Old dog, new trick: Trivalent arsenic as an immunomodulatory drug

Abstract

Trivalent arsenic (As(III)) is recently found to be an immunomodulatory agent. As(III) has therapeutic potential in several autoimmune and inflammatory diseases in vivo. In vitro, it selectively induces apoptosis of immune cells due to different sensitivity. At a non‐toxic level, As(III) shows its multifaceted nature by inducing either pro‐ or anti‐inflammatory functions of immune subsets. These effects are exerted by either As(III)–protein interactions or as a consequence of As(III)‐induced homeostasis imbalance. The immunomodulatory properties also show synergistic effects of As(III) with cancer immunotherapy. In this review, we summarize the immunomodulatory effects of As(III), focusing on the effects of As(III) on immune subsets in vitro, on mouse models of immune‐related diseases, and the role of As(III) in cancer immunotherapy. Updates of the mechanisms of action, the pioneer clinical trials, dosing, and adverse events of therapeutic As(III) are also provided.

Abbreviations

APL

acute promyelocytic leukaemia

As(III)

trivalent arsenic

ATL

adult T‐cell leukaemia/lymphoma

ATRA

all‐trans retinoic acid autoimmune disease

cDC

conventional dendritic cells

CIK

cytokine‐induced killer

iAs

inorganic arsenic compounds

LAK

lymphokine‐activated killer

MDDCs

monocyte‐derived dendritic cells

Nrf2

nuclear factor erythroid 2‐related factor 2

pDCs

plasmacytoid dendritic cells

PML/RARα

promyelocytic leukaemia‐retinoic acid receptor‐α

SLE

systemic lupus erythematosus

1. INTRODUCTION

Inorganic arsenic compounds (iAs) have been used in traditional Chinese and Western medicine for over 2400 years (Emadi & Gore, 2010). Arsenic trioxide (As₂O₃) was rediscovered in the 1970s in the treatment of acute promyelocytic leukaemia (APL) with striking efficacy and good safety profile (Wang & Chen, 2008 ). So far, As₂O_3, together with all‐trans retinoic acid (ATRA), has revolutionized the treatment of APL: The outcome changed from 35% to 45% long‐term overall survival by chemotherapy to a complete remission rate of over 95% and a long‐term survival rate up to 90% with a chemotherapy free approach (Cicconi & Lo‐Coco, 2016; Lo‐Coco et al., 2013). Furthermore, As₂O₃ has also shown promising results in many other malignancies such as adult T‐cell leukaemia/lymphoma (ATL), and NPM1 mutant acute myeloid leukaemia (El Hajj et al., 2015; Kchour et al., 2009).

Modulation of immunity by iAs has been studied and put in clinical use with trivalent arsenicals (As(III)) including As₂O₃ and sodium arsenite (NaAsO₂). As(III) has high affinity for sulfhydryl groups and can bind to cysteine residues in peptides and proteins, leading to protein conformational change and malfunction (Shen, Li, Cullen, Weinfeld, & Le, 2013). As(III) is also a potent ROS inducer (Chou et al., 2004). Moreover, many key proteins are indirectly regulated by As(III) through post‐translational regulations such as sumoylation (Lallemand‐Breitenbach et al., 2001; mechanisms reviewed by Shen et al., 2013).

The iAs are regarded as having general toxic effects on the immune system (Dangleben, Skibola, & Smith, 2013). However, in vitro studies have provided a more detailed picture, revealing the multifaceted effects of As(III) on different immune subsets (Baysan, Yel, Gollapudi, Su, & Gupta, 2007; Gupta et al., 2003). Recent reports on animal models have shown the efficacy of As(III) on several autoimmune and inflammatory diseases (Bobe, Bonardelle, Benihoud, Opolon, & Chelbi‐Alix, 2006; Kavian, Marut, Servettaz, Laude, et al., 2012). In addition, in the era of cancer immunotherapy, emerging evidence indicates As(III) as a promising immunoadjuvant for haematological malignancies and solid tumours (Deaglio et al., 2001; Thomas‐Schoemann et al., 2012). However, during As₂O₃ treatment, several typical adverse events including cardiac and liver toxicities have been reported (Sanz et al., 2019). These adverse events, even though manageable, must be taken into account by medical practitioners.

In this review, in order to promote the better use of this double‐edged sword in immunotherapy, we summarize the mechanisms of action of As(III), the modulation on each immune cell subset, the use of As(III) on preclinical mouse models of immune‐mediated diseases, and the pioneer studies of As(III) as an adjuvant for tumour immunotherapy. We also summarize the standard dosing and therapeutic schedules of As(III) and highlight the possible short‐ and long‐term adverse events that require vigilance by clinicians.

2. PHARMACOLOGICAL MECHANISMS OF ACTION

2.1. As(III) biochemistry

Both As₂O₃ and NaAsO₂ transform to As(OH)₃ in aqueous solution. After being taken up by cells through the https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=119#696 transmembrane protein, As(III) exerts its biochemical effects intracellularly (Leung, Pang, Yuen, Kwong, & Tse, 2007). Interaction with the thiol (or sulfhydryl) groups (–SH) of proteins with a high cysteine content constitutes the basic biochemical reaction of As(III) (Emadi & Gore, 2010), which alters protein conformation, resulting in loss of their function, and affects their recruitment and interaction with other proteins and DNA (Shen et al., 2013). Apart from the direct arsenic‐protein binding, recent studies revealed that many key proteins are modulated through more complicated post‐translational stepwise regulations (de Thé, 2018).

2.2. PML and PML nuclear body regulation

It was initially found that the APL promoter, promyelocytic leukaemia‐retinoic acid receptor‐α (PML/https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=590) was especially sensitive to As₂O₃‐induced degradation, which was the critical step of disease eradication (Chen et al., 1996). The subsequent comprehensive studies by Lallemand‐Breitenbach and de Thé (2018) revealed that As₂O₃ specifically targets the PML moiety. The PML protein forms nuclear bodies (NBs),which recruit and sumoylate dozens of partner proteins, leading to a variety of biological processes (Lallemand‐Breitenbach et al., 2001; Sahin et al., 2014). Notably, As₂O₃‐induced oxidative stress could enhance formation of PML NBs, leading to p53 activation in vivo in normal mice (Niwa‐Kawakita et al., 2017).

In APL, through both ROS production and direct binding, As₂O₃ exerts its dual‐targeting effects (Jeanne et al., 2010). On one hand, As₂O₃ induces PML/RARα sumoylation, proteasomal degradation, and APL cell differentiation (Fasci, Anania, Lill, & Salvesen, 2015; Lallemand‐Breitenbach & de Thé, 2018). On the other hand, As₂O₃ targets the wild‐type PML proteins, leading to re‐formation of PML NBs, subsequent p53 activation, and ultimately APL clearance (Ablain et al., 2014; Niwa‐Kawakita et al., 2017). Also, in ATL, As₂O₃, together with https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=957 induced complete remission of disease, both in mice and in humans, through degradation of the disease driver oncoprotein Tax (El Hajj et al., 2010; El‐Sabban et al., 2000; Kchour et al., 2009). Interestingly, this process was also mediated through a As₂O₃ enforcement of PML NBs formation, Tax sumoylation, and proteasomal degradation (Dassouki et al., 2015). In addition, the As₂O₃/ATRA combination significantly decreased NPM1‐mutant acute myeloid leukaemia blasts in patients though oxidative stress generation, p53 activation, and ultimately mutant NPM1 degradation (El Hajj et al., 2015; Martelli et al., 2015).

Apart from tumour suppression, PML and PML NBs were recently found to play key role in mediating innate immune responses (Hsu & Kao, 2018; Lallemand‐Breitenbach et al., 2001; Scherer & Stamminger, 2016). Studies have identified PML as a direct, positive regulator of IFN type I signalling (Kim & Ahn, 2015) and is implicated in the regulation of and extended spectrum of cytokines such as the pro‐inflammatory https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4974 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4998 (Lo et al., 2013; Scherer & Stamminger, 2016). Thus, it is important to find out if modulation of PML and PML NBs could explain the effectiveness of As₂O₃ in autoimmune and inflammatory diseases with a type I IFN signature and abnormal cytokine profile.

2.3. Apoptosis induction

As(III) can induce immune cell apoptosis through both the mitochondrial‐mediated and the receptor‐mediated pathways (Gupta et al., 2003; Yu, Chen, Liao, Chang, & Yu, 2002). The pro‐apoptotic mechanisms include generation of oxidative stress, caspase activation, alteration of the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2844 family proteins, and up‐regulation/down‐regulation of several survival‐related signalling pathways such as NF‐κB, https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=289/https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=519, and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1870 apoptotic signalling pathways (Gupta et al., 2003; Lemarie, Morzadec, Merino et al., 2006; Lemarie, Morzadec, Bourdonnay, Fardel, & Vernhet, 2006; Yu et al., 2002). Within its therapeutic range, As(III) is able to induce apoptosis of specific types of sensitive immune cells (Bobe et al., 2006; Thomas‐Schoemann et al., 2012). In a mouse model of colon cancer, regulatory T cells are preferentially depleted, leading to immunomodulatory effects (Thomas‐Schoemann et al., 2012). The in vitro EC₅₀ of As(III) on different immune subsets and the possible mechanisms of apoptosis induction are summarized in Table 1.

TABLE 1.

Trivalent arsenic induces immune cell apoptosis: sensitivity and mechanisms of action

Cell subtype	Species	Type of As(III)	EC50 (culture time)	Possible mechanism	References
PBMC	Human	NaAsO2	5 μmol·L−1 (48 and 72 hr)	N/A	(Yu et al., 2002)
Neutrophil	Human	As2O3	<5 μmol·L−1 (22 hr)	H2O2 generation Activation of caspases De novo protein synthesis Syk kinase activation	(Antoine et al., 2010; Binet et al., 2006)
Monocyte
Monocyte (during macrophagic differentiation with GM‐CSF or M‐CSF)	Human	As2O3	~1 μmol·L−1 (6 days)	Inhibition of NF‐κB‐related survival pathways	(Lemarie, Morzadec, Merino, et al., 2006)
Promonocytic U937 cells	Human	As2O3	>4 μmol·L−1 (4 days)
Dendritic cell (DC)
Monocyte‐derived DCs	Human	NaAsO2	>2 μmol·L−1 (6 days)	N/A	(Macoch et al., 2013)
T cell
Primary CD4+ T cell	Human	As2O3	>5 μmol·L−1 (48 hr)	ROS generation	(Gupta et al., 2003)
Primary CD8+ T cell	Human	As2O3	>5 μmol·L−1 (48 hr)	2. Regulation of the Bcl‐2 family proteins
CD4+ T cell within PBMCs	Human	NaAsO2	N/A	TNF‐R1 apoptotic signalling	(Yu et al., 2002)
CD4+CD25+ Treg	Human	As2O3	~1 μmol·L−1 (38 h)	ROS and RNS accumulation High sensitivity to oxydative stress	(Thomas‐Schoemann et al., 2012)
CD4+CD25− effector T cell	Human	As2O3	~2.5 μmol·L−1 (38 hr)	N/A	(Thomas‐Schoemann et al., 2012)
B cell
Ramos cell line	Human	As2O3	>10 μmol·L−1 (24 hr)	ROS generation Up‐regulation of Bax and Bim expression	(Baysan et al., 2007)

Abbreviations: As₂O₃, arsenic trioxide; As(III), trivalent arsenic; EC₅₀, half maximal effective concentration; H₂O₂, hydrogen peroxide; M‐CSF, macrophage colony‐stimulating factor; NaAsO₂, sodium arsenite; PBMC, peripheral blood mononuclear cell; RNS, reactive nitrogen species.

2.4. ROS

ROS accumulation leads to oxidative stress, causing damage to nucleic acids, proteins, and lipids, which can lead to changes in cell signalling, and, ultimately, apoptosis (Schieber & Chandel, 2014). As(III) induces immune cell intracellular ROS accumulation via inhibition of antioxidants such as https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6737 GSH peroxidase, and GSH reductase (Gupta et al., 2003; Singh, Kumar, Lal, Raisuddin, & Sahu, 2010) and activation of enzymes generating ROS such as https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=993 (Lemarie, Bourdonnay, Morzadec, Fardel, & Vernhet, 2008). As a consequence, expressions of redox‐sensitive genes such as HMOX1, NQO1, and GCLM are up‐regulated (Bourdonnay, Morzadec, Fardel, & Vernhet, 2009). Especially, nuclear factor erythroid 2‐related factor 2 (Nrf2), a stress‐activated transcription factor responsible for inducing a battery of cytoprotective genes, is activated by As(III) within mouse splenocytes, human T cells, human primary macrophages, and human monocyte‐derived dendritic cells (MDDCs; Bourdonnay, Morzadec, Fardel, & Vernhet, 2009; Duan et al., 2017; Macoch et al., 2015; Morzadec et al., 2014). The Nrf2 modulation is probably due to As(III)‐induced Nrf2 sumoylation, recruitment to the PML NBs, and degradation by RNF4‐mediated proteolysis (Malloy et al., 2013).

2.5. Signalling pathway regulation

Due to the different affinity of different proteins to As(III), and the potential post‐translational modulation by other structures such as PML NBs, As(III) has the potential to selectively target specific signalling pathways. In macrophages, for example, non‐toxic concentrations of As₂O₃ induce up‐regulation of 32 and depression of 91 genes respectively, indicating a global multi‐directional change of the cell‐signalling (Bourdonnay et al., 2009). Moreover, As(III) may inactivate up to 200 enzymes, and many are crucial regulators of important signalling pathways (Shen et al., 2013). As(III) inhibits IĸB kinase, whose integrity is key to the activation of the NF‐κB pathway (Lemarie, Morzadec, Merino, et al., 2006). Other pathways affected include the Rho‐kinase/p38‐kinase pathway and https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=518 (Binet & Girard, 2008; Lemarie et al., 2008) which will be described in detail in the next section.

3. THE MULTIFACETED EFFECTS OF TRIVALENT ARSENIC ON IMMUNE CELLS

3.1. Systemic immunomodulation

Chronic arsenic exposure leads to systemic immunosuppression (Dangleben et al., 2013). However, clinical use of As₂O₃ has shown a good safety profile, with limited side effects (Shen et al., 1997). No long‐term, treatment‐related, immune‐mediated disease or tumourigenesis is reported. During the As₂O₃ single‐agent treatment for APL, there are inhibitory effects on haematopoietic progenitor cells, and it takes 145 and 265 days for circulating T and B cells and 655 days for NK cells to achieve the median normal levels (Alex et al., 2018). The detailed effects of As(III) on each immune subset are described in the following sections.

3.2. Trivalent arsenic and granulocytes

The multifaceted effects of As(III) on different subsets of immnuocytes are summarized in Figure 1. High concentrations of As₂O₃ induce apoptosis of human neutrophils via generation of H₂O₂, de novo protein synthesis, caspase activation, and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2230 activation (Antoine, Ennaciri, & Girard, 2010; Binet, Cavalli, Moisan, & Girard, 2006). The As₂O₃‐induced de novo synthesized proteins include annexin‐1 and heat shock proteins (Binet, Chiasson, & Girard, 2008). Moreover, As₂O₃ induces endoplasmic reticulum stress within human neutrophils, which elicits either self‐protective mechanisms via the activation of unfolded protein response (UPR) or cell apoptosis, independent of caspase‐4 activation (Binet, Chiasson, & Girard, 2010a; Binet, Chiasson, & Girard, 2010b). Importantly, As₂O₃ recruits the MAPKs, activates p38 and JNK, and ultimately enhances the major neutrophil functions including adhesion, migration, degranulation, and phagocytosis of opsonized sheep red blood cells (Binet & Girard, 2008). Syk activation also helps in this agonistic process (Antoine et al., 2010). In addition, NaAsO₂ promotes the formation of neutrophil extracellular traps, which play an important role in defence against pathogen infection (Wei et al., 2018). Moreover, in an acute As₂O₃‐exposed mouse model the neutrophil cell numbers were increased in the broncho‐alveolar lavage fluid (BALF; Li et al., 2017). Collectively, neutrophils are recruited and functionally enhanced during As(III) treatment, leading to a pro‐inflammatory response.

FIGURE 1.

Diverse functions of As(III) on immune cells. Compounds comprising trivalent arsenic (As(III)) can drive both promoting (top of figure in green) and suppressive (bottom of figure in blue) immune responses in different immune cell subsets

For eosinophils, in a mouse model of asthma, eosinophil recruitment and a reduction of chemotaxis level in BALF were detected after As₂O₃ treatment (Chu et al., 2010; Zhou et al., 2006). Therefore, eosinophil functions may be impaired during As(III) exposure. For basophils, the direct effects of As(III) have not yet been investigated. However, activation of basophils may be impaired in vivo during As(III) administration, because levels of IgE, an important driver of basophil activation, were decreased in the BALF of As₂O_3‐treated mice (Chu et al., 2010; Zhou et al., 2006). For mast cells, it was reported that NaAsO₂ inhibits anti‐IgE stimulated degranulation via suppression of early tyrosine phosphorylation (Hutchinson et al., 2011). Therefore, As(III) may impair mast cell activation, especially that during the process of allergy (Shim et al., 2016).

3.3. Trivalent arsenic and monocytes/macrophages

Recent studies have shown that monocytes are not simply macrophage precursors but can acquire inflammatory effector functions, distinct from those of macrophages. (Guilliams, Mildner, & Yona, 2018). As₂O₃ induces human monocyte apoptosis during macrophage differentiation through down‐regulation of the NF‐κB‐related pathway (Lemarie, Morzadec, Merino, et al., 2006). Moreover, As₂O₃ increases LPS‐dependent expression of the inflammatory IL‐8 gene, by stimulating a redox‐sensitive pathway that strengthens p38‐kinase activation (Bourdonnay, Morzadec, Fardel, & Vernhet, 2011).

For macrophages, high concentration of As₂O₃ induces cell apoptosis through a mitochondrial‐dependent pathway (Sengupta & Bishayi, 2002; Srivastava et al., 2016). Also, it changes human monocyte‐derived macrophage morphology, reduces their adhesive capacity, decreases macrophagic surface marker expressions and impairs phagocytosis of E. coli in vitro (Lemarie, Morzadec, Bourdonnay, et al., 2006). Interestingly, As₂O₃ potentiates macrophage secretion of inflammatory https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5074, IL‐1α, and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4382 and the induction of allogeneic or autologous T‐cell responses (Lemarie et al., 2008; Sakurai, Ohta, & Fujiwara, 2005). These in vitro observations were confirmed by ex vivo data (Banerjee et al., 2009; Bishayi & Sengupta, 2003). The functional changes are probably due to superoxide generation, activation of the Rho‐kinase/p38‐kinase pathway (Lemarie, Morzadec, Merino, et al., 2006), and modulation of the UPR signalling (Srivastava et al., 2013). Especially, activating transcription factor 4 protein, a UPR transcription factor, plays a key role in As₂O₃‐mediated regulation of macrophage functions (Srivastava et al., 2016). In addition, As₂O₃ also globally regulates redox‐sensitive gene expression in human macrophages (Bourdonnay, Morzadec, Fardel, & Vernhet, 2009; Bourdonnay, Morzadec, Sparfel, et al., 2009). Therefore, As(III) exerts dual effects on macrophages by impairing their clearance capacity, while enhancing their pro‐inflammatory functions.

3.4. Trivalent arsenic and dendritic cells

Two types of dendritic cells (DCs), namely, conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDCs), can be distinguished in vivo, even though MDDCs are widely used for research in vitro, because they are more readily available. NaAsO₂ inhibits dendritic differentiation of monocytes in vitro (Bahari & Salmani, 2017). It also decreases MDDCs viability, maturation, phagocytic capacity, and their ability to secrete the pro‐inflammatory cytokines https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4977 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4978 and to stimulate T helper (Th) cells to secrete https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4968 (Macoch, Morzadec, Fardel, & Vernhet, 2013). The inhibition of IL‐12 is mediated by the induced expression of Nrf2 (Malloy et al., 2013). Moreover, As₂O₃ inhibits IFN‐α secretion by murine pDCs (Kavian, Marut, Servettaz, Laude, et al., 2012).

3.5. Trivalent arsenic and T cells

T cells play a central role in https://en.wikipedia.org/wiki/Cell-mediated_immunity. High concentrations of As(III) induce apoptosis of T helper cells (CD4⁺ T cell), cytotoxic T cells (CD8⁺ T cell), and regulatory T cells (Treg; Gupta et al., 2003; Tenorio & Saavedra, 2005; Thomas‐Schoemann et al., 2012). Both mitochondrial‐mediated and the receptor‐mediated pathways are involved in T‐cell apoptosis. Gupta et al. (2003) revealed that As₂O₃ induces apoptosis of CD4⁺ and CD8⁺ T cells via the mitochondrial pathway by enhancing the generation of oxidative stress and by regulating the expression of Bcl‐2 family proteins. When CD4⁺ T cells were investigated within human peripheral blood mononuclear cells, TNF‐α released from other mononuclear cells after NaAsO₂ exposure induced CD4⁺ T‐cell apoptosis through TNFR1 apoptotic signalling pathway (Yu et al., 2002).

There is different sensitivity to As(III) toxicity among the T‐cell subsets. CD4⁺ are more sensitive than CD8⁺ T cells to the pro‐apoptotic toxicity of NaAsO₂, as demonstrated both in vitro and in vivo in human and mouse model (Duan et al., 2017; Vega, Montes de Oca, Saavedra, & Ostrosky‐Wegman, 2004; Yu et al., 2002). This difference leads to a reduced CD4⁺/CD8⁺ ratio, which is associated with immune dysfunction (Lu et al., 2015). Tregs are important CD4⁺ T cells mediating immune tolerance. in vitro tests show that As₂O_3, at the same concentration, preferentially induces apoptosis of human purified CD4⁺CD25⁺ Tregs than CD4⁺CD25⁻ effector T cells and decreases the Treg frequency in APL patients' peripheral blood (Xu et al., 2018). As₂O₃‐mediated selective Treg depletion was also reported in vivo in a mouse model (Thomas‐Schoemann et al., 2012). As₂O₃ induces Treg apoptosis through ROS and reactive nitrogen species generation_, and the differential effect of As₂O₃ on Treg versus other CD4⁺ cells may be related to differences in the cells' redox status (Thomas‐Schoemann et al., 2012). However, some other studies reported opposite results (Tohyama, Tanaka, Onda, Sugiyama, & Hirano, 2013; Zhao, Yang, et al., 2018). After exposure to NaAsO₂, the mRNA level of the Treg specific transcription factor Foxp3 is up‐regulated in the spleen and thymus of the treated mice (Duan et al., 2017). Interestingly, Tohyama et al. (2013) reported that in mitogen‐activated human peripheral blood mononuclear cell, 5‐μM As₂O₃ decreases the Treg frequency after treatment for 48 hr and, conversely, increases its frequency after 96 hr of culture. These results indicate that short‐term exposure to As(III) may deplete Treg, in contrast to long‐term exposure leading to Treg increase.

Upon activation, naïve CD4⁺ T cells proliferate and differentiate into fully functional effector T cells, which lead to either inflammatory responses or immune suppression (Wan & Flavell, 2009). It is reported that As(III) exposure inhibits the proliferation of both human‐ and murine‐activated T cells, by affecting the initial activation step of T‐cell receptor signalling, and by inhibiting IL‐2 expression, at both the protein and mRNA levels (Conde, Acosta‐Saavedra, Goytia‐Acevedo, & Calderon‐Aranda, 2007; Morzadec, Bouezzedine, Macoch, Fardel, & Vernhet, 2012; Soto‐Pena & Vega, 2008; Vega et al., 1999). In addition, NaAsO₂ blocks T cells in the G1 phase and, thus, delays the entry into cell cycle (Galicia, Leyva, Tenorio, Ostrosky‐Wegman, & Saavedra, 2003). More importantly, As(III) significantly alters Th cell differentiation. As₂O₃ inhibits IFN‐γ expression, the characteristic Th1 cytokine, at both mRNA and protein levels during the activation with anti‐CD3/anti‐CD28 of both human and murine T cells (Duan et al., 2017; VanDenBerg et al., 2017). However, As(III) does not change and sometimes increases the secretion of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4996 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4980, the characteristic Th2 cytokines, from activated T cells (Galicia et al., 2003; Soto‐Pena & Vega, 2008). These observations indicate that As(III) probably alters the Th1/Th2 balance towards a Th2 response. However, in ATL patients treated with arsenic/IFN/https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4825, this combination therapy returns the Treg/Th2 cytokine profile at diagnosis towards a Th1 profile, with a diminution of IL‐10 (Kchour et al., 2013). This effect is probably due to the depletion of ATL leukaemia cells that disrupt the immune system initially.

Moreover, As(III) was found to be a potent inhibitor of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4982. At a concentration which is not able to affect IFN‐γ secretion by Th1 cells, NaAsO₂ almost totally blocks the IL‐17 secretion by human Th17 cells, via mRNA reduction of the retinoic‐related orphan receptor (ROR)C gene which encodes RORγt, the key transcription factor of Th17 (Morzadec et al., 2012). As the Th17/Treg balance is critical in keeping immune homeostasis, As(III) treatment may lead to a regulatory response due to the potent inhibition of Th17. The effects of As₂O₃ on apoptosis and functions of CD4⁺ T cells are summarized in Figure 2.

FIGURE 2.

Mechanisms of action underlying the effects of As₂O₃ on CD4⁺ T cells

3.6. Trivalent arsenic and B cells

Baysan et al. showed that As₂O₃ induces apoptosis of the Bcl‐2 negative human B‐cell line Ramos via the mitochondrial pathway, by up‐regulating the expression of pro‐apoptotic proteins Bax and Bim (Dangleben et al., 2013). Moreover, it also blocks the mitogen‐mediated B‐cell differentiation towards plasmacytes and their IgM secretion (Rousselot et al., 1999). In addition, As₂O₃, together with https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6825, reduces the deposition and expression of IgG and IgM in both the xenograft and recipient sera in a heart xenotransplant model (Jiao et al., 2016). Collectively, As(III) treatment probably leads to a global suppression of the humoral immune system.

3.7. Trivalent arsenic and NK cells

As₂O₃ reduces NK cells differentiation from haematopoietic stem cell precursors, leading to a delayed NK cell reconstitution in treated APL (Alex et al., 2018). As₂O₃ also facilitates NK cell‐mediated cytotoxicity towards several cancer cell lines, through modulation of both NK cell receptors and malignant cell ligand profile (Alex et al., 2018; Kim et al., 2008). Collectively, As₂O₃ may enhance the cytotoxicity of NK cells.

3.8. Trivalent arsenic and inflammasomes

In the human monocyte cell line THP‐1 and mouse bone marrow‐derived macrophages, As₂O₃ inhibits the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1770 inflammasome and subsequent IL‐1β and IL‐18 secretion by targeting PML (Ahn et al., 2018; Lo et al., 2013). Moreover, As₂O₃ inhibits https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1768, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2793/https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1782 inflammasomes in the same subset of cells (Maier, Crown, Liu, Leppla, & Moayeri, 2014). However, in the human keratinocyte cell line and mouse skin tissue, NaAsO₂ promotes IL‐1β and IL‐18 secretion via https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2923 inflammasome activation (Zhang et al., 2016). In addition, the inflammasome https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1769 polymorphism is associated with arsenic‐induced skin lesions in humans (Bhattacharjee et al., 2013). Therefore, As(III) exerts bidirectional regulation on inflammasomes, which is probably tissue dependent.

4. TRIVALENT ARSENIC IN MOUSE MODELS OF IMMUNE‐MEDIATED DISEASES

4.1. Autoimmune diseases and asthma

Recently, As(III) was found to have therapeutic efficacy in several mouse models of autoimmune and inflammatory diseases (summarized in Table 2). As₂O₃ induces the regression of almost all the lesions in MRL/lpr mice, a mouse model of systemic lupus erythematosus (SLE) and lymphoproliferative syndrome, by elimination of the unusually activated T lymphocytes, and reduction of the related autoantibodies (Bobe et al., 2006). In the 2,4,6‐trinitrobenzene sulfonic acid‐induced murine model of inflammatory bowel disease, As₂O₃ reduces the induced colitis via NF‐κB down‐regulation and caspase‐3 activation (Singer, Trugnan, & Chelbi‐Alix, 2011). In a murine model of systemic sclerosis constructed by intradermal injections of hypochlorous acid (HOCl), As₂O₃ improves skin and lung fibrosis through ROS‐mediated killing of activated fibroblasts (Kavian, Marut, Servettaz, Nicco, et al., 2012). Moreover, As₂O₃ improves the pathological changes in a rat model of rheumatoid arthritis through a pro‐apoptotic effect on rheumatoid arthritis fibroblast‐like synoviocytes (Mei et al., 2011). In addition, NaAsO₂ decreases the disease incidence and delays its onset in non‐obese diabetic mouse, a mouse model of autoimmune type 1 diabetes, which is due to the reduced proliferation and activation of T cells (Lee et al., 2015).

TABLE 2.

Use of trivalent arsenic in mouse models of immune‐mediated diseases

Investigated disease	Mouse strain	Optimal therapeutic regime	Clinical efficacy	Possible mechanism	References
Autoimmune diseases
SLE/lymphoproliferative syndrome	MRL/lpr mice	As2O3 (i.p) 5 μg·g−1·day−1 for 2 months starting from 2 months of age	1. Prolong survival 2. Quasi‐total regression of antibody‐ and cell‐mediated manifestations	1. Anti‐DNA autoantibody↓RF↓IL‐18↓IFN‐γ↓NO metabolite↓TNF‐α↓Fas ligand↓IL‐10↓ in serum 2. Reduction of immune‐complex deposits in glomeruli	(Bobe et al., 2006)
Inflammatory bowel diseases	TNBS‐induced colitis murine model (BALB/c)	As2O3 (i.p) Prevention: 5 μg·g−1·day−1 from 4 days before TNBS administration Treatment: 5 μg·g−1·day−1 when the disease was noted	1. Prolong survival 2. Reduced the induced colitis as assessed by macroscopic and microscopic scores	1. NF‐κB inhibition 2. TNF‐α↓IL‐1β↓IL‐12↓IL‐17↓IL‐18↓IL‐23↓ expressions in colonic extracts 3. Elimination of inflamed cells through apoptosis	(Singer et al., 2011)
Systemic sclerosis	SSc induced by intradermal injections of HOCl (BALB/c)	As2O3 (i.p) 5 μg·g−1·day−1 for 6 weeks, simultaneously with HOCl injection	1. Reduction in skin and lung fibrosis 2. prevention of endothelial injuries	1. ROS generation that selectively kills activated fibroblasts 2. Autoantibody↓IL‐4↓ and IL‐13↓ from activated T cells	(Kavian, Marut, Servettaz, Nicco, et al., 2012)
Rheumatoid arthritis	Collagen‐induced arthritis rat model	As2O3 (N/A)	Inhibition of synovial hyperplasia and inflammation	1. Apoptosis induction of RA fibroblast‐like synoviocytes through NF‐κB signalling pathway and caspase cascade	(Mei et al., 2011)
Type I diabetes	Non‐obese diabetic mice	NaAsO2 (oral) 5 μg·g−1·day−1 for 8 weeks from 8 weeks of age	1. Decrease of incidence and disease onset delay	1. Reduction of infiltration of immunocytes in islets 2. Inhibition of T cell proliferation/activation	(Lee et al., 2015)
Airway chronic inflammatory disorder
Asthma	Chicken OVA‐challenged murine model of asthma (BALB/c)	As2O3 (i.p) 4 μg·g−1·day−1 for 7 days, 30 min before each challenge	1. Amelioration of the allergen‐driven airway hyperresponsiveness	1. Attenuation of airway eosinophils chemotaxin and recruitment in bronchoalveolar lavage fluid (BALF) 2. IkBα expression increase and NF‐κB decrease	(Chu et al., 2010; Zhou et al., 2006)
Allograft rejection and graft‐versus‐host disease (GVHD)
Cardiac allograft rejection	Model 1. C3H → C57BL/6 Model 2. BALB/c → C57BL/6 Model 3. BALB/c → C57BL/6 (allo‐primed T cells pre‐transferred)	Model 1. As2O3 (i.p) (1 μg·g−1·day−1, Days 3–10) + CsA Model 2. As2O3 (i.p) (5 μg·g−1·day−1, Days 0–10) + (anti‐CD154 and anti‐LFA‐1) Model 3. As2O3 (i.p) (3 μg·g−1·day−1, days 0‐10) + (anti‐CD154 and anti‐LFA‐1)	1. Prolong allograft survival	1. Inhibition of graft lymphocyte infiltration 2. IFN‐γ↓IL‐2↓TGF‐β↑ in both recipient serum and the graft 3. CD4+ Tm (%)↑ CD8+ Tm (%)↓ but Treg (%)↑ in the spleen of recipients	(Yan et al., 2009; Xu et al., 2010; Lin et al., 2011)
Cardiac allograft rejection	Model 1. C57BL/6 → nude mouse (CD4+ Tm pre‐transferred) Model 2. C57BL/6 → nude mouse (allo‐primed CD8+ Tm pre‐transferred)	Models 1 and 2. As2O3 (i.p) (3 μg·g−1·day−1, Days 0–10)	1. Prolong accelerated allograft rejection mediated by CD4 and CD8 memory T cells	1. CD4+ Tm (%)↓ and CD8+ Tm (%)↓ in recipient spleen and lymph nodes 2. IFN‐γ↓ IL‐2↓ and TGF‐β↑ IL‐10↑ in both recipient serum and allograft	(Yan et al., 2013; Li et al., 2015)
Islet allgraft rejection	Model 1. BALB/→C57BL/6 Model 2. Lewis rats→C57BL/6	Model 1. As2O3 (i.p) (2 μg·g−1·day−1, Days 0–9) Model 2. As2O3 (i.p) (10 μg·g−1·day−1, Days 0–9)	1. Prolong islet allograft and xenograft survival	1.CD4+ T (%)↓ CD8+ T (%)↓and Foxp3+ Treg (%)↑ in recipient spleen and lymph nodes 2.IFN‐γ↓IL‐2↓ and TGF‐β↑ in recipient serum and allograft	(Gao et al., 2015; Zhao, Xia, et al., 2018)
Chronic GVHD	B10.D2 → BALB/c	As2O3 (i.p) (5 μg·g−1·day−1, 3 weeks from Day 7 post‐transplantation)	1. Reduction of the fibrotic changes and prevention of the clinical symptoms	1. Activated CD4+ T cells (%)↓ and pDCs (%)↓ in splenocytes through apoptosis 2. Splenic IL‐4↓, IL‐17↓ and serum Anti‐DNA‐topoisomerase 1 autoantibody↓	(Kavian et al, 2012)

Abbreviations: As₂O₃, arsenic trioxide; HOCl, hypochlorous acid; IkBα,nuclear factor of κ light polypeptide gene enhancer in B‐cell inhibitor, α; NaAsO₂, sodium arsenite; OVA, ovalbumin; pDC, plasmacytoid dendritic cell; RA, rheumatoid arthritis; RF, rheumatoid factor; TNBS, 2,4,6‐trinitrobenzene sulfonic acid; SSc, systemic sclerosis; SLE, systemic lupus erythematosus; Tm, memory T cell.

Asthma is a T‐helper type 2 (Th2) lymphocyte‐mediated chronic inflammatory disorder characterized by airway eosinophilia and airway hyperresponsiveness. As₂O₃ is observed to ameliorate the allergen‐driven airway hyperresponsiveness in a mouse model of asthma, through modulation of the NF‐κB pathway, airway eosinophil recruitment, and functions (Chu et al., 2010; Zhou et al., 2006).

Overall, As(III) has therapeutic potential on several autoimmune and inflammatory diseases through a pro‐apoptotic effect and effector‐cell functional modulation.

4.2. Allogeneic organ/stem cell transplantation

During organ or allogeneic stem cell transplantation, human leukocyte antigen mismatches lead to severe T‐ and B‐cell‐mediated alloreactivity, which may eventually lead to severe inflammatory transplantation complications of allograft rejection or graft‐versus‐host disease (GVHD) respectively. As₂O₃ has been shown to prolong the allograft survival in immunocompetent mouse heart transplant models, with reduction of the proportions of CD4⁺ and CD8⁺ memory T cells and increase of Tregs in recipient spleen and lymph nodes (Lin et al., 2011; Xu et al., 2010; Yan et al., 2009). Meanwhile, IFN‐γ expression was reduced, and TGF‐β expression was increased in both the recipient serum and the graft (Lin et al., 2011; Xu et al., 2010). Further studies have shown that As₂O₃ inhibits accelerated allograft rejection mediated by alloreactive CD8⁺ and/or CD4⁺ memory T cells and prolongs allograft survival (Li et al., 2015; Yan et al., 2013). Moreover, in two models (one allo‐ and one xeno‐) of islet transplantation, As₂O₃ was able to prolong the graft survival by inhibiting inflammatory reactions and T‐cell responses (Gao et al., 2015; Zhao, Xia, et al., 2018). GVHD occurs when immunocompetent T cells in the graft recognize the recipient as foreign and attacks the target organs. A study revealed that As₂O₃ can prevent disease occurrence in a murine sclerodermatous GVHD model mediated by overproduction of H₂O₂ which kills activated CD4⁺ T cells and pDCs (Kavian, Marut, Servettaz, Laude, et al., 2012). To summarize, As(III) could be a promising drug for alloreactivity through effector‐cell modulations.

4.3. Clinical trials of trivalent arsenic on immune‐mediated diseases

Given the discoveries in basic research and preclinical models, interests grow on As(III) as a clinical therapeutic agent in immune‐mediated diseases. An ongoing randomized clinical trial (NCT02966301) is currently testing As₂O₃ as first‐line treatment of chronic GVHD. Results of a phase 2a randomized clinical trial (NCT01738360) evaluating the therapeutic efficacy of As₂O₃ in SLE are expected soon.

5. TRIVALENT ARSENIC AND TUMOUR IMMUNOTHERAPY

The direct effects of As(III) on tumour cells are not discussed here but are reviewed by Emadi and Gore (2010). The past decade has witnessed breakthroughs which bring immunotherapy to the forefront of cancer therapy. As(III) acts in cellular immunotherapy in two ways (summarized in Table 3). On the one hand, it selectively depletes tumour promoting cells. The ratio of effector to regulatory T cells (Teff/Treg) is of great importance in immune modulation, and targeting Tregs to enhance anti‐tumour immune responses has become an important strategy in cancer immunology (Roychoudhuri, Eil, & Restifo, 2015). It was observed that As₂O₃ reduces the increased Treg numbers and Foxp3 mRNA levels in ex vivo malignant ascites (Hu et al., 2018). In two murine models of colon and liver cancer, As₂O₃ exerted its anti‐tumour effect in both in situ carcinoma and colon cancer lung metastasis through selective depletion of the infiltrated Tregs (Thomas‐Schoemann et al., 2012; Wang et al., 2017; Wang, Hu, Xu, & Liu, 2016). On the other hand, As(III) enhances cytotoxicity of tumour‐killing cells. It has been shown that As₂O₃ increases the cytotoxic activity of cytokine‐induced killer (CIK) cells and the IFN‐γ secretion in vitro, which may help in tumour control (Wang et al., 2016; Wang et al., 2017). Moreover, exposure of myeloma cell lines to As₂O₃ increases lymphokine‐activated killer (LAK)‐mediated killing by up‐regulation of CD38 and CD54 on the myeloma cells, and increased expression of CD31 (CD38 ligand) and CD11a (CD54 ligand) on LAKs, suggesting that the improved killing is mediated by increased adhesion (Deaglio et al., 2001). In addition, exposure of NK and leukemic cells to low doses of As₂O₃ modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell‐mediated cytolytic activity (Alex et al., 2018; Kim et al., 2008). This effect was proved in a mouse model of APL, where the As₂O₃ + NK treatment promoted longer survival as compared with As₂O₃ alone (Alex et al., 2018). Overall, As₂O₃ could work as an effective adjuvant for cellular immunotherapy in both haematological malignancies and solid tumours.

TABLE 3.

Trivalent arsenic facilitates tumour immunotherapy

Investigated disease	Ex vivo sample/mouse model	Optimal therapeutic regimen	Clinical efficacy	Possible mechanism	References
Malignant ascites	TILs derived from ascites of gastric cancer patients	As2O3 (in vitro, 5 or 10 μM for 48 hr)	N/A	CD4+ T cell (%)↓ CD8+ T cell (%)↑ Treg (%)↓ with Foxp3 expression↓ in TILs 2. IL‐10↓ TGF‐β↓IFN‐γ↑ TIL cytotoxicity↑	(Hu et al., 2018)
Colon cancer	Model 1. Colon cancer‐bearing mouse (BALB/c, injection s.c. of CT‐26 colon cancer cell) Model 2. Lung metastasis model of colon cancer (BALB/c, injection i.v. of CT‐26)	Model 1. As2O3 (i.p) 1 μg·g−1 once at Day 10 of tumour injection Model 2. As2O3 (i.v) 6 μg·g−1 every 2 days for 2 weeks from Day 3 of tumour injection	1. Delay tumour growth 2. Inhibition of lung metastasis and prolong mouse survival	1. Selective Treg depletion with Foxp3 expression↓ in spleen and tumour tissue, due to ROS and RNS accumulation in Tregs. 2. Cytokine induced killer cells (in vitro): cytotoxicity↑IFN‐γ↑	(Thomas‐Schoemann et al., 2012; Wang et al., 2016)
Hepatic cancer	Hepatic cancer bearing mouse (KM strain, H22 hepatic cancer cell liver implantation)	As2O3 (i.v) 6 μg·g−1 every 2 days for 2 weeks right after liver tumour implantation	1. Prolong mouse survival 2. Inhibition of tumour growth 3. Reduction of abdominal adhesion and ascites	1. Selective Treg depletion 2. Serum FN‐γ↑IL‐10↓TGF‐β↓ 3. IOD of CD3+ T cell↑Foxp3+ cell↓ in tumour	(Wang et al., 2017)
Multiple myeloma	Co‐culture of LAK and myeloma cell	As2O3 (in vitro, 0.5 μM for 72 hr)	N/A	1. Increase of LAK‐mediated lysis via up‐regulation of expressions of CD54 and CD38 on myeloma cells, and CD31(CD38 ligand) and CD11a (CD54 ligand) on LAKs	(Deaglio et al., 2001)
Acute promyelocytic leukaemia	APL bearing mouse model (FVB/N, APL blast cells from MRP8‐PML‐RARa transgenic FVB/N mouse)	As2O3 (i.p) (5 μg·g−1·day−1, Days 8 to 35 of tumour injection) and Syngeneic NK cells (i.v) (5 * 105 cells/dose, Days 8, 18, and 28 of tumour injection)	1. Prolong survival comparing As2O3 + NK group and As2O3 group alone	1. Enhancement of NK cell cytolytic activity 2. Alteration of NK cell receptor and ligand Profile on tumour cell	(Alex et al., 2018; Kim et al., 2008)
Hepatic cancer	Hepatic cancer bearing mouse (BALB/c, H22 hepatic cancer cell)	Intratumoral injection of 100 μg B7H3 plasmid (on Day 14 of tumour injection) and 5 μg As2O3 (every 2 days) until tumour disappear	1. Transplanted hepatoma eradication and regression of distant tumour nodules	1. Generation of anti‐tumour immunity relies largely on CD8+ T cells and NK cells 2. Serum IFN‐γ↑ and CTL activity↑	(Luo et al., 2006)
Bladder cancer	Orthotopic bladder cancer model (C3H/HeN, human bladder cancer cells 5637)	Intravesical instillation of 0.2‐ml BCG (600 mg·L−1) and 0.2 ml of As2O3 (30 μmol·L−1·week−1) for 4 weeks	1. Reduction of tumour weight and volume	1. Apoptosis of tumour cells↑ via IER3/Nrf2 pathway↓ 2. DC (%) in cancer tissue↑Expression of CD83/CD86↑IL‐6/IL‐8 in DCs↑	(Mao et al., 2018)

Abbreviations: APL, acute promyelocytic leukaemia; As₂O₃, arsenic trioxide; BCG, bacillus Calmette‐Guerin; IOD, integrated OD; LAK, lymphokine activated killers; TIL, tumour‐infiltrating lymphocytes; RNS, reactive nitrogen species.

Moreover, As(III) can synergize with B7‐H3 (CD276), an important immune checkpoint member of the B7 and CD28 families, to eradicate hepatocellular carcinoma in a mouse model, along with the generation of potent systemic antitumor immunity mediated by CD8⁺ T and NK cells (Luo et al., 2006). As(III) was also used in a mouse model of bladder cancer to facilitate intravesical bacillus Calmette‐Guerin immunotherapy, by targeting the IER3/Nrf2 pathway (Mao et al., 2018).

6. DOSING OF THERAPEUTIC AS(III)

Two different As₂O₃ dosing schedules have been assessed in Phase 3 clinical trials for APL, with the protocol of 0.15 mg·kg⁻¹·day⁻¹ daily until complete remission (up to 140 doses of 0.15 mg·kg⁻¹) in the Italian‐German trial (Lo‐Coco et al., 2013) and the protocol of 0.3 mg·kg⁻¹ on Days 1 to 5 in Week 1 followed by 0.25 mg·kg⁻¹ twice weekly for 7 weeks (63 doses of 0.25–0.30 mg·kg⁻¹) in the U.K. National Cancer Research Institute trial (Burnett et al., 2015). Despite the discrepancy on the frequency of use, the intensity of As₂O₃ in the 2 schedules is, however, almost identical with respect to total As₂O₃ dose in milligrams per kilogram.

The appropriate dosing of As₂O₃ in the treatment of immune‐mediated diseases is being assessed in clinical trials. In the Phase 2a study evaluating As₂O₃ in SLE (NCT01738360), patients received 10 doses of As₂O₃ up to 0.30 mg·kg⁻¹·day⁻¹ within a period of 25 days. In the ongoing Phase 2 trial testing As₂O₃ in chronic GVHD (NCT02966301), each patient received 11 doses of As₂O₃ (0.15 mg·kg⁻¹·day⁻¹) over a 4‐week period (one cycle) and one more cycle for consolidation when patients achieved a partial response. Therefore, As₂O₃ is used in immune‐mediated diseases with similar daily doses but smaller total intensity as compared with that in APL.

Notably, the use of arsenic derivatives is highly embryotoxic and is contraindicated at any stage of pregnancy (Sanz et al., 2019), while As₂O₃ at 0.15 mg·kg⁻¹·day⁻¹ (standard dose) was well tolerated by paediatric patients with APL and achieved satisfactory outcomes in clinical trials (Kutny et al., 2017). The dose of As₂O₃ in elderly and obese patients may need to be attenuated while no consensus has been published due to limited experience (Hickey et al., 2019; Lengfelder, Hofmann, & Nolte, 2013).

The pharmacokinetics of As₂O₃ as measured by plasma concentrations were comparable in different studies (Shen et al., 1997., Fox et al., 2008). In a Phase 1 clinical trial assessing single‐agent As₂O₃ in APL, at 0.15 mg·kg⁻¹·day⁻¹, total arsenic in the plasma reached the maximum concentration (Cmax) of 0.28 μM (0.11–0.37 μM) within 2 hr after completion of administration and was quickly eliminated below the lower limit of detection (0.07 μM) in 10 hr (Fox et al., 2008). Notably, repeated administration did not alter the pharmacokinetic behaviours of As₂O₃ (Shen et al., 1997; Fox et al., 2008), indicating no or minimal plasma drug accumulation. Moreover, the pharmacokinetics for As(III) in the bone marrow seems to be similar as compared with peripheral blood (Iriyama et al., 2012).

Concerning the acute adverse events of As(III), despite the therapeutic superiority for ATRA plus As₂O₃ versus ATRA plus chemotherapy in APL, the former regimen led to less haematological toxicity and fewer infections, but more common cardiac and hepatic toxicities in the two Phase 3 clinical trials (Burnett et al., 2015; Lo‐Coco et al., 2013). Indeed, cardiac toxicity manifesting as QT interval prolongation seems to be the leading adverse event of As₂O₃ (Roboz et al., 2014). As₂O₃ induces cardiotoxicity through multiple mechanisms including ROS generation and disruption of intracellular calcium homeostasis (Haybar, Shahrabi, Rezaeeyan, Jodat, & Saki, 2019). Between 11% and 26% of patients receiving As₂O₃ experienced QT prolongation in different studies (Burnett et al., 2015; Lo‐Coco et al., 2013; Roboz et al., 2014), and it was recommended to withhold As₂O₃ when QT interval is prolonged longer than 500 ms (Sanz et al., 2019). However, clinically relevant arrhythmias are rare and can be avoided with appropriate precautions such as electrolyte repletion (Roboz et al., 2014). In addition, As₂O₃‐induced hepatic toxicity featuring mainly as elevated liver alanine transaminase could occur in between 25% and 63% of patients receiving ATRA plus As₂O₃ and seems to be highly manageable with temporary discontinuation of the treatment (Burnett et al., 2015; Lo‐Coco et al., 2013).

7. ARSENIC RETENTION AND CHRONIC ADVERSE EVENTS

Arsenic (including different arsenic species such as As(III) and As(V)) retention and possible chronic adverse events are concerns surrounding the use of As₂O₃. In a 5‐year follow‐up study including 85 APL patients treated with As₂O₃ + ATRA, Hu et al. observed that after cessation of As₂O₃, the plasma and the urine arsenic concentrations were rapidly decreased to levels below the safety limit recommended by the U.S. Agency for Toxic Substances and Disease Registry even though the concentrations remained slightly greater than in healthy controls. For the nail and hair arsenic contents representing the long‐term exposure and in vivo accumulation of arsenic, the levels were only slightly higher than those in healthy controls. Meanwhile, no adverse events such as secondary carcinoma were observed (Hu et al., 2009). In another 6‐year follow‐up study which enrolled 72 APL patients receiving single‐agent As₂O₃, no additional toxicities developed after completion of the course of As₂O₃, and there was no significant difference in the arsenic retention in hair and nail samples of controls and patients who had completed therapy at least two years earlier. Moreover, seven patients (four women and three men) have had eight normal babies without report of abortions, indicating safety profile for fertility after treatment cessation (Mathews et al., 2010). Importantly, in a 12‐year follow‐up study including 265 APL patients treated with As₂O₃ + ATRA, Zhu et al. reported that plasma and urine, as well as the delayed increased hair and nail arsenic levels all returned to normal after 6 months. Interestingly, the plasma and urine arsenic levels were not higher and even lower than healthy controls on cessation of As₂O₃. However, incidence of mild liver dysfunction (15.2%) and hepatic steatosis (42.9%) was reported. In addition, only one patient developed breast cancer 3 years after termination of As₂O₃ (Zhu et al., 2016). Collectively, no significant arsenic retention was observed in these studies, and chronic adverse events, especially secondary carcinoma, are rare.

However, since the longest published follow‐up data for APL patients was 12 years, the very long‐term adverse effects especially tumourigenesis remain to be better observed. Indeed, chronic ingestion of arsenic through drinking contaminated water or inhalation results in the accumulation of arsenic in vital organs, which may lead to diabetes, atherosclerosis, hypertension, ischaemic heart diseases, hepatotoxicity, nephrotoxicity, skin lesions, and cancer of the skin, bladder, and lungs (see Khairul, Wang, Jiang, Wang, & Naranmandura, 2017).

Oxidative stress has been suggested as an important mechanism for arsenic‐induced carcinogenesis (Kitchin & Conolly, 2010). In rat models, As(III) caused an elevation of 8‐hydroxydeoxyguanosine by generation of ROS, stimulated cell proliferation, and induced liver and bladder carcinogenicity (Kinoshita, Wanibuchi, Wei, Yunoki, & Fukushima, 2007; Suzuki, Arnold, Pennington, Kakiuchi‐Kiyota, & Cohen, 2009). In addition, intracellular signal transduction and transcription factors activation are related to arsenic‐induced carcinogenicity (Khairul et al., 2017). Moreover, As(III) interferes with key cellular processes such as DNA damage‐repair and chromosomal structure, leading to genomic instability. At the epigenetic level, As(III) affects global hypomethylation and hypermethylation of specific gene promoters, leading to deregulation of both oncogenic and tumour‐suppressive genes (Sage et al., 2017). These factors may operate together and lead to abnormal cell proliferation, mutations accumulation, and finally development of cancers. Nevertheless, As(III) may compromise the immune system's ability to rid the host of pathogens and tumours (Dangleben et al., 2013). Arsenic‐exposed mice demonstrated depressed humoral and cellular immunity and displayed significantly impaired resistance against B16F10 melanoma, which resulted in sevenfold increased tumour burden (Sikorski, Mccay, White, Bradley, & Munson, 1989). Collectively, attentions should be paid to patients with immune deficiency or defective DNA repair machinery when considering As(III) treatment as it may lead to higher risk of infection and carcinogenesis in the long term.

Millions of people worldwide are exposed to arsenic on a regular basis. However, as compared with environmental exposure which is low in concentration (mostly below 10 μg·kg⁻¹·day⁻¹) and very long in duration (decades; Dangleben et al., 2013), the standard As₂O₃ treatment is much higher in concentration (0.15 mg·kg⁻¹·day⁻¹) and shorter in duration (weeks). Arsenic‐induced toxicity may differ between these two types of exposure (Zhang, Zhang, Wang, Li, & Zhang, 2018). Moreover, risks of accumulated toxicity due to drug plus environmental exposure may exist and require further studies.

8. CONCLUSIONS AND PERSPECTIVES

As(III), when used in a clinically relevant dose, that is, between 0.07 and 0.28 μM, as found in the plasma of APL patients treated with As₂O₃ (Fox et al., 2008), can target specific immune cell subsets and exert immunomodulatory effects. The efficacy of As(III) on mouse models of autoimmune and inflammatory diseases highlights its therapeutic potential in humans. It emerges as a promising adjuvant to cancer immunotherapy agents in the treatment of both haematological and solid tumours.

It is noteworthy that the As(III) effect is dose, cellular, and tissue dependent, which requires deeper understanding of this drug in vitro, in vivo, and in clinical practice. For this purpose, comprehensive genomic, proteomic, and metabolomic profiling will be critical for identifying and validating potential molecular targets of As(III) for future therapeutic use.

Several clinical trials with As(III) in autoimmune and inflammatory diseases are ongoing to prove the “bench to bedside” indication of As(III). Notably, during As₂O₃ use in APL, adverse events including cardiac and hepatic toxicities have been observed, which require precautions also during its usage in immune‐mediated diseases.

8.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in https://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/2020 (Alexander, Cidlowski et al., 2019; Alexander, Fabbro et al., 2019a, 2019b; Alexander, Kelly et al., 2019; Alexander, Mathie et al., 2019).

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Ye Y, Gaugler B, Mohty M, Malard F. Old dog, new trick: Trivalent arsenic as an immunomodulatory drug. Br J Pharmacol. 2020;177:2199–2214. 10.1111/bph.15011