Arsenic Trioxide – An Old Drug Rediscovered

Abstract

Over the last 17 years, clinical trials conducted worldwide have demonstrated the efficacy of arsenic trioxide (As₂O₃) in the treatment of relapsed acute promyelocytic leukemia (APL). Currently, the role of As₂O₃ in front-line therapy is under investigation. Recent trials in the US have demonstrated that the addition of As₂O₃ to standard treatment regimens improves survival outcomes in patients with APL and may allow a reduction in cytotoxic chemotherapy exposure. As₂O₃ has also shown efficacy in other malignancies, particularly multiple myeloma and myelodysplastic syndromes. Therapeutic doses of As₂O₃ are well tolerated, with no evidence of long-term toxicity. Adverse events include APL differentiation syndrome, electrocardiographic abnormalities, and mild elevations in liver enzymes. This review highlights trials investigating the role of As₂O₃ in induction and consolidation for newly diagnosed APL, as well as its role in other hematologic malignancies. The chemistry, mechanisms of action, and clinical side effects of As₂O₃ are also discussed.

Introduction

Rediscovery of an old drug

Arsenic has been used in medicine for more than 2400 years for a variety of ailments including ulcers, the plague, and malaria.¹ In 1878, potassium arsenite was reported to have an anti-leukemic effect and was used for this purpose in the late 19th and early 20th centuries until it was replaced by busulfan in the 1950s.^2–4

In the modern era, interest in arsenic as a chemotherapy was rekindled after it was identified as an active ingredient in traditional medicines in China.⁵ Researchers evaluated arsenic compounds for the treatment of various cancers and in 1992 published the results of a trial in which intravenous administration of arsenic trioxide (As₂O₃) produced a complete response (CR) in 21 (66%) of 32 patients with acute promyelocytic leukemia (APL).^5,6 In two subsequent studies, Zhang et al reported that As₂O₃ induced a CR in 22 (73%) of 30 newly diagnosed and 22 (52%) of 42 relapsed APL patients,^5,7 and Shen et al. observed a CR in nine (90%) of 10 relapsed APL patients.⁸

As₂O₃ for relapsed APL

Based on the results from the Chinese studies, a US pilot study was conducted in patients who had relapsed after one or more courses of all-trans-retinoic acid (ATRA) and anthracycline-based chemotherapy.⁹ Most patients had experienced multiple relapses and 58% were resistant to retinoid therapy. Eleven of 12 patients achieved a CR with As₂O₃ alone. Eight of these 11 responders also achieved a molecular remission, as defined by the absence of detectable PML-RARα fusion gene expression.

Following the success of the pilot study, a larger, multicenter, single-arm trial was conducted in patients who had relapsed after ATRA-based therapy. More than one third of these patients had multiple relapses and were heavily pretreated (including five patients with prior BMT). CR was achieved in 34 (85%) of 40 patients (Table 1).¹⁰ All patients who achieved a CR also showed evidence of elimination of the t(15;17), as measured either directly by traditional cytogenetics or by assays using FISH or RT-PCR for PML/RAR-α. Many of the remissions were durable. The estimated two-year overall survival (OS) and relapse-free survival (RFS) rates for patients in first relapse were 77% and 58%, respectively. These results are somewhat confounded because eleven patients underwent bone marrow transplant in remission after induction or consolidation with As₂O₃. Among the 27 patients who did not receive transplant, one-third remained alive with duration of remission ranging 22 to 42 months. This pivotal trial provided support for the approval of As₂O₃ in patients with APL who failed to respond to or relapsed following ATRA/anthracycline therapy.

Table 1.

Current therapies for APL

Study	N	Regimen	CR, %	CIR, %	EFS, %	DFS, %	OS, %
Initial therapy
PETHEMA – LPA9911	251	I: ATRA + CT (IDA)	90	8.7, 3-yr	NR	90, 3-yr	85, 3-yr
		C: 3 courses CT (IDA, MXN, IDA; low-risk pts) or 3   courses ATRA + CT (intermediate/high-risk pts)
		M: Low-dose CT (MCP/ MTX) + intermittent ATRA   (every 3 mos) for 2 yrs
European APL Group – APL 200012	340	I: ATRA + CT (DNR ± ara-C)	96	8.4, 2-yr	84.5, 2-yr	NR	91.9, 2-yr
		C: 2 courses CT (DNR ± ara-C)
		M: Low-dose CT (MCP/ MTX) + intermittent ATRA   (every 3 mos) for 2 yrs
JALSG – APL9713	283	I: ATRA (low-risk pts) or ATRA + CT (IDA + ara-C;   intermediate/high-risk pts)	94	NR	NR	68.5, 6-yr	83.9, 6-yr
		C: 3 courses CT (MXN + ara-C, ara-C+ etoposide +   DNR, ara-C + IDA)
		M: Randomized to observation or intensified   maintenance CT (6 courses CT every 6 wks)
Second-line therapy
Soignet et al.10	40	I: As2O3 (daily until BM remission or 60 days)	85	NR	NR	56, 1.5-yr (RFS)	66, 1.5-yr
		C: As2O3 (25 doses in 35 days)
		M: Up to 4 additional consolidation courses of As2O3

Abbreviations: APL, acute promyelocytic leukemia; ara-C, cytarabine; As₂O₃, arsenic trioxide; ATRA, all-trans-retinoic acid; BM, bone marrow; C, consolidation therapy; CIR, cumulative incidence of relapse; CR, complete response; CT, chemotherapy; DFS, disease-free survival; DNR, daunorubicin; EFS, event-free survival; I, induction therapy; IDA, idarubicin; JALSG, Japan Adult Leukemia Study Group; M, maintenance therapy; MCP, mercaptopurine; MXN, mitoxantrone; MTX, methotrexate; NR, not reported; OS, overall survival; PETHEMA, Programme de Estudio y Tratamiento de las Hamopatias Malignas; RFS, relapse-free survival.

Mechanisms of action

As₂O₃ affects multiple cellular functions via different molecular targets (summarized in Fig. 1). Although the fundamental mechanism is the favorable chemical reaction between arsenic and thiol groups within a protein, the final outcome depends on the cell type as well as the dose and duration of arsenite exposure. For example, in APL cells, As₂O₃ at low concentrations (<0.5 µM) induces differentiation; at higher concentrations (0.5–2.0 µM) it causes apoptosis.^14,15

Figure 1. As₂O₃ targets multiple cellular pathways.

In APL cells, arsenic trioxide (As₂O₃) restores differentiation by degrading the PML-RARα fusion protein. However, As₂O₃ has additional targets that are present in multiple cancer cell types. As₂O₃ targets the mitochondria, decreasing the mitochondrial membrane potential (ΔΨm) via multiple specific targets including Bcl-2 and the PTPC. This change in potential results in the release of cytochrome C, which activates the caspase cascade. It also results in increased release of ROS from the mitochondria. ROS levels are increased further by As₂O₃ inhibition of the antioxidant enzyme GPx. As₂O₃ also inhibits activation of the cell-survival factor NFκB via inhibition of IKK, the kinase responsible for releasing NFκB that is sequestered in the cytoplasm.

Abbreviations: APAF, apoptotic peptidase activating factor; GP_x, glutathione peroxidase; IKK, IκB kinase; PTPC, permeability transition pore complex; ROS, reactive oxygen species.

Chemistry of As₂O₃

Trivalent arsenic (As^III) in As₂O₃ or arsenite (As[OH]₃) and pentavalent arsenic (As^V) in arsenate (HAsO₄²⁻) are the two biologically significant forms of arsenic. Although As^V disrupts cellular processes as a phosphate (HPO₄²⁻) mimic, the interaction of As^III with the thiol (or sulfhydryl) groups (-SH) of proteins with a high cysteine content is the basic reaction that underlies the multiple mechanisms of action of this chemotherapeutic agent.¹⁶ In this reaction, the valence orbitals of arsenic (As) have a better overlap and energy match with those of sulfur (S) than with those of oxygen (O), leading to the formation of an As^III-thiolate bond and the release of water, as demonstrated in the following equation¹⁷:

$${\text{As}}^{\text{III}}-\text{OH}+\text{RSH}\leftrightarrows {\text{As}}^{\text{III}}-\text{SR}+{\mathrm{H}}_2\mathrm{O}$$

Vicinal thiols (i.e., thiols bonded to adjacent carbon atoms) have a high affinity for As^III (Fig. 2). Thus, proteins that contain cysteine residues that are conformationally constrained and favorably positioned will have a higher affinity for As^III.¹⁸

Figure 2.

Reaction of vicinal sulfhydryl groups in a protein structure with trivalent arsenic.

Stimulation of differentiation

Empirically, treatment of APL cells with As₂O₃ leads to their terminal differentiation in vitro and in vivo. APL cells are uniquely sensitive to As₂O₃ due to the expression of the PML-RARα fusion protein; however, the mechanism by which arsenic trioxide treatment induces terminal differentiation remains somewhat speculative. In normal myeloid cells, PML protein is localized to macromolecular structures in the nucleus (nuclear bodies), where PML antagonizes many processes required for the initiation and progression of malignancy.¹⁹ In leukemic cells, the PML-RARα fusion protein blocks the expression of genes required for normal differentiation. The fusion protein disrupts the nuclear bodies, and the PML protein is dispersed into smaller organelles.²⁰ PML contains a cysteine-rich region that is hypothesized to interact with As^III, resulting in the degradation of PML-RARα fusion protein.²¹ Furthermore, As₂O₃-induced histone acetylation has been reported to promote differentiation via alteration in gene transcription.²²

Induction of apoptosis

Caspases are intracellular cysteine proteases that are key components in classic apoptosis. Caspase activation occurs in response to various types of mitochondrial damage and proapoptotic stimuli, which cause cytochrome c (normally sequestered between the mitochondrial inner and outer membranes) to be released into the cytosol, where it binds and activates Apaf-1, which in turn activates procaspase-9.^23–25 Caspase-9 cleaves procaspase-3, and downstream of caspase-3, the apoptotic program branches into a multitude of subprograms, the sum of which results in the ordered dismantling and removal of the cell.²⁶

As₂O₃ induces dose-dependent apoptosis in APL and hematopoietic non-APL as well as tumor and non-malignant cell lines.^27,28 The activation of the caspase cascade, the decrease of the mitochondrial membrane potential ΔΨ_m, and the production of reactive oxygen species (ROS) all play roles in induction of As₂O₃-induced apoptosis.²⁹ As₂O₃ also promotes apoptosis via down-regulation of Bcl-2 expression³⁰ by prolongation of the cell cycle and cell cycle arrest of malignant cells^31,32 and by opening the permeability transition pore complex (PTPC) in mitochondrial membranes by directly binding to thiol groups in the PTPC.³³

Accumulation of ROS

ROS are damaging to DNA, RNA, proteins, and lipids, and include free radicals such as hydroxyl (OH^• ) or superoxide (O₂⁻•) and molecules such as hydrogen peroxide (H₂O₂). Mutations in nuclear or mitochondrial genes encoding components of the mitochondrial electron transport chain can lead to the accumulation of electrons along the chain. These electrons are captured by O₂ to form superoxide, which is converted to hydrogen peroxide. The protective buffering systems against ROS include glutathione (GSH), thioredoxin, superoxide dismutase, catalase, and glutathione peroxidase.³⁴

As₂O₃ can increase cellular levels of ROS via several targets. It can prevent ROS detoxification by inhibiting antioxidant enzymes like glutathione peroxidase, which has a thiol group required for its activation. The level of intracellular GSH, which titrates arsenic by forming As(GS)₃ complexes, as well as the activities of related antioxidant enzymes, are major factors for sensitivity to As₂O₃. The sensitivity of APL to As₂O₃-induced apoptosis is closely related to its relatively poor antioxidant capacity.³⁵ Furthermore, proteins with high sulfhydryl-disulfide oxidation potentials may be particularly susceptible to redox regulation, and the affinity of As₂O₃ for sulfhydryl groups on these redox-sensitive proteins explains many of its ROS-related effects.³⁶ Finally, through the use of gene expression profiling, interference RNA, and genetically engineered cells, Chou and colleagues reported that NADPH oxidase, an enzyme complex required for the normal antibacterial function of white blood cells, is a major target of arsenic-induced ROS production.³⁷

Inhibition of NFκB

NFκB is a transcriptional factor promoting cell survival with an important role in many cancer cells. Activation of NFκB depends on the integrity of the IκB kinase (IKK); upon phosphorylation by IKK, the inhibitory protein IκB releases NFκB for translocation to the nucleus. As₂O₃ has been shown to inhibit IKK by binding to cysteine-179 in the activation loop of the enzyme catalytic subunit. Although cysteine-179 is not located in the vicinity of another cysteine within the IKK primary structure, it has been suggested that another cysteine may come within a critical distance of cysteine-179 upon the folding of the polypeptide chain or the dimerization of the catalytic subunits, thus providing a high-affinity target for arsenite.³⁸

As₂O₃ selectivity for APL cells

To understand the selective cytotoxicity of As₂O₃ against human APL, Dilda et al. screened the yeast deletion strains for sensitivity or resistance to the drug.³⁹ A prominent sensitive mutant was missing Hog1, a MAP kinase. The most resistant mutant lacked the plasma-membrane glycerol and arsenite transporter, Fps1. Hog1 and Fps1 control the response to osmotic stress in yeast through regulation of glycerol production and plasma membrane flux, respectively. Based on these results, the investigators tested the APL cell line NB4 for impaired osmoregulation and found that the APL cells did not produce glycerol in response to osmotic stress and underwent apoptotic cell death. Moreover, the glycerol content of NB4 and differentiated NB4 cells correlated with the level of As₂O₃ uptake and the sensitivity of the cells. Additionally, NB4 cells accumulated more As₂O₃ than did non-APL cells and were generally more sensitive to the drug. The investigators concluded that the selectivity of As₂O₃ for APL cells relates, at least in part, to impaired osmoregulation and control of drug uptake.

Efficacy of As₂O₃ in newly diagnosed APL

The standard of care for newly diagnosed APL is differentiation therapy with ATRA plus anthracycline-based chemotherapy.⁴⁰ This combination results in high response rates and prolonged survival (Table 1).^11–13 Incorporating As₂O₃ into the initial treatment of APL may further reduce the relapse rate and provide a more tolerable treatment option for patients who are not candidates for chemotherapy.

As₂O₃ as induction therapy

As₂O₃ has demonstrated clinical activity in patients with newly diagnosed APL, producing CR in 83–86% of patients and 3-year OS rates of 79–86% (Table 2).^41,42 Demonstrated efficacy, combined with evidence of synergy between As₂O₃ and ATRA, has fueled interest in combination therapy. A small randomized study in China evaluated the efficacy and safety of induction with ATRA, As₂O₃, or ATRA plus As₂O₃, followed by consolidation with three courses of conventional chemotherapy (Table 2).⁴³ Response rates were similar between treatment groups; however, relapse was significantly less frequent in patients receiving combination therapy as compared to either agent alone (P = .038). This improved response translated into a 2-year disease-free survival (DFS) advantage. After completion of the randomized phase of the study, the ATRA-plus-As₂O₃ arm remained open and long-term efficacy and safety data were collected (Table 2).⁴⁴ Among 80 patients who achieved CR, four (5%) had relapsed after a median follow-up of 70 months; all four were central nervous system (CNS) relapses.

Table 2.

Clinical studies of arsenic trioxide in first-line therapy for APL^a

Study	N	Regimen	CR	EFS	DFS (or RFS)	OS
As2O3 in induction
Mathews et al.41	72	I: As2O3 (daily until CR or 60 days)	86%	75%, 3-yr	87%, 3-yr	86%, 3-yr
		C: As2O3 (daily for 4 wks)
		M: As2O3 10 d per mo for 6 mos
Ghavamzadeh et al.42	193	I: As2O3 (daily until CR or 60 days)	83%	NR	69%, 3-yr	79%, 3-yr
		C and M: 1 or 4 courses As2O3 (daily for 4 wks)
Shen et al.43	61	I: (A) ATRA or (B) As2O3 or (C) ATRA + As2O3 until CR	A: 95% B: 90% C: 95%	NR	A: 68%, 2-yr B: 89%, 2-yr C: 100%, 2-yr	NR
		C: (all patients) 3 courses CTb
		M: (A) ATRA or (B) As2O3 or (C) ATRA then As2O3 followed    by low-dose CT (MCP or MTX) for 5 cycles
Hu et al.44	85	I: ATRA + As2O3 (daily until CR)	94%	89%, 5-yr	95%, 5-yr (RFS)	92%, 5-yr
		C: 3 courses CTb
		M: ATRA then As2O3 followed by low-dose CT (MCP or    MTX) for 5 cycles
Ravandi et al.45	82	I: ATRA + As2O3 (+ GO in high-risk patients) until BM    remission or 85 days	91% (CR/CRi)	83%, 5-yr	NR	84%, 5-yr
		C and M: 7 cycles ATRA + 4 cycles As2O3
As2O3 in consolidation
Powell et al.46 (C9710)	481	I: ATRA + CT (DNR + ara-C)	A: 89% B: 89%	A: 81%, 3-yr B: 66%, 3-yr P = .0007	NR	A: 86%, 3-yr B: 79%,3-yr P = .063
		C: (randomized) (A) 2 courses As2O3, then 2 courses ATRA +    CT (DNR) or (B) 2 courses ATRA + CT (DNR)
		M: (randomized) ATRA ± low-dose CT (MCP + MTX)
Gore et al.47	45	I: ATRA + CT (DNR) for 60 days	91%	76%, 2.7-yr	90%, 2.7-yr	88%, 2.7-yr
		C: 1 course As2O3 + CT (DNR + ara-C)
		M: ATRA (low/intermediate-risk patients) or ATRA + low-    dose CT (MCP + MTX) (high-risk patients)

Abbreviations: APL, acute promyelocytic leukemia; As₂O₃, arsenic trioxide; ATRA, all-trans-retinoic acid; ara-C, cytarabine; BM, bone marrow; C, consolidation therapy; CR, complete response; CRi, complete response without full platelet recovery; CT, chemotherapy; DFS, disease-free survival; DNR, daunorubicin; EFS, event-free survival; GO, gemtuzumab ozogamicin; I, induction therapy; M, maintenance therapy; MCP, mercaptopurine; MTX, methotrexate; NR, not reported; OS, overall survival; RFS, relapse-free survival.

^aAll patients were newly diagnosed with APL.

^bEach course consisted of 3 consecutive regimens: DA (DNA + ara-C), ara-C pulse, HA (homogarringtonine + ara-C).

A US phase 2 trial examined whether combination therapy with ATRA and As₂O₃ could replace anthracycline-based chemotherapy for newly diagnosed APL.⁴⁵ Low-risk patients (white blood cell [WBC] count <10 × 10⁹/L) received ATRA plus As₂O₃ as induction and post-remission therapy; high-risk patients (WBC count ≥10 × 10⁹/L) also received a single dose of gemtuzumab ozogamicin. It was hypothesized that this approach might be advantageous for patients who are unable to tolerate conventional chemotherapy, particularly elderly patients. Outcomes were similar to those observed with standard ATRA and chemotherapy combinations (Table 2). The overall response rate was 92% and responses were durable. After a median follow-up period of 23 months, relapse was documented in three (4%) of 75 responders, all of whom had high-risk disease. In patients aged 60 years and older, a CR rate of 83% was observed, compared with 95% in patients younger than 60 years (P = .17).

As₂O₃ in consolidation

The only phase 3 trial of As₂O₃ (the North American Intergroup protocol C9710, presented to date only in abstract form) evaluated the addition of As₂O₃ in first CR prior to standard consolidation therapy for newly diagnosed 537 eligible adults and pediatrics patients with APL.⁴⁶ This study demonstrated that administration of As₂O₃ (0.15 mg/kg/d for 5 days each week for 5 week for two cycles, cycle 2 after two weeks rest), as the first consolidation, prior to subsequent consolidation with ATRA (45 mg/m² × 7d) and chemotherapy (daunorubicin 50 mg/m² × 3d; 2d for age < 15 yr) significantly improved event-free survival (EFS) (81% vs 66%, p=0.0007) in adults compared to consolidation with ATRA and chemotherapy only. Three-year OS was higher in the As₂O₃ group, albeit not statistically significant (86% vs 79%, p=0.063). These improvements were presumably due to a decrease in the relapse rate, although DFS has not yet been reported. It is noteworthy to mention that in this study patients who did not receive As₂O₃ appear to have had lower EFS and OS than historical controls treated with ATRA and chemotherapy; indeed, the survival rate in the As2O3 arm was similar to the best published data using ATRA plus chemotherapy. Full analysis of this critical study will require publication of the final manuscript.

Based on the preliminary report, there was remarkably no significant difference in DFS between patients with WBC count greater or less than 10,000/mL in As₂O₃ group. On the other hand, patients with WBC > 10,000/mL, who did not receive As₂O₃ had significantly worse DFS compared with patients with WBC < 10,000/mL (p=0.0016). This finding suggests a major advantage of As₂O₃-based consolidation compared to non-As₂O₃ containing regimens in which patients with high WBC are much more likely to relapse. There were no differences in grade 3 or 4 hematologic or non-hematologic toxicities between the two groups.

A recent phase 2 trial in the US assessed whether the incorporation As₂O₃ into consolidation therapy would allow a reduction in chemotherapy exposure without compromising patient outcomes.⁴⁷ Enrolled patients (45 analyzed) received a single consolidation cycle with As₂O₃ (0.15 mg/kg/day, Monday – Friday, beginning on day 8, for 30 doses)_, daunorubicin (60 mg/m²/day days 1–3), and cytarabine (0.667 mg/m²/day continuous infusion days 1–3) after achieving CR with ATRA plus chemotherapy. Survival outcomes (EFS, DFS, and OS) were comparable to other treatment regimens that included more extensive chemotherapy, including the As₂O₃ treatment arm of the C9710 phase 3 trial (Table 2). Of 37 patients who received consolidation therapy, only one (3%) patient suffered a relapse (in the CNS) after a median follow-up of 1.8 years. Thus, OS was 88% ± 5% and leukemia-free survival was 90 ± 6%. Secondary myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) were not reported, although the longest follow-up in this study is only 5.5 years and median follow-up 2.7 years. These results indicate that a reduction in anthracycline exposure and associated toxicities may be possible while maintaining a low incidence of relapse.

Based on the current data from the As₂O₃-based clinical trials, which indicate that first-line arsenic trioxide therapy markedly decreases the incidence of relapse in de novo APL, and high DFS can be achieved with much lower doses of anthracycline than currently being used, we strongly recommend the routine incorporation of As₂O₃ into the first-line consolidation therapy of de novo APL.

As₂O₃ in maintenance

In the study by Soignet et al ¹⁰, those patients who remained in CR after receiving their consolidation course of As₂O₃ were given the option to receive up to four additional cycles of As₂O₃ therapy on a dose schedule similar to consolidation on a separate protocol. Eighteen patients received additional As₂O₃ as maintenance. Nevertheless, to date there is no published randomized trial evaluating the effect of As₂O₃ in maintenance setting. Meanwhile, until further studies are designed and completed, maintenance therapy for 1 to 2 years with intermittent ATRA with or without 6-mercaptopurine and methotrexate is recommended by us and many others, particularly in patients with high-risk disease.

Efficacy of As₂O₃ in other malignancies

Multiple myeloma (MM)

In preclinical studies, As₂O₃ inhibited the proliferation of MM cell lines at pharmacological (micromolar) concentrations.^31,48 As₂O₃ treatment of cultured bone marrow mononuclear cells from MM patients selectively induced apoptosis in myeloma cells while sparing most myeloid cells.⁴⁸ Initial clinical studies of As₂O₃ as a single agent in heavily pretreated relapsed or refractory patients demonstrated a ≥25% reduction in serum levels of M-protein in 21–33% of patients, with durations of response ranging from 1 to 74+ weeks.^49–51 Disease stabilization occurred in an additional 7–40% of patients. This modest clinical efficacy of As₂O₃ as a single agent prompted trials in combination with other therapies.

As discussed in the mechanism of action section, sensitivity to As₂O₃ in vitro is inversely proportional to cellular levels of the antioxidant GSH, which can attenuate the effects of increased ROS.³⁵ Pharmacological depletion of GSH by ascorbic acid (AA) enhances the cytotoxic effects of As₂O₃ in MM cell lines, providing a rationale for combination therapy.⁵² A phase 1 trial demonstrated that As₂O₃ plus AA was tolerable and demonstrated potential clinical activity (two of six patients had partial responses [PR]).⁵³ Further clinical studies are necessary to determine the efficacy of this combination, particularly in light of more recent in vitro evidence suggesting that AA may protect cells against arsenic cytotoxicity.⁵⁴

Several trials have evaluated As₂O₃ in combination with existing MM therapies, including melphalan, dexamethasone, and bortezomib, in relapsed patients. In a small study of 10 patients treated with As₂O₃, ascorbic acid, and melphalan (MAC), a response (≥25% reduction in serum M-protein) was observed in all patients, with treatment durations of 13–104 weeks.⁵⁵ Notably, six of these 10 patients had received melphalan previously: four as conditioning for stem cell transplantation (SCT) and two as part of a melphalan-prednisone regimen during which disease progression occurred. In a subsequent phase 2 study, MAC treatment produced a response in 31 (48%) of 65 patients who had failed at least two prior regimens; two CR, 15 PR, and 14 minor responses were observed.⁵⁶ Median progression-free survival (PFS) was 7 months and median OS was 19 months. In another randomized phase 2 trial, the addition of As₂O₃ to AA and high-dose melphalan was safe and well tolerated as a preparative regimen for autologous hematopoietic stem cell transplantation.⁵⁷ This study was not powered to detect differences in efficacy between arms; overall, a response was observed in 41 (85%) of 48 patients including 12 (25%) CR, with a median PFS of 25 months. The combination of As₂O₃, AA, and dexamethasone has demonstrated efficacy in patients with relapsed or refractory MM in two phase 2 trials. In a US study, six (30%) of 20 patients achieved a PR, including two near-CR (>90% decrease in M protein); 80% of patients demonstrated at least stable disease.⁵⁸ Similarly, a European trial showed a response in eight (40%) of 20 patients, including two PR.⁵⁹ Median PFS was 4 months and median OS was 11 months. The combination of As₂O₃, AA, and the proteasome inhibitor bortezomib was examined in a phase 2 trial for relapsed MM patients, following a report of in vitro synergy.^60,61 Six (27%) of 22 patients showed a clinical response to the combination, including two PR; an additional nine (41%) patients had stable disease. Median PFS was 5 months, with 1-year PFS and OS rates of 34% and 74%, respectively. The specific contribution of As₂O₃ to the efficacy of these combination therapies cannot be isolated without randomized trials.

Myelodysplastic syndromes (MDS)

Several in vitro studies have demonstrated apoptosis in MDS cells exposed to As₂O₃. ^27,62,63 MDS cells are under increased oxidative stress,⁶⁴ which may confer As₂O₃ sensitivity. In two phase 2 clinical studies involving 191 patients, single-agent As₂O₃ was associated with hematologic improvement in 26–34% of patients with lower-risk (low or intermediate-1 international prognostic scoring system [IPSS] risk groups) MDS and 6–17% of those with higher-risk (intermediate-2 or high IPSS risk groups) MDS.^65,66 Schiller et al. observed one (3%) CR and Vey et al. observed one (<1%) CR and one (<1%) PR in higher-risk patients. Major responses were observed in all hematologic lineages in both trials.

As₂O₃ plus thalidomide was investigated based on the hypothesis that this combination would target both the MDS clone and the bone marrow microenvironment. This combination was evaluated in 28 patients with MDS (12 lower-risk, 16 higher-risk).⁶⁷ Seven patients (25%) responded, including one CR and two trilineage responses. Three of five patients with high baseline levels of the poor prognostic marker EVI1 responded to the combination; this observation was supported by experiments in vitro, which demonstrated greater As₂O₃ sensitivity in cells expressing high levels of EVI1. A subsequent trial of As₂O₃, thalidomide, and retinoic acid in 21 higher-risk patients demonstrated responses in 10 patients (48%), including one CR and one PR.⁶⁸

Other hematologic malignancies

Several trials have demonstrated promising results in AML and human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia-lymphoma (ATL). A phase 1/2 trial examined As₂O₃ in combination with low-dose cytarabine in 61 previously untreated AML patients aged 60 years or older.⁶⁹ A CR was achieved in 21 patients (34%), including 15 (30%) of 50 patients with secondary AML. Median survival was 5.2 months, with a median follow-up of 9.8 months. A randomized phase 3 trial of this combination versus low-dose cytarabine alone is under way (www.clinicaltrials.gov). For relapsed/refractory ATL patients, As₂O₃ in combination with interferon alpha (IFNα) was examined in a small (N = 7) phase 2 trial.⁷⁰ Despite significant toxicity that prevented completion of therapy, one patient achieved a CR lasting more than 32 months, and three patients achieved transient PR (median duration 1 month). In a separate report involving four patients with relapsed/refractory ATL, As₂O₃ plus IFNα produced one PR (duration of 8 months) and one stable disease, while As₂O₃ alone produced no response.⁷¹ In a recent phase 2 trial of As₂O₃ and IFNα plus zidovudine in 10 newly diagnosed ATL patients, a 100% response rate was observed, including seven CR.⁷² Most toxicities were grade 1 or 2; grade 3 neutropenia occurred in 3 patients and grade 3 thrombocytopenia was observed in 2 patients. As₂O₃ has demonstrated limited activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia,⁷³ and no clinical benefit in patients with acute lymphoblastic leukemia.⁷⁴

Solid tumors

As₂O₃ is under investigation as treatment for a variety of solid tumors including bladder cancer, glioma, breast cancer, hepatocellular carcinoma (HCC), cervical cancer, colorectal cancer, esophageal cancer, germ cell tumors, liver cancer, lung cancer, and melanoma (www.clinicaltrials.gov). Limited clinical activity as a single agent has been reported in a small number of patients with HCC,⁷⁵ melanoma,^76,77 and renal cell carcinoma⁷⁸; As₂O₃ in combination with chemotherapy has shown promising activity in osteosarcoma and Ewing sarcoma.⁷⁹

Safety and toxicity

Arsenic is well known as a toxic agent. Inorganic arsenic has been classified by the US Department of Health and Human Services, the International Agency for Research on Cancer, and the US Environmental Protection Agency as a known carcinogen. Chronic exposure to low levels of environmental arsenic has been reported to increase the incidence of skin, liver, bladder, and lung cancers.⁸⁰ Other potential signs of arsenic poisoning include peripheral neuropathy, cardiomyopathy, and renal failure.⁸¹

Despite its reputation as a poison, as a therapeutic entity As₂O₃ has been generally well tolerated. When administered intravenously at a dosage of 0.15 mg/kg/day, leukocytosis, gastrointestinal disorders (e.g., nausea, vomiting, diarrhea), fatigue, fever, headache, cough, and dyspnea are commonly observed. Common potentially serious toxicities include APL differentiation syndrome (APLDS) and electrocardiogram (ECG) abnormalities.⁸²

APL differentiation syndrome

Previously known as “retinoic-acid syndrome,” APLDS may present during remission induction with ATRA or As₂O₃ therapies as a complex of signs and symptoms, including fever, dyspnea, hypotension, weight gain, acute renal failure, and lung infiltrates, and is usually treated with high-dose corticosteroids.⁸³ In the pivotal trial of As₂O₃ in APL, APLDS occurred in 10 (25%) patients; in three of these patients, APLDS was considered to be serious.¹⁰ Therapy with As₂O₃ was briefly interrupted (for 1–5 days) in eight patients. Notably, all patients affected by APLDS achieved a CR. A similar incidence and severity of APLDS was reported with combined ATRA and As₂O₃; in the M. D. Anderson phase 2 trial, 13 patients (16%) developed APLDS, and all cases were successfully managed by withholding ATRA and administering corticosteroids.⁴⁵ In a study comparing As₂O₃, ATRA, and the combination, the incidence of APLDS-associated hyperleukocytosis was not increased with As₂O₃ plus ATRA; however, one death in the combination arm was attributed to APLDS.^43,44

Cardiac events

ECG abnormalities, including prolonged QT interval and complete atrioventricular block, have been reported with As₂O₃ treatment. QT prolongation (defined as ≥450 msec for males and ≥470 msec for females) was seen in 63% of patients in the pivotal trial and led to temporary discontinuation of As₂O₃ therapy in one patient (3%).¹⁰ In the phase 2 study by Ravandi et al., As₂O₃ was discontinued in five patients (6%) due to adverse cardiac events including atrial arrhythmias and myocardial infarction.⁴⁵ ECG and electrolyte monitoring is recommended prior to and during arsenic therapy. Serum potassium levels should be kept above 4 meq/L and magnesium concentrations above 1.8 mg/dL.

Liver and kidney impairment

No significant hepatotoxicity was reported in the pivotal trial in relapsed patients.¹⁰ Hu et al. reported transient grade 1 or 2 liver dysfunction in 75% of patients during induction with ATRA and As₂O₃; no grade 3 or 4 toxicity was observed, and treatment was not discontinued in any patient.⁴⁴ Ravandi et al. noted grade 3 elevations in liver enzymes in two of 82 patients (2%), but treatment was not discontinued.⁴⁵ Grade 3 renal failure occurred in one (3%) patient during the pivotal trial¹⁰ and was reported in four (5%) patients by Ravandi et al.⁴⁵

Secondary malignancies

Carcinogenesis is a major concern associated with long-term exposure to arsenic. In the study published in December 2008 in the Proceedings of the National Academy of Sciences (PNAS) on long-term efficacy and safety of ATRA/As₂O₃-based therapy in newly diagnosed APL patients who started being accrued to the trial in 2001⁴⁴, no secondary carcinoma, including skin cancer, was observed. One male transiently tested positive for carcinoembryogenic antigen (CEA), and a mild, unsustained increase in CA125 in a female patient was recorded. Moreover, arsenic concentrations in the urine of patients who had ceased As₂O₃ treatment for 24 months were below the safety limits recommended by government agencies in several countries or regions.

Secondary malignancies were not reported in the short-term trials US registration trial¹⁰ or the phase 2 trial of As₂O₃ plus ATRA.⁴⁵ Reduced exposure to or avoidance of chemotherapy in As₂O₃ patients may reduce the risk of secondary MDS and AML, which have been previously associated with APL treatment.⁸⁴

Arsenic retention and long-term toxicity

The trial by Hu et al. included extensive screening of APL patients for long-term effects of arsenic exposure following completion of therapy.⁴⁴ Laboratory studies (blood counts, electrolyte panels, urinalysis, liver function tests, and serum tumor marker analysis) and physical examinations (ECGs, echocardiograms, chest x-rays, dermatologic and neurologic consults, and nerve conduction velocity tests) were performed in 33 patients during therapy and at a minimum of 2 years after receiving the last dose of As₂O₃. At the final follow-up, physical exam and laboratory results were similar between patients and healthy controls. No tumors or skin lesions were detected. ECGs, echocardiograms, and chest x-rays were normal. Toxicology analyses in these patients found that As₂O₃ levels in plasma and urine dropped after termination of As₂O₃ treatment to levels only slightly higher than those in healthy controls, and were within recommended US safety limits.

Pharmacokinetics

In a study of 12 Japanese patients receiving As₂O₃ (0.15 mg/kg IV over 2 hours), the mean maximum plasma concentration of inorganic arsenic (As^III and As^V) of 22.6 ± 11.4 ng/mL occurred at completion of the infusion, then declined biphasically, with an initial distribution phase followed by an elimination phase with a terminal half-life of 17 hours.⁸⁵ The volume of distribution was large (55.9 L/kg), indicating extensive distribution throughout the body. In a separate case report of a patient with relapsed APL and meningeal infiltration, As₂O₃ was observed to cross the blood-brain barrier when administered intravenously; arsenic levels in cerebrospinal fluid were approximately 14% of levels in blood.⁸⁶

The major arsenic metabolites identified in humans are the methylated arsenics, methylarsonic acid (MAA^V) and dimethylarsinic acid (DMAA^V).^87–89 In the Japanese study, plasma levels of MAA^V and DMAA^V increased gradually over 24 hours after the first administration of As₂O₃, reaching peak levels of 3.1 ± 1.6 ng/mL and 5.4 ± 2.9 ng/mL, respectively.⁸⁵ The contribution of these metabolites to the therapeutic effect of As₂O₃ is unknown. In APL, chronic lymphocytic leukemia, and leukemia/lymphoma cell lines, methylated arsenic derivates were shown to induce apoptosis, though they did not induce differentiation in APL cells.⁹⁰ Plasma concentrations of inorganic arsenic did not increase with repeated administration; however, the concentrations of both major metabolites were approximately four times higher during week 4 of treatment compared with day 1.⁸⁵ Arsenic content in hair and nails also increased gradually with repeated treatment and reached levels 5–7 times higher than pretreatment levels.⁸

Approximately 60% of the daily dose of arsenic is excreted in the urine, including inorganic and methylated arsenic; other pathways of excretion, such as biliary, may also contribute to As₂O₃ elimination.^85,87

Conclusions

As₂O₃ has demonstrated remarkable efficacy in APL. The use of As₂O₃ in induction or consolidation strategies reduces the relapse rate and improves survival in patients with APL, especially in high-risk patients.^43,44,46 The addition of As₂O₃ to ATRA/chemotherapy regimens may allow a reduction in chemotherapy exposure and associated toxicities without compromising cure rates.⁴⁷ In select patients, chemotherapy may be eliminated altogether, providing an alternative treatment option for patients who cannot tolerate anthracyclines.⁴⁵ Although As₂O₃ has demonstrated less robust efficacy in other malignancies, promising activity has been demonstrated when As₂O₃ is combined with other agents. As₂O₃ is generally well tolerated, both as a single agent and in combination, with manageable adverse effects and no documented evidence of long-term toxicities. Additional comparative trials will be necessary to determine which combinations of available therapies provide the greatest benefit with the least toxicity in patients with APL.

Practice Points

At the Sidney Kimmel Comprehensive Cancer Center (SKCCC) at Johns Hopkins University, patients with APL are treated as follows:

• Whenever possible, they are enrolled in clinical trials.

• Outside of clinical trials, patients should be treated according to a published treatment protocol, from induction through maintenance and post-maintenance monitoring.

• Remission can be achieved with ATRA plus an anthracycline. The use of 60 days of ATRA (rather than administration of ATRA until normalization of hemogram) may lead to a higher level of PML/RARα molecular negativity.

• If WBC ≥20 × 10⁹/L, hydroxyurea is added to maintain WBC <20 × 10⁹/L, with dexamethasone (10 mg IV, twice daily for 14 days) as prophylaxis against APLDS.

• At SKCCC, we believe that an As₂O₃-containing post-remission (i.e., consolidation) chemotherapy regimen results in high relapse-free and overall survivals (Table 2).

• In patients unable to tolerate anthracyclines, remission can be induced with ATRA plus As₂O₃ daily until absence of leukemia in bone marrow is documented, or for a maximum of 60 days.

• Maintenance therapy appears important and should be completed according to the elected treatment protocol.

• Treatment of relapsed disease (determined by two consecutive positive PML/RARα findings by qualitative RT-PCR within 4 weeks) should be individualized per patient.

Research Agenda

• There are 87 recently completed or ongoing clinical trials listed on www.clinicaltrials.gov evaluating As₂O₃ alone or in combination with other agents for treatment of APL, AML, multiple myeloma, MDS, primary myelofibrosis, hepatocellular carcinoma, metastatic melanoma, CNS tumors, and breast, lung, colorectal, and kidney cancers.

• In APL, the role of chemotherapeutic CNS prophylaxis, in all patients or high-risk patients, needs to be addressed in clinical trials.

• In APL, the role of maintenance therapy, particularly in patients who are treated with As₂O₃, also needs to be addressed by future research.