The synthesis of doxorubicin-conjugated magnetite nanoparticles and their magnetic resonance and cytotoxic properties

The possibility of increasing the effectiveness of antitumor drugs such as doxorubicin by preparing its complex with ultrafine magnetic iron oxide nanoparticles is considered. A method for binding doxorubicin molecules to magnetic nanoparticles via citric acid is proposed. The main magnetic properties of the obtained conjugates were studied by proton relaxometry and Mössbauer spectroscopy, while their cytotoxic activity was evaluated via spectrophotometric MTT assay in HeLa cells. It was shown that the conjugates of magnetite nanoparticles with doxorubicin are characterized by a high level of contrast in magnetic resonance imaging. The magnetic properties of doxorubicin-free and bound magnetite nanoparticles are mainly determined by the average size of nanoobjects and the phase composition and slightly depend on the composition of the stabilizing shell. The cytotoxic effect of the synthesized conjugates of magnetite nanoparticles with doxorubicin is higher than that of unbound doxorubicin. This makes it possible to increase the antitumor effect of doxorubicin and control the dynamics of its delivery in the form of a conjugate into the disease focus due to the magnetic contrast properties of nanoparticles.     

New conjugates of antitumor antibiotic doxorubicin with water-soluble galactomannan: synthesis and biological activity

New water-soluble conjugates in the form of Schiff bases (DGM-1 and DGM-2) were prepared by the interaction of water-soluble periodate-oxidized galactomannan with doxorubicin or N-(L-lysyl)doxorubicin, respectively. The water-soluble galactomannan (DAVANAT a commercial product of Pro-Pharmaceuticals company) was obtained by partial acidic hydrolysis of high-molecular-mass galactomannan from Cyamopsis tetragonoloba (guar gum) seeds. The conjugate stability was studied in aqueous solutions. The DGM-1 antiproliferative activity was comparable with that of doxorubicin on three models: cell lines of murine melanoma B 16-F1, human breast cancer MCF-7 (HTB-22), and human colon cancer HT-29 (HTB-38). DGM-2 was poorly active in all the three tests. DGM- 1 can thus be regarded as a high-molecular-mass depot form of doxorubicin.     

Design, synthesis and evaluation of D-galactose-beta-cyclodextrin conjugates as drug-carrying molecules

Several kinds of D-galactose-beta-cyclodextrin conjugates having a phenyl group in the spacers between the D-galactose and beta-cyclodextrin were designed and synthesized as drug-carrying molecules. Their evaluation as drug-carrying molecules was done by measuring the molecular interactions with the anticancer agent, doxorubicin, and with the d-galactose-binding peanut lectin using an SPR optical biosensor. The SPR analyses showed that these conjugates had remarkably high inclusion associations of 10(5) approximately 10(7)M(-1) levels for the immobilized doxorubicin. Their association constants for immobilized peanut lectin were at the level of 10(4) approximately 10(5)M(-1), as we expected. These conjugates will be useful drug-carrying models which can site-specifically carry doxorubicin to the cells containing D-galactose-binding lectin.     

Novel cleavable cell‐penetrating peptide–drug conjugates: synthesis and characterization

We report the first drug conjugate with a negatively charged amphipathic cell‐penetrating peptide. Furthermore, we compare two different doxorubicin cell‐penetrating peptide conjugates, which are both unique in their properties, due to their net charge at physiological pH, namely the positively charged octaarginine and the negatively charged proline‐rich amphipathic peptide. These conjugates were prepared exploiting a novel heterobifunctional crosslinker to join the N‐terminal cysteine residue of the peptides with the aliphatic ketone of doxorubicin. This small linker contains an activated thiol as well as aminooxy functionality, capable of generating a stable oxime bond with the C‐13 carbonyl group of doxorubicin. The disulfide bond formed between the peptide and doxorubicin enables the release of the drug in the cytosol, as confirmed by drug‐release studies performed in the presence of glutathione. Additionally, the cytotoxicity as well as the cellular uptake and distribution of this tripartite drug delivery system was investigated in MCF‐7 and HT‐29 cell lines. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Design, synthesis, and initial biological evaluation of a steroidal anti-estrogen-doxorubicin bioconjugate for targeting estrogen receptor-positive breast cancer cells

As part of our program to develop breast cancer specific therapeutic agents, we have synthesized a conjugate agent that is a conjugate of the steroidal anti-estrogen and the potent cytotoxin doxorubicin. In this effort, we employed a modular assembly approach to prepare a novel 11β-substituted steroidal anti-estrogen functionalized with an azido-tetraethylene glycol moiety, which could be coupled to a complementary doxorubicin benzoyl hydrazone functionalized with a propargyl tetraethylene glycol moiety. Huisgen [3 + 2] cycloaddition chemistry gave the final hybrid that was evaluated for selective uptake and cytotoxicity in ER(+)-MCF-7 and ER(-)-MDA-MB-231 breast cancer cell lines. The results demonstrated that the presence of the anti-estrogenic component in the hybrid compound was critical for selectivity and cytotoxicity in ER(+)-MCF-7 human breast cancer cells as the hybrid was ~70-fold more potent than doxorubicin in inhibition of cell proliferation and promoting cell death.     

Suppression of DNA synthesis in mice by the anthracycline antibiotics daunorubicin, carminomycin and doxorubicin

Inhibition of DNA synthesis by rubomycin (daunorubicin), carminomycin and doxorubicin in the spleen, liver, kidneys and heart was studied on mice. The antibiotics were administered intravenously in a dose of 0.3 LD50. The inhibition level was estimated by incorporation of 3H-thymidine. The time courses of DNA synthesis inhibition by daunorubicin, carminomycin and doxorubicin markedly differed, whereas the patterns of their inhibition curves for all the organs were close. The maximum inhibition of DNA synthesis by carminomycin was observed in 6 hours. After that period it gradually restored. Doxorubicin induced the maximum inhibition of DNA synthesis in 24-48 hours after its administration. Daunorubicin induced two maxima in inhibition of DNA synthesis i. e. in 6 and 48 hours. Definite correlation between the levels of DNA synthesis inhibition by the antibiotics and their toxic action was shown.     

Pteroyl-gamma-glutamate-cysteine synthesis and its application in folate receptor-mediated cancer cell targeting using folate-tethered liposomes

Cell membrane-associated folate receptors are selectively overexpressed in certain human tumors. The high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. Folate-tethered liposomes bearing pteroyl-gamma-glutamate-cysteine-polyethylene glycol (PEG)-distearoylphosphatidylethanolamine (DSPE) as the targeting component are under investigation as mediators of drug and gene delivery to cancer cells that overexpress folate receptors. Pteroyl-gamma-glutamate-cysteine synthesis is one of the crucial starting steps in the preparation of pteroyl-gamma-glutamate-cysteine-PEG-DSPE. However, published methods for the synthesis of pteroyl-gamma-glutamate-cysteine provide low yields and are not easily reproducible. Therefore, we developed a modified synthetic method for the removal of the N(10)-trifluoroacetyl group after cleavage/deprotection that is reliable, is easily reproducible, and has high yield (38%) compared with an unreliable yield of 3-20% with the earlier methods. Folate-tethered liposomes containing calcein or doxorubicin were prepared using pteroyl-gamma-glutamate-cysteine-PEG-DSPE as the targeting component along with nontargeted liposomes with PEG-DSPE. The results of the uptake of calcein and cytotoxicity of doxorubicin in human cervical cancer HeLa-IU(1) cells and human colon cancer Caco-2 cells demonstrated that folate-tethered liposomes were efficient in selective delivery to cancer cells overexpressing folate receptors. The improvement in yield of the targeting component can significantly facilitate "scale up" of folate receptor-mediated liposomal cancer therapy to the preclinical and clinical levels of investigations.     

Click chemistry based synthesis,cytotoxic activity and molecular docking of novel triazole-thienopyrimidine hybrid glycosides targeting EGFR

In the current study, new thienopyrimidine conjugates bearing 1,2,3-triazole core and different sugar moieties have been designed and synthesized by Cu(I)-catalysed click dipolar cycloaddition. The cytotoxic activity of the synthesised conjugates 2, 5, 7, and 13–18 was studied against HCT-116 and MCF-7 cell lines by the MTT assay. The triazole glycosides 16 and 18 provided significant cytotoxic activities against HCT-116 cell lines comparable to that of doxorubicin and other studied compounds. The cytotoxic behaviour against MCF-7 exhibited that all the investigated compounds were more potent than doxorubicin. Moreover, all screened targets were evaluated against mutant EGFR kinase type L858R and the results revealed that the acetylated 1,2,3-triazole glycosides 13–18 exhibited excellent EGFR inhibitory activity in comparison with gefitinib. Furthermore, molecular modelling studies were performed to investigate the binding affinity of the most active compounds to EGFR enzyme.     

Novel hydrogels via click chemistry: synthesis and potential biomedical applications

A novel procedure for the in situ rapid chemical gelation of aqueous solutions of hyaluronan has been employed. In brief, water-soluble polysaccharide derivatives bearing side chains endowed with either azide or alkyne terminal functionality have been prepared. When the latter two types of derivatives are mixed together in aqueous solution they give rise to a 1,3-dipolar cycloaddition reaction resulting in fast gelation (in the presence of catalytic amounts of Cu(I)) at room temperature. Gel formation has been characterized rheologically and could also be followed qualitatively by means of IR spectroscopy. The resulting gels have been studied in terms of swelling properties and, in particular, NMR spectral features. Carrying out the gelation process in aqueous solutions of benzidamine and doxorubicin, respectively, the polysaccharide networks acted as drug reservoirs. The doxorubicin release resulted in well controllable acting upon the gels degree of cross-linking. Finally, formation of the click-gels using aqueous suspensions of Saccharomices cerevisiae yeast cells allowed the obtainment of scaffolds inside which cells were homogeneously distributed and smoothly adhered to the inner pores surfaces, according to SEM analysis. After 24 h about 60% of the entrapped cells exhibited proliferating activity. Click-gels prepared as detailed herein do have a number of positive features that make them, in perspective, materials of choice for drug release and tissue engineering manipulations.     

Doxorubicin inhibits oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by a lactoperoxidase/H(2)O(2) system by reacting with ABTS-derived radical

The effect of doxorubicin on oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by lactoperoxidase and hydrogen peroxide has been investigated. It was found that: (1) oxidation of ABTS to its radical cation (ABTS*(+)) is inhibited by doxorubicin as evidenced by its induction of a lag period, duration of which depends on doxorubicin concentration; (2) the inhibition is due to doxorubicin hydroquinone reducing the ABTS*(+) radical (stoichiometry 1: 1.8); (3) concomitant with the ABTS*(+) reduction is oxidation of doxorubicin; only when the doxorubicin concentration decreases to a near zero level, net oxidation of ABTS could be detected; (4) oxidation of doxorubicin leads to its degradation to 3-methoxysalicylic acid and 3-methoxyphthalic acid; (5) the efficacy of doxorubicin to quench ABTS*(+) is similar to the efficacy of p-hydroquinone, glutathione and Trolox C. These observations support the assertion that under certain conditions doxorubicin can function as an antioxidant. They also suggest that interaction of doxorubicin with oxidants may lead to its oxidative degradation.     

A stable bis-allyloxycarbonyl biotin aldehyde derivative for biotinylation via reductive alkylation: application to the synthesis of a biotinylated doxorubicin derivative

A novel, stable, biotin aldehyde derivative is reported in which the biotin moiety is N1,N3-protected by the allyloxycarbonyl group. The derivative is stable to sodium cyanoborohydride mediated reductive alkylation and is cleaved under mild Pd [0] catalysis. This novel biotin aldehyde should have wide application in avidin- and streptavidin-based detection systems and bioassays. The derivative is utilized in the synthesis of a biotinylated doxorubicin analogue that retains topoisomerase activity.     

A switching mechanism in doxorubicin bioactivation can be exploited to control doxorubicin toxicity

Although doxorubicin toxicity in cancer cells is multifactorial, the enzymatic bioactivation of the drug can significantly contribute to its cytotoxicity. Previous research has identified most of the components that comprise the doxorubicin bioactivation network; however, adaptation of the network to changes in doxorubicin treatment or to patient-specific changes in network components is much less understood. To investigate the properties of the coupled reduction/oxidation reactions of the doxorubicin bioactivation network, we analyzed metabolic differences between two patient-derived acute lymphoblastic leukemia (ALL) cell lines exhibiting varied doxorubicin sensitivities. We developed computational models that accurately predicted doxorubicin bioactivation in both ALL cell lines at high and low doxorubicin concentrations. Oxygen-dependent redox cycling promoted superoxide accumulation while NADPH-dependent reductive conversion promoted semiquinone doxorubicin. This fundamental switch in control is observed between doxorubicin sensitive and insensitive ALL cells and between high and low doxorubicin concentrations. We demonstrate that pharmacological intervention strategies can be employed to either enhance or impede doxorubicin cytotoxicity in ALL cells due to the switching that occurs between oxygen-dependent superoxide generation and NADPH-dependent doxorubicin semiquinone formation.     

A Fickian diffusion transport process with features of transport catalysis. Doxorubicin transport in human red blood cells

The transport of the antineoplastic drug doxorubicin (Adriamycin) in human red blood cells was investigated by measuring the net efflux from loaded cells. Previous data indicated that doxorubicin transport was a Fickian diffusion transport process of the electrically neutral molecule through the lipid domain of the cell membrane (Dalmark, 1981 [In press]). However, doxorubicin transport showed saturation kinetics and a concentration-dependent temperature dependence with nonlinear Arrhenius plots. The two phenomena were related to the doxorubicin partition coefficient between 1-octanol and a water phase. This relationship indicated that the two phenomena were caused by changes in the physiochemical properties of doxorubicin in the aqueous phase and were not caused by interaction of doxorubicin with cell membrane components. The physicochemical properties of doxorubicin varied with concentration and temperature because of the ability of doxorubicin to form polymers by self-association in aqueous solution like other planar aromatic molecules through pi-electron orbital interaction. The hypothesis is proposed that doxorubicin transport across cell membranes takes place by simple Fickian diffusion.     

Albumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacy

We have recently validated a macromolecular prodrug strategy for improved cancer chemotherapy based on two features: (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin and (b) acid-sensitive promoted or enzymatic release of the drug at the tumor site [Kratz, F., Warnecke, A., Scheuemann, K., Stockmar, C., Schwab, J., Lazar, P., Druckes, P., Esser, N., Drevs, J., Rognan, D., Bissantz, C., Hinderling, C., Folkers, G., Fichtner, I., and Unger, C. (2002) J. Med. Chem. 45, 5523-33]. In the present work, we developed water-soluble camptothecin (CPT) and doxorubicin (DOXO) prodrugs that incorporate the peptide linker Ala-Leu-Ala-Leu that serves as a substrate for the tumor-associated protease, cathepsin B, which is overexpressed in several solid tumors. Consequently, two albumin-binding prodrugs were synthesized [EMC-Arg-Arg-Ala-Leu-Ala-Leu-Ala-CPT (1) and EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOXO (2) (EMC = 6-maleimidocaproic acid)]. Both prodrugs exhibited excellent water-solubility and bound rapidly and selectively to the cysteine-34 position of endogenous albumin. Further in vitro studies showed that the albumin-bound form of the prodrugs was cleaved specifically by cathepsin B as well as in human tumor homogenates. Major cleavage products were CPT-peptide derivatives and CPT for the CPT prodrug and H-Leu-Ala-Leu-DOXO, H-Leu-DOXO, and DOXO for the doxorubicin prodrug. In vivo, 1 was superior to free camptothecin in an HT-29 human colon xenograft model; the antitumor efficacy of prodrug 2 was comparable to that of free doxorubicin in the M-3366 mamma carcinoma xenograft model at equimolar doses.     

Stage-specific inhibition of deoxyribonucleic acid synthesis and induction of apoptosis by antracyclines in cultured rat spermatogenic cells

A rapid in vitro method has been developed to detect early effects of cytostatic drugs on rat spermatogenesis. The induction of programmed cell death (apoptosis) and changes in DNA synthesis induced by doxorubicin and idarubicin were measured in specific stages of the cycle of seminiferous epithelium including mitotic (stage V) and meiotic (stage VIII-IX) S-phase cells. The model was used to investigate the protective effect of an organic thiophosphate, amifostine, against the toxicity of antracyclines. Premitotic DNA synthesis was found to be more sensitive than premeiotic DNA synthesis to antracyclines. Idarubicin was more toxic than doxorubicin to germ cells in inducing apoptosis and suppressing DNA synthesis. Amifostine had no protective effect against doxorubicin- or idarubicin-induced inhibition of DNA synthesis. In contrast, a significant stimulation of DNA synthesis in premitotic cells by amifostine was found, suggesting that this compound may have a stimulative effect on spermatogenic stem cells. These data show that stage-specific dissection of the seminiferous tubules and their in vitro exposure to predetermined doses of drugs may give us a unique possibility to detect drug action and protection against the cytotoxicity of antineoplastic agents at the cellular level of the spermatogenic cycle.     

Doxorubicin directly binds to the cardiac-type ryanodine receptor

The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.     

Non-pegylated doxorubicin (Myocet®) as the less cardiotoxic alternative of free doxorubicin

Anthracyclines have probably been considered to be the most active agents for the treatment of breast cancer and some other solid tumors and hematological malignancies. However, they are associated with dose-related cardiotoxicity, which can lead to progressive myocardial damage and limits the maximal cumulative dose that can be given. This review focuses on the non-pegylated liposome-encapsulated doxorubicin (Myocet?), which has been developed to increase the therapeutic index of free doxorubicin. The encapsulation of doxorubicin within a macromolecular vector, such as a liposome ("nanoparticle based drug delivery system") reduces its distribution volume, diminishing its toxicity for healthy tissues while increasing the concentration within the neoplastic tissue. The most common adverse event is neutropenia, which is consistent with previous experience with free doxorubicin. Available evidence suggests that the incidence of hematological toxicity is lower than with conventional doxorubicin. Myocet? both as a single agent and in combination is effective and safe with an associated reduction in incidence and severity of cardiac events. Nagykálnai T. Non-pegylated doxorubicin (Myocet?) as the less cardiotoxic alternative of free doxorubicin.     

Gambogic Acid Sensitizes Ovarian Cancer Cells to Doxorubicin Through ROS-Mediated Apoptosis

Ovarian cancer is one human malignancy which has response portly to doxorubicin. The anti-cancer activity of gambogic acid has been tested in in vitro and in vivo studies. In this study, we showed that gambogic acid, a natural compound, could potentiate the anticancer activity of doxorubicin in ovarian cancer through ROS-mediated apoptosis. Platinum-resistant human ovarian cancer cell line (SKOV-3) was treated with gambogic acid, doxorubicin, or the combination of both to investigate cell proliferation and apoptosis. We found that the combination of gambogic acid and doxorubicin causes synergistic loss of cell viability in SKOV-3 cells and this synergistic effect correlated with increased cellular ROS accumulation. Moreover, in vivo results showed that gambogic acid and doxorubicin combination resulted in a synergistic suppressing effect on tumor growth in ovarian cancer mice model. Taken together, the results suggested that doxorubicin in combination with gambogic acid might provide a promising therapeutic strategy to enhance chemosensitivity of ovarian cancer to doxorubicin.     

Oxidative and non-oxidative mechanisms in the inactivation of cardiac mitochondrial electron transport chain components by doxorubicin.

The quinonoid anthracycline, doxorubicin (Adriamycin) is a potent anti-neoplastic agent whose clinical use is limited by severe cardiotoxicity. Mitochondrial damage is a major component of this cardiotoxicity, and rival oxidative and non-oxidative mechanisms for inactivation of the electron transport chain have been proposed. Using bovine heart submitochondrial preparations (SMP) we have now found that both oxidative and non-oxidative mechanisms occur in vitro, depending solely on the concentration of doxorubicin employed. Redox cycling of doxorubicin by Complex I of the respiratory chain (which generates doxorubicin semiquinone radicals, O2-, H2O2, and .OH) caused a 70% decrease in the Vmax. for NADH dehydrogenase during 15 min incubation of SMP, and an 80% decrease in NADH oxidase activity after 2 h incubation. This inactivation required only 25-50 microM-doxorubicin and represents true oxidative damage, since both NADH (for doxorubicin redox cycling) and oxygen were obligatory participants. The damage appears localized between the NADH dehydrogenase flavin (site of doxorubicin reduction) and iron-sulphur centre N-1. Succinate dehydrogenase, succinate oxidase, and cytochrome c oxidase activities were strongly inhibited by higher doxorubicin concentrations, but this phenomenon did not involve doxorubicin redox cycling (no NADH or oxygen requirement). Doxorubicin concentrations of 0.5 mM were required for 50% decreases in these activities, except for cytochrome c oxidase which was only 30% inhibited following incubation with even 1.0 mM-doxorubicin. Our results indicate that low concentrations of doxorubicin (50 microM or less) can catalyse a site-specific oxidative damage to the NADH oxidation pathway. In contrast, ten-fold higher doxorubicin concentrations (or more) are required for non-oxidative inactivation of the electron transport chain; probably via binding to cardiolipin and/or generalized membrane chaotropic effects. The development of agents to block doxorubicin toxicity in vivo will clearly require detailed clinical studies of doxorubicin uptake in the heart.     

Characterization of the interaction of doxorubicin with (poly)phosphoinositides in model systems. Evidence for specific interaction with phosphatidylinositol-monophosphate and -diphosphate.

The anticancer drug doxorubicin penetrates into Langmuir monolayers containing phosphoinositides. Upon binding of doxorubicin to phosphoinositide-containing SUV, its fluorescence is self-quenched due to self-association. As compared to other anionic phospholipids, as much as 2- to 3-fold larger effects were obtained with PIP and PIP2, in mixtures of these lipids with DOPC. Doxorubicin competes efficiently with the non-penetrating antibiotic neomycin for binding to PIP2. According to its penetration, specific binding of doxorubicin was half-maximal at 5-15 microM. It is likely that also in biological membranes doxorubicin binds specifically to PIP and PIP2.