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Contains fulltext : 251339.pdf (Publisher’s version ) (Open Access) OBJECTIVE: To compare CollaboRATE and SDM-Q-9 questionnaires when appreciating patient-perceived level of shared decision-making (SDM) in doctor-patient consultations. METHODS: Data were harvested from five separate studies on SDM, conducted in three university and one large community hospital in the Netherlands, using Dutch versions of both questionnaires. CollaboRATE and SDM-Q-9 scores were expressed as percentages. Correlation was assessed using Spearman's Rho coefficient. Bland&Altman analysis was used to assess the degree of agreement. Top scores were calculated to assess possible ceiling effects. RESULTS: The five studies included 442 patients. Median CollaboRATE scores (88.9%, IQR 81.5-100%) were significantly higher (p

Background Choosing an antipsychotic medication is an important medical decision in the treatment of schizophrenia. This decision requires risk-benefit assessments of antipsychotics, and thus, shared-decision making between physician and patients is strongly encouraged. Although the efficacy and side-effect profiles of antipsychotics are well-established, there is no clear framework for the communication of the evidence between physicians and patients. For this reason, we developed an evidence-based shared-decision making assistant (SDM-assistant) that presents high-quality evidence from network meta-analysis on the efficacy and side-effect profile of antipsychotics and can be used as a basis for shared-decision making between physicians and patients when selecting antipsychotic medications. Methods The planned matched-pair cluster-randomised trial will be conducted in acute psychiatric wards (n = 14 wards planned) and will include adult inpatients with schizophrenia or schizophrenia-like disorders (N = 252 participants planned). On the intervention wards, patients and their treating physicians will use the SDM-assistant, whenever a decision on choosing an antipsychotic is warranted. On the control wards, antipsychotics will be chosen according to treatment-as-usual. The primary outcome will be patients’ perceived involvement in the decision-making during the inpatient stay as measured with the SDM-Q-9. We will also assess therapeutic alliance, symptom severity, side-effects, treatment satisfaction, adherence, quality of life, functioning and rehospitalizations as secondary outcomes. Outcomes could be analysed at discharge and at follow-up after three months from discharge. The analysis will be conducted per-protocol using mixed-effects linear regression models for continuous outcomes and logistic regression models using generalised estimating equations for dichotomous outcomes. Barriers and facilitators in the implementation of the intervention will also be examined using a qualitative content analysis. Discussion This is the first trial to examine a decision assistant specifically designed to facilitate shared-decision making for choosing antipsychotic medications, i.e., SDM-assistant, in acutely ill inpatients with schizophrenia. If the intervention can be successfully implemented, SDM-assistant could advance evidence-based medicine in schizophrenia by putting medical evidence on antipsychotics into the context of patient preferences and values. This could subsequently lead to a higher involvement of the patients in decision-making and better therapy decisions. Trial registration German Clinical Trials Register (ID: DRKS00027316, registration date 26.01.2022).

Purpose Central serous chorioretinopathy (CSCR) has been associated with oxidative stress–related risk factors. The objective of this study was to optimize an analytical method for evaluating the oxidative stress biomarker malondialdehyde (MDA) in human tears and determine its level in the tears of patients with CSCR. Methods In this pilot study, tear samples were obtained from 34 healthy donors and 31 treatment-naïve CSCR male patients (eight with acute CSCR and 23 with chronic CSCR). Two analytical methods based on high-performance liquid chromatography followed by fluorescence detection were evaluated, with either 2-thiobarbituric derivative (TBA) or 2-aminoacridone (2-AA). Activity of CSCR was defined by the serous retinal detachment (SRD) height, which was measured by two independent observers on spectral-domain optical coherence tomography. Results The 2-AA method showed higher sensitivity and precision compared to the TBA method. When the 2-AA method was applied to tears from healthy donors, the levels of MDA were statistically significantly higher in men compared to women (mean ± standard deviation, SD: 9,914 nM ± 6,126 versus 4,635 nM ± 1,173, p = 0.006). No difference was found in tear MDA levels between male patients with CSCR and age-matched control men (p = 0.17). However, MDA levels were statistically significantly higher in acute compared to chronic CSCR cases (mean ± SD: 12,295 nM ± 8,495 versus 6,790 ± 3,969 nM, p = 0.03). Additionally, there was a correlation between MDA levels and RPE leakage, quantified by the height of the serous retinal detachment (p = 0.02, r = 0.40). Conclusions Levels of MDA in tears, measured with an optimized analytical method, correlate with RPE leakage in CSCR.

Background Social robots (SRs) have been used for improving anxiety in children in stressful clinical situations, such as during painful procedures. However, no studies have yet been performed to assess their effect in children while waiting for emergency room consultations. Objective This study aims to assess the impact of SRs on managing stress in children waiting for an emergency room procedure through the assessment of salivary cortisol levels. Methods This was an open randomized clinical trial in children attending a pediatric emergency department. Children accessing the emergency room were randomized to 1 of 3 groups: (1) playing with a NAO SR, (2) playing with a study nurse, or (3) waiting with parents. The salivary cortisol levels of all children were measured through a swab. Salivary cortisol levels before and after the intervention were compared in the 3 groups. We calculated the effect size of our interventions through the Cohen d-based effect size correlation (r). Results A total of 109 children aged 3-10 years were enrolled in the study, and 94 (86.2%) had complete data for the analyses. Salivary cortisol levels significantly decreased more in the group exposed to robot interaction than in the other two groups (r=0.75). Cortisol levels decreased more in girls (r=0.92) than in boys (r=0.57). Conclusions SRs are efficacious in decreasing stress in children accessing the emergency room and may be considered a tool for improving emotional perceptions of children and their families in such a critical setting. Trial Registration ClinicalTrials.gov NCT04627909; https://clinicaltrials.gov/ct2/show/study/NCT04627909

Objective: Ready for SDM was developed in Norway as a comprehensive modularized curriculum for health care providers (HCP). The current study evaluated the efficacy of one of the modules, a 2-hour interprofessional SDM training designed to enhance SDM competencies. Methods: A cluster randomized controlled trial was conducted with eight District Psychiatric Centres randomized to wait-list control (CG) or intervention group (IG). Participants and trainers were not blinded to their allocation. The IG received a 2-hour didactic and interactive training, using video examples. The primary outcome was the agreement between the participants’ and an expert assessment of patient involvement in a video recorded consultation. The SDM-knowledge score was a secondary outcome. Results: Compared to the CG (n = 65), the IG (n = 69) judged involvement behavior in a communication example more accurately (mean difference of weighted T, adjusted for age and gender:=− 0.098, p = 0.028) and demonstrated better knowledge (mean difference=− 0.58; p = 0.014). A sensitivity analysis entering a random effect for cluster turned out not significant. Conclusion: The interprofessional group training can improve HCPs’ SDM-competencies. Practice implications: Addressing interprofessional teams using SDM communication training could supplement existing SDM training approaches. More research is needed to evaluate the training module’s effects as a component of large-scale implementation of SDM.

The reliance of number processing on sensorimotor mechanisms involved in hand action has been extensively documented by behavioural studies. Nonetheless, where and how the computations of number and hand action interact in the brain has received limited attention. In this study we investigated the brain networks underlying symbolic number comparison and the hand action of reaching and grasping, capitalizing on functional imaging studies meta-analyzed with the seed-based d mapping with permutation of subject images meta-analytic method (SDM-PSI). The main objective was to test whether and to what extent symbolic number processing recruits the same sensorimotor network involved in the hand action of reaching and grasping. We included 42 studies (756 participants) adopting symbolic number comparison tasks and 58 studies (867 participants) investigating hand reaching and hand grasping. The conjunction analysis of brain networks common to number processing, reaching, and grasping revealed spatial convergence over frontoparietal areas. Specifically, four clusters were identified, in and around the left and right intraparietal sulci, in the left precentral gyrus, and in the supplementary motor area. The degree of overlap was extensive, since the reach/grasp network mostly included the number areas. A qualitative analysis of functional characterization capitalizing on the Neurosynth database depicted a strong multifunctionality of the regions of overlap between numbers and hand action: these brain areas were also associated to a variety of functions within the domains of memory and imagery, visuospatial attention, and language. Overall, these results characterize the neuroanatomical substrate of the interaction between reaching, grasping, and symbolic number comparison.

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 μg mL−1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect.

This study aims to test the psychometric properties of the Malay, English, and Chinese 9-Item Shared Decision Making Questionnaire (SDM-Q-9) in breast cancer patients making treatment decisions. The original German SDM-Q-9 was translated to Malay using the back-translation method. A total of 222 newly diagnosed breast cancer patients making treatment decisions were sampled conveniently from three breast clinics between August 2015 and February 2016. A total of 66 patients answered the SDM-Q-9 in Malay, 87 in English, and 69 in Chinese. Data were analyzed using SPSS and AMOS software. SDM-Q-9 demonstrated good reliability in the three translations. All the items correlated well except for Item 1 in English. The factor loadings were within acceptable range except for Item 1 in Malay, Items 1 and 2 in English, and Items 7 and 9 in Chinese SDM-Q-9. However, no items were deleted in accordance with experts’ opinions and the previous SDM-Q-9 validation studies. The Malay, English, and Chinese SDM-Q-9 demonstrated good reliability and validity.

An intriguing challenge in the human–robot interaction field is the prospect of endowing robots with emotional intelligence to make the interaction more genuine, intuitive, and natural. A crucial aspect in achieving this goal is the robot’s capability to infer and interpret human emotions. Thanks to its design and open programming platform, the NAO humanoid robot is one of the most widely used agents for human interaction. As with person-to-person communication, facial expressions are the privileged channel for recognizing the interlocutor’s emotional expressions. Although NAO is equipped with a facial expression recognition module, specific use cases may require additional features and affective computing capabilities that are not currently available. This study proposes a highly accurate convolutional-neural-network-based facial expression recognition model that is able to further enhance the NAO robot’ awareness of human facial expressions and provide the robot with an interlocutor’s arousal level detection capability. Indeed, the model tested during human–robot interactions was 91% and 90% accurate in recognizing happy and sad facial expressions, respectively, 75% accurate in recognizing surprised and scared expressions, and less accurate in recognizing neutral and angry expressions. Finally, the model was successfully integrated into the NAO SDK, thus allowing for high-performing facial expression classification with an inference time of 0.34 ± 0.04 s.

Shared decision making (SDM) is on the international healthcare agenda among patient advocacy groups and decision makers in healthcare worldwide [1–5]. In Denmark, implementation of SDM with all ca...

To quantify the synaptic vesicle glycoprotein 2A (SV2A) changes in the whole central nervous system (CNS) under pathophysiological conditions, a high affinity SV2A PET radiotracer with improved in vivo stability is desirable to minimize the potential confounding effect of radiometabolites. The aim of this study was to develop such a PET tracer based on the molecular scaffold of UCB-A, and evaluate its pharmacokinetics, in vivo stability, specific binding, and nonspecific binding signals in nonhuman primate brains, in comparison with [The racemic SDM-16 (4-(3,5-difluorophenyl)-1-((2-methyl-1H-imidazol-1-yl)methyl)pyrrolidin-2-one) and its two enantiomers were synthesized and assayed for in vitro binding affinities to human SV2A. We synthesized the enantiopure [SDM-16 was synthesized in 3 steps with 44% overall yield and has the highest affinity (KWe have successfully synthesized a novel SV2A PET tracer [

Transitive inference (TI) is a critical capacity involving the integration of relevant information into prior knowledge structure for drawing novel inferences on unobserved relationships. To date, the neural correlates of TI remain unclear due to the small sample size and heterogeneity of various experimental tasks from individual studies. Here, the meta-analysis on 32 fMRI studies was performed to detect brain activation patterns of TI and its three paradigms (spatial inference, hierarchical inference, and associative inference). We found the hippocampus, prefrontal cortex (PFC), putamen, posterior parietal cortex (PPC), retrosplenial cortex (RSC), supplementary motor area (SMA), precentral gyrus (PreCG), and median cingulate cortex (MCC) were engaged in TI. Specifically, the RSC was implicated in the associative inference, whereas PPC, SMA, PreCG, and MCC were implicated in the hierarchical inference. In addition, the hierarchical inference and associative inference both evoked activation in the hippocampus, medial PFC, and PCC. Although the meta-analysis on spatial inference did not generate a reliable result due to insufficient amount of investigations, the present work still offers a new insight for better understanding the neural basis underlying TI.

Objective Shared decision making integrates health care provider expertise with patient values and preferences. The MAPPIN’SDM is a recently developed measurement instrument that incorporates physician, patient, and observer perspectives during medical consultations. This review sought to critically appraise the development, sensibility, reliability, and validity of the MAPPIN’SDM and to determine in which settings it has been used. Methods This critical appraisal was performed through a targeted review of the literature. Articles outlining the development or measurement property assessment of the MAPPIN’SDM or that used the instrument for predictor or outcome purposes were identified. Results Thirteen studies were included. The MAPPIN’SDM was developed by both adapting and building on previous shared decision making measurement instruments, as well as through creation of novel items. Content validity, face validity, and item quality of the MAPPIN’SDM are adequate. Internal consistency ranged from 0.91 to 0.94 and agreement statistics from 0.41 to 0.92. The MAPPIN’SDM has been evaluated in several populations and settings, ranging from chronic disease to acute oncological settings. Limitations include high reading levels required for self-administered patient questionnaires and the small number of studies that have employed the instrument to date. Conclusion The MAPPIN’SDM generally shows adequate development, sensibility, reliability, and validity in preliminary testing and holds promise for shared decision making research integrating multiple perspectives. Further research is needed to develop its use in other patient populations and to assess patient understanding of complex item wording.

The use of humanoid robots as assistants in therapy processes is not new. Several projects in the past several years have achieved promising results when combining human-robot interaction with standard techniques. Moreover, there are multiple screening systems for autism; one of the most used systems is the Quantitative Checklist for Autism in Toddlers (Q-CHAT-10), which includes ten questions to be answered by the parents or caregivers of a child. We present Q-CHAT-NAO, an observation-based autism screening system supported by a NAO robot. It includes the six questions of the Q-CHAT-10 that can be adapted to work in a robotic context; unlike the original system, it obtains information from the toddler instead of from an indirect source. The detection results obtained after applying machine learning models to the six questions in the Autistic Spectrum Disorder Screening Data for Toddlers dataset were almost equivalent to those of the original version with ten questions. These findings indicate that the Q-CHAT-NAO could be a screening option that would exploit all the benefits related to human-robot interaction.

Background Among the studied antitumor acridinone derivatives developed in our laboratory, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) exhibited cytotoxic and antitumor properties against several cancer types and were selected to be evaluated in preclinical and early-phase clinical trials. In the present work, we investigated the impact of C-1305 and C-1311 on UDP-glucuronosyltransferase (UGT) activity. Methods Enzyme activity modulation was studied using HPLC by analyzing standard UGT substrate metabolism in the presence and absence of antitumor drugs. The investigations were performed in two model systems: (i) under noncellular conditions, including human liver microsomes (HLM) and recombinant UGT1A1, 1A9 and 1A10 isoenzymes and (ii) in tumor cells. Results There was observed a slight impact of studied drugs on enzyme activity. Only UGT1A1 action was altered by both compounds. The modulatory effects of UGT activity in cellular systems depended on the tumor cell type. In the case of HepG2, C-1305 and C-1311 strongly induced UGT activity, particularly for C-1311, at concentrations significantly lower than the EC 50 . This effect contradicted irinotecan mediated UGT inhibition. HT29 colon tumor cells were less sensitive than HepG2 to enzyme modulation in the presence of the studied compounds, particularly C-1305, where enzymatic inhibition similar to that of irinotecan was observed. Conclusions The results demonstrated that UGT activity modulation should be expected in the case of antitumor therapy with C-1305 or/and C-1311. Analysis of the results indicated that these modulations would occur via cellular regulatory pathways not by direct drug-enzyme interactions.

Socially assistive robots have shown potential benefits in therapy of child and elderly patients with social and cognitive deficits. In particular, for autistic children, humanoid robots could enhance engagement and attention, thanks to their simplified toy-like appearance and the reduced set of possible movements and expressions. The recent focus on autism-related motor impairments has increased the interest on developing new robotic tools aimed at improving not only the social capabilities but also the motor skills of autistic children. To this purpose, we have designed two embodied mirroring setups using the NAO humanoid robot. Two different tracking systems were used and compared: Inertial Measurement Units and the Microsoft Kinect, a marker-less vision based system. Both platforms were able to mirror upper limb basic movements of two healthy subjects, an adult and a child. However, despite the lower accuracy, the Kinect-based setup was chosen as the best candidate for embodied mirroring in autism treatment, thanks to the lower intrusiveness and reduced setup time. A prototype of an interactive mirroring game was developed and successfully tested with the Kinect-based platform, paving the way to the development of a versatile and powerful tool for clinical use with autistic children.

The metabolism of antitumor-active 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) has been investigated widely over the last decade but some aspects of molecular mechanisms of its metabolic transformation are still not explained. In the current work, we have reported a direct and rapid analytical tool for better prediction of C-1311 metabolism which is based on electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS). Simulation of the oxidative phase I metabolism of the compound was achieved in a simple electrochemical thin-layer cell consisting of three electrodes (ROXY™, Antec Leyden, the Netherlands). We demonstrated that the formation of the products of N-dealkylation reactions can be easily simulated using purely instrumental approach. Newly reported products of oxidative transformations like hydroxylated or oxygenated derivatives become accessible. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass ion data and tandem mass spectrometry experiments. In silico prediction of main sites of C-1311 metabolism was performed using MetaSite software. The compound was evaluated for cytochrome P450 1A2-, 3A4-, and 2D6-mediated reactions. The results obtained by EC were also compared and correlated with those of reported earlier for conventional in vitro enzymatic studies in the presence of liver microsomes and in the model peroxidase system. The in vitro experimental approach and the in silico metabolism findings showed a quite good agreement with the data from EC/ESI-MS analysis. Thus, we conclude here that the electrochemical technique provides the promising platform for the simple evaluation of drug metabolism and the reaction mechanism studies, giving first clues to the metabolic transformation of pharmaceuticals in the human body.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311), a promising antitumor agent that is also active against autoimmune diseases, was determined to be a selective inhibitor of the cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1311 might modulate the effectiveness of other drugs used in multidrug therapy. The present work aimed to identify the mechanism of the observed C-1311-mediated inactivation of CYP1A2 and CYP3A4.The inactivation experiments were performed in vitro using the human recombinant CYP1A2 and CYP3A4 (Bactosomes). CYP isoenzyme activities were determined using the CYP-specific reactions, 7-ethoxycoumarin O-deethylation (CYP1A2) and testosterone 6β-hydroxylation (CYP3A4). The concentrations of CYP-specific substrates and their metabolites formed by CYP isoenzymes were measured by RP-HPLC with UV-Vis detection.The inhibition of CYPs by C-1311 was time-, concentration- and NADPH-dependent, which suggested a mechanism-based mode of action. Using a 10-fold dilution method and potassium ferricyanide we demonstrated the irreversible nature of the inhibition. In addition, the inhibition was attenuated by the presence of alternate substrates (alternative active site ligands) but not by a nucleophilic trapping agent (glutathione) or a reactive oxygen scavenger (catalase), which further supported a mechanism-based action. Substrate depletion partition ratios of 299 and 985 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.Our results indicated that C-1311 is a potent mechanism-based inactivator of CYP1A2 and CYP3A4. This finding provided new insights into the mechanism of C-1311 antitumor action, particularly in relation to potential pharmacokinetic drug-drug interactions between C-1311 and/or its derivatives and the substrates of CYP isoforms.

Activity modulation of drug metabolism enzymes can change the biotransformation of chemotherapeutics and cellular responses induced by them. As a result, drug-drug interactions can be modified. Acridinone derivatives, represented here by C-1305 and C-1311, are potent anticancer drugs. Previous studies in non-cellular systems showed that they are mechanism-based inhibitors of cytochrome P4503A4 and undergo glucuronidation via UDP-glucuronosyltranspherase 1A10 isoenzyme (UGT1A10). Therefore, we investigated the potency of these compounds to modulate P4503A4 and UGT1A10 activity in breast MCF-7 and colon HCT116 cancer cells and their influence on cytotoxicity and cellular response in cells with different expression levels of studied isoenzymes. We show that C-1305 and C-1311 are inducers of not only P4503A4 but also UGT1A10 activity. MCF-7 and HCT116 cells with high P4503A4 activity are more sensitive to acridinone derivatives and undergo apoptosis/necrosis to a greater extent. UGT1A10 was demonstrated to be responsible for C-1305 and C-1311 glucuronidation in cancer cells and glucuronide products were excreted outside the cell very fast. Finally, we show that glucuronidation of C-1305 antitumor agent enhances its pro-apoptotic properties in HCT116 cells, while the cytotoxicity and cellular response induced by C-1311 did not change after drug glucuronidation in both cell lines.

Nowadays, necessity is shown up concerning modeling of certain ecological processes actually emerging in various biotops and hence step-by-step study of development of biofilms on solid surfaces (glass and plastic). To implement such kind of studies the light and luminescent microscopy are the most available and rather informative tools. The study used strains V.cholerae O1 and O139 serogroups with different genetic characteristics. The biofilms were developed on cover glass and/or plastic plates which were placed in bottles with tap autoclave water in vertical position in special device. The lifetime visualization of stages of development of biofilm was implemented using luminescent microscopy by coloring biofilm with solution of acridine yellow. The biofilms colored with Congo read and fuchsine were analyzed using light microscopy. The light and luminescent microscopy permit investigating biofilms in dynamics at various stages of development, to evaluate condition of its physiological functioning in quantitative and qualitative dimensions and to trace synthesis of exopolysaccharides in different serogroups of vibrios that has significance in prognosis of velocity of occurrence of biofilms on abiotic objects.

Altres ajuts: FA/AAP-2013-65-101349 Altres ajuts: ANR/ANR-12-BS07-0008-01 Altres ajuts: ANR/ANR-11-LABX-0029 Symptomatic treatment of myasthenia gravis is based on the use of peripherally-acting acetylcholinesterase (AChE) inhibitors that, in some cases, must be discontinued due to the occurrence of a number of side-effects. Thus, new AChE inhibitors are being developed and investigated for their potential use against this disease. Here, we have explored two alternative approaches to get access to peripherally-acting AChE inhibitors as new agents against myasthenia gravis, by structural modification of the brain permeable anti-Alzheimer AChE inhibitors tacrine, 6-chlorotacrine, and huprine Y. Both quaternization upon methylation of the quinoline nitrogen atom, and tethering of a triazole ring, with, in some cases, the additional incorporation of a polyphenol-like moiety, result in more polar compounds with higher inhibitory activity against human AChE (up to 190-fold) and butyrylcholinesterase (up to 40-fold) than pyridostigmine, the standard drug for symptomatic treatment of myasthenia gravis. The novel compounds are furthermore devoid of brain permeability, thereby emerging as interesting leads against myasthenia gravis.

Drugs targeting receptor tyrosine kinase FLT3 are of particular interest since activating FLT3-internal tandem duplication (ITD) mutations abundantly occur in fatal acute myeloid leukemias (AMLs). Imidazoacridinone C-1311, a DNA-reactive inhibitor of topoisomerase II, has been previously shown to be a potent and selective inhibitor of recombinant FLT3. Here, we expand those findings by studying its effect on leukemia cells with wild-type FLT3, FLT3-ITD mutant and no FLT3 receptor. While brief C-1311 exposure blocked wild-type and FLT3-ITD activity, profound and sustained inhibition was achieved only for FLT3-ITD mutants. C-1311 inhibited FLT3 downstream pathways (MAPK and AKT) independent of FLT3 status, yet translation to decreased viability was significant in FLT3-ITD cells. RNA interference against FLT3-ITD reduced cytotoxic effect and apoptosis induced by C-1311, indicating selective inhibition of FLT3-ITD crucial for high efficacy of drug against activated leukemia cells. Cellular responses in treated FLT3-ITD mutants included G1 and G2/M phase arrest, moderate inhibition of Bcl-2, caspase-3 activation, PARP cleavage, and depolarization of mitochondria. Consistent with selective decrease in FLT3-ITD activity, C-1311 remarkably reduced antiapoptotic survivin mRNA and protein expression, correlating well with enhanced apoptosis of FLT3-ITD cells. No survivin decrease and respectively lower level of apoptosis was found in wild-type and null-FLT3 cells. Combination of C-1311 with cytarabine or doxorubicin again showed distinct synergistic activity in FLT3-ITD-positive cells. The ability of C-1311 to selectively target constitutively active FLT3, suggests a favorable therapeutic index for AML carrying FLT3-ITD mutations. Thus further preclinical and clinical studies addressing its potency against FLT3-ITD kinase is well justified.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 μg GAG. Limits of detection and quantitation were 0.02–0.15 and 0.07–0.31 μg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

// Anna Skwarska 1, 2 , Shaliny Ramachandran 1 , Grzegorz Dobrynin 1 , Katarzyna B. Leszczynska 1 , Ester M. Hammond 1 1 Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, The University of Oxford, Oxford, UK 2 Department of Pharmaceutical Technology and Biochemistry, Chemical Faculty, Gdansk University of Technology, Gdansk, Poland Correspondence to: Anna Skwarska, email: annskwar@pg.gda.pl Ester M. Hammond, email: ester.hammond@oncology.ox.ac.uk Keywords: p53, C-1311/Symadex, radiation, senescence, apoptosis Received: January 27, 2017 Accepted: March 01, 2017 Published: March 10, 2017 ABSTRACT C-1311 is a small molecule, which has shown promise in a number of pre-clinical and clinical studies. However, the biological response to C-1311 exposure is complicated and has been reported to involve a number of cell fates. Here, we investigated the molecular signaling which determines the response to C-1311 in both cancer and non-cancer cell lines. For the first time we demonstrate that the tumor suppressor, p53 plays a key role in cell fate determination after C-1311 treatment. In the presence of wild-type p53, cells exposed to C-1311 entered senescence. In contrast, cells lines without functional p53 underwent mitotic catastrophe and apoptosis. C-1311 also induced autophagy in a non-p53-dependent manner. Cells in hypoxic conditions also responded to C-1311 in a p53-dependent manner, suggesting that our observations are physiologically relevant. Most importantly, we show that C-1311 can be effectively combined with radiation to improve the radiosensitivity of a panel of cancer cell lines. Together, our data suggest that C-1311 warrants further clinical testing in combination with radiotherapy for the treatment of solid tumors.

During this study, 9-aminoacridine and artemisinin–acridine hybrid compounds were synthesized and the in vitro for antimalarial activity against both the chloroquine sensitive but also gametocytocidal strain (NF54), and chloroquine resistant (Dd2) strains of Plasmodium falciparum was determined. In vitro cytotoxicity against CHO cells, apoptosis of HepG2 and SH-SY5Y as well as anticancer activity against HeLa cell lines were assessed. The hybrids were synthesized, using a microwave-assisted radiation method by covalently linking artemisinin and acridine pharmacophores by means of a liable, aminoethyl ether linker. The synthesized compounds were found active against both the Plasmodium strains and displayed superior selective toxicity towards the parasitic cells. Hybrid 7, however, containing ethylenediamine linker, proved the most active of all of the synthesized compounds. It had seven-fold higher antigametocytocidal activity compared to chloroquine and was also found to be seven-fold more potent than chloroquine against the Dd2 strain, with highly selective action towards the parasitic cells. This hybrid also showed favourable anti-cancer activity against the HeLa cells, three- and eight-fold higher than those of chloroquine and melphalan, respectively. This hybrid may therefore stand as drug candidate for further investigation in the search for new and effective drugs against malaria and cervical cancer http://dx.doi.org/10.1016/j.ejps.2014.01.014 http://www.journals.elsevier.com/european-journal-of-pharmaceutical-sciences/ Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2014.01.014 National Research Foundation (NRF) and the North-West University

Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.

Cardiolipin (CL) is a unique phospholipid found in mitochondrial inner membrane. It is a key component for mitochondrial function in both respiration and apoptosis. The level of CL is an important parameter for investigating these intracellular events and is a critical indicator of a number of diseases associated with mitochondrial respiratory functions. 10-Nonyl acridine orange (NAO) is the only fluorescent dye currently available for CL detection. However, the performance of NAO is far from satisfactory in terms of selectivity and sensitivity. In this work, we report an aggregation-induced emission-active fluorogen, TTAPE-Me, for CL detection and quantification. With improved sensitivity and excellent selectivity to CL over other major mitochondrial membrane lipids, TTAPE-Me could serve as a valuable fluorescent sensor for CL quantification. The use of TTAPE-Me for the quantification of isolated mitochondria is also demonstrated.

Attempting to unravel mechanisms in optical probing of proteins, we have performed pilot calculations of two cationic chromophores—acridine yellow and proflavin—located at different binding sites within human serum albumin, including the two primary drug binding sites as well as a heme binding site. The computational scheme adopted involves classical molecular dynamics simulations of the ligands bound to the protein and subsequent linear response polarizable embedding density functional theory calculations of the excitation energies. A polarizable embedding potential consisting of point charges fitted to reproduce the electrostatic potential and isotropic atomic polarizabilities computed individually for every residue of the protein was used in the linear response calculations. Comparing the calculated aqueous solution-to-protein shifts of maximum absorption energies to available experimental data, we concluded that the cationic proflavin chromophore is likely not to bind albumin at its drug binding site 1 nor at its heme binding site. Although agreement with experimental data could only be obtained in qualitative terms, our results clearly indicate that the difference in optical response of the two probes is due to deprotonation, and not, as earlier suggested, to different binding sites. The ramifications of this finding for design of molecular probes targeting albumin or other proteins is briefly discussed.

Novel 9-aminoacridine derivatives were synthesized by linking the heteroaromatic core to different cinnamic acids through an aminobutyl chain. The test compounds demonstrated mid-nanomolar in vitro activity against erythrocytic stages of the chloroquine-resistant W2 strain of the human malaria parasite Plasmodium falciparum. Two of the most active derivatives also showed in vitro activity against liver-stage Plasmodium berghei, with activity greater than that of the reference liver-stage antimalarial primaquine. The compounds were not toxic to human hepatoma cells at concentrations up to 5 mu M. Hence, 9-(N-cinnamoylbutyl)aminoacridines are a new class of leads for prevention and treatment of malaria. (c) 2012 Elsevier Ltd. All rights reserved.

The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311 (NSC-645809), is an antitumor agent shown to be effective against breast cancer in phase II clinical trials. A similar compound, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, shows high activity against experimental tumors and is expected to have even more beneficial pharmacological properties than C-1311. Previously published studies showed that these compounds are not substrates for cytochrome P450s; however, they do contain functional groups that are common targets for glucuronidation. Therefore, the aim of this work was to identify the human UDP-glucuronosyltransferases (UGTs) able to glucuronidate these two compounds. High-performance liquid chromatography analysis was used to examine the activities of human recombinant UGT1A and UGT2B isoforms and microsomes from human liver [human liver microsomes (HLM)], whole human intestinal mucosa [human intestinal microsomes (HIM)], and seven isolated segments of human gastrointestinal tract. Recombinant extrahepatic UGT1A10 glucuronidated 8-hydroxyl groups with the highest catalytic efficiency compared with other recombinant UGTs, V(max)/K(m) = 27.2 and 8.8 μl · min⁻¹ · mg protein⁻¹, for C-1305 and C-1311, respectively. In human hepatic and intestinal microsomes (HLM and HIM, respectively), high variability in UGT activities was observed among donors and for different regions of intestinal tract. However, both compounds underwent UGT-mediated metabolism to 8-O-glucuronides by microsomes from both sources with comparable efficiency; V(max)/K(m) values were from 4.0 to 5.5 μl · min⁻¹ · mg protein⁻¹. In summary, these studies suggest that imid azoacridinone and triazoloacridinone drugs are glucuronidated in human liver and intestine in vivo and may form the basis for future translational studies of the potential role of UGTs in resistance to these drugs.

The ability of small molecules to target DNA forms the basis of many clinically used antitumour agents. This study examines the effects of novel 9-aminoacridine carboxamides, synthesised by click chemistry based upon the reactions of either 9-(2-azidoethyl)amino or 9-propargylaminoacridine compounds, on various types of DNA tertiary structures. This gave either monomeric or dimeric compounds, the dimeric derivatives being the first unsymmetrical acridine dimers to be described. The compounds were assayed for duplex DNA, quadruplex DNA and four-way junction DNA binding. Their antiproliferative activity in the Human promyelocytic leukaemia cell line, HL60, was also assessed. Although for some of the compounds, notably the acridine 4-carboxamides, activity correlated with DNA binding affinity, for others it did not, with the rigidly linked dimers in particular showing a complicated relationship between 3- and 4-carboxamide structure and biological activity. The monomeric 3-carboxamides were more effective at stabilising G-quadruplex structures and also gave more hits in the four-way junction stabilisation assay. There is clear evidence from the binding of the 3-carboxamides that these compounds destabilise the open X form of the junction at lower concentrations and stabilise the X-stacked at higher concentrations. This might have implications for the biological activity of these compounds against proteins that bind to the Holliday junction (HJ).

Cardiolipin is a mitochondria-specific phospholipid known to be intimately involved with numerous mitochondrial functions. Accordingly, quantitative determination of cardiolipin provides a valuable aspect for assessing mitochondrial content and function. The current study was undertaken to develop a simple and reliable method for direct analysis of cardiolipin with particular application for the assessment of mitochondrial number of HepG2 cells. The method presented is based on the online 10-N-nonyl acridine orange (NAO) interaction with cardiolipin using CE with LIF detection. An aqueous-organic solvent system composed of 10% H(2) O-40% methanol-50% ACN (all v/v) containing 20 μM NAO provides both short analysis time within 2 min and a definite fluorescence enhancement at 525 nm for NAO-cardiolipin complex as compared with NAO alone. Under the optimum condition, a calibration curve between the peak area and the concentration of cardiolipin was established in the range of 0.1-200 μM with a correlation coefficient of 0.9955. The detection limit is 9 nM. The proposed method was successfully applied to the analysis of cardiolipin in mitochondria from HepG2 cells. A new biochemical method estimating the mitochondrial number per cell was developed and used together with the proposed method for cardiolipin per cell measurement and cardiolipin per mitochondrion reported before.

Antitumor imidazoacridinone C-1311 is a DNA-reactive topoisomerase II and FLT3 receptor tyrosine kinase inhibitor. Here, we demonstrate the mechanism of C-1311 inhibitory action on novel targets: hypoxia-inducible factor-1α (HIF-1α), vascular-endothelial growth factor (VEGF), and angiogenesis. In a cell-free system, C-1311 prevented HIF-1α binding to an oligonucleotide encompassing a canonical hypoxia-responsive element (HRE), but did not directly interfere with HIF-1α protein. Whereas C-1311 did not affect HIF-1α transcription, at higher concentrations (10 µM), it decreased HIF-1α protein accumulation. Furthermore, C-1311 potently reduced the transcription of HIF-1-dependent reporter gene as well as endogenous HIF-1 target gene encoding vascular endothelial growth factor. In colon cancer HCT116 and murine melanoma B16/F10 cells, C-1311-induced down-regulation of VEGF, resulted in decreased VEGF protein expression. C-1311 did not alter normoxic VEGF secretion. Consistent with VEGF depletion, C-1311 significantly affected angiogenesis in vivo. A single dose of C-1311 (40 mg/kg), inhibited angiogenesis by 70%, as measured by vascularization of Matrigel plugs in mice. Continuous low C-1311 dosing (8 mg/kg/day for 5 days) gave similar inhibition of angiogenesis (80%), suggesting that low-dose, frequent C-1311 administration may be more effective in terms of reducing toxicity. Anti-angiogenic activity of C-1311 was further demonstrated in an experimental model in which B16/F10 cells were encapsulated in alginate beads and implanted into mice. C-1311 (8 mg/kg/day for 9 days), completely abrogated vascularization of the alginate implants. Our findings indicate that C-1311 is an effective inhibitor of HIF-1α, VEGF, and angiogenesis and provide new perspectives into the mechanism of its anticancer activity.

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P(FMO), which is identified as an N-oxide derivative, was identical to P3(R) of liver microsomes. P3(R) was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3(R) = P(FMO). Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.

A series of novel N(10)-substituted acridone derivatives bearing alkyl side-chain with tertiary amine groups at the terminal position were evaluated for their in vitro cytotoxic effects against drug sensitive and resistant cancer cell lines. All the molecules were designed on the basis of hydrogen bond acceptors, carbonyl, fluoro groups with precise spatial separation and structural features of lipophilicity, positive charge at neutral pH and presence of aromatic rings. The in vitro cytotoxic effects in comparison with reference drugs doxorubicin (DX) and C(1311) against cancer cell lines SW 1573, SW 1573 2R 160 (Pg-P expressing) which are non-small cell lung cancer cells, human embryo kidney cells HEK 293, HEK 293 MRP4, HEK 293 MRP5i, human promyelocytic leukemia sensitive cell line HL-60, including its multidrug cross-resistant of two main (P-gp and MRP) phenotype sublines vincristine resistant HL-60/VINC and doxorubicin resistant HL-60/DX cancer cell lines are presented. Compounds 14, 15 and 16 exhibited highest cytotoxicity among the derivatives. On the other hand, the in vitro cytotoxic activity of compound 14 (with butyl side-chain and tertiary amino group β-hydroxy ethyl piperizine) against resistant cancer cell lines indicate that it might be a promising new hit for further development as an anti-MDR agent. The non-covalent interaction of these molecules with DNA duplexes have been investigated by ESI-MS technique. The results indicate, these acridone derivatives interact with duplex DNA by intercalation, possesses higher affinity to GC than AT base pairs of the DNA and they could not interact non-covalently with the minor grooves of the DNA. The ability of acridones to inhibit calmodulin dependent cAMP phosphodiesterase has been determined. The results suggest that acridones inhibit the Ca(2+)/calmodulin stimulated cAMP-phosphodiesterase activity and have no direct effects on the enzyme itself and a strong correlation between calmodulin inhibition and cytotoxicity against HL-60/VINC and HL-60/DX MDR cancer cell lines.

Malondialdehyde (MDA) has become a well-established biomarker for oxidative stress. The most commonly used way to determine urinary MDA levels is the thiobarbituric acid (TBA) assay, which suffers from several drawbacks. In this manuscript, we describe a novel derivatization strategy for the highly sensitive and selective fluorescence-based determination of MDA in urinary samples. The methodology is based on the mild labeling of MDA with 2-aminoacridone, which can be carried out in aqueous citrate buffer at 40°C, yielding a highly fluorescent substance. No further sample preparation than mixing with the necessary chemicals is necessary. The formed MDA derivative can conveniently be separated from the label itself and matrix constituents by gradient LC in less than 5 minutes on a cyano-based reversed-phase material. The method was validated with respect to matrix effects, linearity, selectivity and sensitivity (values as low as 1.8 nM for the LOD and 5.8 nM for the LOQ could be achieved). Standard addition quantitation was applied for the determination of MDA in human urine samples. Additionally, the protocol was applied to the measurement of a stability indicating analysis of MDA in urine at different storage conditions. © 2011 The Royal Society of Chemistry.

Conventional spectroscopic tools such as absorption, fluorescence, and circular dichroism spectroscopy used in the study of photoinduced drug-protein interactions can yield useful information about ground-state and excited-state phenomena. However, photoinduced electron transfer (PET) may be a possible phenomenon in the drug-protein interaction, which may go unnoticed if only conventional spectroscopic observations are taken into account. Laser flash photolysis coupled with an external magnetic field can be utilized to confirm the occurrence of PET and authenticate the spin states of the radicals/radical ions formed. In the study of interaction of the model protein human serum albumin (HSA) with acridine derivatives, acridine yellow (AY) and proflavin (PF(+)), conventional spectroscopic tools along with docking study have been used to decipher the binding mechanism, and laser flash photolysis technique with an associated magnetic field (MF) has been used to explore PET. The results of fluorescence study indicate that fluorescence resonance energy transfer takes place from the protein to the acridine-based drugs. Docking study unveils the crucial role of Ser 232 residue of HSA in explaining the differential behavior of the two drugs towards the model protein. Laser flash photolysis experiments help to identify the radicals/radical ions formed in the due course of PET (PF(•), AY(•-), TrpH(•+), Trp(•)), and the application of an external MF has been used to characterize their initial spin-state. Owing to its distance dependence, MF effect gives an idea about the proximity of the radicals/radical ions during interaction in the system and also helps to elucidate the reaction mechanisms. A prominent MF effect is observed in homogeneous buffer medium owing to the pseudoconfinement of the radicals/radical ions provided by the complex structure of the protein.

Purpose C-1311 is a member of the novel imidazoacridinone family of anticancer agents. This phase 1 trial was designed to investigate the safety, tolerability and preliminary anti-tumour activity of C-1311. Patients and methods This was a phase 1, inter-subject dose escalating and pharmacokinetic study of intravenous (IV) C-1311, administered weekly during 3 consecutive weeks followed by 1 week rest (constituting 1 cycle) in subjects with advanced solid tumours. Results Twenty-two (22) patients were treated with C-1311, the highest dose given was 640 mg/m 2 . All subjects experienced one or more treatment-related adverse events (AEs). The most frequently observed treatment-related AEs were neutropaenia and nausea (50% each), followed by vomiting (27%), anaemia (23%), asthenia (23%) and diarrhoea (18%). Most treatment-related AEs were of Common Terminology Criteria for Adverse Events (CTCAE) grades 1–2, except for the blood and lymphatic system disorders, which were primarily of grades 3–4. The recommended dose (RD) of C-1311 administered as once weekly IV infusions for 3 weeks every 4 weeks is 480 mg/m 2 , with the dose limiting toxicity (DLT) being grade 4 neutropaenia lasting more than 7 days. Treatment at this dose offers a predictable safety profile and excellent tolerability. Conclusion The safety profile and preliminary anti-tumour efficacy of C-1311, observed in this broad-phase dose-finding study, warrants further evaluation of the compound.

In this study, we developed an on-line reverse-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO(4) at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-(Fp)-ESI-MS.

A small set of 9-aminoacridine-3- and 4-carboxamides were synthesized efficiently using the benzyne/azide click chemistry. The products bind to duplex DNA but have different antitumour activity in the HL60 cell line.

SUMMARY Fifty two patients (aged 15–57 years) with primarily failed and relapsed acute myelogenous (AML) or lymphoblastic leukemia (ALL) and with high-grade non-Hodgkin's lymphoma (NHL) have been treated with intermediate dose cytosine arabinoside. Ara-C (1 g/m2 q 12 h × 12) with 3 d of m-AMSA, 115 mg/m2 daily was given to 18 patients, with daunorubicin (3 patients) 45 mg/m2 daily for 3 days to 3 patients and in 14 patients 1 day of m-AMSA (115 mg/m2) was administered. In 17 patients ara-C was combined with VP16.213 120 mg/m2 i v. on day 1 and 7, m-AMSA 115 mg/m2 i.v. on day 1 and 7 and prednisone 60 mg/m2 orally on days 1–7, these patients received methotrexate 10 mg/m2 intrathecally on day 1. Thirty three patients obtained a complete remission (AML 18/24, ALL 7/9, NHL 8/18). 17 patients received consolidation treatment with 1–3 courses comprising 4 d of ara-C (3 g/m2 q 12 h × 8). in 9 patients combined with m-AMSA 115 mg/m2 i.v. on day 5 and in 8 patients with VP16.213 120 mg/m2 on day 1 and 5, m-AMSA 115 mg/m2 on day 1 and 5 and prednisone 60 mg/m2 orally days 1–5 and methotrexate 10 mg/m2 intrathecally on day 1. Three patients underwent allogeneic bone marrow transplantation, all other patients received no further treatment. Median remission duration was 3 months for those patients who received no treatment after remission induction. Five patients who received consolidation treatment relapsed after 4 months (2 ALL), 6 months (NHL), 9 months (AML) and 16 months (AML). Thirteen patients still are in remission at 2+ - 21+ months. In addition to vomiting, fever, skin rashes, conjunctivitis and myalgia toxicity included diarrhoea in 35.8 per cent of patients and 10 patients developed diffuse interstitial pneumonia without clear cause and probably due to ara-C administration. Fifty-eight septicemic episodes were observed, in 65% caused by Streptococcus viridans. The pancytopenic period ranged from 16–25 days (median 21 days) after the remission induction and 14–21 days (median 19 days) after the consolidation course. Intermediate dose ara-C is active in primary refractory and relapsed acute leukemia and lymphoblastic non-Hodgkin's lymphoma. Side effects are acceptable although interstitial lung-involvement remains to be further investigated.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Current chemotherapy regimes include the topoisomerase II poison etoposide and the transcription inhibitor actinomycin D. Poor clinical response necessitate identification of new agents to improve patient outcomes.We assessed the in vitro cytotoxicity (MTT assay) of DNA intercalating agents in five established human RMS cell lines. These include novel classes of transcription inhibitors and topoisomerase poisons, previously shown to have potential as anti-cancer agents.Amongst the former agents, bisintercalating bis(9-aminoacridine-4-carboxamides) linked through the 9-position, and bis(phenazine-1-carboxamides) linked via their side chains, are compared with established transcription inhibitors. Amongst the latter, monofunctional acridine-4-carboxamides related to N-[2-(dimethylamino)ethyl]acridine-4-carboxamide, DACA, are compared with established topoisomerase poisons.Our findings specifically highlight the topoisomerase poison 9-amino-DACA, its 5-methylsulphone derivative, AS-DACA, and the bis(phenazine-1-carboxamide) transcription inhibitor MLN944/XR5944, currently in phase I trial, as candidates for further research into new agents for the treatment of RMS.

Symadex is the lead acridine compound of a novel class of imidazoacridinones (IAs) currently undergoing phase II clinical trials for the treatment of various cancers. Recently, we have shown that Symadex is extruded by ABCG2-overexpressing lung cancer A549/K1.5 cells, thereby resulting in a marked resistance to certain IAs. To identify the IA residues essential for substrate recognition by ABCG2, we here explored the ability of ABCG2 to extrude and confer resistance to a series of 23 IAs differing at defined residue(s) surrounding their common 10-azaanthracene structure. Taking advantage of the inherent fluorescent properties of IAs, ABCG2-dependent efflux and drug resistance were determined in A549/K1.5 cells using flow cytometry in the presence or absence of fumitremorgin C, a specific ABCG2 transport inhibitor. We find that a hydroxyl group at one of the R1, R2, or R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent IA resistance. Moreover, elongation of the common distal aliphatic side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Hence, the current study offers novel molecular insight into the structural determinants that facilitate ABCG2-mediated drug efflux and consequent drug resistance using a unique platform of fluorescent IAs. Moreover, these results establish that the IA determinants mediating cytotoxicity are precisely those that facilitate ABCG2-dependent drug efflux and IA resistance. The possible clinical implications for the future design of novel acridines that overcome ABCG2-dependent multidrug resistance are discussed.

Synthesis and properties of a new fluorescent/fluorogenic substrate Ac-DEVD-AMAC for caspase-3 are reported. The substrate is obtained by conventional Fmoc-based solid phase peptide synthesis and its properties are investigated with regard to fluorescence, sensitivity, applicability and kinetic constants. A non-traditional approach to assay the proteases activity using 2-aminoacridone labeled peptides is proposed. This approach utilizes the decrease of fluorescence intensity of a sample as a measure for the enzyme activity.