Amsacrine downregulates BCL2L1 expression and triggers apoptosis in human chronic myeloid leukemia cells through the SIDT2/NOX4/ERK/HuR pathway.

Accumulating evidence indicates that the anticancer activity of acridine derivatives is mediated through the regulation of anti-apoptotic and pro-apoptotic BCL2 protein expression. Therefore, we investigated whether the cytotoxicity of amsacrine with an acridine structural scaffold in human chronic myeloid leukemia (CML) K562 cells was mediated by BCL2 family proteins. Amsacrine induced apoptosis, mitochondrial depolarization, and BCL2L1 (also known as BCL-XL) downregulation in K562 cells. BCL2L1 overexpression inhibited amsacrine-induced cell death and mitochondrial depolarization. Amsacrine treatment triggered SIDT2-mediated miR-25 downregulation, leading to increased NOX4-mediated ROS production. ROS-mediated inactivation of ERK triggered miR-22 expression, leading to increased HuR mRNA decay. As HuR is involved in stabilizing BCL2L1 mRNA, downregulation of BCL2L1 was noted in K562 cells after amsacrine treatment. In contrast, amsacrine-induced BCL2L1 downregulation was alleviated by restoring ERK phosphorylation and HuR expression. Altogether, the results of this study suggest that amsacrine triggers apoptosis in K562 cells by inhibiting BCL2L1 expression through the SIDT2/NOX4/ERK-mediated downregulation of HuR. Furthermore, a similar pathway also explains the cytotoxicity of amsacrine in CML MEG-01 and KU812 cells.

Amsacrine-based induction therapy in AML patients with cardiac comorbidities: a retrospective single-center analysis.

Intensive chemotherapy is the backbone of induction treatment in patients with acute myeloid leukemia (AML). However, AML patients with concomitant cardiac disease may not be eligible for anthracycline-based therapies. In a small cohort of patients, we have previously shown that anthracycline-free, amsacrine-based chemotherapy TAA (thioguanine, cytarabine, amsacrine) may be as effective as cytarabine/daunorubicin for induction therapy in these patients. In this systematic retrospective single-center analysis, we documented the outcome of 31 patients with significant cardiac comorbidities including coronary heart disease or cardiomyopathy receiving TAA as induction chemotherapy. Median (range) ejection fraction (EF) was 48% (30-67%) in this cohort. Patients with EF below 30% were considered unfit for intensive induction therapy. Event-free survival (EFS), overall survival (OS), and relapse-free survival (RFS) were 1.61, 5.46, and 13.6 months respectively. Poor outcome was primarily related to a high early mortality rate within the first 30 days of therapy, mainly caused by infectious complications. TAA cannot be recommended as a substitute of standard induction for AML patients with significant concomitant cardiac disease. In the era of novel agents, alternative strategies (e.g., hypomethylating agents plus venetoclax) should be considered when anthracycline-based regimens are not suitable.

Instantaneous amplitude: Association of ventricular fibrillation waveform measures at time of shock with outcome in out-of-hospital cardiac arrest.

BACKGROUND: Prompt defibrillation is key to successful resuscitation from ventricular fibrillation out-of-hospital cardiac arrest (VF-OHCA). Preliminary evidence suggests that the timing of shock relative to the amplitude of the VF ECG waveform may affect the likelihood of resuscitation. We investigated whether the VF waveform amplitude at the time of shock (instantaneous amplitude) predicts outcome independent of other validated waveform measures. METHODS: We conducted a retrospective study of VF-OHCA patients ≥18 old. We evaluated three VF waveform measures for each shock: instantaneous amplitude at the time of shock, and maximum amplitude and amplitude spectrum area (AMSA) over a 3-s window preceding the shock. Linear mixed-effects modeling was used to determine whether instantaneous amplitude was associated with shock-specific return of organized rhythm (ROR) or return of spontaneous circulation (ROSC) independent of maximum amplitude or AMSA. RESULTS: The 566 eligible patients received 1513 shocks, resulting in ROR of 62.0% (938/1513) and ROSC of 22.3% (337/1513). In unadjusted regression, an interquartile increase in instantaneous amplitude was associated with ROR (Odds ratio [OR] [95% confidence interval] = 1.27 [1.11-1.45]) and ROSC (OR = 1.27 [1.14-1.42]). However, instantaneous amplitude was not associated with ROR (OR = 1.13 [0.97-1.30]) after accounting for maximum amplitude, nor with ROR (OR = 1.00 [0.87-1.15]) or ROSC (OR = 1.05 [0.93-1.18]) after accounting for AMSA. By contrast, AMSA and maximum amplitude remained independently associated with ROR and ROSC. CONCLUSIONS: We did not observe an independent association between instantaneous amplitude and shock-specific outcomes. Efforts to time shock to the maximal amplitude of the VF waveform are unlikely to affect resuscitation outcome.

Resolution of Otitis Media with Effusion in Adults after a Three-Day Course of Treatment with a Manosonic Nebulizer-A Pilot Study.

Background and Objectives: Aerosol drug administration is the primary treatment modality of otitis media with effusion (OME). An automatic manosonic aerosol generator (AMSA) delivers, with an acoustic overpressure, a therapeutic dosage of a drug by inhalation of the aerosol. However, available studies confirming their efficacy, especially in adults, are limited. Therefore, this pilot single-arm trial aimed to analyze changes in adults with OME following AMSA treatment. Materials and Methods: A group of 36 patients (mean age 51.4 years) with OME underwent a three-day treatment with inhaled mucolytic and steroids administered by AMSA. Tympanometry (tympanogram type, volume, compliance, pressure, and gradient) was performed to measure middle ear effusion before and after the intervention. Results: Following the intervention, partial and complete OME remission was observed in, respectively, 29 (81%) and 14 (39%) patients. The tympanogram type of the affected ears differed between baseline and after intervention measurements (p < 0.001). Tympanometry-based normalization, improvement deterioration and no change were observed in, respectively, 34 (68%), 1 (2%) 2 (4%), and 13 (26%) affected ears. Following the intervention, we observed an increase in continuously assessed middle ear volume (∆median 0.19 mL, p = 0.002) and pressure (∆median 142 daPa, p < 0.001), as well as a higher proportion of patients achieving categorical normalization of compliance (16% vs. 54%, p < 0.001) and pressure (28 vs. 64%, p < 0.001). Conclusions: Treatment efficacy was not affected by age, sex, or season of recruitment (all p > 0.05). The results of this pilot study are encouraging, however, the use of AMSA management of OME in adults needs to be verified in future studies.

Amsacrine combined with etoposide and methylprednisolone is a feasible and safe component in first-line intensified treatment of pediatric patients with high-risk acute lymphoblastic leukemia in CoALL08-09 trial.

BACKGROUND: The prognosis of children with acute lymphoblastic leukemia (ALL) has improved considerably over the past five decades. However, to achieve cure in patients with refractory or relapsed disease, novel treatment options are necessary. METHODS: In the multicenter trial Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (CoALL)08-09, one additional treatment element consisting of the rarely used chemotherapeutic agent amsacrine combined with etoposide and methylprednisolone (AEP) (amsacrine 2 × 100 mg/m2 , etoposide 2 × 500 mg/m2 , and methylprednisolone 4 × 1000 mg/m2 ) was incorporated into the first-line treatment of pediatric patients with poor treatment responses at the end of induction (EOI), measured by minimal residual disease (MRD). These patients were stratified into a high-risk intensified arm (HR-I), including an AEP element at the end of consolidation. Patients with induction failure (IF), that is, with lack of cytomorphological remission EOI, were eligible for hematopoietic stem cell transplantation (HSCT) after remission had been reached. These patients received AEP as a part of their MRD-guided bridging-to-transplant treatments. RESULTS: A significant improvement in probability of overall survival (pOS) was noted for the CoALL08-09 HR-I patients compared to MRD-matched patients from the preceding CoALL07-03 trial in the absence of severe or persistent treatment-related toxicities. Relapse rate and probability of event-free survival (pEFS) did not differ significantly between trials. In patients with IF, stable or improved MRD responses after AEP were observed without severe or persistent treatment-related toxicities. CONCLUSION: In conclusion, AEP is well tolerated as a component of the HR treatment and is useful in bridging-to-transplant settings.

Design, synthesis, and biological evaluation of novel pyrido-dipyrimidines as dual topoisomerase II/FLT3 inhibitors in leukemia cells.

Dual inhibition of topoisomerase (topo) II and FLT3 kinase, as in the case of C-1311, was shown to overcome the shortcomings of using topo II inhibitors solely. In the present study, we designed and synthesized two series of pyrido-dipyrimidine- and pseudo-pyrido-acridone-containing compounds. The two series were evaluated against topo II and FLT3 as well as the HL-60 promyelocytic leukemia cell line in vitro. Compounds 6, 7, and 20 showed higher potency against topo II than the standard amsacrine (AMSA), whereas compounds 19 and 20 were stronger FLT3 inhibitors than the standard DACA. Compounds 19 and 20 showed to be dual inhibitors of both enzymes. Compounds 6, 7, 19, and 20 were more potent inhibitors of the HL-60 cell line than the standard AMSA. The results of the in vitro DNA flow cytometry analysis assay and Annexin V-FITC apoptosis analysis showed that 19 and 20 induced cell cycle arrest at the G2/M phase, significantly higher total percentage of apoptosis, and late-stage apoptosis in HL-60 cell lines than AMSA. Furthermore, 19 and 20 upregulated several apoptosis biomarkers such as p53, TNFα, caspase 3/7 and increased the Bax/Bcl-2 ratio. These results showed that 19 and 20 deserve further evaluation of their antiproliferative activities, particularly in leukemia. Molecular docking studies were performed for selected compounds against topo II and FLT3 enzymes to investigate their binding patterns. Compound 19 exerted dual fitting inside the active site of both enzymes.

Acridine derivatives as inhibitors/poisons of topoisomerase II.

The potential of acridines (amsacrine) as a topoisomerase II inhibitor or poison was first discovered in 1984, and since then, a considerable number of acridine derivatives have been tested as topoisomerase inhibitors/poisons, containing different substituents on the acridine chromophore. This review will discuss a series of studies published over the course of the last decade, which have investigated various novel acridine derivatives against topoisomerase II activity.

Biomechanical properties of a suture anchor system from human allogenic mineralized cortical bone matrix for rotator cuff repair.

BACKGROUND: Suture anchors (SAs) made of human allogenic mineralized cortical bone matrix are among the newest developments in orthopaedic and trauma surgery. Biomechanical properties of an allogenic mineralized suture anchor (AMSA) are not investigated until now. The primary objective was the biomechanical investigation of AMSA and comparing it to a metallic suture anchor (MSA) and a bioabsorbable suture anchor (BSA) placed at the greater tuberosity of the humeral head of cadaver humeri. Additionally, we assessed the biomechanical properties of the SAs with bone microarchitecture parameters. METHODS: First, bone microarchitecture of 12 fresh frozen human cadaver humeri from six donors was analyzed by high-resolution peripheral quantitative computed tomography. In total, 18 AMSAs, 9 MSAs, and 9 BSAs were implanted at a 60° angle. All three SA systems were systematically implanted alternating in three positions within the greater tuberosity (position 1: anterior, position 2: central, position 3: posterior) with a distance of 15 mm to each other. Biomechanical load to failure was measured in a uniaxial direction at 135°. RESULTS: Mean age of all specimens was 53.6 ± 9.1 years. For all bone microarchitecture measurements, linear regression slope estimates were negative which implies decreasing values with increasing age of specimens. Positioning of all three SA systems at the greater tuberosity was equally distributed (p = 0.827). Mean load to failure rates were higher for AMSA compared to MSA and BSA without reaching statistical significance between the groups (p = 0.427). Anchor displacement was comparable for all three SA systems, while there were significant differences regarding failure mode between all three SA systems (p < 0.001). Maximum load to failure was reached in all cases for AMSA, in 44.4% for MSA, and in 55.6% for BSA. Suture tear was observed in 55.6% for MSA and in 22.2% for BSA. Anchor breakage was solely seen for BSA (22.2%). No correlations were observed between bone microarchitecture parameters and load to failure rates of all three suture anchor systems. CONCLUSIONS: The AMSA showed promising biomechanical properties for initial fixation strength for RCR. Since reduced BMD is an important issue for patients with chronic rotator cuff lesions, the AMSA is an interesting alternative to MSA and BSA. Also, the AMSA could improve healing of the enthesis.

DNA-Binding Anticancer Drugs: One Target, Two Actions.

Amsacrine, an anticancer drug first synthesised in 1970 by Professor Cain and colleagues, showed excellent preclinical activity and underwent clinical trial in 1978 under the auspices of the US National Cancer Institute, showing activity against acute lymphoblastic leukaemia. In 1984, the enzyme DNA topoisomerase II was identified as a molecular target for amsacrine, acting to poison this enzyme and to induce DNA double-strand breaks. One of the main challenges in the 1980s was to determine whether amsacrine analogues could be developed with activity against solid tumours. A multidisciplinary team was assembled in Auckland, and Professor Denny played a leading role in this approach. Among a large number of drugs developed in the programme, N-[2-(dimethylamino)-ethyl]-acridine-4-carboxamide (DACA), first synthesised by Professor Denny, showed excellent activity against a mouse lung adenocarcinoma. It underwent clinical trial, but dose escalation was prevented by ion channel toxicity. Subsequent work led to the DACA derivative SN 28049, which had increased potency and reduced ion channel toxicity. Mode of action studies suggested that both amsacrine and DACA target the enzyme DNA topoisomerase II but with a different balance of cellular consequences. As primarily a topoisomerase II poison, amsacrine acts to turn the enzyme into a DNA-damaging agent. As primarily topoisomerase II catalytic inhibitors, DACA and SN 28049 act to inhibit the segregation of daughter chromatids during anaphase. The balance between these two actions, one cell cycle phase specific and the other nonspecific, together with pharmacokinetic, cytokinetic and immunogenic considerations, provides links between the actions of acridine derivatives and anthracyclines such as doxorubicin. They also provide insights into the action of cytotoxic DNA-binding drugs.

The FLAMSA concept-past and future.

The FLAMSA reduced intensity (RIC) concept, also known as "sequential therapy", is a conceptual platform for the treatment of leukemia separated in several parts: induction therapy, a sequence of antileukemic and immunosuppressive conditioning for allogeneic stem cell transplantation, and immune restitution supported by donor lymphocyte transfusions. The antileukemic part consists of fludarabine, cytosine arabinoside, and amsacrine (FLAMSA); non-cross reactive agents like fludarabine and amsacrine have been successfully used in cases of refractoriness and relapse. Immunosuppressive conditioning and transplantation follow after only 3 days of rest. This way, the toxicity of allogeneic transplantation could be reduced and the anti-leukemia effects by using allogeneic immune cells could be optimized. This review summarizes available data on efficacy and toxicity of this approach. Further, possible strategies for improvements are discussed in order to provide better chances for elderly and frail patients and patients with advanced and high-risk disease. Among others, several new agents are available that target molecular changes of leukemia for induction of remission and allow for bridging the time after transplantation until adoptive immunotherapy becomes safe and effective.

A synthetic lethal approach for compound and target identification in Staphylococcus aureus.

The majority of bacterial proteins are dispensable for growth in the laboratory but nevertheless have important physiological roles. There are no systematic approaches to identify cell-permeable small-molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small-molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery that has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of major bacterial processes that can be adapted to numerous organisms.

Optimization of Weight Ratio for DSPE-PEG/TPGS Hybrid Micelles to Improve Drug Retention and Tumor Penetration.

PURPOSE: To enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40 nm were developed to improve drug retention and tumor penetration. METHODS: ASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymers. Stability of micelles was optimized in terms of critical micelle concentration (CMC) and drug release properties. The encapsulation efficiency (EE) and drug loading were determined using an established dialysis-mathematic fitting method. Multicellular spheroids (MCTS) penetration and cytotoxicity were investigated on MCF-7 cell line. Pharmacokinetics of ASL-micelles was evaluated in rats with ASL-solution as control. RESULTS: The ASL-micelles prepared with DSPE-PEG2000 and TPGS (1:1, w/w) exhibited small size (~18.5 nm), higher EE (~94.12%), better sustained in vitro drug release with lower CMC which may be ascribed to the interaction between drug and carriers. Compared to free ASL, ASL-micelles showed better MCTS penetration capacity and more potent cytotoxicity. Pharmacokinetic studies demonstrated that the half-life and AUC values of ASL-micelles were approximately 1.37-fold and 3.49-fold greater than that of free ASL. CONCLUSIONS: The optimized DSPE-PEG2000/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.

Sequential Intensified Conditioning Regimen Allogeneic Hematopoietic Stem Cell Transplantation in Adult Patients with Intermediate- or High-Risk Acute Myeloid Leukemia in Complete Remission: A Study from the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation.

Post-transplant relapse is the leading cause of treatment failure in acute myeloid leukemia (AML) patients after reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation (allo-HSCT). To improve their outcome, we evaluated the outcome of a sequential intermediate-intensity conditioning regimen combining fludarabine, cytosine arabinoside, amsacrine, cyclophosphamide, and either total body irradiation or busulfan (FLAMSA) in patients with intermediate or high-risk AML in first or second complete remission (CR). A total of 265 patients (median age, 55 years; range, 19 to 76) with AML who underwent allo-HSCT using a FLAMSA regimen were included. At the time of transplant, 216 (81.5%) were in CR1 and 49 (18.5%) in CR2. Cytogenetic was intermediate in 114 (43%) and poor in 42 (15.8%) patients, whereas 109 patients (41.1%) had a secondary AML. With a median follow-up of 46 months (range, 1 to 145), the Kaplan-Meier estimate of overall and leukemia-free survival at 2 years were 56.1% (95% CI, 49.7% to 62.6%) and 52.8% (95% CI, 46.4% to 59.2%), respectively. At 2 years, the cumulative incidences of relapse and nonrelapse mortality were 22.8% (95% CI, 17.6% to 28.4%) and 24.0% (95% CI, 18.8% to 29.5%), respectively. In multivariate analysis, patient age and cytogenetics were the only parameters with a significant impact on overall survival. These data suggest that the FLAMSA sequential intermediate conditioning regimen provides an efficient disease control in intermediate- and high-risk AML patients, including those in CR2 and with secondary AML.

Amsacrine-induced apoptosis of human leukemia U937 cells is mediated by the inhibition of AKT- and ERK-induced stabilization of MCL1.

Previous studies have attributed the anticancer activity of amsacrine to its inhibitory effect on topoisomerase II. However, 9-aminoacridine derivatives, which have the same structural scaffold as amsacrine, induce cancer cell apoptosis by altering the expression of BCL2 family proteins. Therefore, in the present study, we assessed whether BCL2 family proteins mediated the cytotoxic effects of amsacrine on human leukemia U937 cells. Amsacrine-induced apoptosis of U937 cells was characterized by caspase-9 and caspase-3 activation, increased intracellular Ca2+ concentration, mitochondrial depolarization, and MCL1 down-regulation. Amsacrine induced MCL1 down-regulation by decreasing its stability. Further, amsacrine-treated U937 cells showed AKT degradation and Ca2+-mediated ERK inactivation. Blockade of ERK-mediated phosphorylation of MCL1 inhibited the effect of Pin1 on the stabilization of MCL1, and AKT degradation promoted GSK3ß-mediated degradation of MCL1. Restoration of ERK phosphorylation and AKT expression abrogated amsacrine-induced MCL1 down-regulation. Moreover, MCL1 over-expression inhibited amsacrine-induced depolarization of mitochondria membrane and increased the viability of amsacrine-treated cells. Taken together, our data indicate that amsacrine abolishes ERK- and Pin1-mediated stabilization of MCL1 and promotes GSK3ß-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells.

Novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-ones act as covalent poisons of human topoisomerase IIα.

A number of topoisomerase II-targeted anticancer drugs, including amsacrine, utilize an acridine or related aromatic core as a scaffold. Therefore, to further explore the potential of acridine-related compounds to act as topoisomerase II poisons, we synthesized a series of novel trifluoromethylated 9-amino-3,4-dihydroacridin-1(2H)-one derivatives and examined their ability to enhance DNA cleavage mediated by human topoisomerase IIα. Derivatives containing a H, Cl, F, and Br at C7 enhanced enzyme-mediated double-stranded DNA cleavage ∼5.5- to 8.5-fold over baseline, but were less potent than amsacrine. The inclusion of an amino group at C9 was critical for activity. The compounds lost their activity against topoisomerase IIα in the presence of a reducing agent, displayed no activity against the catalytic core of topoisomerase IIα, and inhibited DNA cleavage when incubated with the enzyme prior to the addition of DNA. These findings strongly suggest that the compounds act as covalent, rather than interfacial, topoisomerase II poisons.

Quantitative determination of intracellular Asulacrine in MCF-7 breast cancer cells by liquid chromatography-mass spectrometry and its application to cellular pharmacokinetic studies of P188 modified liposomes.

Asulacrine (ASL), an analogue of amsacrine, has shown higher anti-breast and anti-lung cancer activity. Hereby, a new sensitive and selective liquid chromatography-mass spectrometry (LC/MS) method was developed to determine intracellular asulacrine. The chromatographic separation was performed on an Agilent Zorbax Extend-C18 column (2.1 mm i.d. × 50 mm, 5 µm) using gradient elution with water (2 mmol/L ammonium acetate and 0.1% acetic acid) and acetonitrile as the mobile phase. The detection was achieved with selected ion monitoring mode using electrospray ionization in positive mode with target ions at m/z 465.3 and m/z 326.1 for asulacrine and midazolam, respectively. The standard curve showed a good linearity with the lower limit of quantification of 1 ng/mL, as a result of which, the trace concentration of ASL in cell suspension could be quantified. The intra- and inter-day accuracy ranged from -5.28 to 6.5% and from -6.32 to 1.05%, and the intra- and inter-day precisions were no more than 7.65% and 11.71%, respectively. Additionally, no degradation of asulacrine was observed during stability evaluation. The method was proved to be powerful and practical to determine and compare the intracellular distribution and kinetics of ASL under different formulations in MCF-7 breast cancer cells.

Phase II, multicenter, randomized trial of CPX-351 (cytarabine:daunorubicin) liposome injection versus intensive salvage therapy in adults with first relapse AML.

BACKGROUND: CPX-351 is a liposome-encapsulated fixed-molar-ratio formulation of cytarabine and daunorubicin that exploits molar ratio-dependent drug-drug synergy to enhance antileukemic efficacy. METHODS: This phase II study randomized 125 patients 2:1 to CPX-351 or investigators' choice of first salvage chemotherapy. Patients with acute myeloid leukemia (AML) in first relapse after initial Complete Remission (CR) lasting ≥1 month were stratified per the European Prognostic Index (EPI) into favorable, intermediate, and poor-risk groups based on duration of first CR, cytogenetics, age, and transplant history. Control salvage treatment was usually based on cytarabine and anthracycline, often with 1 or more additional agents. Survival at 1 year was the primary efficacy end point. RESULTS: Patient characteristics were well balanced between the 2 study arms. Improvements in efficacy outcomes were observed following CPX-351, but did not meet prospectively defined statistical criteria for 1-year survival improvement in the overall population. Subset analyses of the EPI-defined poor-risk strata demonstrated higher response rates (39.3% vs 27.6%) and improvements in event-free survival (HR, 0.63; P = .08) and overall survival (HR, 0.55; P = .02). Also, 60-day mortality was lower in the CPX-351 study arm for poor-risk patients (16.1% vs 24.1%). CONCLUSIONS: Taken together, the data suggest possible improved outcomes in CPX-351-treated first relapse AML patients with EPI-defined poor-risk disease.

Strategies to maximize liposomal drug loading for a poorly water-soluble anticancer drug.

PURPOSE: To develop a liposomal system with high drug loading (DL) for intravenous (i.v.) delivery of a poorly water-soluble basic drug, asulacrine (ASL). METHODS: A thin-film hydration and extrusion method was used to fabricate the PEGylated liposomal membranes followed by a freeze and thaw process. A novel active drug loading method was developed using ammonium sulphate gradient as an influx driving force of ASL solubilized with sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD). DL was maximized by optimizing liposomal preparation and loading conditions. Pharmacokinetics was evaluated following i.v. infusion in rabbits. RESULTS: Freeze-thaw resulted in unilamellar liposome formation (180 nm) free of micelles. Higher DL was obtained when dialysis was used to remove the untrapped ammonium sulphate compared to ultracentrifuge. The pH and SBE-ß-CD level in the loading solution played key roles in enhancing DL. High DL ASL-liposomes (8.9%w/w, drug-to-lipid mole ratio 26%) were obtained with some drug "bundles" in the liposomal cores and were stable in a 5% glucose solution for >80 days with minimal leakage (<2%). Surprisingly, following administration of ASL-liposomes prepared with or without SBE-ß-CD, the half-lives were similar to the drug solution despite an increased area under the curve, indicating drug leakage from the carriers. CONCLUSIONS: High liposomal DL was achieved with multiple strategies for a poorly-water soluble weak base. However, the liposomal permeability needed to be tailored to improve drug retention.

On the structural basis and design guidelines for type II topoisomerase-targeting anticancer drugs.

Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme-DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 ß-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3'-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex. Analysis of drug-binding modes and the conformational landscapes of the drug-binding pockets provide rationalization of the drugs' structural-activity relationships and explain why Top2 mutants exhibit differential effects toward each drug. Drug design guidelines were proposed to facilitate the development of isoform-specific Top2-targeting anticancer agents.

Amsacrine suppresses matrix metalloproteinase-2 (MMP-2)/MMP-9 expression in human leukemia cells.

This study explores the suppression mechanism of amsacrine (4-(9-Acridinylamino)-N-(methanesulfonyl)-m-anisidine hydrochloride) on matrix metalloproteinase-2 (MMP-2) and MMP-9 expression in human leukemia cells. Amsacrine attenuated cell invasion with decreased MMP-2/MMP-9 protein expression and mRNA levels in U937, Jurkat, HL-60, K562, KU812, and MEG-01 cells. Moreover, amsacrine reduced both MMP-2/MMP-9 promoter luciferase activity and MMP-2/MMP-9 mRNA stability in leukemia cells. Studies on amsacrine-treated U937 cells revealed that amsacrine-elicited ROS generation induced JNK and p38 MAPK activation but reduced the phospho-ERK level. Amsacrine-induced ERK inactivation and p38 MAPK/JNK activation were demonstrated to suppress MMP-2/MMP-9 promoter luciferase activity and promote MMP-2/MMP-9 mRNA decay, respectively. p38 MAPK/JNK activation led to up-regulation of protein phosphatase 2A catalytic subunit α (PP2Acα) in amsacrine-treated U937 cells. Okadaic acid (PP2A inhibitor) treatment increased MMP-2/MMP-9 mRNA stability in amsacrine-treated cells, whereas PP2Acα over-expression increased MMP-2/MMP-9 mRNA decay. Amsacrine-induced MMP-2/MMP-9 down-regulation was also related to PP2Acα up-regulation on Jurkat, HL-60, K562, KU812, and MEG-01 cells. Collectively, our data indicate that amsacrine induces MMP-2/MMP-9 down-regulation via simultaneous suppression of genetic transcription and mRNA stability in human leukemia cells.