the ability of AA to deplete intracellular GSH in vivo, and the targets (topoisomerase II enzyme mutations, tubulin gene mu- tation, glucocorticoid receptor down-regulation), and up-regu-

lation of antiapoptotic genes (Bcl-2, Bcl-xL, Mcl-1) have also been reported as chemoresistant mechanisms in MM (6–10). Median survival for chemoresistant relapsed/refractory Received 4/1/02; revised 7/17/02; accepted 8/1/02. MM is predictably short, in the order of months (6). Chemo- The costs of publication of this article were defrayed in part by the therapy regimens for refractory and relapsed MM (e.g., VAD in payment of page charges. This article must therefore be hereby marked ␣ advertisement in accordance with 18 U.S.C. Section 1734 solely to melphalan-resistant disease, high-dose steroids, IFN- , cyclo- indicate this fact. phosphamide ϩ topotecan, and so forth) typically have 10–30% 1 Supported in part by Senior Research Grant from the Multiple My- response rates of short duration (11). The failure of “traditional” eloma Research Foundation (to L. H. B.), an American Society of Clin- chemotherapy in relapsed and refractory MM is the rationale for ical Oncology (ASCO) Young Investigator Award (to N. J. B.), an American Institute of Cancer Research fellowship (to J. M-G.), and the testing novel agents that may act via unique tumoricidal mech- Sylvester Comprehensive Cancer Center Developmental Award (to K. P. L. and L. H. B.) and Postdoctoral Fellowship Award (to N. J. B.). 2 Present address: Division of Hematology and Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH 44106. 3 Present address: Section of Hematology and Oncology, Department of 5 The abbreviations used are: MM, multiple myeloma; NCI, National Medicine, University of Oklahoma Health Sciences Center, Oklahoma Cancer Institute; CR, complete remission; APL, acute promyelocytic City, OK 73104. leukemia; ROS, reactive oxygen species; GSH, glutathione; AA, ascor- 4 To whom requests for reprints should be addressed, at Papanicolaou bic acid; BSO, buthionine sulfoximine; DLT, dose-limiting toxicity; Building, Room 211, 1550 10th NW Avenue, Miami, FL 33156. E-mail: CTEP, Cancer Therapy Evaluation Program; PR, partial response; [email protected]. PBMC, peripheral blood mononuclear cell; EKG, electrocardiogram. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2002 American Association for Cancer Research. Clinical Cancer Research 3659

anisms. The antiangiogenic agent thalidomide has demonstrated and AA in relapsed and refractory MM. We now report the significant promise in refractory myeloma with an overall re- results of the Phase I component of this trial. sponse rate of 32% (12), and is currently in wide clinical use for this cohort of patients. Material and Methods The antitumor activity of arsenic trioxide (As203) was Clinical Trial Design. This study (NCI 43/SCCC 20010) rediscovered in the modern era when Chinese groups reported was designed as a combined Phase I/II trial. The goal of the Ͼ that As203 induced CRs in 50% of APL patients that failed Phase I component was to establish a dose of As203 (0.15 versus all-trans retinoic acid therapy (13, 14, 15), followed by United 0.25 mg/kg/day) that could be safely combined with AA (500 or States clinical studies (using 0.06–0.2 mg/kg/day and cumula- 1000 mg/day). This dose would then be used in the Phase II

tive doses of 160–515 mg) in APL demonstrating an 80–90% component of the trial. Because the highest dose of As2O3 (0.25 CR rate (16, 17). On the basis of these studies, As203 was mg/kg) tested has already been shown to be safe as a single approved by the Food and Drug Administration for APL, with agent (in APL studies), the primary objective of the Phase I the current recommended induction regimen of 0.15 mg/kg/day component was to determine whether the addition of AA sig-

i.v. until CR or a maximum of 60 doses is reached. In APL, the nificantly changed the toxicity profile of As203 alone. The

reported As203-related toxicities were relatively mild, the most 1000-mg AA dose was predicted to give a plasma concentration ␮ prevalent being skin rash, fatigue, nausea/vomiting/diarrhea, of 100 mol/liter, a level we found synergistic with As203 in musculoskeletal pain, leukocytosis, liver function abnormalities, vitro. Escalation of AA beyond 1000 mg is not likely to further

and “retinoic acid” syndrome. Although As203 does not appear increase efficacy because plasma ascorbate levels plateau at a to cause cardiomyopathy, 69% (18) to 100% (19) of patients had dose between 500 to 1000 mg, and intracellular levels saturate QTc prolongation during arsenic treatment, which was associ- at an even lower dose (41). ated in some patients with nonsustained ventricular arrhythmias. The Phase I study was designed only to examine specific Other groups have reported complete atrioventricular block and dose levels and was not intended to establish the maximum torsade de pointes, and sudden death (20–22). tolerated dose (MTD). According to a “3 ϩ 3” Phase I study design, a minimum of 6 patients (maximum of 18) were required In MM, As203 has been shown to induce apoptosis in several cell lines (23) that was not blocked by the addition of the to determine an As2O3 and AA dose level for which the inci- prosurvival cytokine interleukin-6 (24). We have also found that dence of DLT was less than 33%. DLT was defined as grade 3 or higher nonhematological toxicity or a grade 4 hematological As203 induces apoptosis in the drug-resistant myeloma cell lines toxicity occurring within the first course of treatment. Three despite bcl-xL or mdr1 gene overexpression (10). In a small Phase II study of As 0 alone in heavily pretreated refractory patients were to be enrolled at the first dose level of 0.15 2 3 ϩ myeloma, Munshi et al. have reported a 23% response rate using mg/kg/day As2O3 1000 mg/day AA. If no DLT were seen, three patients were to be enrolled at the next dose level of 0.25 As 0 alone (0.15 mg/kg/day for 60 days) in nine patients (25). 2 3 mg/kg/day As O ϩ 1000 mg/day AA. The intermediate dose of The induction of apoptosis by As 0 is likely mediated via 2 3 2 3 As O , 0.20 mg/kg/day, and/or de-escalation of AA to 500 the production of ROS that damage mitochondria (26, 27), 2 3 mg/day would occur only if more than 33% of patients treated because alterations in cellular antioxidant status modulate at the highest doses of each agent experienced DLT.6 arsenic-induced cell death (28, 29). In addition to reducing ROS, This trial was approved by the University of Miami Insti- the critical intracellular antioxidant GSH is also directly conju- tutional Review Board and the CTEP, NCI. Informed consent gated to arsenic and subsequently transported out of the cell by was obtained from each subject. multidrug resistance efflux pumps (30–32). It follows that Treatment. Patients were treated with As 0 (provided agents that deplete GSH will sensitize cells to As 0 -induced 2 3 2 3 by NCI/Division of Cancer Treatment and Diagnosis) as an i.v. apoptosis. One such agent is AA. Although heralded as an infusion over 1–2 h followed by a 15-min i.v. infusion of AA antioxidant, AA also has prooxidant activities. In the plasma, it (American Regent, Shirley, NY). Although AA can be given is rapidly oxidized to monohydroascorbyl and, further, to dehy- p.o., i.v. administration avoids the decreased gastrointestinal droascorbic acid (DHA), which is then transported into the cell. absorption that can be induced by As203 (diarrhea, mucositis, AA is then regenerated from DHA via glutaredoxin, which and so forth), yields higher peak-plasma levels, and allows for converts GSH to GSH disulfide (33). This depletes intracellular more accurate sequencing of AA-mediated GSH depletion with GSH and increases cellular susceptibility to oxidative stress (34, peak As203 levels. Treatment was administered on an outpatient 35). We have reported that AA-mediated GSH depletion aug- basis except for the 1st week of the first cycle (to allow for ments As203-induced apoptosis in myeloma cells in vitro (10), initial toxicity assessment and pharmacokinetic studies). Both and other groups have reported similar findings in in vitro and drugs were given Monday through Friday for 5 consecutive animal models of lymphoma and leukemia (29, 36, 37). Of weeks followed by 2 weeks of rest (25 days of treatment over a particular relevance is the selective enhancement of As203- 35-day time period constituted one cycle). Patients were allowed induced apoptosis in tumor cells (versus normal cells) by AA in to receive a maximum of six cycles. ϩ vitro and, similarly, the lack of additional toxicity of As203 Patients were followed twice weekly for complete blood AA compared with As203 alone in animal studies. The minimal toxicity of AA compares favorably with other GSH-depleting agents like BSO (38, 39, 40). On the basis of these promising preclinical data, we initi- 6 The full description of the escalation/de-escalation algorithm can be ated a NCI/CTEP-sponsored Phase I/II clinical trial of As203 found in the complete clinical protocol at www.sylvester.org.

Table 1 Patient characteristics No. Cumulative ␤ M protein 2M As2O3 AA of cycles/ As2O3 dose Patient Age Sex Stage PSa (mg/dl) (mg/liter) Previous therapies (mg/kg/day) (mg/day) total days (mg) TP1 59 F IIIA 70 IgA 5,260 7.7 MP, VAD, cyclo, HD dex 0.15 1,000 2/44 312 TP2 52 F IIIA 70 Nonsecretory 2.53 MP, VAD, cyclo, HD dex, radiation 0.15 1,000 3/75 663 TP3 52 M IIIA 70 IgG 10,300 4.8 MP, VAD, thalidomide, cyclo, 0.15 1,000 5/125 1,250 etoposide TP4 40 M IIIA 70 IgG 8,480 6.0 MP, VAD, radiation, thalidomide, 0.25 1,000 1/25 500 cyclo TP5 65 M IIIA 80 IgG 3,800 3.4 MP, VAD, dex 0.25 1,000 4/100 1,800 TP6 60 F IIIA 80 IgG 5,150 4.3 MP, VAD, cyclo, thalidomide 0.25 1,000 6/150 2,450 a PS, performance status (Karnofsky scale); ␤2M, ␤2 microglobulin; MP, melphalan ϩ prednisone; cyclo, cyclophosphamide; VAD, vincris- tine ϩ Adriamycin ϩ dexamethasone; HD dex, high dose dexamethasone; dex, dexamethasone.

count with differential, electrolyte panel, calcium, magnesium, within 7 days after the completion of each cycle. In vitro blood urea nitrogen, creatinine, glucose, total protein, albumin, sensitivity of bone marrow mononuclear cells after 48-h expo- ϩ ϩ hepatic transaminases, bilirubin, urine analysis. Serum K and sure to As203,As203 AA, or etoposide was determined and ϩ Mg2 levels were rigorously maintained above 3.8 and 1.8 analyzed as described previously (10). Briefly, bone marrow mM/liter, respectively. Twelve lead EKGs were done twice mononuclear cells were isolated from aspirates by discontinuous weekly to determine the QTc interval and monitor for arrhyth- density-gradient centrifugation. Cells (4 ϫ 105) were cultured in mias or conduction defects. All of the toxicities were graded as ␮ Ϯ ␮ the presence or absence of As2O3 (2 M) AA (100 M)or defined by the NCI Common Toxicity Criteria (CTC v2.0). of etoposide (10 ␮g/ml). After 48 h, cells were triple stained Additional studies for intracellular GSH, serum AA, and plasma with a phycoerythrin-conjugated mouse antihuman CD38 anti- elemental arsenic levels are detailed below. Serum protein elec- body, a CyChrome-conjugated mouse antihuman CD45 anti- trophoresis and bone marrow biopsy were performed within 1 body (both from Becton-Dickinson, San Jose, CA), and FITC- week of the completion of each cycle. conjugated annexin V (Biovision, Mountain View, CA), or Disease response was defined as reported previously (16): isotype-matched control antibodies. Plasma cells were identified CR was defined as the absence of detectable M protein and on flow cytometry by their CD38high/CD45dim phenotype (6). Ͻ5% plasma cells in the bone marrow. PR was defined as a Non-plasma cells were defined by low or no CD38 expression Ͼ25% decrease in the amount of M protein or a 50% reduction Ϫ (CD38 ). Apoptotic cells were identified by annexin-V positiv- in the plasma cells in the bone marrow. Responses were graded ity. Flow cytometry was done on a FACScan cytometer (Bec- as a good PR if there was Ͼ50% reduction in M protein, or as ton-Dickenson) using CellQuest software. Because there was a poor PR if there was Ͻ50% reduction. Stable disease was patient-to-patient variation in the percentage of apoptotic my- defined as a Ͻ25% change in M protein. A Ͼ25% increase in M eloma cells in the untreated conditions [17.85 Ϯ 7.5% (range, protein was defined as progressive disease. 6.1–31.5%)], the percentage apoptotic cells in the untreated Intracellular GSH Measurement. GSH levels were de- conditions (background apoptosis) was subtracted from the termined as described previously (10) in PBMCs on day 1 (preinfusion and 1 h posttreatment), day 8 and day 33 (both drug-treated cultures results to generate the percentage apoptotic postinfusion) of each cycle. GSH was measured in discontinu- cells specifically attributable to treatment. Similar analysis was ous density-gradient-purified PBMCs using the Calbiochem performed for apoptosis induced in normal bone marrow cells. (San Diego, CA) GSH assay kit per the manufacturer’s instruc- tions. Intracellular GSH was normalized to total cellular protein Results content (DC Protein Assay; Bio-Rad, Richmond, CA). AA and Elemental Arsenic Plasma Levels. Serum AA Patient Characteristics levels were determined by capillary electrophoresis at the Mayo According to the escalation/de-escalation study design, six Clinic reference laboratory (Rochester, MN). Elemental arsenic patients were required to achieve the primary objective of the blood levels were measured by mass spectrometry at Nichols Phase I component of this trial. All of the patients had failed or Laboratory (San Juan, CA), with a lower limit of detection of 10 relapsed after multiple chemotherapy regimens, including three ␮g/liter. AA levels were measured on day 1 at 0, 1, 5 and 24 h patients who had thalidomide-refractory disease. The median postinfusion, on day 8, and on day 33. Elemental arsenic levels age was 54 (range, 40–65 years). All of the patients were stage were measured on day 1 at 0 and 1, 5, and 24 h postinfusion, on IIIA (Durie-Salmon) with a mean ␤2-microglobulin of 4.78 day 8, and on day 33. Calculations of the serum concentration of mg/liter (range, 2.53–7.70). A total of three patients were treated ϩ the trioxide form of arsenic were done using the assumptions of at the 0.15-mg/kg/day As203 1000-mg/day AA dose level, ϩ Westervelt et al. (22), that the only source of measured plasma and three patients were treated at the 0.25-mg/kg/day As203

elemental arsenic is the administered As203 and that elemental 1000-mg/day AA dose level. Patient demographics, disease arsenic is 76% of As2O3 by weight. characteristics, total dose of As203, and the number of cycles ؎ In Vitro Sensitivity to As203 AA. Patient bone mar- received are detailed in Table 1. A total of 21 cycles of ϩ row aspirates were obtained immediately before cycle 1 and As203 AA were administered with a mean of 3.3 cycles/

Fig. 1 Elemental arsenic plasma levels. A, postinfusion elemental arsenic plasma levels on day 1/cycle 1. Blood samples were drawn at the times indicated and assayed for elemental arsenic. ϩ Arrows,As203 AA infusions. B, preinfusion arsenic levels over cycle 1 and 2. Plateau elemental arsenic levels immediately preceding the infu-

sion of As203 on days 1, 8, and 33 of each cycle. C, postinfusion arsenic levels over the first and second cycles. Plasma elemental arsenic levels

were measured 1 h after the infusion of As203 as above.

patient. Of note, four patients have exceeded the current recom- day 1/cycle 3 (cycle 3 data not shown in Fig. 1). The mean

mended total As203 dose for APL. postinfusion plasma elemental arsenic levels for cycle 2 and 3 were similar to those for cycle 1, with day-1/1-h values of 61 Plasma Elemental Arsenic Pharmacokinetics (cycle 2) and 81 Ϯ 7 ␮g/liter (cycle 3), day-8 values of 133.5 Ϯ Ϯ ␮ The pharmacokinetics of As203 were investigated at the 21.9 (cycle 2) and 115 4 g/liter (cycle 3), and day-33 values ϩ Ϯ Ϯ ␮ 0.25 As203 mg/kg/day 1000-mg/day AA dose level. Because of 156 46 (cycle 2) and 149.5 6 g/liter (cycle 3). there are no standard clinical assays for plasma As203 levels, we It is possible that any clinical effect of AA coadministra- measured elemental arsenic plasma levels. The mean elemental tion is mediated indirectly by effects on arsenic pharmacokinet-

arsenic levels on day 1/cycle 1 at 1, 5, and 24 h post-As203- ics, as opposed to a direct effect of AA on intracellular GSH infusion were 60.3 Ϯ 19, 46 Ϯ 12, and 47 Ϯ 5.7 ␮g/liter, levels. In our study, the 1-h postinfusion elemental arsenic ␮ respectively (Fig. 1A). As indicated in Fig. 1, B and C, there is levels (C1-h postinfusion) had a mean value of 60.3 g/liter on an accumulation of elemental arsenic in the blood as the treat- day 1/cycle 1. A steady accumulation of elemental arsenic in ment continues. On cycle 1/day 8, the mean elemental arsenic the blood during treatment was observed, reaching a mean ␮ levels preinfusion (Fig. 1B) and 1 h postinfusion (Fig. 1C) were C1-h postinfusion of 174.7 g/liter by day 33. The calculated mean Ϯ ␮ Ϯ ␮ 68.3 11.6 g/liter and 119.5 7.8 g/liter, respectively, and C1-h postinfusion for the trioxide form of arsenic on days 1 and 33 on day 33, were 137 Ϯ 36.8 ␮g/liter and 174.7 Ϯ 28.7 ␮g/liter, are 79 ␮g/liter and 229 ␮g/liter, respectively, similar to values respectively. After 2 weeks off treatment, the elemental arsenic in the studies administering arsenic alone (14, 22). These find- levels were still detectable with mean preinfusion values of ings suggest that the concomitant administration of AA does not Ϯ ␮ Ϯ ␮ 40.5 27.6 g/liter on day 1/cycle 2, and 31.5 12 g/liter on significantly alter plasma levels of As203.

Fig. 2 PBMC intracellular GSH levels and blood AA levels. Intracellular GSH levels in PBMC and plasma AA levels were measured on day 1 (pretreatment), 8, and 33 (1 h postinfusion) of each cycle. GSH (____, left axis), AA (...... , right axis).

The Effect of AA Administration on Intracellular respectively. The mean AA level 1 h postinfusion on day 2 was GSH Levels 145.6 ␮mol/liter, on day 8 was 191.8 ␮mol/liter (62.5 before the Although in vitro and animal studies demonstrate that infusion), and on day 33 was 145.2 ␮mol/liter. Increasing the

AA can act as a chemosensitizer by depleting intracellular As203 dose did not seem to affect AA pharmacokinetics, be-

GSH, this ability (a central hypothesis of this trial) has never cause there were no significant differences in AA C1-h postinfusion been clinically tested. Reliably measuring GSH changes in (P Ͼ 0.5 for all time points) in patients receiving the 0.15-mg/

the myeloma cells themselves is not feasible with current kg/day versus the 0.25-mg/kg/day As203 dose. assays, given the inadequate number of myeloma cells that The effect of AA administration on intracellular GSH can be purified from a bone marrow aspirate, the effect of the during the first three cycles is shown in Fig. 2. Overall, intra- cell purification process itself on GSH levels, and the inva- cellular GSH decreased from day 1 to day 33 in 14 of the 19 siveness of doing multiple bone marrow biopsies per cycle cycles that we have complete data on. The mean percentage for serial assessment. However, previous studies using the decline in GSH during cycle one; (from day 1 to day 33) was GSH-depleting agent BSO have demonstrated that changes in 13.1 Ϯ 32.6%; during cycle two; was 60.7 Ϯ 42.3%; and during the intracellular GSH levels in PBMCs paralleled changes in cycle 3, was 64.1 Ϯ 34%. The decline was most prominent biopsied tumor cells (40), although this has not been specif- when starting GSH levels were Ͼ25.5 nmol/mg total protein. ically demonstrated for myeloma. We, therefore, measured Only patient 4 did not display this behavior. When GSH levels intracellular GSH levels in PBMCs as a correlate to bone on day 1 were Ͻ25.5 nmol/mg total protein, increased AA marrow myeloma cell GSH levels. Plasma AA levels were levels had little effect on GSH through the rest of the cycle. drawn simultaneously with the PBMCs for GSH assay. Interestingly, GSH levels appeared to increase between cycles, All six of the patients received the 1000-mg AA dose. On significantly in some patients (patients 2, 5, and 6). However, day 1/cycle 1, the mean plasma AA levels at 0, 1, 5, and 24 h these elevated GSH levels were substantially lowered by AA on after the infusion were 49.8, 180, 66, and 46.5 ␮mol/liter, day 8 and 33 of the subsequent treatment cycle.

Table 2 Toxicitya during the first cycle of treatment Hematological Toxicity. In the first cycle, grade 2 leu- % of patients in each treatment group of kopenia was observed in five patients and grade 3 leukopenia in 3 patients that experienced one patient. This was more prevalent at the 0.25-mg/kg As203 the specific toxicity dosage and typically developed in the 3rd week of a cycle. ϩ ϩ Grade 1–2 anemia was noted at both dose levels, but no trans- As2O3 0.15 mg/kg As2O3 0.25 mg/kg Toxicity AA 1000 mg AA 1000 mg fusions were required at the completion of the first cycle. No General thrombocytopenia was observed. Fatigue 100% (grade 1–2) 100% (grade 1–2) Nonhematological/Noncardiac Toxicity. Mild fatigue Insomnia 0% 33% (grade 2) was seen in all of the patients treated. However, this did not Peripheral edema 0% 33% (grade 2) seem to significantly impact the patients’ quality of life, and Rigors 0% 33% (grade 1) Gastrointestinal patients who were working before trial enrollment continued to Nausea 33% (grade 1) 66% (grade 1–2) do so during treatment. Mild-to-moderate nausea, dysgeusia Anorexia 66% (grade 1) 66% (grade 1–2) with a metallic taste, and anorexia were the most common Diarrhea 0% 66% (grade 1–2) gastrointestinal toxicities. Reversible erectile dysfunction and Constipation 33% (grade 2) 0% Dysgeusia 0% 66% (grade 1–2) loss of libido were observed in two patients receiving 0.25 Neurological mg/kg of As203. In both of these patients, serum testosterone Sensory neuropathy 66% (grade 1–2) 66% (grade 1–2) levels were less than 60 mg/dl but returned to normal range after Hearing loss 0% 33% (grade 2) completion of the cycle. Mild-to-moderate sensory neuropathy Cardiac Prolonged QTc 33% (grade 1) 100% (grade 1) was noted in two patients in each dosing group. Dry skin, Hematological pruritis, and diffuse maculopapular rash were the most frequent Anemia 66% (grade 1–2) 100% (grade 1–2) dermatological toxicities and were self-limited in the majority of Leukopenia 100% (grade 1–2) 66% (grade 1–2) the cases or were relieved with topical steroids. Patient 5 de- 33% (grade 3) Infection 33% (grade 2) 0% veloped oliguric renal failure during his fourth cycle while Skin receiving vancomycin for a nonneutropenic port infection. He Pruritis 66% (grade 1–2) 66% (grade 1–2) subsequently required permanent hemodialysis and was taken Dry skin 66% (grade 1) 66% (grade 1–2) off the study. Finally, patient 6 developed unilateral grade 2 Genito-urinary sensineural hearing loss during her sixth cycle of therapy. Erectile impotence 0% 66% (grade 1–2) Decreased libido 0% 66% (grade 1) Cardiac Toxicity. QTc prolongation has been consis- Eye tently seen in the majority of APL patients treated with As203, Excessive tearing 0% 33% (grade 1) with serious arrhythmias (particularly torsade de pointes) noted a Toxicity is graded by the NCI Common Toxicity Criteria v2.0. in some studies (21, 22, 42). Because the heart may be subjected ϩ to both acute and chronic toxicity from As203 AA administration, we have followed each trial patient with twice-weekly EKGs for QTc prolongation (Fig. 3). Grade 1 QTc interval Toxicity prolongation was observed in four patients. No dose reduction Compared with the experience with As203 alone in APL, was necessary in any patients, and no treatment interruption was factors that may have altered the toxicity profile in our trial required. The QTc intervals returned to normal before the start included the addition of AA, the increased As203 dose (in the of the next cycle, and there was no lengthening of baseline QTc 0.25-mg/kg/day group), the schedule (25 days of treatment over intervals with cumulative cycles suggestive of chronic toxicity. a 35-day time period versus 60 consecutive days for APL), and The average QTc prolongation was 23.77 Ϯ 2.95 ms for the the duration of treatment (a maximum of 150 total days of 0.15-mg/kg/day group, and 35.79 Ϯ 3.68 ms for the 0.25-mg/ treatment versus 60 days for APL). All of the toxicities encoun- kg/day group. This compares favorably with the mean QTc tered for each dose group during the first cycle are shown in prolongation of 63 Ϯ 13 ms seen in APL patients treated with Table 2. No grade 3 or 4 nonhematological or grade 4 hemato- As203 alone (18). In 21 administered cycles, no episodes of logical toxicity occurred during cycle 1 in any of the patients. supraventricular or ventricular arrhythmias were observed. All of the toxicities resolved in the 2 weeks between cycles, except for the incomplete resolution of sensory neuropathy in ؉ patients 3, 4, and 5. However, these patients had preexisting Serial Assessment of in Vitro As203 AA Sensitivity sensory neuropathy secondary to prior therapy. Treatment was To determine whether resistance to arsenic Ϯ AA devel- withheld during cycle 2 in patient 1 because of nonneutropenic oped over multiple cycles of treatment, we conducted serial in sepsis from a surgical wound infection, which led to her death. vitro assessments of patient bone marrow plasma cells apoptosis Ϯ We have subsequently treated eight additional subjects in the induced by As2O3 AA or by etoposide. Bone marrow was Phase II component of this trial (using the 0.25-mg/kg/day obtained before treatment and then after every cycle (we were ϩ As203 1000 mg AA dose), and have found the same toxicity unable to obtain bone marrow from patient 3). As seen in Fig. 4, profile.7 primary plasma cells from all of the patients were initially sensitive to arsenic in vitro (20–40% apoptosis after 48 h). The

sensitivity of plasma cells from patients 5 and 6 to As203 was decreased after cycle 1 but was restored by the addition of AA 7 N. J. Bahlis et al., unpublished observations. at 100 ␮mol/liter. Over subsequent cycles, we did not see

Fig. 3 QTc intervals. EKGs were performed twice weekly, and QTc was plotted for each patient and cycle. A, ϩ 0.15 mg/kg/day As203 1000 mg/day ϩ AA. B, 0.25 mg/kg/day As203 1000 mg/day AA.

ϩ further development of resistance to As203 AA by these in started on thalidomide. Patient 5 had his major decrease in M vitro assays. protein by cycle 2 with stable M proteins through cycle 4, but Consistent with previous findings by our group and others developed vancomycin-induced renal failure and was taken off (10, 26), normal bone marrow cells (CD38Ϫ) were less sensitive the study. He had stable disease on thalidomide but suffered a ϩ than the myeloma cells to arsenic or the As203 AA treatment. fatal myocardial infarction (he had pre-existing coronary artery For AA treatment alone, the mean percentage of apoptotic cells disease and had had a bypass) 8 months after going off the in the nonmalignant cell population was 1.10 Ϯ 3.43% (range, study. Ϫ5.27 to 6.23%), for arsenic treatment alone, the value was Patient 2 initially had ␬ light chain disease that later Ϯ Ϫ ϩ 5.96 7.78% (range, 3.1 to 27.95%), for As203 AA, it was evolved into nonsecretory myeloma with 40% bone marrow Ϯ Ϫ 12.75 12.29% (range, 3.18 to 37.28), and for etoposide, it plasma cells before trial enrollment. After three cycles of As203 was 7.70 Ϯ 8.37% (range, Ϫ1.6 to 25.43). ϩ AA, the patient a stable degree of plasma cell infiltration in her bone marrow and no detectable urine ␬ chains. The patient Response and Current Status opted to go off study after her third cycle because of transpor- Although our Phase I trial was not designed to look at tation problems and was started on pulse steroid therapy and efficacy, the evaluation of response may give some indication of thalidomide (200 mg). Her disease has subsequently progressed ϩ ␬ As203 AA’s activity against refractory/relapsed myeloma. As with a reappearance of urine light chains. She expired 3 seen in Table 3, two patients (patients 3 and 6) achieved major months after she was taken off study while still on thalidomide. responses with 47.5 and 58% decreases, respectively, in their M protein. Neither had previously responded to thalidomide. Pa- Discussion

tient 3 had a decrease in M protein from 10.3 mg/dl to 5.6 mg/dl As203 alone has been shown in vitro and in vivo to be by cycle 3 but subsequently progressed after cycle 5 and was active against chemotherapy-resistant MM. We and others have taken off the study. He did not receive further treatment and died demonstrated in vitro that AA can deplete intracellular GSH,

4 weeks after being taken off the study. Patient 6, after two increasing the efficacy of As203 by decreasing the cell’s ability cycles, had a decrease of M protein from 5.15 to 2.18 mg/dl, to directly detoxify arsenic (via conjugation to GSH and export) which remained stable at this level for her remaining four and/or reducing the arsenic-generated ROS that cause cellular cycles, such that she completed the protocol (six cycles) without damage and induce apoptosis. These studies form the rationale evidence of disease progression. Because she had residual dis- for our NCI/CTEP Phase I/II trial using AA to modulate intra- ϩ ease, she is being maintained on thalidomide dexamethasone. cellular GSH and increase the efficacy of As203 in patients with Three other patients (patients 1, 4, and 5) had stable disease refractory/relapsed MM. To our knowledge, this is the first with less than 25% decrease in their M protein (Ϫ22%, ϩ4%, clinical trial using AA as a GSH-depleting and chemosensitizing and Ϫ17%, respectively). Patient 1 developed nonneutropenic agent. The primary objective of the Phase I component of this

systemic candidiasis from an open surgical wound during her study was to determine whether the addition of AA to As203 (at second cycle, which was the cause of her death. Patient 4 doses previously reported to have acceptable toxicity) signifi- decided not to continue on the protocol after one cycle and was cantly altered the acute and chronic toxicity profile reported for

Fig. 4 Serial in vitro assessment of apoptosis induced in patient bone marrow Ϯ plasma cells by As2O3 AA or by etoposide. Patient bone mononuclear cells were obtained at the time points indicated, and cultured in the absence or ϩ presence of As2O3 (As), As2O3 AA (AsϩAA), or etoposide (Etop). The CD38high/CD45dim/annexin Vϩ phenotype was used to quantitate the degree of plasma cell apoptosis.

Table 3 Response and current status M protein (mg/dl) Best response Patient Pretreatment (cycle achieved) % change Response Current status TP1 5,260 4,060 (2) Ϫ22% SDa Expired (on-study, sepsis) TP2 Nonsecretory Expired (off study, relapsed MM) TP3 10,300 5,400 (3) Ϫ47.5% PRb Expired (on study, relapsed MM) TP4 8,480 8,840 (1) ϩ4% SD Off study (on thalidomide) TP5 3,800 3,020 (4) Ϫ22% SD Expired (off study, MI)a TP6 5,150 2,180 (6) Ϫ58% PRb Completed protocol (6 cycles) (on thalidomide ϩ dexamethasone) a SD, stable disease (Ͻ25% change in M protein); MI, myocardial infarction. b Poor PR, 25–50% reduction in M protein; good PR, Ն50% reduction in M protein.

As203 alone, and if so, how? Central to this objective were attempting to establish a maximally tolerated dose, this allowed correlative studies determining whether coadministration of AA us to enroll a small number of patients to achieve this objective. significantly changed the pharmacokinetics of As203 (compared Our second objective was to test the hypothesis that AA admin- with what has been reported for arsenic alone). The established istration would deplete intracellular GSH in patients and to ϩ acceptable dose of As203 AA would then be used in the Phase determine the characteristics of this depletion. The degree of II component of the trial. Because this Phase I component was GSH depletion may also correlate to toxicity and response. Our designed to examine only two doses of arsenic and was not last objective was to initially assess the clinical efficacy of this

combination, and conversely to assess the development (and patients, this suggests it is the degree of AA-mediated GSH

mechanisms) of As203 resistance in vitro. depletion that may be the clinically relevant parameter. Further Compared with the experience of As203 alone in APL, correlation of GSH depletion (e.g., a threshold value or percent- factors that may alter the toxicity profile in our myeloma trial age depletion) to specific toxicity and clinical response is being include the disease itself, the addition of AA coadministration, evaluated in the Phase II component of this trial. ϩ increased As203 dosage (in the 0.25 mg/kg/day group), sched- The toxicities of As203 AA at both the 0.15-mg/kg and

ule, and duration of treatment. All of these factors may alter the 0.25-mg/kg As203 doses were largely limited and reversible. plasma concentration of As203 and, thus, toxicity. To assess this, Compared with the reported toxicities of As203 alone (e.g., 0.15 we determined arsenic pharmacokinetics in the 0.25-mg/kg/day mg/kg/day for APL), we did not see evidence of increased

As203 group. Our mean C1-h postinfusion As2O3 on days 1 and 33 toxicity attributable to AA coadministration. Only erectile im- were 79 ␮g/liter (0.4 ␮mol/liter) and 229 ␮g/liter (1.16 ␮mol/ potence appears to be unique to our trial. Consistent with the liter), respectively. Similarly, in APL patients, Shen et al. (14) APL studies, the major toxicity was fatigue, although this was

reported day 1/cycle 1 C1-h postinfusion values ranging from 1.8 to generally mild. No grade 3 or 4 nonhematological toxicity was ␮ 2.2 mol/liter after a single 10-mg i.v. dose of As2O3 (0.14 observed in any patient. Grade 3 myelosuppression (asymptom- mg/kg for a 70-kg patient). In contrast to our findings, this study atic) was seen in only one patient at the end of the first cycle. did not demonstrate an accumulation of plasma arsenic by day Interestingly, in our trial some of the toxicities reported in APL 30/cycle 1. In another study, however, Westervelt et al. (22) patients were either absent [dyspnea, chest pain, hyperglycemia, recently reported that a dosing regimen of 0.10 mg/kg/day in liver dysfunction, ventricular arrhythmias, leukocytosis, and APL patients resulted in barely detectable day 1/cycle 1 mean retinoic acid syndrome (these last two may be unique to APL)] Ͻ ␮ Cmax As203 levels of 0.26 mol/liter, which rose to a mean or diminished in incidence and/or severity (fluid retention/ ␮ Cmax of 0.95 mol/liter by day 28, indicating an accumulation edema, QTc prolongation, fever, headache, abdominal pain, and of blood arsenic over time. Thus our As2O3 plasma levels cough). appear to be similar to those in previous studies, with the caveats Cardiac arrhythmia and sudden death were recently re-

that we used a higher dose but a “discontinuous” (weekdays ported in two small studies of As203 alone in APL patients (21, only) schedule, and our small patient numbers may have limited 22) but not in a larger multicenter trial (17). We have not the ability to discern even relatively large (50%) changes in drug observed any arrhythmias to date. Asymptomatic QTc prolon- disposition. Our findings are also similar to trials in solid tumors gation (all Ͻ500 ms) was seen in four patients and did not 8 using As203 alone at doses from 0.15 to 0.30 mg/kg/day for require dose modification or treatment interruption. Rigorous both the mean day-1 postinfusion plasma levels and the accu- efforts to avoid hypokalemia and hypomagnesemia may have mulation of plasma arsenic over time. Together, these findings contributed to the absence of cardiac arrhythmias detected in our suggest that the concomitant administration of AA does not study. significantly alter the plasma pharmacokinetics of arsenic com- With a median follow-up of 6.6 months (2–12 months), the

pared with the administration of As203 alone. Additional phar- only evidence of chronic/cumulative toxicity in the four patients macokinetic studies are ongoing in our Phase II trial. who have received three or more cycles has been one case of We found that i.v. administration of 1000 mg of AA mild sensineural hearing loss. This has been reported as an ␮ yielded a mean plasma level of 180 mol/liter at 1 h, above the uncommon toxicity of the medical use of As203 (22) and is more 100 ␮mol/liter concentration that depletes intracellular GSH in frequently associated with chronic environmental exposure (44). our in vitro studies (10). Correlative measurements in PBMCs Three of these patients have received Ͼ1200 mg total dose of ϳ demonstrated that AA administration can deplete intracellular As203 (versus 600 mg recommended total dose for APL). In GSH levels, most significantly when starting levels are particular, we have not seen clinical or EKG evidence of cumu- Ͼ25.5 nmol/mg total protein. These results suggest that AA lative cardiac toxicity. Together, these findings indicate that

does not further deplete beyond a lower threshold (making an coadministration of AA does not increase the toxicity of As203 ϩ arsenic-sensitive cell more sensitive) but can deplete elevated and that As203 AA is well tolerated. Clearly, longer follow-up ϩ GSH levels back to this threshold and maintain arsenic sensi- is needed to determine any serious chronic toxicity of As203 tivity. AA administration was also able to deplete the elevated AA, although the lack of evidence for this in the patients who GSH levels seen at the beginning of cycle 2 and/or cycle 3. The have received Ͼ1200 mg of arsenic is promising. cause of this elevated GSH is currently being studied, but may To assess potential correlates and mechanisms of response

represent a compensatory cellular response to GSH depletion or resistance to As203, we serially examined the in vitro sensi- ϩ during the previous cycle. It has been reported that GSH deple- tivity of patient bone marrow plasma cells to As203,As203 tion by BSO induces ␥-glutamylcysteine synthetase gene ex- AA, or etoposide (as an unrelated control). In all six of the pression, the rate-limiting enzyme in GSH synthesis (43). Inter- pre-cycle-1 samples, the addition of AA did not substantially

estingly, the one patient who had high starting levels of GSH enhance As203-induced apoptosis. However, in the four patients that were not affected by AA administration (patient 4) was also who demonstrated decreased plasma cell sensitivity to As203 the least responsive to treatment. Because there was no clear after cycle 1 (however slight), the addition of AA restored

evidence that pretreatment GSH levels predicted response in our As203-induced apoptosis to (or above) pretreatment levels. In comparison, the one patient whose myeloma cells remained sensitive to arsenic (patient 2) continued to have relatively little augmentation by AA. If arsenic resistance is mediated primarily 8 S. Soignet, personal communication. through increased intracellular GSH, these findings would sug-

gest that a major clinical benefit of AA is preventing this 8. Kickhoefer, V. A., Rajavel, K. S., Scheffer, G. L., Dalton, W. S., resistance. By these in vitro assays, we did not see evidence for Scheper, R. J., and Rome, L. H. Vaults are up-regulated in multidrug- the development of arsenic resistance at the myeloma cell level resistant cancer cell lines. J. Biol. Chem., 273: 8971–8974, 1998. over multiple courses of treatment. This is largely consistent 9. Zawydiwski, R., Harmon, J. M., and Thompson, E. B. Glucocorti- coid-resistant human acute lymphoblastic leukemic cell line with func- with our clinic responses, in which only one of five patients tional receptor. Cancer Res., 43: 3865–3873, 1983. ϩ receiving more than one cycle relapsed while receiving As203 10. Grad, J. M., Bahlis, N. J., Reis, I., Oshiro, M. M., Dalton, W. S., and AA (patient 3, receiving the 0.15-mg/kg/day As203 dose). Un- Boise, L. H. Ascorbic acid enhances arsenic trioxide-induced cytotox- fortunately, we were unable to obtain marrow samples from this icity in multiple myeloma cells. Blood, 98: 805–813, 2001. patient at the time of relapse to assess the nature of the resist- 11. Buzaid, A. C., and Durie, B. G. Management of refractory my- ance. eloma: a review. J. Clin. Oncol., 6: 889–905, 1988. Overall, two of six patients (both with thalidomide-refrac- 12. Singhal, S., Mehta, J., Desikan, R., Ayers, D., Roberson, P., Ed- tory disease) achieved a PR, and four patients had stable disease. dlemon, P., Munshi, N., Anaissie, E., Wilson, C., Dhodapkar, M., ϩ Zeddis, J., and Barlogie, B. Antitumor activity of thalidomide in refrac- These results suggest that As203 AA has activity in heavily tory multiple myeloma. N. Engl. J. Med., 341: 1565–1571, 1999. pretreated myeloma patients and, of note, in patients with 13. Zhang, P., Wang, S., Hu, L., Shi, F., Qiu, F., Hong, L., Han, X., thalidomide-resistant disease. Interestingly, three of the four Yang, H., Song, Y., Liu, Y., Zhou, J., Li, X., Xiao, Y., and Jin, Z. patients who had an initial decrease in M protein maintained this Treatment of acute promyelocytic leukemia with intravenous arsenic decrease as long as they were on treatment. The fact that the trioxide. Chinese J. Hemat., 17: 58–60, 1996. disease response plateaued indicates that an equilibrium be- 14. Shen, Z. X., Chen, G. Q., Ni, J. H., Li, X. S., Xiong, S. M., Qiu, tween arsenic response and resistance was established. Because Q. Y., Zhu, J., Tang, W., Sun, G. L., Yang, K. Q., Chen, Y., Zhou, L., Fang, Z. W., Wang, Y. T., Ma, J., Zhang, P., Zhang, T. D., Chen, S. J., our in vitro assays did not find evidence that the myeloma cells Chen, Z., and Wang, Z. Y. Use of arsenic trioxide (As2O3)inthe themselves became resistant, this suggests that host factors may treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy be involved. 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Nizar J. Bahlis, Jennifer McCafferty-Grad, Ileana Jordan-McMurry, et al.

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