Separation methods for anthraquinone related anti-cancer drugs

The quinoid anthracycline-related anti-cancer agents represent an important group of anti-tumour drugs with a wide spectrum of activity. We review here some of the separation techniques used for the analysis of anthracyclines and related compounds. In this review we have covered a range of compounds from the early anthracycline antibiotics such as doxorubicin to the more recent anthracenediones and anthrapyrazoles such as mitoxantrone and losoxantrone, respectively. We also include novel compounds such as AQ4N and C1311, both awaiting clinical trial. Separations of the anthraquinone related anti-cancer agents are predominantly by HPLC. These separation techniques have been used for a variety of applications including drug stability, protein binding and therapeutic drug monitoring as well as detailed pharmacokinetic and metabolic studies. Pharmacokinetics, and therefore drug analysis, plays a central role in both the development of new agents and also leads to a better understanding of clinically established agents in this class. Sample preparation and extraction methods including solid-phase and liquid–liquid extraction have also been highlighted. Many anthraquinone related compounds are highly coloured and fluoresce. They are suitable for a range of detection methods including UV–Vis, electrochemical and fluorescence. The methods described are used for sometimes complex separations that are needed for the evaluation of such compounds in biological samples.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid–liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative–reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS–MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.     

Mitoxantrone and ametantrone inhibit hydroperoxide-dependent initiation and propagation reactions in fatty acid peroxidation

The anthracenedione antineoplastic agents mitoxantrone and ametantrone are potent inhibitors of basal and drug-stimulated lipid peroxidation in a variety of subcellular systems (Kharasch, E. D., and Novak, R. F. (1983) J. Pharmacol. Exp. Ther. 226, 500-506). The mechanism by which these compounds function as antioxidants has been investigated using enzymic and chemical systems. Mitoxantrone and ametantrone inhibited NADPH-cytochrome P-450 reductase- and xanthine oxidase-catalyzed conjugated diene formation from linoleic acid in a concentration-dependent manner with half-maximal inhibition achieved at approximately 0.5 microM anthracenedione. Inhibition of linoleic acid peroxidation was not attributable to a decrease in P-450 reductase activity, hydroxyl radical scavenging, or iron chelation by the anthracenediones. Nonenzymic fatty acid peroxidation was also inhibited by the anthracenediones. Linoleic acid oxidation initiated by superoxide (ferrous iron autoxidation) or by hydroxyl radicals (Fenton's reagent) was diminished by mitoxantrone and ametantrone after a brief delay, suggesting an effect subsequent to activated oxygen-dependent initiation. In contrast, linoleic acid oxidation initiated by iron-dependent hydroperoxide decomposition was inhibited immediately. Reinitiation of linoleic acid oxidation in an anthracenedione-inhibited system was accomplished only by superoxide generation, but not by fatty acid hydroperoxide decomposition. These results suggest the anthracenediones diminished neither oxygen radical formation nor oxygen radical-dependent initiation of peroxidation. Rather, inhibition of fatty acid peroxidation by mitoxantrone and ametantrone results from the inhibition of hydroperoxide-dependent initiation and propagation reactions.     

Bis(alkylamino)anthracenedione antineoplastic agent metabolic activation by NADPH-cytochrome P-450 reductase and NADH dehydrogenase: diminished activity relative to anthracyclines

Stimulation of the rates of NAD(P)H oxidation, superoxide generation, and hydrogen peroxide formation by three anthracenedione antineoplastic agents in the presence of NADPH-cytochrome P-450 reductase, NADH dehydrogenase, or rabbit hepatic microsomes was studied and the results compared with those obtained for the anthracyclines Adriamycin and daunorubicin. In all cases the anthracenediones, including mitoxantrone and ametantrone, were significantly (5- to 20-fold) less effective than the anthracyclines in stimulating NAD(P)H oxidation, superoxide formation, or hydrogen peroxide production. Of the three anthracenediones studied, the ring-monohydroxylated compound showed the greatest activity followed by the ring-dihydroxylated derivative (mitoxantrone). In contrast, the non-ring-hydroxylated anthracenedione (ametantrone) was a relatively ineffective electron acceptor and inhibited the reduction of more effective acceptors such as Adriamycin. Michaelis-Menten kinetic constants were determined by analysis of the rates of NADPH oxidation. NADP+ and 2'-AMP inhibited the reduction of the ring-hydroxylated anthracenediones and anthracyclines, demonstrating the enzymatic nature of the reaction. The non-ring-hydroxylated anthracenedione inhibited the reduction of Adriamycin by both P-450 reductase and NADH dehydrogenase with 50% inhibition achieved at approximately 300 microM. Thus, there appears to exist a structural relationship between anthracenedione ring hydroxylation and metabolic activation. These results also suggest that the relative inability of the anthracenediones to function as artificial electron acceptors in comparison to the anthracyclines may be correlated with diminished anthracenedione cardiotoxicity.     

Formaldehyde-activated WEHI-150 induces DNA interstrand crosslinks with unique structural features

Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent nature, these DNA lesions are relatively labile. It was recently established that analogues of mitoxantrone featuring extended side-chains terminating in primary amino groups typically yielded high levels of stable DNA adducts following their activation by formaldehyde. In this study we describe the DNA sequence-specific binding properties of the mitoxantrone analogue WEHI-150 which is the first anthracenedione to form apparent DNA crosslinks mediated by formaldehyde. The utility of this compound lies in the versatility of the covalent binding modes displayed. Unlike other anthracenediones described to date, WEHI-150 can mediate covalent adducts that are independent of interactions with the N-2 of guanine and is capable of adduct formation at novel DNA sequences. Moreover, these covalent adducts incorporate more than one formaldehyde-mediated bond with DNA, thus facilitating the formation of highly lethal DNA crosslinks. The versatility of binding observed is anticipated to allow the next generation of anthracenediones to interact with a broader spectrum of nucleic acid species than previously demonstrated by the parent compounds, thus allowing for more diverse biological activities.     

Design,synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA–topoisomerase IIα covalent complex indicating that they acted as topoisomerase IIα poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.     

Design, synthesis, and biological evaluation of a novel series of bisintercalating DNA-binding piperazine-linked bisanthrapyrazole compounds as anticancer agents

A series of bisintercalating DNA binding bisanthrapyrazole compounds containing piperazine linkers were designed by molecular modeling and docking techniques. Because the anthrapyrazoles are not quinones they are unable to be reductively activated like doxorubicin and other anthracyclines and thus they should not be cardiotoxic. The concentration dependent increase in DNA melting temperature was used to determine the strength of DNA binding and the bisintercalation potential of the compounds. Compounds with more than a three-carbon linker that could span four DNA base pairs achieved bisintercalation. All of the bisanthrapyrazoles inhibited human erythroleukemic K562 cell growth in the low to submicromolar concentration range. They also strongly inhibited the decatenation activity of topoisomerase IIα and the relaxation activity of topoisomerase I. However, as measured by their ability to induce double strand breaks in plasmid DNA, the bisanthrapyrazole compounds did not act as topoisomerase IIα poisons. In conclusion, a novel group of bisanthrapyrazole compounds were designed, synthesized, and biologically evaluated as potential anticancer agents.     

Effects of anthrapyrazole antineoplastic agents on lipid peroxidation

The effects of three anthrapyrazoles and an aminoacridine derivative on doxorubicin- and iron-stimulated lipid peroxidation in rabbit hepatic microsomes have been characterized. Two anthrapyrazoles, CI-937 and CI-942, were potent inhibitors of lipid peroxidation with 15 microM drug inhibiting the rate of peroxidation 70 to 90%. In contrast CI-941 was relatively ineffective in inhibiting lipid peroxidation with only 35% inhibition occurring at 100 microM drug. CI-921, an aminoacridine derivative, diminished lipid peroxidation by 65% at 15 microM. All four drugs failed to decrease the rate of doxorubicin-stimulated NADPH oxidation at concentrations less than 50 microM, suggesting that inhibition of lipid peroxidation was not the result of diminished enzyme activity. CI-937 formed a 2:1 complex with ferric ion, KD = 47 microM, which was reversible with EDTA.     

Spectroscopic evidence for anthracenedione antineoplastic agent self-association and complex formation with flavin nucleotides

Dihydroxyanthraquinone (DHAQ) and ametantrone (anthraquinone) are two new anthracenedione antineoplastic agents which were found by proton NMR spectroscopy to self-associate in aqueous media. Self-association was consistent with a bimolecular model, with average association constant values of 3400 and 2900 m^?1 determined for DHAQ and ametantrone, respectively. Both anthracenediones interacted with the flavin nucleotides FMN and FAD to produce concentration-dependent upfield shifts of the flavin isoalloxazine ring proton signals, as observed by proton NMR spectroscopy. Average association constant values obtained for FMN-DHAQ, FAD-DHAQ, FMN-metantrone, and FAD-ametantrone complexation were 5100, 2600, 4300, and 1600 m^?1, respectively. Optical difference spectroscopy confirmed FMN-DHAQ complexation, which resulted in a hyperchromic, bathochromic shift of the DHAQ spectrum following addition of FMN. These results were consistent with the formation of a ππ bimolecular ring-stacking complex. Information obtained on anthracenedione self-association and complexation with flavins may be of consequence in the interpretation of anthracenedione-DNA binding data and flavoprotein-mediated anthracenedione metabolic activation.     

The structure-based design, synthesis and biological evaluation of DNA-binding bisintercalating bisanthrapyrazole anticancer compounds

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of ester-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should not be cardiotoxic like the anthracyclines. Based on the docking scores of a series of bisanthrapyrazoles with different numbers of methylene linkers (n) that were docked into an X-ray structure of double-stranded DNA, five bisanthrapyrazoles (n=1-5) were selected for synthesis and physical and biological evaluation. The synthesized compounds were evaluated for DNA binding and bisintercalation by measuring the DNA melting temperature increase, for growth inhibitory effects on the human erythroleukemic K562 cell line, and for DNA topoisomerase IIalpha-mediated cleavage of DNA and inhibition of DNA topoisomerase IIalpha decatenation activities. The results suggest that the bisanthrapyrazoles with n=2-5 formed bisintercalation complexes with DNA. In conclusion, a novel group of bisintercalating anthrapyrazole compounds have been designed, synthesized and biologically evaluated as possible anticancer agents.     

Engineered macromolecular Toll-like receptor agents and assemblies

Macromolecular Toll-like receptor (TLR) agents have been utilized as agonists and inhibitors in preclinical and clinical settings. These agents interface with the TLR class of innate immune receptors which recognize macromolecular ligands that are characteristic of pathogenic material. As such, many agents that have been historically investigated are derived from the natural macromolecules which activate or inhibit TLRs. This review covers recent research and clinically available TLR agents that are macromolecular or polymeric. Synthetic materials that have been found to interface with TLRs are also discussed. Assemblies of these materials are investigated in the context of improving stability or efficacy of ligands. Attention is given to strategies which modify or enhance the current agents and to future outlooks on the development of these agents.     

Amide bond direction modulates G-quadruplex recognition and telomerase inhibition by 2,6 and 2,7 bis-substituted anthracenedione derivatives

G-quadruplex structures of DNA represent a potentially useful target for anticancer drugs. Stabilisation of this arrangement at the ends of chromosomes may inhibit the action of telomerase, an enzyme involved in immortalization of cancer cells. Appropriately substituted amido anthracenediones are effective G-quadruplex stabilizers, but no information is available as yet on the possible modulation of G-quadruplex recognition and telomerase inhibition produced by the direction of the amide bond. To understand the basis of amido anthracenedione selectivity, we have synthesized a number of derivatives bearing the -CO-NH- or -NH-CO- group linked to the planar anthraquinone (AQ) moiety at 2,6 and 2,7 positions. The various isomers were tested in terms of telomerase inhibition, determined by the TRAP assay, G-quadruplex stabilisation measured by the increase in melting temperature of the appropriately folded oligonucleotide using FRET, and conformational and G4 binding properties examined by molecular modelling techniques. In all cases, enzymatic inhibition and G-quadruplex stabilization were directly related, which strongly supports the proposed molecular mechanism of telomerase interference. Interestingly, the AQ-NH-CO- arrangement performs invariantly better than the AQ-CO-NH- arrangement, showing a clear preference among isomeric derivatives. Theoretical calculations suggest that the former amide arrangement is co-planar with the aromatic system, whereas the latter is tilted by about 30 degrees when considering the most stable conformation. A more extended planar surface would allow more efficient stacking interactions with the quadruplex structure, hence more effective telomerase inhibition.     

Mechanistically comparing reproductive manipulations caused by selfish chromosomes and bacterial symbionts

Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.Subject terms: Development, Genomic instability     

Microtubules: a dynamic target in cancer therapy

The tubulin/microtubule system is an important target for anticancer therapy. Two of the most clinically valuable groups of these agents are the vinca alkaloids and taxanes. In recent years, new tubulin-binding agents have been under preclinical or clinical development. One of these classes of agents, epothilones, has shown great promise in phase III clinical trials. What all these agents share in common, is that they bind to beta-tubulin and disrupt microtubule function during mitosis which in turn leads to mitotic arrest and cell death. In addition, these agents can inhibit angiogenesis. Not withstanding their effectiveness, drug resistance can pose a major clinical problem. This review provides an overview of the mechanisms mediating resistance to tubulin-binding agents related to the cellular target and discusses strategies to overcome this important clinical problem.     

The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds.

The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.     

Using Antifungal Pharmacodynamics to Improve Patient Outcomes

In vitro and in vivo studies of available and investigational antifungals have broadened our understanding of the pharmacodynamics of these agents as well as the pharmacokinetic/pharmacodynamic characteristics that are associated with efficacy. These data are increasingly being used as surrogate means to answer questions about dosing and administration of antimicrobial agents in order to improve outcomes in patients with invasive fungal infections, as these questions are difficult to answer in clinical trials. The objective of this article is to review the pharmacodynamic activity of widely used classes of antifungal agents, including the azoles, amphotericin B, and the echinocandins, discuss the pharmacokinetic/pharmacodynamic parameters associated with efficacy of these agents in preclinical studies, and describe how this information is being translated into the clinical arena to optimize patient outcomes.     

Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.     

Switchable MRI contrast agents based on morphological changes of pH-responsive polymers

Magnetic resonance imaging (MRI) contrast agents are effective tools in both medical diagnosis and life science research. Various smart contrast agents have been developed for the visualization of biological phenomena. These contrast agents have molecular switches that increase or reduce MRI signal intensity in response to the target biological reaction. Therefore, novel approaches to the design of molecular switches for versatile in vivo studies using MRI are eagerly anticipated. Here, we report one such approach for the development of molecular switches based on morphological changes of pH-responsive polymers. We designed and synthesized three types of contrast agents based on a linear homopolymer or spherical copolymers with two different cross-linking degrees. The relaxivity measurements showed that these agents have molecular switches that respond to pH changes, and fluorescence studies indicated that these switches are based on the alteration of the molecular tumbling caused by pH-responsive morphological changes. As a result, the spherical polymers possess promising characteristics for the development of switchable MRI contrast agents.     

RNA viruses as vectors for the expression of heterologous proteins

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.