Dose Of Arsenic Trioxide Research Articles

Arsenite medicinal use, metabolism, pharmacokinetics and monitoring in human hair

Acute promyelocytic leukaemia (APL) is a distinctive subtype of acute myeloid leukaemias. Even through this human disease can be treated by the intravenous administration of all-trans retinoic acid (ATRA), 25% of patients typically relapse after the first treatment. In this context, the intravenous administration of APL patients with an aqueous solution of arsenic trioxide has also been demonstrated to be successful despite the established mammalian toxicity of this arsenic compound. Accordingly, the administration of a therapeutic dose of arsenic trioxide has resulted in an improved patient survival in both relapsing as well newly diagnosed APL patients. We present here a mini-review of the medicinal use of arsenite, its mammalian metabolism (with an emphasis on biomethylation pathways), its elimination and pharmacokinetics and the novel application of hair analysis as a biomonitoring material. This mini-review also introduces our own results on the analysis of hair of patients receiving arsenic trioxide therapy. In this work, instead of quantifying arsenic content in bulk hair, we performed longitudinal analysis in order to use hair as a marker of arsenic exposure correlated to a time scale. Taking into account the hair growth rate, the longitudinal analysis of hair is demonstrated to provide a chronological record of the treatment of patients with arsenic trioxide. The small quantity of material to be analysed required the use of Synchrotron radiation based X-ray fluorescence (SXRF) spectroscopy. The hair arsenic content was well correlated with the clinical background of patients and reflected the intake of arsenic trioxide. In particular, the onset of arsenic trioxide therapy and interruptions during therapy were reflected by total arsenic content, which suggested rapid elimination. Another type of experiment, micro-XRF cartography on thin hair slices, allowed us to obtain distribution maps of arsenic, which demonstrated that arsenic is located at the periphery of hair. Micro-XANES spectra recorded at the periphery of hair, suggest that inorganic arsenic is incorporated in hair in its trivalent oxidation state, in agreement with previous results.

Characterization of cytotoxicity induced by arsenic trioxide (a potent anti-APL drug) in rat cardiac myocytes

Arsenic, a known environmental toxicant, is ubiquitously present in the environment. Arsenic trioxide (ATO), an anti-acute promyelocytic leukemia (APL) drug, is associated with cardiac toxicity. It is reported to induce cardiac arrhythmia via altering various ion channels involved in the repolarization phase of cardiac action potential. The exact molecular mechanism of cardiovascular adverse effect due to ATO exposure has not been fully elucidated except for alteration on ion channels. To evaluate the cytotoxic effect of ATO on cardiac myocytes, primary culture of myocytes was treated with different doses (30, 60 and 90 μM) of ATO for various periods (24, 48 and 72 h). Cardiac toxicity was assessed by monitoring cell viability, mitochondrial and deoxyribonucleic acid (DNA) integrity, reactive oxygen species (ROS) generation, calcium overload and apoptosis. ATO exposure caused alteration in mitochondrial integrity, generation of ROS, calcium overload and apoptosis in cardiac cells in dose- and duration-dependent manner. There was no DNA fragmentation. Hence our results show that ATO causes apoptosis in cardiomyocytes by generation of ROS and the induction of calcium overload.

Antitumor Activity of Arsenic Trioxide on Retinoblastoma: Cell Differentiation and Apoptosis Depending on Arsenic Trioxide Concentration

Arsenic trioxide (ATO) targets multiple pathways in malignant cells, resulting in the promotion of differentiation or in the induction of apoptosis. The antitumor activity of ATO on retinoblastoma was investigated. Human retinoblastoma cells were incubated with various ATO concentrations. The antiproliferative effect of ATO was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the effect of ATO on cell-cycle progression was validated by flow cytometry. At a low concentration, the ATO-induced differentiation of retinoblastoma cells was evaluated by neurofilament expression and extracellular signal-regulated kinase (ERK)1/2 activation, which was confirmed by the inhibition of ERK1/2. At a high concentration, ATO-induced H(2)O(2) production was investigated with the cell-permeable fluorescent dye 2'7'-dichlorofluorescein-diacetate, and the relationship of ATO-induced H(2)O(2) production with caspase-3-dependent apoptosis was validated by Western blot and 4'6-diamidino-2-phenolindole staining, which was confirmed by reactive oxygen species (ROS) inhibition. The effect of ATO on tumor formation was assessed with an orthotopic animal model of retinoblastoma. The antitumor activity of ATO in retinoblastoma was related to two main mechanisms, differentiation and apoptosis, which were determined by the level of ATO. At a low dose (<or= 1 microM), ATO induced the differentiation of retinoblastoma cells through ERK1/2 activation, whereas ROS generation by a high dose (>or= 2 microM) of ATO induced apoptosis in retinoblastoma cells. Moreover, ATO at low and high doses effectively inhibited tumor formation. These results suggest that ATO can be used as an effective alternative therapeutic for the treatment of retinoblastoma.

A Randomized Phase II Trial of High-Dose Melphalan, Ascorbic Acid and Arsenic Trioxide with or without Bortezomib in Multiple Myeloma

Background: There is a role of novel preparative regimens to further improve the outcome after high-dose chemotherapy and autologous hematopoietic stem cell transplantation (ASCT) in multiple myeloma. Arsenic trioxide (ATO) has synergistic activity with melphalan and ascorbic acid (AA) both in vitro and in vivo, and this 3-drug combination was found to be safe and feasible in both conventional and high-dose settings. Bortezomib, a proteasome inhibitor, is an active agent in newly diagnosed or relapsed multiple myeloma in conventional therapy setting. We conducted a randomized phase II trial to determine the safety and efficacy of a combination of ATO, AA and melphalan with or without bortezomib as preparative regimen in patients with myeloma.Methods: Sixty patients were enrolled and 58 patient received ASCT between October 2006 and September 2007. One patient developed respiratory failure and another developed extensive pulmonary embolism after enrollment and did not proceed to ASCT. All 58 evaluable patients received melphalan 100 mg/m2 IV on days -4 and -3, AA 1000 mg/day IV on days -9 to -3 and ATO 0.25 mg/kg IV on days -9 to -3. Patients were randomized to 3 arms; no bortezomib (arm 1), bortezomib 1 mg/m2 on days -9, -6 and -3 (arm 2), and bortezomib 1.5 mg/m2 on days -9, -6 and -3 (arm 3). Patients were also asked to fill out a standardized Quality of Life (QOL) questionnaire at enrollment and at 6 months post-ASCT.Results: Median age of patients in arms 1, 2 and 3 were 61, 59 and 64 years, respectively (p=0.08). Median interval between diagnosis and ASCT were 12.2, 9.6 and 8.8 months, respectively (p=0.3). Cytogenetic abnormalities were detected in 2, 5 and 8 patients in arms 1, 2 and 3, respectively (p=0.08). With a median follow up of 11.4 months (range 5 to 20) post ASCT in surviving patients, cumulative non-relapse mortality was1.7%. Grade 3–4 toxicity was seen in 6 patients in arm 1 (mucositis 3, dyspnea 1, acute renal failure 1, pleural effusion 1), 6 patients in arm 2 (mucositis 3, diarrhea 1, pneumonia 1 and hydronephrosis 1) and 6 patients in arm 3 (pulmonary edema 2, mucositis 1, intestinal obstruction 1, low back pain 1, elevated transaminases 1) (p=0.9). The most common adverse events were nausea, diarrhea and pedal edema. Grade 1–2 weight gain due to fluid retention was seen in 84, 70 and 95% of patients in arms 1, 2 and 3, respectively (p= 0.1). Median time to neutrophil engraftment (ANC >500/dl) was 10 days in each arm. Complete response rates in arms 1, 2 and 3 were 26, 10 and 16%, respectively (0=0.4). Kaplan-Meiers estimates of progression-free survival (PFS) and overall survival (OS) are shown in Figures 1 and 2. Median PFS and OS have not been reached. There was no significant difference in CR, PFS or OS between the 3 arms (p = 0.4, 0.62 and 0.4, respectively). Relapsed disease at ASCT (p=0.003), >12 months interval between diagnosis and ASCT (p=0.05), and cytogenetic abnormalities at diagnosis (p=0.005) predicted a shorter PFS. On a subset analysis, there was no significant difference in PFS or OS between the 3 arms in patients undergoing ASCT in first remission. Fifty-patients submitted their responses to the QOL questionnaire. More patients at 6-month post ASCT reported better overall health (56% vs.12%, p=0.0001). In contrast, more patients at study enrollment reported moderate to severe pain (44% vs. 27%, p=0.09) and debilitating emotional problems (42% vs. 23%, p=0.07).Conclusions: Adding bortezomib to ATO, AA and high dose melphalan is safe and well tolerated as preparative regimen for ASCT in patients with multiple myeloma. Patients with chromosomal abnormalities were predominantly treated in the bortezomib arms without an adverse impact on PFS or OS. Overall QOL indicators showed improvement at 6-month post ASCT. Longer follow up is needed to assess the efficacy of this combination. [Display omitted] [Display omitted]

Arsenic Trioxide Sensitizes Human Glioma Cells, but not Normal Astrocytes, to TRAIL-Induced Apoptosis via CCAAT/Enhancer-Binding Protein Homologous Protein–Dependent DR5 Up-regulation

The current study shows that treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells with a combination of TRAIL and subtoxic doses of arsenic trioxide (As(2)O(3)) induces rapid apoptosis. Whereas TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in glioma cells, treatment with As(2)O(3) efficiently recovered TRAIL-induced activation of caspases. We also found that As(2)O(3) treatment of glioma cells significantly up-regulated DR5, a death receptor of TRAIL. Furthermore, suppression of DR5 expression by small interfering RNA (siRNA) inhibited As(2)O(3)/TRAIL-induced apoptosis of U87MG glioma cells, suggesting that DR5 up-regulation is critical for As(2)O(3)-induced sensitization of glioma cells to TRAIL-mediated apoptosis. Our results also indicate that an increase in CCAAT/enhancer binding protein homologous protein (CHOP) protein levels precedes As(2)O(3)-induced DR5 up-regulation. The involvement of CHOP in this process was confirmed by siRNA-mediated CHOP suppression, which not only attenuated As(2)O(3)-induced DR5 up-regulation but also inhibited the As(2)O(3)-stimulated TRAIL-induced apoptosis. These results therefore suggest that the CHOP-mediated DR5 up-regulation, brought about by As(2)O(3), stimulates the TRAIL-mediated signaling pathway. This in turn leads to complete proteolytic processing of caspase-3, which is partially primed by TRAIL in glioma cells. In contrast to human glioma cells, astrocytes were very resistant to the combined administration of As(2)O(3) and TRAIL, demonstrating the safety of this treatment. In addition, As(2)O(3)-mediated up-regulation of CHOP and DR5, as well as partial proteolytic processing of procaspase-3 by TRAIL, was not induced in astrocytes. Taken together, the present results suggest that the combined treatment of glioma cells with As(2)O(3) plus TRAIL may provide an effective and selective therapeutic strategy.

Analytical artefacts in the speciation of arsenic in clinical samples

Urine and blood samples of cancer patients, treated with high doses of arsenic trioxide were analysed for arsenic species using HPLC-HGAFS and, in some cases, HPLC-ICPMS. Total arsenic was determined with either flow injection-HGAFS in urine or radiochemical neutron activation analysis in blood fractions (in serum/plasma, blood cells). The total arsenic concentrations (during prolonged, daily/weekly arsenic trioxide therapy) were in the μg mL −1 range for urine and in the ng g −1 range for blood fractions. The main arsenic species found in urine were As(III), MA and DMA and in blood As(V), MA and DMA. With proper sample preparation and storage of urine (no preservation agents/storage in liquid nitrogen) no analytical artefacts were observed and absence of significant amounts of alleged trivalent metabolites was proven. On the contrary, in blood samples a certain amount of arsenic can get lost in the speciation procedure what was especially noticeable for the blood cells although also plasma/serum gave rise to some disappearance of arsenic. The latter losses may be attributed to precipitation of As(III)-containing proteins/peptides during the methanol/water extraction procedure whereas the former losses were due to loss of specific As(III)-complexing proteins/peptides (e.g. cysteine, metallothionein, reduced GSH, ferritin) on the column (Hamilton PRP-X100) during the separation procedure. Contemporary analytical protocols are not able to completely avoid artefacts due to losses from the sampling to the detection stage so that it is recommended to be careful with the explanation of results, particularly regarding metabolic and pharmacokinetic interpretations, and always aim to compare the sum of species with the total arsenic concentration determined independently.

Arsenic trioxide, ascorbic acid, filgrastim and erythropoietin for the treatment of myelodysplastic syndromes

17519 Background: Arsenic trioxide (ATO) has been reported to have activity when used as monotherapy for the myelodysplastic syndromes (MDS). Herein we report the results of MDS patients treated with ATO as a differentiating agent coupled with ascorbic acid and cytokine pharmacotherapy. Methods: Nine patients with MDS, regardless of IPSS category and prior treatment, were enrolled in this study. Patients with the proliferative variant of CMML and those with significant cardiac dysfunction or a prolonged QT interval were excluded. They received ATO and ascorbic acid combined with growth factors. All patients received induction therapy with ATO at 0.25 mg/kg IV infusion daily x 5 for one week, followed by twice weekly for seven weeks. Responders were eligible for one consolidation cycle of twice weekly ATO for eight weeks. Four patients received one cycle of consolidation. G-CSF 300 mcg was given thrice weekly and EPO 40,000 units twice weekly. Ascorbic acid 1 gm was given intravenously 30 minutes after each dose of ATO. Response to treatment was assessed using the International Working Group (IWG) criteria for MDS. Results: Median age of patients was 69.5 years (range = 56 to 83 years). IPSS scores were: Int-1 = 5, Int-2 = 2, High = 2. Patients had received prior therapy with azacitidine (n = 8), cytokines (n = 4), and transfusions alone (n = 1). The overall response rate was 56% (5/9): partial response (PR) = 2, hematologic improvement (HI) = 3. Platelet responses were seen in 44% of patients (4/9). Four patients had stable disease (44%). The average duration of response for PR and HI was 122 days and 70 days, respectively. The overall median duration of HI was 3.5 months (range = 0.6 to 4.1 months). There were no cytogenetic responses noted in four evaluable patients. Four patients died during the study. Two patients died due to disease related illness and two due to complications of their comorbid conditions. No treatment related mortality was seen. Conclusions: Arsenic trioxide in combination with ascorbic acid and cytokines has activity in patients with MDS. Larger studies are necessary to establish the efficacy of this regimen earlier in the treatment schema of MDS. No significant financial relationships to disclose.

Arsenic Trioxide Affects Early Stage Angiogenesis through Inhibition of CD14 Monocyte Transdifferentiation into Endothelial Cells Induced by Pleiotrophin and mCSF.

We have discovered a novel mechanism leading to blood vessel formation involving transdifferentiation of monocytes into endothelial cells by tumor cell production of pleiotrophin (PTN), a protein highly produced by myeloma (H. Chen et al, Blood, 2005; Yeh et al BJH, 2006). Arsenic trioxide (ATO) induces apoptosis of cancer cells directly through a number of mechanisms, and this drug has also been shown to inhibit angiogenesis. However, it remains unknown whether ATO affects the earliest stages of angiogenesis and vasculogenesis important in tumor development. We purified human monocytes (CD14+) and cultured these cells on collagen I-coated dishes. mCSF was added to the cells after 1 hour of culture. PTN was added twice to the culture, once after 24 hours and again after 5 days with or without ATO or bortezomib. FLK-1 expression (VEGFR-2) showed that the cells incubated on collagen I without drugs formed tube-like structures in the presence of PTN and mCSF. However, the tube-like structures disappeared after adding either the IC50 (5x10−6M) dose or low (5x10−7M) dose of ATO. FLK-1 staining remains in the tube-like structures with low doses (3x10−12M) of bortezomib. In order to examine whether ATO or bortezomib affects endothelial gene expression when monocytes are induced to transdifferentiate in the presence of these cytokines, we also examined expression using RT-PCR on endothelial cell genes (vascular endothelial growth factor receptor-2 (Flk-1), Tie-2 and von Willebrand factor (vWF)) and Western blot analysis for protein expression. The results of both RT-PCR and Western blot analysis showed that the expression of endothelial markers was blocked at both the higher (5x10−6M) and lower (5x10−7M) doses of ATO. In contrast, the expression of endothelial markers was not reduced by adding low dose bortezomib (3x10−12M). We further examined the effects of ATO and bortezomib on early stage angiogenesis in vivo using the chorioallantoic membrane (CAM) assay. Fertilized chick eggs were incubated horizontally at 38°C in a humidified incubator, windowed by day 3 of incubation and processed by day 8. The tested micro-sponge with ATO (5x10−6M) or bortezomib (3x10−11M) or control reagents was implanted on the CAM. The eggs were sealed with adhesive tape and returned to the incubator for 48 hours. The assay scored positive when two independent observers reported a significant reduction of vessels in the treated area. The results of the CAM assay showed that compared to saline, ATO significantly reduced new macroscopic and microscopic vessel formation. In contrast, bortezomib did not affect angiogenesis in the CAM assay. These experiments define a previously unrecognized novel mechanism by which ATO may have anti-angiogenetic effects in cancer patients-preventing the transdifferentiation of monocytes into endothelial cells by PTN. They also suggest ATO as a potential new specific agent to inhibit angiogenesis resulting from transdifferentiation of monocytes into vascular endothelial cells driven by pleiotrophin and mCSF. These results suggest a novel way by which anti-cancer agents may impact angiogenesis.

Mdm2 Inhibitor Nutlin-3 Enhances the Cytotoxic Synergism Induced by the Combination of MEK1 Inhibitor and Arsenic Trioxide (ATO) in AML Cells.

We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, sensitizes primary acute myelogenous leukemia (AML) to ATO-induced apoptosis via p73, a p53 paralogue, and Bad pro-apoptotic pathways activation (Blood 107: 4549–4554, 2006). We also demonstrated that high doses of ATO (2μM) induced p53 accumulation (more than two-fold increase compared with control) in 11 out of 21 patients (52%), indicating a possible contribution of p53 pathway in apoptosis induction of dual treated blasts. TP53 mutations are quite rare in AML (5–10%) and MDM2 (murine double minute 2), its principal negative regulator, has been found to be frequently overexpressed in AML, a process that can actively enhance tumorigenic potential and resistance to apoptosis. The aim of this study was to investigate whether Nutlin-3, a potent and selective small-molecule MDM2 antagonist, can potentiate the apoptotic effect of the PD plus ATO combination in AML cells that retain a functional p53. Apoptosis was evaluated, after 24 and 48 hours of treatment, by measurement of sub-G1 DNA content, annexin V binding and mitochondrial transmembrane potential assays. We first analyzed the pharmacologic interactions between Nutlin-3, PD and ATO (0, 0.25, 0.5, 1, 2, 5, or 10 μM) using a fixed-ratio (1:1:1) experimental design in OCI-AML-3 cell line that has wild-type p53. We found that the three-drugs combination showed cytotoxic synergism stronger than PD plus ATO combination indicating that the inhibition of the p53-MDM2 interaction can positively influence the pro-apoptotic efficacy of dual-treated (PD plus ATO) cells: the averaged Combination Index (CI) values calculated from the ED50 (50% effective dose), ED75 and ED90, in Nutlin-3 plus PD plus ATO versus PD plus ATO treated cells were 0.36± 0.03 and 0.66± 0.02 respectively (CI values equal to 0.85 or less indicate synergism). In order to investigate the molecular effectors involved in Nutlin-3-PD-ATO or PD-ATO-induced apoptosis we first studied the kinetics (2h, 12h, 24h and 48h) of p53, p73 and phospho-Bad at Ser112 in OCI-AML-3. In the absence of Nutlin-3 ATO, even at high doses, did not promote a p53 accumulation whereas modulated the expression of the p73 gene by inducing the pro-apoptotic and anti-proliferative TAp73 and the anti-apoptotic and pro-proliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD reduced the level of ΔNp73 and blunted the ATO-mediated up-regulation of ΔNp73 thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. In the presence of Nutlin-3, p53 accumulated and the triple combination of Nutlin-3, PD and ATO enhanced the loss of mitochondrial membrane potential occurred in PD plus ATO treatment. Furthermore, pretreatment with MEK1 inhibitor strongly increased the expression of dephosphorylated Bad and blunted the ATO-mediated phosphorylation of Bad at Ser112, thereby activating its pro-apoptotic functions. These findings suggest that the pro-apoptotic p73 and Bad pathways, both involved in PD plus ATO efficacy, can be potentiated by the rescue of p53 pathway in AML cells that possess a functional p53 pathway.

Arsenic Trioxide Sensitizes Glucocorticoid-Resistant T-Cell Acute Lymphoblastic Leukemia to Dexamethasone through Inactivation of AKT and Downregulation of XIAP.

Arsenic trioxide (ATO) is a very effective agent for the treatment of acute promyelocytic leukemia (APL). Because of its pro-apoptotic activity already at low dosage, ATO may be beneficial for combination treatment of drug-resistant disease to augment the effect of other chemotherapeutic agents. In childhood acute lymphoblastic leukemia (ALL), de novo resistance to glucocorticoids (GC) is markedly associated with poor outcome. As a mechanism of action, GCs induce apoptosis in steroid-sensitive leukemic cells. Recent evidence suggests that GC-resistance is associated with altered levels of pro- and anti-apoptotic regulatory proteins. We therefore evaluated the potential of ATO as a chemo-sensitizer in a model of GC-resistant ALL. As expected, ATO effectively induced cell death in ALL cell lines and in primary ALL patient samples at clinically relevant concentrations (IC50 values between 0.4–1.2 uM). Interestingly, ATO re-sensitized the GC-resistant ALL lines CEM C1-15, MOLT-4 and Jurkat to dexamethasone at doses that do not affect cell survival as single agent. The same effect was observed using primary cells from two patients with GC-resistant T-cell ALL. The sensitizing effect of ATO could not be observed in combination with the standard ALL chemotherapeutic agents daunorubicin, asparaginase and vincristine, suggesting that a mechanism specifically relevant for GC-resistance is involved. ATO, alone or at lower dose in combination with dexamethasone, induced cell death by activating caspase-3 and −8, which resulted in PARP cleavage. We then investigated the MAPK and PI3K/AKT pathways as candidate mechanisms of action with key apoptosis regulators such as Bad and Bcl-2 as targets. The MEK1/2 inhibitor PD98059 only marginally augmented the response to dexamethasone, suggesting that this pathway would not play a major role for GC sensitization. In contrast, we found that low dose ATO treatment resulted in a rapid decrease of AKT phosphorylation at the activating serine 473 phosphorylation site, while AKT levels remained unaffected. Concomitantly, the AKT targets Bad and XIAP appeared to be modulated. The pro-apoptotic regulator Bad was induced, and its phosphorylation was decreased. Downregulation of Bad mediated by siRNA partly rescued cells from ATO-induced cell death indicating that Bad might have a sensitizing role in ATO-mediated apoptosis. Moreover, ATO induced downregulation of the NF-kB target XIAP. The protein levels of other NF-kB targets such as Bcl-2 and Bcl-xL were unchanged. Downregulation of XIAP could be due to decreased protein stability resulting from reduced XIAP phosphorylation by AKT. The effects of low dose ATO on AKT phosphorylation as well as Bad and XIAP levels were more pronounced when cells were treated in combination with dexamethasone. Ongoing experiments will further define the role of XIAP and NF-kB as specific therapeutic targets in this model. These results indicate that resistance to GCs is - at least in part - mediated through apoptosis regulators in ALL. Our data provide evidence that ATO may be beneficial as sensitizing agent in defined subgroups of ALL patients, next to its role in APL and multiple myeloma treatment.

PK11195, a Ligand of the Peripheral Benzodiazepine Receptor, Inhibits Myeloma Cell Growth In Vitro and Chemosensitizes Myeloma Cells In Vivo in SCID-hu Models of Human Multiple Myeloma.

The peripheral benzodiazepine receptor (mPBR) appears to be a potential target to induce apoptosis in tumor cells. The expression of this receptor has been linked to a poor prognosis in cancer patients. PK11195 may represent a new, well-tolerated potent chemosensitizing agent that affects multiple resistance mechanisms within malignant cells. We have evaluated whether PK11195 inhibits multiple myeloma (MM) cell growth in vitro; and, furthermore, whether this drug can chemosensitize a melphalan resistant human MM tumor, LAGλ-1 (Campbell et al, International Journal of Oncology 2006), to arsenic trioxide (ATO) and melphalan using an in vivo SCID-hu model. The MM cell lines RPMI8226 and U266 were treated with varying concentrations of PK11195 (1 – 100 mM). After incubating with PK11195 for 24 hours, cell growth was measured by MTT assay. Those cells treated with PK11195 showed decreased proliferation at concentrations as low as 1 mM compared to the untreated cells. Next, we investigated the chemosensitizing effects of PK11195 using an in vivo model of human MM. To accomplish this, each immunodeficient (SCID) mouse was implanted with a 2.0 – 4.0 mm3 LAGλ-1 tumor fragment into the left superficial gluteal muscle. The tumors were allowed to grow for 14 days at which time human IgG levels were detectable in the mouse serum or when tumors became palpable (21 days) and mice were blindly assigned into treatment groups. PK11195 (10, 50 and 100 mg/kg) was administered via oral gavage once weekly when combined with melphalan and once daily five times per week when combined with ATO. Melphalan (3 mg/kg) was administered once weekly via intraperitoneal (i.p.) injection. ATO (1.25 mg/kg) was administered once daily five times per week via i.p. injection. Mice receiving the combination of PK11195 and melphalan (3 mg/kg) showed marked inhibition of tumor growth (PK11195 10 mg/kg, P = 0.03; PK11195 50 mg/kg, P = 0.02; PK11195 200 mg/kg, P < 0.01) compared to mice receiving no therapy. Animals treated with melphalan, as a single agent, did show minimal tumor growth inhibition and reduced paraprotein levels whereas mice treated with single agent PK11195 showed tumor growth similar to the control mice. Mice receiving the combination of PK11195 and low dose ATO (1.25 mg/kg) also showed inhibition of tumor growth (PK11195 200 mg/kg, P < 0.01) whereas treatment with either single agent PK11195 or ATO demonstrated growth similar to the control groups. Treatment with the highest dose of PK11195 (200 mg/kg) was not associated with any observed toxicity suggesting that high doses can be safely administered and are well tolerated. In this study, we showed PK11195 inhibits MM cell growth in vitro at very low concentrations and can chemosensitize drug resistant tumor cells in vivo at doses that have no observable toxicity. We are further evaluating PK11195 as a single agent and in combination therapy both in vitro and in vivo..

Sensitivity of leukemic cell line HL-60 to combination of neferine and arsenic

ObjectiveTo determine whether neferine (Nef) enhances the sensitivity of human myeloid leukemia HL-60 cells to arsenic trioxide (ATO).MethodsApoptosis was detected by DNA electrophoresis. Giemsa staining was used to observe the apoptotic cells under microscope. The apoptotic rates of cells were analyzed using flow cytometry. The inhibitory rates of cell growth were assayed by MTT, and the expression of P-gp was determined by flow cytometry.ResultsLow doses of ATO (1.0 μmol/L) only partially inhibitory percentage of cell growth at 72 h was (9.92±3.03) % (P>0.05, vs control). The combination of 1.0 μmol/L ATO with 2.0 μmol/L Nef inhibited (45.27±4.93) % of cell growth, and induced apoptosis in leukemic cells more significantly than wither ATO or Nef on their own (P<0.01). Moreover, ATO-induced expression of P-gp in leukemic cells was inhibited significantly by Nef.ConclusionThese results indicate that Nef significantly increases sensitivity of leukemic cells to ATO, which might be associated with inhibitory expression of P-gp gene. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.

MEK1 inhibition sensitizes primary acute myelogenous leukemia to arsenic trioxide–induced apoptosis

AbstractWe found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73 gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD184352 reduces the level of ΔNp73 and blunts the arsenic-mediated up-regulation of ΔNp73, thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1 (p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.

Optimization of combination therapy of arsenic trioxide and fractionated radiotherapy for malignant glioma

The primary objective was to optimize the combined treatment regimen using arsenic trioxide (ATO) and fractionated radiotherapy for the treatment of malignant glioma. Nude mice with human glioma xenograft tumors were treated with fractionated local tumor radiation of 250 cGy/fraction/day and 5 mg/kg ATO for 5-10 days. Time course experiments demonstrated that maximal tumor growth delay occurred when ATO was administered between 0 and 4 h after radiation. The combination treatment of ATO and radiation synergistically inhibited tumor growth and produced a tumor growth delay time of 13.2 days, compared with 1.4 days and 6.5 days for ATO and radiation alone (p < 0.01), respectively. The use of concurrent therapy of radiation and ATO initially, followed by ATO as maintenance therapy, was superior to the use of preloading with ATO before combined therapy and produced a tumor growth delay time of 22.7 days as compared with 11.7 days for the ATO preloading regimen (p < 0.01). The maintenance dose of ATO after concurrent therapy was effective and important for continued inhibition of tumor growth. The combined use of fractionated radiation and ATO is effective for the treatment of glioma xenograft tumors. ATO was most effective when administered 0-4 h after radiation without pretreatment with ATO. These results have important implications for the optimization of treatment regimen using ATO and fractionated radiotherapy for the treatment of brain tumors.

Use of Arsenic Trioxide in Acute Promyelocytic Leukemia Leads to Cardiotoxicity in African-American Patients.

Arsenic trioxide (ATO) is an effective therapy for acute promyelocytic leukemia (APL), but cardiac side effects have been described. We reviewed the charts of all 77 patients given ATO at M.D. Anderson from 1998 to present. Our objectives were to: (1) identify patients at relatively high risk of developing arrhythmias after receiving ATO and (2) evaluate the sensitivity and specificity of the electrocardiographic QTc interval in predicting subsequent arrhythmias. The median age of the patients was 54 years. 5% were African-American. 70% received ATO for treatment of APL, 18% for myelodysplastic syndrome, and 12% for other hematological malignancies. The dose of ATO was 0.15 mg/kg daily for 60 days. If, after this time, response was observed ATO was given for an additional 6 months, on a 2 weeks on, 2 weeks off schedule. The dose was lowered from 0.15 mg/kg in the event of acute toxicity, however the African-American patients discussed below were on the standard 0.15 mg/kg dose when they experienced arrhythmias.Arrhythmias were observed in 4 of the 77 patients (5%, exact 95% CI 1– 13%). Of these 4 patients, 2 developed ventricular tachycardia and 2 atrial tachycardia. In 3 of the 4 patients, these arrhythmias led to discontinuation of ATO and its replacement by mylotarg. Three of the 4 African-Americans given ATO developed arrhythmias vs. only 1/73 non African-Americans (Fisher exact p value, 0.002). The patients who developed arrhythmias were younger (median age = 32) than those who did not (median age = 57) and had no history of cardiac disease, electrolyte abnormalities, or use of medications that would alter their response to ATO relative to the other patients. African-Americans tended to be younger than non African-Americans: 3/4 were age < 40 vs. 19/73 non African-Americans. However, since the incidence of arrhythmias in patients age < 40 was 3/3 for African-Americans vs. 0/19 for others (p= 0.0006), it seems that race, not age, is the principal predictor of arrhythmias. Similarly, although all 4 African-Americans had APL vs. 50/73 others, the respective incidence of arrhythmias in African-American APL patients was 3/4 vs. 1/50 in non African-American APL patients (p = 0.0006). Furthermore, arrhythmias were seen on average 8 days after ATO induction in the 3 African-Americans, while the Caucasian patient did not develop arrhythmias until 45 days after ATO induction. Although all patients were on weekly to bimonthly ECG monitoring, QTc interval prolongation (> 430 ms) was not observed in the patients who developed serious cardiac arrhythmias. None of the 3 patients with QTc prolongation had a documented arrhythmia. Thus, QTc monitoring had zero sensitivity and zero specificity for eventual arrhythmia development. Despite uncertainty as to mechanism, physicians should be aware that ATO may cause arrhythmias in young African Americans. Our data also calls into question the value of QTc monitoring, at least in patients given ATO.

Treatment of Non-APL Acute Myelogenous Leukemia with Intravenous Arsenic Trioxide Plus Ascorbic Acid.

Introduction: Arsenic trioxide (ATO) is an exceptionally active drug in acute promyelocytic leukemia (APL), inducing complete remissions (CR) in 85% of relapsed patients. ATO also has clinical activity in myelodysplastic syndromes (MDS). In non-APL AML cells lines, ATO induces apoptosis in vitro; however, in a small study of 11 non-APL AML patients, ATO showed no activity (Parmer et al Leuk Res 28:090, 2004). ATO-induced apoptosis has been shown to correlate inversely with the level of intracellular reduced glutathione (GSH) via generation of reactive oxygen species; cells with high concentrations of GSH are more resistant to ATO. Ascorbic acid (AA) increases apoptosis and overcomes resistance to ATO in multiple myeloma, non-APL AML and other cell lines by reducing intracellular GSH levels. AA alone has no activity in these cells. We therefore administered ATO plus AA to patients with non-APL AML.Methods: ATO 0.25 mg/kg was administrated intravenously over 1-4 hours with 1 gram of intravenous AA given 30 minutes after ATO daily for five days a week (five days on/2 days off) for five weeks (25 doses=one cycle). These doses were based on a phase I/II trial of ATO + AA in patients with multiple myeloma (Bahlis et al Clin Cancer Res 8:3658, 2002)). Responding patients received an additional consolidation cycle of 25 doses followed by maintenance including ATO + AA two weeks of every month for 4 cycles. Patients who failed to respond after two cycles were considered treatment failures.Results: Nine patients aged 36–84 years (median: 66 years) were treated: 5 pts. had refractory AML or relapsed after chemotherapy; 4 elderly pts,. aged 66–84, with antecedent MDS were previously untreated. Activity was seen in 5 (56%) patients, including all 4 of the previously untreated pts: CR in 1 pt. (patient with an early relapse), CRp in 1 pt., decrease in bone marrow blasts to <5% with reduced transfusion requirements in 1 pt, and drop in peripheral blood blasts from 84% and 51% to <5% in 2 patients. Responses were observed after the first treatment cycle. Duration of responses were 6 months in the CR and CRp pts., and 1 week, 2 months. and 4 months in the other responders. Four pts had progressive disease; one patient died on study from progressive disease and infection. Grade 3 /4 toxicity included: infection in 8 pts., and anorexia, sensory neuropathy, elevated bilirubin in 1 pt. each. One responding patient developed shortness of breath with severe hypoxemia, reminiscent of the APL differentiation syndrome, which responded immediately to dexamethasone.Conclusions: ATO + AA has anti-leukemic activity in non-APL AML patients. Despite the high doses of ATO, higher than used in APL and MDS, toxicity was tolerable. This combination could be a less toxic alternative front-line approach to intensive chemotherapy in elderly patients with non-APL AML. Further study of ATO + AA is warranted either alone or in combination with other agents in an attempt to further improve the results.

Phase II trial of arsenic trioxide (ATO) and ascorbic acid (AA) with temozolomide (TMZ) in patients with metastatic melanoma (MM) with or without CNS metastases

7553 Background: TMZ is commonly used to treat MM but has a response rate of only 10 to 15%. Melanoma resistance to TMZ may occur through upregulation of NF-κB and inhibition of apoptosis. ATO downregulates NF-κB activation and can arrest cancer cell growth and induce apoptosis. AA augments the effects of ATO by depleting glutathione stores. We therefore wanted to determine whether treatment with ATO and AA would augment the response rate to TMZ by increasing melanoma sensitivity to apoptosis. As both ATO and TMZ cross the blood-brain barrier, we also wanted to determine whether this regimen was active in MM patients with CNS disease. Methods: We developed a phase II trial combining TMZ with ATO/AA. The design called for the enrollment of 40 patients with stage IV melanoma and an ECOG PS of 0–2, and included early stopping rules for low response rate or toxicity. Prior therapy other than TMZ or DTIC was permitted. ATO was administered IV as a loading dose at 0.25 mg/kg/d x 5 days during week 0, and then twice weekly at 0.35 mg/kg x 8 weeks. Each dose of ATO was followed by a 1000 mg IV dose of AA. During each 8-week cycle, oral TMZ was given at 200 mg/m2/d x 5d on weeks 1 and 5. Results: Eleven patients have been enrolled to date, including 5 with CNS metastases, and 9 were evaluable for response. Common grade 1 and 2 side effects included edema (6), fatigue (4), rash (3), and elevated AST or ALT (3). Grade 3 and 4 side effects were nausea and vomiting (4), elevated AST or ALT (2), seizure (1), and renal failure (1). All 9 patients had progressive disease after 1 cycle of therapy. Conclusions: ATO and AA can be administered safely with TMZ on an outpatient basis with acceptable toxicity. No responses have been observed among the first 9 melanoma patients. The lack of activity will trigger early closure of the trial if the tenth patient currently on study does not have a major response. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cell Therapeutics

Perfusion and diffusion MRI in the assessment of the antivascular effect of arsenic trioxide combined with radiotherapy for glioblastoma multiforme: NABTT phase I study

1538 Background: Significant tumor control and prolongation of survival have been demonstrated by combined use of radiosurgery and antivascular agent, Arsenic Trioxide (ATO), in rodent gliosarcoma model in rats. This agent is now undergoing clinical evaluation in the NABTT Brain Tumor Consortium in a Phase I trial with ATO dose escalation in combination with fractionated radiation therapy. Since this agent showed rapid effect on the tumor vasculature in the animal studies, MRI perfusion and diffusion studies were performed shortly following ATO administration. Methods: The patients were treated with external beam radiotherapy 60 Gy in 6 weeks with ATO at a starting dose of 0.25 mg/kg once a week during the course radiotherapy. ATO dose was escalated by 0.05 mg. The perfusion images were acquired using a Echo Planar Image (EPI) sequence. Perfusion parameters were Mean Transit Time (MTT), relative Cerebral Blood Volume (rCBV), and relative Cerebral Blood Flow (rCBF). The perfusion parameter values under regions of interest (ROI) from the enhancing and non-enhancing portions of the lesion were then followed serially during treatment. Results: There was a delay on mean transit time (MTT) and relative cerebral flow (rCBF) by ATO and radiotherapy, compared to counterpart normal brain. This effect was apparent within 24 hours of ATO infusion. The extent and duration of these changes will be correlated with conventional measures of outcome, such as lesion volumes, response, and time to tumor progression (TTP). There were no dose limiting toxicities with doses of ATO as high as 0.35 mg/kg once a week. Conclusions: A total of 12 patients were treated with this phase I trial. At the doses studied thus far, ATO combined with radiotherapy appears safe in the treatment of glioblastoma multiforme. ATO induced a differential delay of blood flow in the tumor compared to the normal brain. ATO showed the immediate effects and serial evaluation highlights the dynamic nature of the intratumoral vasculature in response to ATO, antivascular agent. No significant financial relationships to disclose.

Targeting signal transducer and activator of transcription 3 protein with arsenic trioxide: A phase I clinical trial

6538 Background: Constitutive signal transducer and activator of transcription (STAT) 3 activity has been shown to be present in acute myeloid leukemia (AML) cells from 50% of patients and to correlate with shorter disease-free survival. We hypothesized that down-regulating constitutive STAT3 activity might improve outcome. In the absence of a specific STAT3 inhibitor, we studied arsenic trioxide (ATO) in vitro and showed that ATO at a concentration of 0.91 μM achieved 50% down-regulation of constitutive STAT3 activity in AML cells within 6 hours. We then conducted a phase I clinical trial to determine the biologically effective dose (BED) and/or the maximally tolerated dose of ATO in vivo in conjunction with induction chemotherapy. Methods: Untreated AML patients younger than age 60 received a single one-hour ATO infusion in escalating doses in 3-patient cohorts prior to high-dose cytarabine [3 gm/m2 (1.5 mg/m2 for age ≥ 50) every 12 hrs x 12 doses] and idarubicin (12 mg/m2 x 3 doses). The BED was defined by a 50% reduction in constitutive STAT3 activity in leukemia cells in vivo as determined by western blotting. ATO doses ranged from 0.01 to 0.25 mg/kg, the latter equivalent to 4 μM, the effective concentration in patients with acute promyelocytic leukemia. Results: Twenty-seven patients (17:10 female:male) were accrued, including 2 with secondary AML. Median age was 46 (range 22–57) years, and median white blood cell count at diagnosis was 13.2 (range 1.6–95.1) x 109/L. Karyotype was favorable in 3, normal in 14 and unfavorable in 10. Eighteen patients achieved complete remission, 5 had refractory disease, 3 were unevaluable and 1 is too early to assess. The BED was achieved at 0.25 mg/kg without dose-limiting toxicity and we are currently accruing 7 more patients at this dose level to study apoptosis and gene expression profiling in pre- and post-ATO samples. Conclusions: ATO down-regulates constitutive STAT3 activity in vivo in clinically tolerable doses. Its impact on treatment outcome will be studied in future trials. No significant financial relationships to disclose.

Extended stability of ascorbic acid in 5% dextrose injection and 0.9% sodium chloride injection

Arsenic trioxide induces apoptosis in multiple myeloma cells.[1][1] Ascorbic acid 1000 mg, given intravenously over 15 minutes, has been administered after doses of arsenic trioxide to decrease the intracellular levels of glutathione.[2][2]–[6][3] Elevated glutathione levels are thought to be the