Realgar-Indigo Naturalis Formula Research Articles

Oral arsenic plus imatinib versus imatinib solely for newly diagnosed chronic myeloid leukemia: a randomized phase 3 trial with 5-year outcomes

PurposeThe synergistic effects of combining arsenic compounds with imatinib against chronic myeloid leukemia (CML) have been established using in vitro data. We conducted a clinical trial to compare the efficacy of the arsenic realgar–indigo naturalis formula (RIF) plus imatinib with that of imatinib monotherapy in patients with newly diagnosed chronic phase CML (CP-CML).MethodsIn this multicenter, randomized, double-blind, phase 3 trial, 191 outpatients with newly diagnosed CP-CML were randomly assigned to receive oral RIF plus imatinib (n = 96) or placebo plus imatinib (n = 95). The primary end point was the major molecular response (MMR) at 6 months. Secondary end points include molecular response 4 (MR4), molecular response 4.5 (MR4.5), progression-free survival (PFS), overall survival (OS), and adverse events.ResultsThe median follow-up duration was 51 months. Due to the COVID-19 pandemic, the recruitment to this study had to be terminated early, on May 28, 2020. The rates of MMR had no significant statistical difference between combination and imatinib arms at 6 months and any other time during the trial. MR4 rates were similar in both arms. However, the 12-month cumulative rates of MR4.5 in the combination and imatinib arms were 20.8% and 10.5%, respectively (p = 0.043). In core treatment since the 2-year analysis, the frequency of MR4.5 was 55.6% in the combination arm and 38.6% in the imatinib arm (p = 0.063). PFS and OS were similar at five years. The safety profiles were similar and serious adverse events were uncommon in both groups.ConclusionThe results of imatinib plus RIF as a first-line treatment of CP-CML compared with imatinib might be more effective for achieving a deeper molecular response (Chinadrugtrials number, CTR20170221).

Research on different compound combinations of Realgar-Indigo naturalis formula to reverse acute promyelocytic leukemia arsenic resistance by regulating autophagy through mTOR pathway

Ethnopharmacological relevanceIn China, the Chinese patent drug Realgar-Indigo naturalis Formula (RIF) is utilized for the therapy of acute promyelocytic leukemia (APL). Comprising four traditional Chinese herb—Realgar, Indigo naturalis, Salvia miltiorrhiza, and Pseudostellaria heterophylla—it notably includes tetra-arsenic tetra-sulfide, indirubin, tanshinone IIa, and total saponins of Radix Pseudostellariae as its primary active components. Due to its arsenic content, RIF distinctly contributes to the therapy for APL. However, the challenge of arsenic resistance in APL patients complicates the clinical use of arsenic agents. Interestingly, RIF demonstrates a high remission rate in APL patients, suggesting that its efficacy is not significantly compromised by arsenic resistance. Yet, the current state of research on RIF's ability to reverse arsenic resistance remains unclear. Aim of the studyTo investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL. Materials and methodsThe present study utilized the arsenic-resistant HL60-PMLA216V-RARα cell line to investigate the effects of various RIF compounds, namely tetra-arsenic tetra-sulfide (A), indirubin (I), tanshinone IIa (T), and total saponins of Radix Pseudostellariae (S). The assessment of cell viability, observation of cell morphology, and evaluation of cell apoptosis were performed. Furthermore, the mitochondrial membrane potential, changes in the levels of PMLA216V-RARα, apoptosis-related factors, and the PI3K/AKT/mTOR pathway were examined, along with autophagy in all experimental groups. Meanwhile, we observed the changes about autophagy after blocking the PI3K or mTOR pathway. ResultsTanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PMLA216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy. ConclusionsWe illustrated that the synergistic effect of different compound combinations of RIF can regulate autophagy through the mTOR pathway, enhance cell apoptosis, and degrade arsenic-resistant PMLA216V-RARα.

Long-term outcome of children with acute promyelocytic leukemia: a randomized study of oral versus intravenous arsenic by SCCLG-APL group

Realgar-Indigo naturalis formula (RIF), an oral traditional Chinese medicine mainly containing Realgar (As4S4), is highly effective in treating adult acute promyelocytic leukemia (APL). However, the treatment efficacy and safety of RIF have not been verified in pediatric patients. SCCLG-APL group conducted a multicenter randomized non-inferiority trial to determine whether intravenous arsenic trioxide (ATO) can be substituted by oral RIF in treating pediatric APL. Of 176 eligible patients enrolled, 91 and 85 were randomized to ATO and RIF groups, respectively. Patients were treated with the risk-adapted protocol. Induction, consolidation, and 96-week maintenance treatment contained all-trans-retinoic acid and low-intensity chemotherapy, and either ATO or RIF. The primary endpoint was 5-year event-free survival (EFS). The secondary endpoints were adverse events and hospital days. After a median 6-year follow-up, the 5-year EFS was 97.6% in both groups. However, the RIF group had significantly shorter hospital stays and lower incidence of infection and tended to have less cardiac toxicity. All 4 relapses occurred within 1.5 years after completion of maintenance therapy. No long-term arsenic retentions were observed in either group. Substituting oral RIF for ATO maintains treatment efficacy while reducing hospitalization and adverse events in treating pediatric APL patients, which may be a future treatment strategy for APL.

Research on Different Compound Combinations of Realgar-Indigo Naturalis Formula to Reverse APL Arsenic Resistance By Regulating Autophagy through mTOR Pathway

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia, and 90% of APL patients carry positive fusion gene of the promyelocytic leukemia protein (PML)- retinoic acid receptor α (RARα), which drives the development of APL. All-trans retinoic acid and arsenic trioxide is able to cure 90% of APL patients by targeting PML-RARα. However, there are still 5%-10% of patients who will relapse due to drug resistance. Compound realgar-indigo naturalis formula (RIF) was developed based on the characteristics of APL pathogenesis. It contains four Chinese herbs: realgar, natural indigo, danshen root and heterophylly falsestarwort root. Clinical studies have confirmed that RIF has a certain efficacy on APL, which shown non-inferiority compared with arsenic trioxide. In addition, RIF combined with all-trans retinoic acid has become the first-line treatment regimen in APL guidelines in China. This study further clarified the efficacy of RIF on arsenic-resistant APL, explored the mechanism, and laid the foundation for further improving the efficacy of APL. O bjective: To investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL. M ethods: The arsenic-resistant cell line HL60-PML A216V-RARα, which could stably express PML A216V-RARα, was established by lentiviral transfection. Among compounds of RIF, the active compound of realgar is tetra-arsenic tetra-sulfide (As 4S 4, A), for natural indigo is indirubin (I), for danshen root is tanshinone IIa (T), and the for heterophylly falsestarwort root is total saponins of Radix Pseudostellariae (S). The four compounds were applied on the HL60-PML A216V-RARα cell line. Cell viability was detected by CCK-8 method. Hoechst staining was used to observe cell morphology, Annexin V/PI double staining was applied to detect cell apoptosis, JC-1 detection was used to detect mitochondrial membrane potential, flow cytometry was used to detect CD33 expression to evaluate cell differentiation, and Western blotting was used to observe the changes in the levels of PML A216V-RARα, apoptosis-related factors, PI3K/AKT/mTOR and autophagy in the control, A, ITS and AITS groups. The PI3K inhibitor LY294002 and the autophagy inhibitor Baf A1 were combined and applied to observe the changes in the levels of PML A216V-RARα, mTOR and p62 after blocking the PI3K or autophagy pathway. Results: The arsenic-resistant cell line HL60-PML A216V-RARα was successfully established. Compared with the control group, A, ITS and AITS groups all inhibited the growth of HL60-PML A216V-RARα cells in a time-dependent manner ( P < 0.0001). The IC50 value of AITS was significantly lower than the other groups ( P < 0.0001), and AITS group exhibited the strongest inhibitory effect, followed by ITS and A. Hoechst staining and JC-1 detection both showed that AITS had the strongest effect in inducing apoptosis. Annxin V/PI detection showed that the apoptosis rate of the control group was 10.04%, 15.19% for A group, 12.46% for ITS group, and 28.05% for AITS group. The differences between the groups were statistically significant ( P < 0.0001). Detection of CD33 expression showed: control group (79.72%), ITS group (79.19%), A group (28.60%), AITS group (24.46%), and there were significant differences between groups ( P < 0.001 or P < 0.0001). AITS could reduce PML A216V-RARα protein, caspase3, Bcl-2 levels more than ITS and A ( P < 0.05 or P < 0.0001), and ITS was better than AITS and A in increasing cleaved PARP-1 levels ( P < 0.0001). AITS decreased the levels of PI3K, mTOR, P-mTOR, AKT, p-AKT and p62 and increased the level of LC3B ( P < 0.001 or P < 0.0001). Electron microscopic detection of autophagy showed that AITS had the highest level of autophagy, AITS combined with LY294002 increased the level of p62 and decreased the level of mTOR ( P < 0.0001) compared to the AITS group; while when AITS was combined with Baf A1, the level of p62 increased ( P < 0.0001) and the level of mTOR also increased ( P < 0.001). Autophagy could induced by serum starvation. As the concentration of fetal bovine serum decreased, the effects of degrading PML A216V-RARα and p62, and LC3B level increasing were stronger. C onclusion: Tanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PML A216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy.

Improved Prevention and Treatment Strategy of Differentiation Syndrome Contribute to Reduce Early Death of Patients with Acute Promyelocytic Leukemia

Purpose: All-trans retinoic acid (ATRA) with arsenic trioxide (ATO) has been the standard of care for acute promyelocytic leukemia (APL) with high efficacy in china.However,early death (ED) still remains the major reason for treatment failure.Severe differentiation syndrome (DS) with no response to full dose dexamethasone is one of direct or indirect important causes of ED.Therefore,to further optimize prevention and treatment strategy of DS is critical for APL therapy success. Patients and Methods： The APL-01 study was a prospective and single-arm clinical trial in patients with newly diagnosed APL from June 2019 to December 2021.111 eligible patients aged between 18 and 70 years classified as Low-risk APL (WBC count at diagnosis＜10*10 9/L n=78) or High-risk APL (WBC count at diagnosis≥10*10 9/L n=33) received induction therapy consisted of ATRA and intravenous arsenic trioxide or oral tetra-sulfide arsenic formulation (Realgar-Indigo naturalis formula,RIF) with chemotherapy-free or chemotherapy-substantially reduced protocol in China. During the course of induction therapy,we used different intravenous doses of dexamethasone to prevent DS according to the WBC count at presentation or after the initiation of ATRA.For example,if WBC counts≥ 5*10 9/l and <10*10 9/l,5mg per day;if ≥10 *10 9/l,10mg per day.And we gave patients dexamethasone 10mg per 12h as preemptive therapy once DS was suspected.Ruxolitinib was administered when steroids therapy were considerd insensitive. We evaluated the effect of selective steroids prophylaxis and preemptive therapy on the incidence and severity of DS,which contributed to increased early mortality rate. The safety and feasibility of Ruxolitinib as second-line therapy for DS was also explored. Results： In this study,41 of 111 patients (36.9%) experienced DS,16 (14.4%) were severe cases and 25 (22.5%) were moderate cases. Univariate analysis identified the following prognostic factors as regards developing DS（P-value<0.1）:age,sex and WBC count were included for exploring the difference between DS and Non-DS patients Increased WBC count (>4*10*109/l )(OR (95% CI)=1.241 (1.131-1.361)) were potential risk factors for patients with DS.We further investigated the difference between severe DS and moderate DS using age,sex,ECOG,WBC and PLT count.We found that only advanced age (over 40 years old)(OR (95% CI)=12.200 (1.640-90.772)) was associated with developing severe DS. Dyspnea (71%),weight gain (85%) and pulmonary infiltrates(78%) were the most common symptoms of patients with DS.Compared with moderate DS,severe DS patients had more frequency of dyspnea (Fishers exact P=0.013),pleuropericardial effusion (χ2=5.577, P=0.018)and acute renal dysfunction (Fishers exact P=0.018). Among the 41 patients with DS,23 patients were treated effectively with dexamethasone 10mg per 12h.18 patients with steroid resistance received Ruxolitinib to control the cytokine release storm（CRS）,12 of which showed immediate remission, however,the situations of the remaining 6 patients did not alleviate until ATRA was suspended temporarily.All of them（6/41）recovered with the help of noninvasive mechanical ventilation. The total 30-day mortality rate was 1.8% (2/111).During induction treatment,no death owing to DS or infection occurred.Two patients with high-risk APL died due to intracranial hemorrhage,one occurred on day 8 and the other on day 9 after ATRA were initiated. Of all the 109 patients evaluable,78 (100%)Low-risk patients and 31 (100%) high-risk patients achieved complete remission (CR)at the end of induction therapy,respectively.The median follow-up time was 34 months,estimated 2-year overall survival (OS) was 98.7% in the Low-risk group and was 93.9% in the High risk group. Conclusions: Our study suggests individualized steroids prevention,preemptive treatment and Ruxolitinib as second-line therapy for DS contribute to control DS,which in verse lessen the discontinuation of ATRA, consequently decreasing early death due to DS and hemorrhage in APL patients.(clinicaltrials.gov NCT04446806) Keywords: acute promyelocytic leukemia;early death;prophylaxis strategy;pulmonary hemorrhage;differentiation syndrome;retinoid acid syndrome;Ruxolitinib;steroid-resistance;cytokine release syndrome;

Recommendations on Minimal Residual Disease Monitoring in Acute Promyelocytic Leukemia Treated with Combination of All- Trans Retinoic Acid and Arsenic Trioxide

Background All- trans retinoic acid (ATRA)-arsenic trioxide (ATO) combination therapy significantly reduces acute promyelocytic leukemia (APL) relapse compared to ATRA combined with chemotherapy, leading to a cure rate of 80-90% in APL patients. The ideal frequency and duration of minimal residual disease (MRD) monitoring needs to be identified in the era of ATO. Methods A total of 1072 APL patients were enrolled in this study, comprising 855 from the APL2012 trial (NCT01987297) and 217 from our center between January 2018 to June 2022 (APL2018 study). The APL2018 protocol was adjusted for consolidation and maintenance therapy based on APL2012 (Chen et. al. PNAS 2021): In consolidation therapy, low-, intermediate-, and high-risk patients were performed following the ATO-group scheme of corresponding risk stratification in the APL2012 study. From December 2019, chemotherapy was no longer administered to high-risk group during consolidation phase. In maintenance treatment, ATO was replaced with oral arsenic realgar-indigo naturalis formula, and methotrexate was removed. RT-PCR or RQ-PCR was routinely used for MRD monitoring, detecting PML::RARA fusion genes in bone marrow before each consolidation and maintenance therapy. MRD testing was conducted every 3 months within the first year, every 6 months within the second year, and annually from 3 to 5 years after treatment. Results A total of 993 patients achieved complete remission (CR) after induction therapy. The negative MRD rates were observed in 61.7% of patients before 1 st consolidation , 95.4% before 2 nd consolidation, 99.3% before 3 rd consolidation, and 99.5% before maintenance therapy , respectively. The median time to achieve negative MRD was 57 (22-301) days with no significant difference between relapsed and non-relapsed patients (p=0.166). A total of 34 patients (30 in the APL2012 trial and 4 in the APL2018 study) relapsed, including 5 at low risk, 9 at intermediate risk, and 20 at high risk. The median recurrence time was 13.5 (3.5-49.0) months after achieving CR, with 94% (32/34) relapsing within 3 years of achieving CR. After induction remission, the risk of relapse decreased with time (44% occurred within the first year, 29% within the second year, 21% within the third year, and only 6% after three years, Figure 1). The median follow-up for the 179 evaluable patients after achieving CR was 39.7 (3.5 -64.6) months in the APL2018 group. The estimated 5-year disease-free survival (DFS) rates were 95.8% (95% CI 92.8-98.9). Prior to the 1 st, 2 nd, and 3 rd consolidation treatment, and before maintenance therapy, the estimated 5-year DFS rates for MRD positive and negative patients were 92.4% (95% CI 86.1-99.2) versus 97.8% (95% CI 94.8-100), p=0.100; 57.1% (95% CI 30.1-100) versus 97.3% (95% CI 94.7-100), p<0.001; 0 versus 96.5% (95% CI 93.1-100), p<0.001; 0 versus 96.8% (95% CI 94.0-99.6), p<0.001, respectively. The results of the APL2012 study were similar to those of APL2018. In the APL2018 study, the estimated 5-year cumulative incidence of relapse (CIR) was 2.51% (95% CI 2.48-2.54), which was 0%, 1.16% (95% CI 1.13-1.19), and 7.83% (95% CI 7.44-8.22) in the low-risk, intermediate-risk, and high-risk patients, respectively (p=0.057). The estimated 5-year CIR for the low to intermediate risk group was 0.74% (95% CI 0.73-0.75), which was statistically different from high-risk patients (p=0.019). The estimated 5-year CIR for MRD positive and negative patients prior to the 1 st and 2 nd consolidation treatments was 4.92% (95% CI 4.76-5.08) versus 1.10% (95% CI 1.08-1.12), p=0.194; 14.29% (95% CI 10.30-18.28) versus 2.08% (95% CI 2.05-2.11), p=0.010, respectively. Conclusion Our results show that MRD after one course of consolidation treatment, rather than before, is a crucial predictive factor for APL relapse. Patients with positive MRD after 1 st consolidation therapy have a high risk of relapse. ATRA-ATO based treatment has significantly reduced APL relapse, so the value of frequent MRD monitoring may be limited. It is recommended to implement an optimized MRD monitoring for risk stratification. For low to intermediate-risk patients and those with negative MRD after a consolidation treatment, the frequency and duration of MRD testing can be minimized. For high-risk patients with sustained MRD negativity for more than 3 years, MRD testing can be considered for discontinuation.

Differentiation syndrome and coagulation disorder — comparison between treatment with oral and intravenous arsenics in pediatric acute promyelocytic leukemia

Realgar-Indigo naturalis formula (RIF), with A4S4 as a major ingredient, is an oral arsenic used in China to treat pediatric acute promyelocytic leukemia (APL). The efficacy of RIF is similar to that of arsenic trioxide (ATO). However, the effects of these two arsenicals on differentiation syndrome (DS) and coagulation disorders, the two main life-threatening events in children with APL, remain unclear. We retrospectively analyzed 68 consecutive children with APL from South China Children Leukemia Group-APL (SCCLG-APL) study. Patients received all-trans retinoic acid (ATRA) on day 1 of induction therapy. ATO 0.16 mg/kg day or RIF 135 mg/kg·day was administrated on day 5, while mitoxantrone was administered on day 3 (non-high-risk) or days 2–4 (high-risk). The incidences of DS were 3.0% and 5.7% in ATO (n = 33) and RIF (n = 35) arms (p = 0.590), and 10.3% and 0% in patients with and without differentiation-related hyperleukocytosis (p = 0.04), respectively. Moreover, in patients with differentiation-related hyperleukocytosis, the incidence of DS was not significantly different between ATO and RIF arms. The dynamic changes of leukocyte count between arms were not statistically different. However, patients with leukocyte count > 2.61 × 109/L or percentage of promyelocytes in peripheral blood > 26.5% tended to develop hyperleukocytosis. The improvement of coagulation indexes in ATO and RIF arms was similar, with fibrinogen and prothrombin time having the quickest recovery rate. This study showed that the incidence of DS and recovery of coagulopathy are similar when treating pediatric APL with RIF or ATO.

Traditional mineral medicine realgar and Realgar-Indigo naturalis formula potentially exerted therapeutic effects by altering the gut microbiota.

Realgar has a long history ofuse in traditional medicines. However, the mechanism through which Realgar or Realgar-Indigo naturalis formula (RIF) exert therapeutic effects is only partially understood. In this study, 60 feces and 60 ileum samples from rats administered with realgar or RIF were collected to examine the gut microbiota. The results showed that realgar and RIF influenced different microbiota in both feces and ileum. Compared with realgar, RIF at low dosage (0.1701 g/3 ml) significantly increased the microbiota diversity. LEfSe and random forest analyses showed that the bacterium Bacteroidales was significantly altered after RIF administration, and it was predicted that these microorganisms contribute to the inorganic arsenic metabolic process. Our results suggest that realgar and RIF may exert their therapeutic effects through influencing microbiota. The low dose of RIF had greater effects on increasing the diversity of microbiota, and Bacteroidales in feces might participate in the inorganic arsenic metabolic process to exert therapeutic effects for realgar.

Oral Realgar-Indigo Naturalis Formula Versus Intravenous Arsenic Trioxide for Non-High-Risk Patients with Acute Promyelocytic Leukemia: Interim Results of a Multicenter Randomized Controlled Trial APL16

Oral Realgar-Indigo Naturalis Formula Versus Intravenous Arsenic Trioxide for Non-High-Risk Patients with Acute Promyelocytic Leukemia: Interim Results of a Multicenter Randomized Controlled Trial APL16

The comparison of plasma arsenic concentration and urinary arsenic excretion during treatment with Realgar-Indigo naturalis formula and arsenic trioxide in children with acute promyelocytic leukemia

Realgar-Indigo naturalis formula (RIF) is a traditional Chinese medicine containing As4S4 and effective in treating acute promyelocytic leukemia (APL). The dose of RIF remains to be determined in pediatric patients. Comparison of plasma arsenic concentrations and toxicity between RIF and arsenic trioxide (ATO) treatment in APL may help to establish an appropriate therapeutic dose of RIF for children. From October 2018 to March 2020, 19 pediatric patients with APL treated with SCCLG-APL protocol were included, 9 in RIF group at 135mg/kg/day orally three times daily, and 10 in ATO group at 0.16mg/kg/day intravenously over 12h daily. Peak and trough plasma arsenic concentrations were assayed at D1, 2, 7 and 14 of induction treatment. Urine arsenic excretions were assessed with spot urine samples and the measurements were adjusted using creatinine. Toxicities were compared between two groups. The plasma arsenic concentration reached steady state at D7 either in the RIF or ATO group, and the mean peak and trough concentrations were similar between two groups (P > 0.05), which were 0.54μmol/L and 0.48μmol/L in RIF group, and 0.63μmol/L and 0.51μmol/L in ATO group, respectively. Urine arsenic excretion rate was positively correlated with the concentration of plasma arsenic. The rates of treatment-related adverse events were similar in two groups. In conclusion, the dose of RIF at 135mg/kg/day may be an appropriate therapeutic dose in children with APL. Urine arsenic level can be used as an indicator to estimate plasma arsenic concentration. Trial registration www.clinicaltrials.gov NCT02200978.

Realgar Indigo Formula Plus ATRA As Postremission Treatment in an Rral and Chemo-Free Model for High-Risk Acute Promyelocytic Leukemia Patients: A Prospective, Multicenter, Phase II Study

▪Background:Low-risk patients with acute promyelocytic leukemia (APL) can be cured using only ATRA and arsenic trioxide (ATO), without chemotherapy(Lo-Coco F, et al. NEJM 2013) .Our group simplified the protocol by replacing iv ATO with oral arsenic, referred to as RIF, allowing for outpatient, oral and chemotherapy-free treatment for an increasing number of APL patients (Zhu HH, et al. NEJM 2014; 371:2239-41;Zhu HH, et al. Lancet Oncol 2018;19:871-879; Zhu HH, et al. Blood 2019;134:597-605). We also reported a promising single-center results using chemo-free postremission treatment for high-risk APL patients (Zhu HH, Blood 2018;131:2987-2989), which need to be confirmed in a well designed multi-center trial.Objective:To evaluate the efficacy of safety Realgar indigo formula plus retinoic acid as postremission treatment in an oral and chemo-free model for high-risk APL patients .Design, setting and participants:a prospective, multicenter, single-arm, phase II clinical trial conducted in the First Affiliated Hospital, Zhejiang University College of Medicine, China. Eligible patients (>18 years old) with newly diagnosed APL and achieved complete remission(CR) were enrolled since May 2019, with final follow-up in July 31,2021.Interventions:The consolidation therapy included realgar-Indigo naturalis formula (60 mg/kg daily in an oral divided dose) in a 4-week-on and4-week-off regimen for 4 cycles and ATRA (25 mg/m 2 daily in anoral divided dose) in a 2-week-on and 2-week-off regimen for 7 cycles. The primary endpoint was the 2-year DFS. Secondary endpoints included complete molecular remission (CMR) defined as the absence of detectable PML-RARA transcripts., event-free survival (EFS), OS, and safety.Main outcomes and measures:38 eligible patients were enrolled including 18 males and 20 females and the median age was 41 years old (18-77 years). The median of WBC count is 25.39（10.2-113.9）×109/L[ >50×10 9/L n=10; (20-50)×10 9/L n=14，(10-20)×10 9/L n=14].All the patients achieved CMR during post-remission treatment phase (7 months). Until now, no molecular, hematologic recurrence,and central nervous system leukemia has happened with median follow-up of 13 months. What's more, this chemotherapy-free, completely oral regimen was well tolerated.Conclusions:These exciting results proved that RIF plus retinoic acid as postremission treatment of high-risk APL was effective, safe and convenient. The study is ongoing, and the effective of this regimen need to be evaluated by more patients and longer time.Figure 1. The overall survival(OS) and disease-free survival (DFS) of high-risk APL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Safety and efficacy of Compound Huangdai Tablets combined with all-trans retinoic acid for treatment of acute promyelocytic leukemia: Clinical evidence and potential mechanisms

ObjectiveTo evaluate the safety and efficacy of Compound Huangdai Tablets (Realgar-Indigo Naturalis formula, RIF) combined with all-trans retinoic acid (ATRA) to treat acute promyelocytic leukemia (APL). MethodsThis study was registered in PROSPERO (CRD42018108118). The relevant literatures on RIF treatment of APL were systematically searched in the following databases: China National Knowledge Infrastructure, Wanfang, VIP Medical Information System, Chinese Biomedical Database, EMBASE, Cochrane Library, and PubMed. The quality of the included studies was evaluated and Review Manager 5.3 software and Stata 13.0 software were used to perform the Meta-analysis. In addition, this study used the method of network pharmacology to conduct a preliminary exploration of the mechanism of RIF on APL. ResultsThe study included 12 studies involving 775 APL patients. The Meta-analysis showed that there was no significant difference (P ＞ 0.05) between the RIF group and the arsenic trioxide (ATO) group for primary outcomes, secondary outcomes apart from liver dysfunction. The incidence of liver dysfunction (P = 0.006) in the RIF group were significantly lower than those in the ATO group. In addition, the cost of maintenance therapy in the RIF group was significantly lower (P ＜ 0.05) than the ATO group. Besides, the active ingredients in RIF mainly act on targets proteins such as ACHE, NCOA2, RXRA, and then play a role in the treatment of APL through regulating multiple molecular mechanisms, such as TP53 regulates transcription of cell cycle genes, nuclear receptor transcription pathway. ConclusionThere was no significant difference in efficacy of oral RIF combined with ATRA compared with intravenous ATO combined with ATRA for the treatment of APL. The oral RIF exposed patients to less risk, offered more convenience and had lower prices. RIF can treat APL by multi-target and multi-pathway interventions that inducing apoptosis of APL cells and inhibiting the proliferation of APL cells, and so on. Therefore, oral RIF in the treatment of APL is worthy of further research and development.

Targeting Systemic Inflammatory Response Syndrome As Initial Therapy and Oral Arsenic Based Consolidation Regimen for Acute Promyelocytic Leukemia

Early life-threatening complications are a hindrance towards the cure of acute promyelocytic leukemia (APL). Some patients could indicate symptoms in line with systemic inflammatory response syndrome (SIRS) at admission or along with induction therapy. Here we designed this multicenter, single-arm APL-SIRS prospective study and enrolled 140 patients to test the efficacy and safety of the early intervention through targeting SIRS as initial therapy. The diagnostic of SIRS based on previous reported criteria. Other systematic manifestations include: weight gain (from capillary leak and soft tissue edema), respiratory distress, radiographic opacities, pleural or pericardial effusion, hypotension, renal failure, abnormal coagulation manifestation, and disseminated intravascular coagulation (DIC) may also be present. We stratified patients with SIRS based on the presence or absence of systematic symptoms, and tailored treatment accordingly. The risk groups were defined as (i) low-risk (LR): Patient diagnosed with SIRS but without systematic manifestations of SIRS or underlying diseases; (ii) medium-risk (MR): Patient diagnosed with SIRS with 1-2 of systematic presentations; (iii) high-risk (HR): Patients diagnosed with SIRS plus with ≥ 3 of the systematic presentations. We used stratified glucocorticoid therapy in addition to ATRA, anthracycline, and ATO in induction. The consolidation regimen consisted of two additional cycles of ATRA and oral tetra-arsenic tetra-sulfide formula, Realgar-Indigo Naturalis Formula (RIF), followed by maintenance therapy with ATRA, oral methotrexate (MTX), and 6-mercaptopurine (6MP). Our results indicated that increased SIRS risk levels are associated with higher WBC counts (P 0.05). This result suggests that though elevated WBCs could reflect an ongoing inflammation cascade initiated by cytokine secretion, appropriate stratified glucocorticoid therapy in addition to standard induction therapy appear to mitigate any potential differences in outcome. We monitored the levels of both pro-inflammatory (TNFα, IL-1β, IL2, IL-6, IL-8) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines on day 1, 8, 15, 22, and 28 and evaluated their prognostic value in different SIRS risk groups. Interestingly, we see differences in cytokine expression level within different SIRS risk groups. However, following glucocorticoid treatment the increased cytokine levels decline by the end of induction, and differences in cytokine levels between groups disappeared. Further statistical analysis indicated there is no difference among different SIRS risk groups at the end of induction in both pro- and anti–inflammatory cytokine levels. We also found that the change of cytokine level happens prior to clinical symptom changes, which make it possible that cytokines may be a sensitive prognostic marker tailoring for APL treatment. In total 126 cases attained CR and all survived throughout the follow-up period. The estimated 10 years overall survival for the whole cohort was 92.14±2.27%. There are 11 people died in SIRS-LR (2/44), -MR (3/44), -HR (6/50), respectively. The overall survival of SIRS-LR, -MR, and -HR group was 95.46±3.14%; 93.48±3.64%; and 88.00±4.60%. During follow up, 15 cases has events (SIRS-LR: 3/44, SIRS-MR: 5/46, SIRS-HR: 7/50) and all of them are in remission again after intervention. No patients received hematopoietic stem cell transplantation. The EFS for SIRS-LR, SIRS-MR, SIRS-HR are 88.64±4.79%, 82.61%±5.59%, 74.00±6.20%, respectively. In short, early diagnosis and intervention of SIRS is likely a key to decreasing the early mortality of APL. The dynamic change of cytokine value could be potentially used as prognostic makers to detect early disease progression of APL. Disclosures No relevant conflicts of interest to declare.

Oral Arsenic and Retinoic Acid for Children with Non High-Risk Acute Promyelocytic Leukemia

ObjectiveAcute promyelocytic leukemia (APL) has now become a highly curable disease with the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) as first-line treatment. Adult non high-risk APL patients (WBC< 10×109/L) can be cured using only ATRA and ATO without chemotherapy. We also provided similar results using a totally oral (oral arsenic and ATRA), chemo-free outpatient protocol (Beijing protocol) for adult patients with non high-risk APL. However, experiences in children with APL are scarce. These prompted us to try to extend the Beijing protocol to children patients with non high-risk APL.MethodsWe conducted a single-centre pilot study to evaluate the efficacy of oral arsenic and ATRA for children patients with non high-risk APL from April 2014 through October 2016. Nine patients with the median age of 16 years old (range 13-18) were enrolled and received oral arsenic realgar-indigo naturalis formula (RIF, 60 mg/kg) and ATRA (25 mg/m2) as induction therapy until hematologic complete remission (CR). Hydroxyurea and/or cytarabine (without anthracyclines) were used to diminish the increased WBC during induction treatment. The post-remission therapy included RIF in a 4-week on and 4-week off , and ATRA in a 2-week on and 2-week off schedule for 7 months. The primary endpoint was the complete molecular response (CMR) post-consolidation, defined as absence of detectable PML-RARA transcripts by quantitative PCR. Secondary endpoints included CR, safety, hospital stay. Median follow-up was 15 months (range 6-36 months) by April 2017.ResultsAll 9 patients achieved hematologic CR after a median time of 30 days. CMR rate was 100% at 3 and 6 months. Grade1,2,3, and 4 liver adverse events was 2,3,1,0, respectively. Differentiation syndrome occurred in 3 patients during induction (Table1). No hematological relapse or molecular relapse occurred at the last follow-up. Both the 2-year estimated EFS and OS were 100%.The total hospital time was 17 days (4-37 days) during the treatment phase. Of note, 100% (9/9) of patients completed the post-remission therapy on an outpatient basis without hospitalization. Patients resumed their usual lifestyle during post-remission therapy and their QoL was nearly normal during post-remission therapy.ConclusionsOur study, which employed a largely home-based treatment protocol with complete oral regimen, chemo-free during induction in children patients with non high-risk APL, suggested to be effective, convenient. However, lager sample studies and long-term outcome are warranted. DisclosuresNo relevant conflicts of interest to declare.

Oral Realgar-Indigo Naturalis Formula Plus Retinoic Acid for Acute Promyelocytic Leukemia.

Treatment paradigm of acute promyelocytic leukemia (APL) is by no mean the most remarkable story of cancer therapy. Recently, the advent of oral arsenic formulations (oral-arsenic trioxide and Realgar-Indigo Naturalis formula (RIF)) based regimens may provide a therapeutic advance by curing APL with two oral agents. Indeed, the oral RIF plus all-trans-retinoic acid (ATRA) without chemotherapy display highly efficacy in patients with APL. The safety profile of RIF plus ATRA make possible to treat APL patients in a home-based manner during postremission therapy. To our knowledge, RIF was the first commercially available oral arsenic agent approved in China. The RIF plus ATRA regimens are becoming a preferred frontline care for APL in China. In this review, we will discuss the history, current evidences and challengers of RIF-based strategies in APL. More and more APL patients may enjoy a cure with a normal quality-of-life after induction in the near future.

Abstract 6265: Combining arsenic and topoisomerase inhibitors synergistically inhibit gastric cancer

Abstract Objectives: Arsenic compounds have cytotoxic activities in gastric cancer cells, however, the underlying mechanism is unclear and the clinical potential need to be explored. This study was aimed to investigate the mechanism of arsenic on gastric cancer and find a novel regimen for the treatment of gastric cancer. Methods: Gastric cancer cell lines were treated with arsenics and/or chemotherapeutic drugs. MTT assay, FACS, Western blotting, q-RT-PCR and CHIP were used to detect proliferation, modulation of proteins and genes. Results: We previously showed that NFATc3 was a target of Arsenic sulfide and its inhibition blocks gastric cancer growth. Here we show that the NFATc3 expression is critically important for the growth and survival of gastric cancer cells because knockdown of NFATc3 resulted in DNA damage and cell cycle arrest. Arsenic sulfide can target NFATc3 in gastric cancer cells and cause cell double-strand DNA breaks (DSB). However, overexpression of NFATc3 can block the DNA damage. Abolishing NFATc3 expression, whether by shRNA-induced silencing or Arsenic sulfide treatment, directly upregulated recombination activating gene 1 (RAG1, an endonuclease which is essential for immunoglobulin V(D)J recombination), leading to fatal DSB. NFATc3 targets the promoter of RAG1 for transcriptional inhibition. Moreover, we find that Arsenic sulfide and topoisomerase inhibitors (Irinotecan and Epirubicin, Doxorubicin, Etoposide and SN38) synergistically inhibit gastric cancer. Arsenic sulfide, as well as Arsenic Trioxide, enhanced the cytotoxic effect of Irinotecan and they synergistically induced NFATc3 degradation, resulted in disability of DNA repair system, as reflected by the decrease in the phosphorylated ATM/CHK2/CHK1 protein level and the dramatic increase in the phosphorylated H2AX protein level. More inspiring, a patient with advanced gastric cancer with liver and lymph-node metastasis who failed in first-line treatment achieved PR with 2 months treatment of Irinotecan combined with Realgar-Indigo Naturalis Formula, the main component of which is Arsenic sulfide. The progression-free survival was 4 months with no additional adverse event. Conclusions: Our research links arsenic compounds to the regulation of DNA damage repair and RAG1 expression as a mechanism for the cytotoxic effect. The combination of arsenic with topoisomerase inhibitors may represent a novel regimen for the treatment of gastric cancer and warrants further studies. Citation Format: Ting Kang, Maolin Ge, Shumin Lu, Qing Wang, Shouhai Zhu, Chuanying Zhu, Leizhen Zheng, Han Liu, Siyu Chen. Combining arsenic and topoisomerase inhibitors synergistically inhibit gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6265.

Treatment of non-high-risk acute promyelocytic leukemia with realgar-indigo naturalis formula (RIF) and all-trans retinoid acid (ATRA): study protocol for a randomized controlled trial

BackgroundAcute promyelocytic leukemia (APL) is a highly curable disease when treated with all-trans retinoid acid (ATRA) and arsenic trioxide (ATO). The combination of ATO and ATRA has become the standard therapeutic protocol for induction therapy in non-high-risk APL. An oral arsenic realgar-indigo naturalis formula (RIF) has also showed high efficacy and it has a more convenient route of administration than the standard intravenous regimen. Unlike in previous trials, the arsenical agent was used simultaneously with ATRA during post-remission therapy in this trial.MethodsThis study was designed as a multicenter, randomized controlled trial. The trial has a non-inferiority design with superiority being explored if non-inferiority is identified. All patients receive ATRA-ATO during the induction therapy. After achieving hematologic complete remission (HCR), patients were randomly assigned (1:1) to receive treatment with ATRA-RIF (experimental group) or ATRA-ATO (control group) as the consolidation therapy. During the consolidation therapy, the two groups receive ATRA plus RIF or intravenous ATO 2 weeks on and 2 to ~ 4 weeks off until molecular complete remission (MCR), then ATRA and oral RIF 2 weeks on and 2 to ~ 4 weeks off giving a total of six courses.DiscussionThis trial aims to compare the efficacy of ATRA-ATO versus ATRA-RIF in non-high-risk patients with APL, to demonstrate that oral RIF application reduces the total hospitalization days and medical costs. The simple schedule was studied in this trial.Trial registrationClinicalTrials.gov, NCT02899169. Registered on 14 September 2016.

Efficacy and the Adverse Effects of Oral Versus Intravenous Arsenic for Acute Promyelocytic Leukemia: A Meta-Analysis of Randomized-Controlled Studies.

Acute promyelocytic leukemia, a subtype of acute myeloid leukemia, is highly curable. In subgroup of patients with non-high-risk acute promyelocytic leukemia, intravenous arsenic trioxide plus all-trans-retinoic acid is considered the preferred regimen for acute promyelocytic leukemia. Recently, there are interests in the use of the oral form of arsenic, named the Realgar-Indigo naturalis formula, but the data on its efficacy and safety are still relatively limited. The current study was conducted with the aims to identify and summarize the results of all available randomized-controlled studies. A systematic review was conducted in the 2 major databases, utilizing the terms for arsenic and acute promyelocytic leukemia. Eligible studies had to be randomized-controlled studies that compared efficacy and/or adverse effects of oral arsenic versus intravenous arsenic for treatment of patients with acute promyelocytic leukemia. The Mantel-Haenszel method was used to pool the effect estimates and 95% confidence intervals of the included studies together. A total of 4 randomized controlled studies with 482 patients with acute promyelocytic leukemia (258 in Realgar-Indigo naturalis formula group and 224 in intravenous arsenic trioxide group) were included in the meta-analysis. The chances of achieving complete remission were numerically higher in the Realgar-Indigo naturalis formula group but the difference was not statistically significant (pooled odds ratio: 4.59, 95% CI: 0.74-28.57, I 2 = 0%). Similarly, other efficacy outcomes, including 30-day mortality rate, overall survival, and event-free survival, also tended to favor the Realgar-Indigo naturalis formula group but the difference was not statistically significant. There was no significant difference in the chance of developing differentiation syndrome, cardiac complications, grades 3 to 4 liver toxicity, grades 3 to 4 renal toxicity, and infection between the 2 groups. The results may suggest that all-trans-retinoic acid plus oral Realgar-Indigo naturalis formula regimen is, at minimum, not a worse alternative to the standard all-trans-retinoic acid plus intravenous intravenous arsenic trioxide regimen for treatment of acute promyelocytic leukemia, especially for patients with low-to-intermediate risk.

Pilot Study of Oral Realgar-Indigo Naturalis Formula in Pediatric Acute Promyelocyticleukemia: Effectiveness and Population Pharmacokinetics

# The first two authors contributed equally * The last two authors contributed equally Background: Realgar-Indigo Naturalis Formula (RIF), the only commercially oral arsenic agent, was launched in China from 2009. It has been recommended as consolidation or maintenance therapy in adults with acute promyelocytic leukemia (APL). Recently, RIF had been off-label used in pediatric APL in China. However, the knowledge of efficacy and dosage of RIF in pediatric was limited until now. Objective: The purpose of this pilot study was to evaluate effectiveness and pharmacokinetics of RIF as consolidation or maintenance therapy for newly diagnosed and relapsed APL in pediatric after induction therapy with intravenous arsenic trioxide. Meanwhile, modeling and simulation techniques were used to evaluate and optimize RIF dosing regimen in pediatric population. Patients and Methods: A total of 11 newly diagnosed APL and 1 relapsed patient (4-14years of age) were included from July 2016 to August 2017.The one relapsed and six newly diagnosed patients were treated with RIF (60 mg/kg/day TID) as maintenance therapy. Five newly diagnosed patients were treated with RIF (60 mg/kg/day TID) as consolidation and maintenance therapy. The treatment protocol and MRD results are shown in Figure 1. Event-free survival and overall survival were used to evaluate the efficacy. The plasma concentration of arsenic was quantified by inductively coupled plasma mass spectrometry (ICP-MS). Population pharmacokinetic analysis was carried out using the nonlinear mixed effects modeling program NONMEM V 7.2 (Icon Development Solutions, USA). Results: All newly diagnosed APL patients were alive as of July 1, 2019 and were in MCR with a median follow-up of 28 months (range, 23 to 37 months). Both the estimated 3-year EFS and OS rates were 100%. Of note, all the patients completed the postremission therapy containing RIF on an outpatient basis. A total of 107 arsenic concentrations were used for population pharmacokinetic model building. The arsenic concentrations of the blood samples ranged from 0.1 to 75.0 µg/L. The steady-state trough plasma arsenic concentration during the RIF treatment was 47.43μg/L (range, 25.74 to 62.97μg/L).A one-compartment model with first-order elimination fitted the data. Body weight was the only significant covariate of CL for the final model development. The developmental pharmacokinetic model was evaluated by goodness-of-fit plots and bootstrap analysis. The mean and variance of NPDE were -0.118 and 0.93, respectively. Conclusions: The effectiveness and pharmacokinetics were evaluated for the first time in pediatricAPL.The developmental population pharmacokinetic model for RIF in children revealed that one-compartment model with first-order elimination fitted the data.The current dosing regimen of 60 mg/kg/day TID resulted higher steady-state through concentration than that in adults (24.4μg/L, range:11.5 to 64μg/L). Disclosures:No relevant conflicts of interest to declare. This trial was conducted in accordance with the Declaration of Helsinki. OffLabel Disclosure: The combination of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) has become the front-line treatment for patients with acute promyelocytic leukemia (APL). Realgar-Indigo Naturalis Formula (RIF), the only commercially oral arsenic agent, was launched in China from 2009. Since then, RIF had been used for over 5000 adults with acute promyelocytic leukemia (APL). One pill of RIF is 270 mg which contains 30 mg of Realgar, 125 mg of Indigo naturalis, 50 mg of Radix salviae miltiorrhizae, 45 mg of Radix pseudostellariae, and 20mg of garment film. However, the pharmacokinetics of RIF in children has not been studied.

Pilot Study on the Safety of Intravenous Arsenic Trioxide and Oral Realgar-Indigo Naturalis Formula Administrated in Children with Acute Promyelocytic Leukemia: A Single-Center Study

Objective Intravenous arsenic trioxide (ATO) and all-trans retinoic acid have become the front-line treatments for patients with acute promyelocytic leukemia (APL). Realgar-Indigo naturalis formula (RIF), an oral arsenic drug, not only shows a clinical efficacy comparable to ATO but also promotes incorporating an outpatient postremission therapy model into clinical practice for both low-risk and high-risk APL patients in China. However, the safety of ATO/RIF used in children with APL is unknown. To assess the safety of arsenic (ATO/RIF) administrated in children with APL, we designed the study. Methods Children with newly diagnosed APL treated with CCLG-APL2016 protocol (ChiCTR-OIN-17011227) in Beijing Children's Hospital from July 2016 to December 2018 were eligible for the study. The arsenic concentrations in different tissues, including plasma, urine, hair and nail, were measured at 10 time points: before ATO/RIF(D0), on the 7th, 14th and 28th day of ATO/RIF administration(D7, D14, D28), on the end of consolidation therapy (Con), during the 10 weeks of maintenance therapy (W10), on the cessation of therapy, after six months, one year and 1.5 years without arsenic administration. Adverse reactions were observed to evaluate the safety of arsenic received in children with APL. Results Nineteen patients with newly diagnosed APL were enrolled, including 9 boys and 7 girls with an onset average age of (10.9±3.7) years. There were 14 patients in low-risk group (white cell count &lt;10,000/ul) and 5 patients in high-risk group (white cell count≥10,000/ul). All of them got hematologic complete remission (HCR) and molecular complete remission (MCR). The time to get HCR and MCR were 31 days (range, 27~74days) and (72.2±16.4) days, respectively. Besides, there were 13 patients getting ATO and 6 patients getting RIF in induction phase, and all patients receiving RIF as an outpatient postremission therapy. It showed that plasma and urine arsenic levels were significantly elevated after ATO/RIF administration. The median plasma arsenic ranged from 38.4 ng/ml to 76.10 ng/ml on D7, D14, D28, Con and W10 while the median urine arsenic retention ranged from 1562.80ng/ml to 3791.00ng/ml on D7, D14, D28, Con and W10. Plasma arsenic level rapidly decreased to 1.01 ng/ml after six months without RIF administration, which was slightly higher than D0 (1.01ng/ml vs 0.60ng/ml, P=0.043) and decreased to normal after 1 year without arsenic administration compared with D0 (0.55 ng/ml vs 0.60ng/ml, P=0.655). Urine arsenic level decreased to normal within 0~6 months off therapy (25.80ng/ml vs 13.74ng/ml, P=0.866). Hair arsenic (6480.95ng/g, range 2616.20-14683.70ng/g) and nail arsenic levels (17896.85ng/g, range 400.00-30334.00ng/g) peaked at the time of cessation of therapy. Hair arsenic level decreased to normal within half a year off arsenic (156.50 ng/g vs 103.45 ng/g, P=0.345) while nail arsenic retention decreased to normal after 1 year off arsenic (P=0.655). In addition, Spearman rank correlation analysis showed that plasma arsenic concentration was positively correlated with urine (r=0.825, P&lt;0.001), hair (r=0.595, P&lt;0.001) and nail (r=0.584, P&lt;0.001) arsenic. Urine arsenic were also positively correlated with hair (r=0.624, P&lt;0.001), and nail (r=0.575, P&lt;0.001). And arsenic concentration in hair was positively correlated with nail (r=0.805, P&lt;0.001), too. Conclusions Through the detection of arsenic concentration in different periods and different tissues, it was found that the plasma arsenic concentration could be maintained within the effective concentration range in each treatment stage, and the arsenic concentration in plasma and hair gradually decreased to normal after six months off arsenic. Urine and nail arsenic went down to normal after 1 year off arsenic. Up to now, with a longest follow-up period of 34.7 months and the mean follow-up time of 19.6 months, the short-term response of arsenic disappeared after symptomatic therapy or arsenic reduction, and no chronic side effects of arsenic were observed. Therefore, the use of ATO/RIF in children with APL is safe, but it still needs long-term follow-up. Disclosures No relevant conflicts of interest to declare.