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1.
Bioorg Chem ; 122: 105752, 2022 05.
Article in English | MEDLINE | ID: mdl-35339926

ABSTRACT

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.


Subject(s)
Antineoplastic Agents , Leukemia, Promyelocytic, Acute , Amsacrine/chemistry , Amsacrine/pharmacology , Antineoplastic Agents/chemistry , Apoptosis , Cell Proliferation , DNA Topoisomerases, Type II/metabolism , Humans , Molecular Docking Simulation , Topoisomerase II Inhibitors , fms-Like Tyrosine Kinase 3
2.
Recent Pat Anticancer Drug Discov ; 17(1): 102-119, 2022.
Article in English | MEDLINE | ID: mdl-34323200

ABSTRACT

BACKGROUND: DNA topoisomerases are a class of enzymes that play a critical role in fundamental biological processes of replication, transcription, recombination, repair and chromatin remodeling. Amsacrine (m-AMSA), the best-known compound of 9-anilinoacridines series, was one of the first DNA-intercalating agents to be considered a Topoisomerase II inhibitor. OBJECTIVES: A series of sulfur-containing 9-anilinoacridines related to amsacrine were synthesized and evaluated for their anticancer activity. METHODS: Cell viability was assessed by the MTT assay. The topoisomerase II inhibitory assay was performed using the Human topoisomerase II Assay kit, and flow cytometry was used to evaluate the effects on the cell cycle of K562 cells. Molecular docking was performed using the Schrödinger Maestro program. RESULTS: Compound 36 was found to be the most cytotoxic of the sulfide series against SW620, K562, and MCF-7. The limited SAR suggested the importance of the methansulfonamidoacetamide side chain functionality, the lipophilicity, and the relative metabolic stability of 36 in contributing to the cytotoxicity. Topoisomerase II α inhibitory activity appeared to be involved in the cytotoxicity of 36 through the inhibition of decatenation of kinetoplast DNA (kDNA) in a concentration- dependent manner. Cell cycle analysis further showed Topo II inhibition through the accumulation of K562 cells in the G2/M phase of the cell cycle. The docking of 36 into the Topo II α-DNA complex suggested that it may be an allosteric inhibitor of Topo II α. CONCLUSION: Compound 36 exhibits anticancer activity by inhibiting topoisomerase II, and it could further be evaluated in in vivo models.


Subject(s)
Amsacrine , Antineoplastic Agents , Amsacrine/analogs & derivatives , Amsacrine/chemistry , Amsacrine/pharmacology , Antineoplastic Agents/chemistry , DNA Topoisomerases, Type II/metabolism , Humans , Molecular Docking Simulation , Sulfur , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology
3.
Molecules ; 26(3)2021 Jan 21.
Article in English | MEDLINE | ID: mdl-33494466

ABSTRACT

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.


Subject(s)
Adenocarcinoma of Lung/drug therapy , Antineoplastic Agents , DNA, Neoplasm/metabolism , Lung Neoplasms/drug therapy , Topoisomerase II Inhibitors , Adenocarcinoma of Lung/history , Adenocarcinoma of Lung/metabolism , Amsacrine/chemistry , Amsacrine/history , Amsacrine/pharmacokinetics , Amsacrine/therapeutic use , Anaphase/drug effects , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/history , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Chromatids/metabolism , Chromosome Segregation/drug effects , DNA Topoisomerases, Type II/metabolism , History, 20th Century , History, 21st Century , Humans , Lung Neoplasms/history , Lung Neoplasms/metabolism , Mice , Naphthyridines/chemistry , Naphthyridines/pharmacokinetics , Naphthyridines/therapeutic use , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacokinetics , Topoisomerase II Inhibitors/therapeutic use
4.
Chem Biol Drug Des ; 97(2): 237-252, 2021 02.
Article in English | MEDLINE | ID: mdl-32772433

ABSTRACT

The efficient synthesis of molecular hybrids including a DNA-intercalating 9-anilinoacridine (9-AnA) core and a methyl triazene DNA-methylating moiety is described. Nucleophilic aromatic substitution (SN Ar) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low micromolar IC50 values in antiproliferative assays performed with various cancer cell lines. In comparison, compound 7b outperformed DNA-intercalating drugs like amsacrine and AHMA. Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA-repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by intercalation of the acridine core.


Subject(s)
Amsacrine/analogs & derivatives , Antineoplastic Agents/chemical synthesis , Triazenes/chemistry , Amsacrine/chemistry , Amsacrine/metabolism , Amsacrine/pharmacology , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , DNA/chemistry , DNA/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/metabolism , Drug Screening Assays, Antitumor , Humans , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Intercalating Agents/pharmacology , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/metabolism , Triazenes/metabolism , Triazenes/pharmacology
5.
Int J Biol Macromol ; 165(Pt A): 1410-1421, 2020 Dec 15.
Article in English | MEDLINE | ID: mdl-33045299

ABSTRACT

Electrospinning technology was applied for the preparation of polyacrylonitrile (PAN) nanofiber membrane in this work. After hot pressing, alkaline hydrolysis and neutralization treatment, a weak acid cation exchange membrane (P-COOH) was prepared. By the covalent coupling reaction between the acidic membrane and aminomethane sulfonic acid (AMSA), a strong acidic nanofiber membrane (P-SO3H) was obtained. The surface morphology, chemical structure, and thermal stability of the prepared ion exchange membranes were analyzed via SEM, FTIR and TGA. Analytical results showed that the membranes were prepared successfully and thermally stable. The ion exchange membrane (IEX) was conducted with the newly designed membrane reactor, and different operating conditions affecting the adsorption efficiency of Toluidine Blue dye (TBO) were investigated by dynamic flow process. The results showed that dynamic binding capacity (DBC) of weak and strong IEX membranes for TBO dye was ~170 mg/g in a dynamic flow process. Simultaneously, the ion exchange membranes were also used for purifying lysozyme from chicken egg white (CEW). Results illustrated that the recovery yield and purification factor of lysozyme were 93.43% and 29.23 times (P-COOH); 90.72% and 36.22 times (P-SO3H), respectively. It was revealed that two type ion exchange membranes were very suitable as an adsorber for use in dye waste treatment and lysozyme purification process. P-SO3H strong ion-exchange membrane was more effective either removal of TBO dye or purification of lysozyme. The ion exchange membranes not only effectively purified lysozyme from CEW solution, but also effectively removed dye from wastewater.


Subject(s)
Amsacrine/chemistry , Coloring Agents/chemistry , Muramidase/chemistry , Nanofibers/chemistry , Acrylic Resins/chemistry , Adsorption/drug effects , Cations/chemistry , Coloring Agents/isolation & purification , Ion Exchange , Membranes, Artificial , Muramidase/isolation & purification
6.
Curr Drug Res Rev ; 11(2): 118-128, 2019.
Article in English | MEDLINE | ID: mdl-31513003

ABSTRACT

BACKGROUND: Human Epidermal development factor Receptor-2 (HER2) is a membrane tyrosine kinase which is overexpressed and gene amplified in human breast cancers. HER2 amplification and overexpression have been linked to important tumor cell proliferation and survival pathways for 20% of instances of breast cancer. 9-aminoacridines are significant DNA-intercalating agents because of their antiproliferative properties. OBJECTIVE: Some novel isoxazole substituted 9-anilinoacridines(1a-z) were designed by in-silico technique for their HER2 inhibitory activity. Docking investigations of compounds 1a-z are performed against HER2 (PDB id-3PP0) by using Schrodinger suit 2016-2. METHODS: Molecular docking study for the designed molecules 1a-z are performed by Glide module, in-silico ADMET screening by QikProp module and binding free energy by Prime-MMGBSA module of Schrodinger suit. The binding affinity of designed molecules 1a-z towards HER2 was chosen based on GLIDE score. RESULTS: Many compounds showed good hydrophobic communications and hydrogen bonding associations to hinder HER2. The compounds 1a-z, aside from 1z have significant Glide scores in the scope of - 4.91 to - 10.59 when compared with the standard Ethacridine (- 4.23) and Tamoxifen (- 3.78). The in-silico ADMET properties are inside the suggested about drug likeness. MM-GBSA binding of the most intense inhibitor is positive. CONCLUSION: The outcomes reveal that this study provides evidence for the consideration of isoxazole substituted 9-aminoacridine derivatives as potential HER2 inhibitors. The compounds, 1s,x,v,a,j,r with significant Glide scores may produce significant anti breast cancer activity and further in vitro and in vivo investigations may prove their therapeutic potential.


Subject(s)
Amsacrine/analogs & derivatives , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Isoxazoles/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Amsacrine/chemistry , Amsacrine/pharmacokinetics , Amsacrine/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Computer Simulation , Drug Design , Ethacridine/pharmacology , Female , Humans , Hydrogen Bonding , Isoxazoles/chemistry , Isoxazoles/pharmacokinetics , Models, Molecular , Molecular Dynamics Simulation , Structure-Activity Relationship , Tamoxifen/pharmacology
7.
Anticancer Agents Med Chem ; 19(11): 1350-1358, 2019.
Article in English | MEDLINE | ID: mdl-30961512

ABSTRACT

BACKGROUND: 9-anilinoacridines are acting as DNA-intercalating agents which plays an important role as antitumor drugs, due to their anti-proliferative properties. Some anticancer agents contain 9- anilinoacridines such as amsacrine (m-AMSA), and nitracrine (Ledakrine) have been already developed. METHODS: In this study, novel 9-anilinoacridines substituted with thiazines 4a-r were designed, synthesized, characterized by physical and spectral data and their cytotoxic activities against DLA cell lines were evaluated. RESULTS: Among those compounds, 4b, c, e, g, i, j, k, m, o, p, q, r exhibited significant short term in vitro cytotoxic activity against Daltons lymphoma ascites (DLA) cells with CTC50 value of 0.18 to 0.31µM. The compounds 4b, c, e, g, i, j, k, m, o, p, q, r are also exhibited significant long term in vitro anti-tumour activity against human tumor cell lines, HEp-2 (laryngeal epithelial carcinoma) by Sulforhodamine B assay with CTC50 value of 0.20 to 0.39µM. The compounds 4b, i, j exhibited significant in vivo antitumor activity with % Increase in Life Span (ILS) 48-82%. CONCLUSION: Results obtained in this study clearly demonstrated that many of the thiazine substituted 9- anilinoacridines exert interesting anti-tumour activity. The compounds 4b, i, j have significant anti-tumour activity and useful drugs after further refinement. The above derivatives will encourage to design future antitumor agents with high therapeutic potentials.


Subject(s)
Amsacrine/analogs & derivatives , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Thiazines/pharmacology , Amsacrine/chemical synthesis , Amsacrine/chemistry , Amsacrine/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Male , Mice , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Structure-Activity Relationship , Thiazines/chemistry , Tumor Cells, Cultured
8.
Pharm Res ; 35(1): 13, 2018 01 04.
Article in English | MEDLINE | ID: mdl-29302821

ABSTRACT

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.


Subject(s)
Amsacrine/analogs & derivatives , Antineoplastic Agents/pharmacokinetics , Micelles , Phosphatidylethanolamines/chemistry , Polyethylene Glycols/chemistry , Amsacrine/chemistry , Amsacrine/pharmacokinetics , Amsacrine/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Culture Techniques , Cell Survival , Delayed-Action Preparations , Drug Carriers/chemistry , Drug Liberation , Drug Stability , Half-Life , Humans , MCF-7 Cells , Male , Nanoparticles/chemistry , Particle Size , Permeability , Rats , Rats, Sprague-Dawley , Surface Properties , Vitamin E/chemistry
9.
J Mol Graph Model ; 72: 209-219, 2017 03.
Article in English | MEDLINE | ID: mdl-28110185

ABSTRACT

Amsacrine is an effective topoisomerase II enzyme inhibitor in acute lymphatic leukemia. Previous experimental studies have successfully identified two important mutations (R487K and E571K) conferring 100 and 25 fold resistance to Amsacrine respectively. Although the reduction of the cleavage ligand-DNA-protein ternary complex has been well thought as the major cause of drug resistance, the detailed energetic, structural and dynamic mechanisms remain to be elusive. In this study, we constructed human topoisomerase II alpha (hTop2α) homology model docked with Amsacrine based on crystal structure of human Top2ß in complex with etoposide. This wild type complex was used to build the ternary complex with R487K and E571K mutants. Three 500ns molecular dynamics simulations were performed on complex systems of wild type and two mutants. The detailed energetic, structural and dynamic analysis were performed on the simulation data. Our binding data indicated a significant impairment of Amsacrine binding energy in the two mutants compared with the wild type. The order of weakening (R487K>E571K) was in agreement with the order of experimental drug resistance fold (R489K>E571K). Our binding energy decomposition further indicated that weakening of the ligand-protein interaction rather than the ligand-DNA interaction was the major contributor of the binding energy difference between R487K and E571K. In addition, key residues contributing to the binding energy (ΔG) or the decrease of the binding energy (ΔΔG) were identified through the energy decomposition analysis. The change in ligand binding pose, dynamics of protein, DNA and ligand upon the mutations were thoroughly analyzed and discussed. Deciphering the molecular basis of drug resistance is crucial to overcome drug resistance using rational drug design.


Subject(s)
Amsacrine/chemistry , DNA Topoisomerases, Type II/genetics , Drug Resistance, Neoplasm , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation/genetics , Solvents/chemistry , Structural Homology, Protein , Amsacrine/pharmacology , DNA/chemistry , Humans , Mutant Proteins/chemistry , Protein Conformation , Thermodynamics
10.
Bioorg Med Chem Lett ; 27(3): 586-589, 2017 02 01.
Article in English | MEDLINE | ID: mdl-27998679

ABSTRACT

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.


Subject(s)
Acridines/chemistry , Acridines/pharmacology , Antigens, Neoplasm/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Amsacrine/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA/metabolism , DNA Cleavage/drug effects , Enzyme Activation/drug effects , Humans , Intercalating Agents/chemistry , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology
11.
Curr Cancer Drug Targets ; 17(7): 657-668, 2017.
Article in English | MEDLINE | ID: mdl-27834128

ABSTRACT

BACKGROUND: DNA topoisomerase II-α (Top2-α), an essential enzyme for the management of DNA during replication, transcription, recombination, and chromatin remodeling, is one of the most important anticancer targets. Numerous molecules have been designed as Top2-α inhibitors. However, several studies have shown that polymorphisms and mutations in Top2 have conferred resistance to most of these anticancer drugs. The aim of this study was to computationally examine the mechanisms by which genomic variations in Top2-α could affect its resistance to Amsacrine and Mitoxantrone as important inhibitors of the enzyme. RESULTS: The results showed that variants K529E, R568H, R568G and T530M could affect Top2-α inhibition by Amsacrine causing possible drug-resistant. Moreover, R487K, and Y481C variants could change the response of the enzyme to Mitoxantrone. CONCLUSION: These results could facilitate the prediction and development of more effective drugs for Top2-α variants, making the cancer chemotherapy more effectiv.


Subject(s)
Amsacrine/chemistry , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Mitoxantrone/chemistry , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/genetics , Polymorphism, Single Nucleotide , Amsacrine/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Computer Simulation , Drug Resistance, Neoplasm/genetics , Humans , Mitoxantrone/pharmacology , Molecular Docking Simulation , Protein Conformation , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacology
12.
J Biomol Struct Dyn ; 35(6): 1260-1271, 2017 May.
Article in English | MEDLINE | ID: mdl-27064820

ABSTRACT

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21 ± .05) × 105 M-1, while that to myoglobin was three times higher (3.59 ± .15) × 105 M-1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridines.


Subject(s)
Amsacrine/chemistry , Calorimetry , Hemoglobins/chemistry , Myoglobin/chemistry , Spectrum Analysis , Amsacrine/metabolism , Calorimetry/methods , Hemoglobins/metabolism , Humans , Ligands , Molecular Structure , Myoglobin/metabolism , Protein Binding , Spectrum Analysis/methods , Thermodynamics
13.
Int J Pharm ; 505(1-2): 194-203, 2016 May 30.
Article in English | MEDLINE | ID: mdl-27021465

ABSTRACT

This paper describes a novel method to improve drug retention in liposomes for the poorly water-soluble (lipophilic) model drug asulacrine (ASL). ASL was loaded in the aqueous phase of liposomes and the effects of aging conditions and drug loading levels on drug retention were investigated using an in vitro bio-relevant drug release test established in this study. The status of intra-liposomal drug was investigated using differential scanning calorimetry (DSC) and cryo-transmission electron microscopy (cryo-TEM). Pharmacokinetics and venous tolerance of the formulations were simultaneously studied in rabbits following one-hour intravenous infusion via the ear vein. The presence of glucose during aging was found to be crucial to accelerate drug precipitation and to stabilize the liposomal membrane with high drug loading (8.9% over 4.5% w/w) as a prerequisite. Although no drug crystals were detected, DSC showed a lower phase-transition peak in the glucose-assisted aged ASL-liposomes, indicating interaction of phospholipids with the sugar. Cryo-TEM revealed more 'coffee bean' like drug precipitate in the ASL-liposomes aged in the glucose solution. In rabbits, these liposomes gave rise to a 1.9 times longer half-life than the fresh liposomes, with no venous irritation observed. Inducing and stabilizing drug precipitation in the liposome cores by aging in the presence of sugar provided an easy approach to improve drug retention in liposomes. The study also highlighted the importance of bio-relevance of in vitro release methods to predict in vivo drug release.


Subject(s)
Amsacrine/analogs & derivatives , Antineoplastic Agents/administration & dosage , Glucose/chemistry , Amsacrine/administration & dosage , Amsacrine/chemistry , Animals , Antineoplastic Agents/chemistry , Calorimetry, Differential Scanning , Chemical Precipitation , Chemistry, Pharmaceutical/methods , Drug Liberation , Half-Life , Infusions, Intravenous , Liposomes , Microscopy, Electron, Transmission , Phase Transition , Rabbits , Solubility
14.
Nat Chem Biol ; 12(1): 40-5, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26619249

ABSTRACT

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.


Subject(s)
Amsacrine/pharmacology , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Staphylococcus aureus/drug effects , Aminoglycosides/pharmacology , Amsacrine/chemistry , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Wall/metabolism , High-Throughput Screening Assays/methods , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Mutation , Small Molecule Libraries/pharmacology , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Staphylococcus aureus/pathogenicity , Teichoic Acids/metabolism
15.
Pharm Res ; 32(4): 1451-61, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25355460

ABSTRACT

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.


Subject(s)
Ammonium Sulfate/chemistry , Amsacrine/analogs & derivatives , Antineoplastic Agents/chemistry , Drug Carriers/chemistry , Technology, Pharmaceutical/methods , beta-Cyclodextrins/chemistry , Amsacrine/administration & dosage , Amsacrine/chemistry , Amsacrine/pharmacokinetics , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Infusions, Intravenous , Liposomes , Molecular Structure , Rabbits , Solubility
16.
Med Chem ; 11(4): 373-82, 2015.
Article in English | MEDLINE | ID: mdl-25524547

ABSTRACT

We tested the antiproliferative activity and mechanism of the action of several novel aminoacridine derivatives. Six different cancer cell lines were used to evaluate the potential cytotoxic effect of eleven aminoacridine-based molecules. A standard MTT assay was used for cell bioavailability analysis. Additionally, the potential cytotoxic effect of the tested compounds on non-cancer cells was investigated in rat skeletal muscle myotubes (L6) and in bovine aortic smooth muscle cells. In order to investigate whether the DNA binding activity of tested compounds correlated with their cytotoxic effect, circular dichroism (CD) measurement and DNA T4 ligase assay were performed. Finally, the potential mutagenic activity of the lead compound 5 was investigated. The cytotoxic effect of compound 5 in cancer cells was obtained in lower concentrations than the well-known: 9- aminoacridine based drug, amsacrine. The lead compound binds to DNA, but in a different mode than the parent molecules. Additionally, compound 5 was not cytotoxic in the effective range of concentrations in non-cancer cells. In identical concentrations, the parent compound (9-aminoacridine) and amsacrine were extremely toxic for both types of these normal cells. Finally, based on CD measurement and T4 ligase assay, it was confirmed that 5 binds to DNA but in different from the parent compounds manner. Important to mention, that compound 5 might have increased mutagenic activity which must be verified in vivo. Based on these in vitro results, we conclude that 5 is a more potent and more selective antiprolifirative compound than amsacrine. Compound 5 was also more effective in HepG2 and P-12 cells. Thus, 5 is suitable for future in vivo biological evaluation and its structure might be used as a basis for developing novel anticancer drugs.


Subject(s)
Aminoacridines/chemical synthesis , Antineoplastic Agents/chemical synthesis , Intercalating Agents/pharmacology , Aminoacridines/pharmacology , Amsacrine/chemistry , Amsacrine/toxicity , Animals , Antineoplastic Agents/pharmacology , Cattle , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA/antagonists & inhibitors , DNA/chemistry , DNA Ligase ATP , DNA Ligases/chemistry , Humans , Intercalating Agents/chemistry , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Organ Specificity , Rats , Structure-Activity Relationship
17.
Int J Pharm ; 473(1-2): 528-35, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25079434

ABSTRACT

To facilitate the development of a liposomal formulation for cancer therapy, the physicochemical properties of asulacrine (ASL), an anticancer drug candidate, were characterized. Nano-liposomes were prepared by thin-film hydration in conjugation with active drug loading using ammonium sulphate and post-insertion with Poloxamer 188. A stability-indicating HPLC assay with diode array detection was developed for the determination of ASL concentrations. The U-shaped pH-solubility profile in aqueous solutions, with a lowest solubility at pH 7.4 (0.843 µg/mL), indicated that ASL is an ampholyte, and dilution or neutralization of acidic drug solutions used in clinical trials with physiological fluids may cause drug precipitation. The basic pKa value measured by absorbance spectroscopy was 6.72. The logD value at pH 3.8 was 1.15 which increased to 3.24 as pH increased to 7.4. ASL was found to be the most stable in acidic conditions and degraded most rapidly in alkaline conditions. An extra-liposomal pH of 5.6 during drug loading was found to be optimal to achieve the highest drug loading (DL) of 4.76% and entrapment efficiency (EE) of 99.9%. At this pH, >90% of ASL was ionized conferring high drug solubility (1mg/mL) and acted as a reservoir of unionized ASL to be transported into liposomal cores. As a suspension the optimized liposomes showed great physicochemical stability for five months at 4°C. In summary, the obtained physicochemical parameters provided insightful information useful to maximise DL into the liposomes, and explain a tendency of drug precipitation of pH-solubilized formulations following intravenous infusion.


Subject(s)
Amsacrine/analogs & derivatives , Antineoplastic Agents/chemistry , Amsacrine/chemistry , Chemistry, Pharmaceutical , Chromatography, High Pressure Liquid , Drug Stability , Hydrogen-Ion Concentration , Liposomes , Solubility
18.
Biochem Biophys Res Commun ; 446(4): 916-20, 2014 Apr 18.
Article in English | MEDLINE | ID: mdl-24642256

ABSTRACT

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme-DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.


Subject(s)
Amsacrine/pharmacology , Antitubercular Agents/pharmacology , DNA Topoisomerases, Type I/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Topoisomerase I Inhibitors/pharmacology , Topoisomerase II Inhibitors/pharmacology , Amsacrine/chemistry , Antitubercular Agents/chemistry , DNA, Bacterial/metabolism , Humans , Molecular Docking Simulation , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/growth & development , Mycobacterium tuberculosis/growth & development , Topoisomerase I Inhibitors/chemistry , Topoisomerase II Inhibitors/chemistry , Tuberculosis/drug therapy , Tuberculosis/microbiology
19.
J Cell Physiol ; 229(5): 588-98, 2014 May.
Article in English | MEDLINE | ID: mdl-24122234

ABSTRACT

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.


Subject(s)
Amsacrine/pharmacology , Enzyme Inhibitors/pharmacology , Leukemia/enzymology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Amsacrine/chemistry , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 9/genetics , Molecular Structure , Protein Phosphatase 2/classification , Protein Phosphatase 2/genetics , Protein Phosphatase 2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , U937 Cells , Up-Regulation , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
20.
Med Chem ; 10(5): 506-11, 2014.
Article in English | MEDLINE | ID: mdl-24070167

ABSTRACT

We have synthesized a series of polyamine-based anilinoacridine derivatives. The preliminary biological evaluation indicated that the 9-anilinoacridine-polyamine derivatives had low or insignificant in vitro cytotoxicity against K562 cell line and K562/ADM, the drug-resistant cell line. However, the evaluation for P-gp modulation showed that they held potent P-gp inhibitory ability. Among them, the effect of compound 7c on P-gp was even greater than that of Verapamil, the known P-gp modulator. The results suggest that 9-anilinoacridine-polyamine derivatives can be employed as effective P-gp modulators.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , Amsacrine/analogs & derivatives , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Polyamines/chemistry , Polyamines/pharmacology , Amsacrine/chemistry , Antineoplastic Agents/chemical synthesis , Humans , K562 Cells , Polyamines/chemical synthesis
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