Speciation of arsenic trioxide metabolites in peripheral blood and bone marrow from an acute promyelocytic leukemia patient

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML) and accounts for approximately 10-15% of all cases of AML in adults [1]. It is also characterized by a specific cytogenetic reciprocal chromosome translocations, t(15;17), generating PML/RARα fusion gene, which is thought to play a central role in the initiation of leukemogenesis [2‐5]. An introduction of all-trans retinoic acid (ATRA) since 1986 has dramatically improved the outcome of treatment of this disease [3]. Nevertheless, an approximately 30% of the patients relapse and often become resistant to the conventional treatment with ATRA alone, or in combination with chemotherapy [5]. On the other hand, investigators from China and the USA have demonstrated that the treatment with arsenic trioxide (ATO, As(III)) could induce complete remission (CR) in 90% of relapsed patients [1, 6, 7].

We have been investigating the effects of As(III) using a unique in vitro system comprising primary cultured chorion and amnion cells prepared from human fetal membranes [8‐10], and demonstrated that aquaporin 9 and multidrug resistance-associated protein 2 are functionally involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As(III) cytotoxicity between these cells [11]. These findings may have broad important implications for revealing the mechanisms underlying the side effects of ATO in patients. Furthermore, in order to understand the mode of actions of ATO and provide an effective treatment protocol for individual APL patients, studies have been conducted on the pharmacokinetics of ATO in APL patient using biological samples such as urine, blood and cerebrospinal fluid [12‐14]. In fact, we recently demonstrated that various arsenic species including inorganic arsenic and methylated arsenic metabolites accumulated in red blood cells (RBCs) in an APL patient [13]. We have also demonstrated for the first time that these arsenic metabolites also existed in cerebrospinal fluid [14], in which the total arsenic concentrations reached levels necessary for differentiation induction [1, 15]. These findings may provide a new insight into clinical applications of ATO, and better therapeutic protocols [16].

It has become clear that like hematopoietic stem cells, leukemia stem cells (LSCs), which acquired limitless self-renewal through oncogenic transformation, also reside in bone marrow (BM) microenvironment, and contribute to maintain the acute myeloid leukemia phenotype [17]. Recent studies have also demonstrated that incomplete eradication of primary LSCs is closely linked to chemotherapy resistance, and consequently contribute to eventual disease relapse [17, 18]. In this regard, it is significant to note that targeting for the PML with ATO could lead to loss of self-renewal capability of LSCs in chronic myeloid leukemia (CML) based on the studies on stem cells isolated from BM of human CML patients and mouse bone marrow transplantation models [18]. These findings suggest that ATO would be a promising candidate for a LSCs-targeted therapy and raise a concern as to the detailed distribution profiles of arsenic in BM. These ideas are based on previous findings indicating that the total arsenic concentrations in the plasma of BM from five relapsed APL patients were close to levels for inducing differentiation, yet the analysis was conducted for the BM sample collected at just only one collection time point [19]. Furthermore, to the best of our knowledge, no speciation analysis of arsenicals in BM from APL patients undergoing long-term administration of ATO has been done before, despite the fact that BM is a vital site for regulating the production of blood cells.

It has been established that biomethylation is a major metabolic pathway for inorganic arsenic in human beings as well as many animal species, in which As(III) is methylated to form trivalent and pentavalent products such as methylarsonous acid (MA(III)), methylarsonic acid (MA(V)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMA(V)) [16]. Furthermore, it has become clear that trivalent arsenicals can interact with biological molecules of importance containing sulfhydryls [20]. In fact, a study on complexes of trivalent arsenicals with proteins has been conducted using organ samples such as livers and kidneys in rats after an intravenous injection of arsenite [21]. However, no such studies have been conducted in biological samples from APL patients undergoing ATO treatment. Furthermore, the biological as well as clinical importance of proteins-bound arsenic complex has been proposed [20, 22], yet the detailed studies have not been preformed.

In this study, we evaluated the clinical efficiency of ATO in a relapsed APL patient undergoing long-term administration of ATO by response assessment. In order to obtain a general view regarding the pharmacokinetic behaviors of ATO in APL patients, we also performed a detailed systematic analysis of the accumulation of ATO in RBCs as well as its metabolites in plasma of peripheral blood (PB). Most importantly, besides total arsenic determination, we further investigated for the first time the arsenic speciation in plasma of BM, and compared the arsenic speciation profiles between PB and BM in order to gain more detailed information on the distribution of arsenic. Furthermore, the total arsenic concentrations of high molecular weight fraction (HMW-F) in BM and PB plasma were determined. Additionally, we collected clinical samples just before (within 30 minutes) the start of daily administration in order to get accurate blood trough levels, since the levels are well known to be closely related to clinical outcomes.

Administration of ATO has demonstrated a remarkable efficacy in the treatment of relapsed and refractory APL patients, who are generally resistant to the combinatory conventional treatment protocol consisting of ATRA and chemotherapy [1, 2, 6, 7, 16, 26]. Consistent with these previous reports, the present study reconfirmed the clinical efficacy of ATO in a relapsed APL patient who achieved CR with ATRA once. Similar to a previous review report in which the median time to achieve CR in patients treated with ATO alone ranged from 28 to 38 days [27], the patient in the present study also achieved CR on day 28 (Table 1). We also demonstrated a substantial induction of APL cells differentiation in the patient after the treatment with ATO as evidenced by the alterations of the expression levels of cell surface antigens associated with myeloid maturation (Table 1). Results of the differentiation induction are in good agreement with other experimental results showing that a relatively lower dose of ATO induces differentiation of freshly isolated APL cells from patients as well as NB4 cells [1, 2, 15, 16]. Of note, there was no requirement of any additional treatment for complications or blood transfusion and no drug discontinuation throughout the remission induction therapy, although some clinical data failed to return to a normal clinical reference range probably due to immediate determination after treatment with ATO. These results suggested that the patient would be a good model for studying the pharmacokinetics as well as the pharmacodynamics of ATO.

In agreement with our previous report [13], our present results clearly demonstrated that the total arsenic concentrations in either PB RBCs or PB plasma increased with time during the consecutive administration, and that the majority of arsenic was present in the RBCs (Figure 1 and Table 2). Therefore, careful attention should be paid to profiles of arsenic species in RBCs. We also demonstrated for the first time a time-dependent increase of the total arsenic concentrations in BM plasma, which was similar to that in PB plasma (Figure 1 and Table 2). Furthermore, the total arsenic concentration levels tended to be higher in the BM plasma than those in the PB plasma, raising clinical concerns and inspiring us to unravel the detailed information on the distribution of arsenic as well as its speciation in these biological samples.

We have previously demonstrated that the PB plasma concentrations of both methylated arsenic metabolites (MA(V) and DMA(V)), and inorganic arsenic (As(V) and As(III)) remarkably increased after the start of administration in a Japanese APL patient undergoing consolidation therapy [13]. Intriguingly, in the current study, the concentrations of MA(V) and DMA(V) increased substantially after the start of administration, while those of As(III) were still kept at a low level until day 10, followed by substantial increase from day 14 (Figure 3(B) and Table 3). It has been reported that in many animal species including human beings, biomethylation is a major metabolic pathway for inorganic arsenic, through which arsenic undergoes metabolic conversion by the reduction of As(V) to As(III) with subsequent methylation, yielding MA(V) then to DMA(V) [16, 28, 29]. Compared to the APL patient in our previous study [13], the patient enrolled in the current study appeared to have relatively higher metabolic efficiency probably due to her relatively young age or without clinical complications. Results from previous studies and our present study suggest that the efficiency of drug metabolism is obviously different in individual patients with different backgrounds, such as age range, with or without organ failure or DIC, which in turn affect clinical outcomes and appearance of side effects.

Of note, a previous study on the pharmacokinetics of ATO in Japanese patients with relapsed or refractory APL [30] demonstrated that the PB plasma concentrations of inorganic arsenic reached the steady state during the consecutive administration, while the methylated arsenic metabolites increased in relation to administration frequency. On the other hand, our recent study involving an APL patient with first relapse demonstrated that the PB plasma concentrations of both inorganic arsenic and methylated arsenic metabolites increased with administration frequency [13]. Interestingly, a trend toward reaching a plateau in these arsenic species was observed in PB plasma in the current study (Figure 3(B) and Table 3). These results clearly suggest that arsenic metabolism among individual patients were different. A similar observation was reported by Wang et al. [12], in which there are interindividual differences in excretion profiles and the relative concentrations of major arsenic species in urine among four Chinese APL patients undergoing ATO treatment. It is significant to note that genetic polymorphisms in arsenic metabolism genes, such as human arsenic methyltransferase (AS3MT) gene and glutathione S-transferase gene, are considered to be related to interindividual variation in the arsenic metabolism [31‐33]. Collectively, continuous efforts to understand the differences in arsenic metabolism and the relationship between these genetic polymorphisms and arsenic metabolism among patients are definitely useful for providing an effective treatment protocol of ATO for individual APL patients, ultimately contribute to reduction of its side effects. Moreover, it should be noted that a close similarity of the arsenic speciation profiles between PB and BM plasma was observed throughout the remission induction therapy (Figure 5 and Table 5). These results thus suggested for the first time that arsenic speciation analysis of PB plasma could be predicative for BM speciation without applying BM aspiration.

We then focused on the total amount of arsenic in HMW-F trapped in a 10-kDa molecular mass cutoff filters and demonstrated that its concentrations were much higher in BM plasma than those in PB plasma (Figure 6 and Table 6). One important biological effects of arsenic has been suggested to be mediated by reaction with closely spaced cysteine residues on critical cell protein [34]. Several proteins such as tubulin, thioredoxin reductase, AS3MT with a high cysteine content and accessible thiol group are candidates for interactions with arsenic [16, 20]. In fact, arsenic bound to high molecular weight proteins (MW > 10-kDa) has been detected in livers and kidneys in rats after an intravenous injection of arsenite [21]. Based on the vital role of BM microenvironment in maintaining the homeostasis of hematopoietic system, we assumed that a higher amount of proteins (MW > 10-kDa)-bound arsenic complex contribute to protection effect from the attack of free arsenic species. Likewise, the patients with low-proteinemia besides liver and/or renal dysfunctions might frequently develop arsenic-mediated side effects. Understandably, further investigation of the detailed information about these proteins is needed.

In conclusion, we clarified the arsenic speciation for the first time in BM plasma, and found that speciation profiles of BM plasma were very similar to those of PB plasma. We also demonstrated that the total arsenic concentrations of HMW-F were much higher in BM plasma than those in PB plasma. These results may further provide not only significance of clinical application of ATO, but also a new insight into host defense mechanisms in APL patients undergoing ATO treatment. Of note, it has been demonstrated that targeting for the PML with ATO could lead to loss of self-renewal capability of LSCs, which is closely linked to chemotherapy resistance and disease relapse, in CML [18], suggesting that ATO would be a promising candidate for a LSCs-targeted therapy [17]. Our results thus may provide new evidence for LSCs-targeted therapy in APL patients based on the following facts: 1) PML-RARα can be detected in more than 95% of APL patients [2], 2) long-term follow-up of newly diagnosed patients with APL treated with single ATO therapy shows a high 5-year disease-free survival (DFS) rate and overall survival (OS) rate [35‐37]. It is well known that the blood trough levels correlated with clinical benefit better than either maximum levels or area under the curve (AUC). Similar to our and other group's previous reports [13, 38], the plasma trough levels of As(III) were much lower compared to a previous report [6]. These results thus leave open a possibility that much lower concentrations of As(III) possess biological effects such as differentiation-inducing activity, although we could not exclude whether other arsenicals, such as As(V) and methylated arsenic metabolites, alone or their combination contribute to the effects based on a previous report showing that methylated arsenic metabolites also possess cytocidal activity against leukemia and lymphoma cells, consequently may contribute to the therapeutic effect of ATO in APL patients [39]. Details of the study are currently underway.