Novel oxazolinoanthracyclines as tumor cell growth inhibitors-Contribution of autophagy and apoptosis in solid tumor cells death.

Chemical modification of known, effective drugs are one method to improve the chemotherapy of tumors. We reported ability of oxazoline analogs of doxorubicin (O-DOX) and daunorubicin (O-DAU) to induce apoptosis and autophagy in ovarian and liver cancer cells. Reactive oxygen and nitrogen species (ROS and RNS, respectively), together with intracellular calcium-mediated downstream signaling, are essential for the anticancer effect of these new anthracycline analogs. The changes of mitochondrial membrane potential and induction of the ceramide pathway suggests that these compounds induce cell death by apoptosis. In addition, a significant increase of autophagosome formation was observed by fluorescence assay and acridine orange staining, indicating that the new analogs also induce autophagic cell death. Compared to free DOX- and DAU-treated cells, we observed inhibition of colony formation and migration, a time-dependency between ROS/RNS levels and a greater fall in mitochondrial membrane potential. Altogether, our research broadens the base of molecular oxazolinoanthracyclines targets and reveals that derivatives mediated oxidative stress, ceramide production and increase in intracellular calcium level by mitochondria. Furthermore, our data highlight the importance of mitochondria that simultaneously assume the role of activator of autophagy and apoptosis signals.

Molecular mechanism of action of oxazolinoanthracyclines in cells derived from human solid tumors. Part 2.

BACKGROUND/AIM: Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are derivatives of anthracyclines (DOX and DAU) with a modified daunosamine moiety. We aimed to clarify their mechanisms of action by investigating intracellular accumulation and effects on the cell cycle, phosphatidylserine externalization, and proteasome 20S activity. MATERIALS AND METHODS: Experimental model consisted of SKOV-3, A549 and HepG2 cells. Compounds were used at the concentration of 80nM. Intracellular accumulation, drug uptake, and proteasome 20S activity were evaluated by fluorimetric methods. The effects on the cell cycle and phosphatidylserine externalization were measured by flow cytometry. RESULTS: O-DOX was equivalent to DOX in terms of inducing G2/M arrest, but O-DAU was less potent in SKOV-3, HepG2, and A549 cells. O-DOX had the greatest effect on initiating apoptosis in all tested cells. Externalization of phosphatidylserine was significantly higher following O-DOX treatment compared with control cells and cells incubated with DOX. The intracellular accumulation and uptake of the derivatives were similar to those of the reference drugs. Tested compounds are able to activate proteasome 20S activity. CONCLUSION: Our results extended the understanding of the toxicity, mechanism of action, and biochemical properties of oxazoline derivatives of doxorubicin and daunorubicin, including their effects on cell cycle, apoptosis and DNA degradation.

In Vitro Antileukemic Activity of Formamidine Epidoxorubicin Analogs.

BACKGROUND/AIM: Epidoxorubicin is an anthracycline agent. The present study was undertaken to compare the antileukemic potential of epidoxorubicin and its two formamidine analogs containing either a morpholine moiety (EPIFmor) or a hexamethyleneimine moiety (EPIFhex) in the amidine group. MATERIALS AND METHODS: The experiments were performed in vitro on MOLT-4 cells using spectrophotometry, Coulter electrical impedance, flow cytometry, and light microscopy methods. RESULTS: The leukemia cell responses to the action of the anthracyclines were manifested in their different viability, count and volume, degree of apoptosis and necrosis, activity of caspases -8, -9, and -3/7, mitochondrial membrane potential, and in the cell-cycle distribution. In general, epidoxorubicin appeared to be the most active, and EPIFmor was more active than EPIFhex against MOLT-4 cells. CONCLUSION: The structural modifications of epidoxorubicin in the amidine group were responsible for the varied action of its formamidine analogs on human acute lymphoblastic leukemia cells.

Effects of structural modification of the daunosamine moiety of anthracycline antibiotics on pKa values determined by capillary zone electrophoresis.

The thermodynamic acid dissociation constants (pKa1 and pKa2) of 16 anthracycline antibiotics, including doxorubicin (DOX) and daunorubicin (DAU), their epimers, epidoxorubicin (EDOX) and epidaunorubicin (EDAU), as well as novel anthracycline derivatives containing piperidine (FPIP), morpholine (FMOR) and hexamethylenoimine (FHEX) rings in the formamidine group of the daunosamine moiety were determined by analysis of the dependence between measured electrophoretic mobility and the pH of the buffer using the capillary zone electrophoresis method. The results obtained confirmed the ampholytic character of anthracyclines with at least two ionization states. The determined values were in the range of 8.36-9.28 and 9.38-11.48 for pKa1 and pKa2 arising from ionization of amino and phenolic groups, respectively. Structural modifications in the daunosamine moiety of the studied anthracyclines affected their pharmacological properties, such as antiproliferative activity.

Identification of the key pathway of oxazolinoanthracyclines mechanism of action in cells derived from human solid tumors.

Oxazolinodoxorubicin (O-DOX) and oxazolinodaunorubicin (O-DAU) are novel anthracycline derivatives with a modified daunosamine moiety. In the present study, we evaluated the cytotoxicities, genotoxicities and abilities of O-DOX and O-DAU to induce apoptosis in cancer cell lines (SKOV-3; A549; HepG2), and compared the results with their parent drugs. We assessed antiproliferative activity by MTT assay. We evaluated apoptosis-inducing ability by double-staining with fluorescent probes (Hoechst 33258/propidium iodide), and by determining expression levels of genes involved in programmed cell death by reverse transcription-polymerase chain reaction. Genotoxicities of the compounds were tested by comet assays. Oxazolinoanthracyclines demonstrated high anti-tumor activity. O-DOX had significantly higher cytotoxicity, apoptosis-inducing ability, and genotoxicity compared with parental doxorubicin (DOX) in all tested conditions, while O-DAU activity differed among cell lines. The mechanism of oxazoline analog action appeared to involve the mitochondrial pathway of programmed cell death. These results provide further information about oxazoline derivatives of commonly used anthracycline chemotherapy agents. O-DOX and O-DAU have the ability to induce apoptosis in tumor cells.

A SIMPLE AND-SENSITIVE STABILITY-INDICATING UHPLC-DAD METHOD FOR THE DETERMINATION OF CEFETAMET PIVOXIL HYDROCHLORIDE.

A fast and sensitive UHPLC-DAD method was developed and subsequently validated for determination of cefetamet pivoxil hydrochloride in the presence of its degradation products. The chromatographic separation was carried out on a Waters Acquity BEH C18, (2.1 x 100 mm, 1.7 µm) column. The mobile phase was composed of 0.1% formic acid and acetonitrile (40 : 60, v/v) at the flow rate 0.7 mL/min. The detection wavelength was 265 nm and the temperature was 30 °C. Cefetamet pivoxil hydrochloride was susceptible to degradation under the influence of sodium hydroxide, hydrochloric acid and in the conditions of increased temperature and relative humidity. However, it was stable after irradiation, in increased temperature in dry air and in the presence of oxidizing agent. The developed UHPLC-DAD method was linear over the concentration range of 10-240 µg/mL (r2 = 0.9999; n = 12). The obtained RSD values were less than 2%, demonstrating that the described procedure is precise. The accuracy was also confirmed (mean recoveries were 97.79-102.08%). Under applied chromatographic conditions LOD and LOQ values were 2.08 mg/mL and 6.29 mg/mL, respectively. The proposed method was successfully applied in determination of cefetamet pivoxil hydrochloride in aqueous solutions as well as in the solid state.

Spectroscopic studies of anthracyclines: Structural characterization and in vitro tracking.

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488nm and 785nm as source wavelengths, with submicrometer spatial resolution, although the cellular accumulation of the drug is different in each case. The high information content of Raman spectra allows studies of the drug-cell interactions, and so the method seems very suitable for monitoring drug uptake and mechanisms of interaction with cellular compartments at the subcellular level.

STUDIES OF THE CRYSTALLINE FORM OF CEFUROXIME AXETIL: IMPLICATIONS FOR ITS COMPATIBILITY WITH EXCIPIENTS.

Amorphous and crystalline forms of cefuroxime axetil were identified and characterized using DSC, XRPD, SEM, FT-IR and Raman spectroscopy. Based on the results of chromatographic studies, changes in the kinetic mechanism and rate of degradation of the crystalline form of cefuroxime axetil in binary systems with excipients were also evaluated. The findings suggest that the mechanism of degradation of cefuroxime axetil in such systems depends on two factors: the applied excipient and storage conditions. Cefuroxime axetil in combination with magnesium stearate, croscarmellose sodium and crospovidone, microcrystalline cellulose, aerosil is decomposed according to the first-order reaction model in dry air as well as at an increased relative air humidity, which may be associated with non-catalytic interactions between the active pharmaceutical ingredient and the excipients. However, in the presence of mannitol, under elevated humidity conditions (RH - 76%), the degradation of cefuroxime axetil follows the autocatalytic model. According to ESP maps, computed binding energies and HOMO - LUMO gaps, differences of degradation curves between cefuroxime axetil - mannitol and other investigated systems were explained. This study of the polymorphic transformation of the crystalline form of cefuroxime axetil and its binary systems with excipients after exposure to increased temperature and humidity indicated a conversion towards the amorphous form or the coexistence of both forms.

Subcellular localization of anthracyclines in cultured rat cardiomyoblasts as possible predictors of cardiotoxicity.

In this study, we compared the cellular uptake, intracellular localization and cytotoxicity of two groups of anthracycline derivatives in cultured H9c2(2-1) rat cardiomyoblasts. The first group consisted of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N = CH-N<) at the C-3' position with a morpholine (DOXM) or a hexamethyleneimine (DOXH) ring. The second group consisted of daunorubicin (DRB) and its derivatives containing a morpholine (DRBM) or a hexamethyleneimine (DRBH) ring. DOXH and DRBH were taken up by cardiomyoblasts more efficiently than estimated for other tested anthracyclines. The cellular uptakes of DOXM and DRBM were reduced compared to those of the parent compounds. Applied structural modifications of DOX and DRB influenced the subcellular localization of the tested derivatives. DOX and DOXH were localized primarily in nuclei, whereas the other anthracyclines were found in the nuclei and cytoplasm. The percentages of the compounds that accumulated in the nuclei were 80.2 and 54.2 % for DOX and DOXH, respectively. The lowest nuclear accumulation values were observed for DRBM (19.9 %), DRBH (21.9 %) and DOXM (23.7 %). The ability of anthracyclines to accumulate in the nuclei correlated with their DNA binding constants (r = 0.858, P = 0.029). A correlation was found between the accumulation of the tested anthracyclines in the nuclei of cardiomyoblasts and their cardiotoxicity in vivo, which was observed in our previous study. We suggest that cytotoxicity and the anthracycline accumulation level in the nuclei of cultured cardiomyoblasts could be used for early prediction of their cardiotoxicity.

Formamidinodoxorubicins are more potent than doxorubicin as apoptosis inducers in human breast cancer cells.

BACKGROUND/AIM: The ability of five formamidinodoxorubicins to induce apoptosis of MCF-7 breast cancer cells was tested. All these compounds were modified at C-3' and contain a formamidine group (-N=CH-NRR), with the rest of the cyclic secondary amine (HNRR) of a gradually increasing ring size. MATERIALS AND METHODS: Cytotoxicity was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To analyze apoptosis, double staining using fluorescence probes Hoechst 33258/propidium iodide (PI) and annexin V- Fluorescein isothiocyanate/PI was carried-out. Additionally, the TdT-mediated dUTP nick-end labelling test and activity of caspase 3 were determined. RESULTS: The four tested derivatives displayed a significant increase in antiproliferative activity in comparison to doxorubicin. All of the tested derivatives induced caspase-dependent apoptosis of MCF-7 cells. CONCLUSION: DOX-F MOR and DOX-F PAZ analogs are more potent apoptosis inducers than doxorubicin.

Application of vibrational spectroscopy supported by theoretical calculations in identification of amorphous and crystalline forms of cefuroxime axetil.

FT-IR and Raman scattering spectra of cefuroxime axetil were proposed for identification studies of its crystalline and amorphous forms. An analysis of experimental spectra was supported by quantum-chemical calculations performed with the use of B3LYP functional and 6-31G(d,p) as a basis set. The geometric structure of a cefuroxime axetil molecule, HOMO and LUMO orbitals, and molecular electrostatic potential were also determined by using DFT (density functional theory). The benefits of applying FT-IR and Raman scattering spectroscopy for characterization of drug subjected to degradation were discussed.

Cytotoxicity and induction of apoptosis by formamidinodoxorubicins in comparison to doxorubicin in human ovarian adenocarcinoma cells.

BACKGROUND/AIM: In this study we investigated the effect of DOX and five of its derivatives containing a formamidine group (NCHNRR) at the 3' position with pyrrolidine (DOX-F PYR), piperidine (DOX-F PIP), morpholine (DOX-F MOR), N-methylpiperazine (DOX-F PAZ) and hexamethyleneimine (DOX-F HEX) ring on SKOV-3 ovarian cancer cells. We have focused on the anti-proliferative activity and the value of apoptosis induced by tested analogues. MATERIALS AND METHODS: The following methods were used: spectrophotometric assay with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); fluorimetric assays - double staining with Hoechst 33258 and propidium iodide (PI), measurement of caspase-3 activity; flow cytometry methods - phosphatidylserine (PS) externalization using Annexin V-FITC and PI fluorochromes, and TUNEL assay. RESULTS: All of the investigated derivatives were considerably more cytotoxic to the SKOV-3 cell line than DOX. The predominant type of cell death induced by the anthracycline analogues was apoptosis. Necrotic cells represented only a small percentage (<5%) of all cells. The number of apoptotic cells was dependent on the compound and the incubation time. Moreover, a significant increase in caspase-3 activity, DNA fragmentation, and morphological changes in ovarian cells were observed predominantly in new DOX analogues. CONCLUSIONS: All new formamidine derivatives of DOX were effective against ovarian cancer cells. They induced mainly the apoptotic pathway of cell death mediated by caspase-3. The most promising results were obtained for DOX-F MOR and DOX-F PAZ. The least potent was DOX-F HEX.

Cell-cycle disturbance and induction of programmed death by new formamidine analogs of daunorubicin.

BACKGROUND/AIM: Structural modifications of daunorubicin are an important way to change its anticancer activity. For this reason, formamidinodaunorubicins have been synthesized. The present study was undertaken to determine and compare the in vitro effects of daunorubicin and its new formamidine derivatives on human acute leukemia MOLT-4 and ML-1 cells. MATERIALS AND METHODS: The experiments were performed on human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells. The study was conducted using flow cytometry and light microscopy methods. RESULTS: The various patterns of temporary changes in the cell cycle and DNA fragmentation, as well as the extent of mitotic catastrophe, apoptosis, and necrosis, were determined. The anti-leukemic activities of the new daunorubicin analogs were weaker than that of daunorubicin. CONCLUSION: The influence of these anthracyclines on cell-cycle progression, DNA damage, and induction of mitotic catastrophe and cell death depended on the agent and its concentration, the time interval after application, and the cell line used. The structural modifications of daunorubicin were responsible for the different cytotoxic effects of the two formamidinodaunorubicins.

Assay of Diastereoisomers of Cefuroxime Axetil in Amorphous and Crystalline Forms Using UHPLC-DAD.

A sensitive UHPLC-DAD method was developed for determination of diastereoisomers of cefuroxime axetil in bulk substance in amorphous and crystalline forms as well as in pharmaceutical preparations. Chromatographic separation was achieved on Kinetex C-18 (100 mm × 2.1 mm, 1.7 µm) column with mobile phase consisting of 0.1 % formic acid:methanol (88:12, v/v), at the flow rate of 0.7 mL min-1 and total run time of 3 min. The wavelength of the DAD detector was set at 278 nm. Inter-day precision (RSD) was less than 3 % and accuracy level ranged between 98.31 and 104.99 %. Degradation products of cefuroxime axetil in aqueous solutions and in the solid state were identified with a EIS-Q-MS mass spectrometer. The solubility of above-mentioned polymorphic forms of cefuroxime axetil in suitable solvents is a crucial factor during preparation of samples and is essential for chromatographic separation of its diastereoisomers.

Stability of [(N-piperidine)methylene]daunorubicin hydrochloride and [(N-pyrrolidine)methylene]daunorubicin hydrochloride in solid state.

The influence of temperature and relative air humidity on the stability of two novel derivatives of daunorubicin: [(N-piperidine)methylene]daunorubicin hydrochloride and [(N-pyrrolidine)methylene]daunorubicin hydrochloride was investigated. The process of degradation was studied by using high-performance liquid chromatography with ultraviolet (UV) detection. The kinetic and thermodynamic parameters of degradation were calculated.

The influence of pH and temperature on the stability of N-[(piperidine)methylene]daunorubicin Hydrochloride and a comparison of the stability of daunorubicin and its four new amidine derivatives in aqueous solutions.

The influence of pH and temperature on the stability of N-[(piperidine)methylene]daunorubicin hydrochloride (PPD) was investigated. Degradation was studied using an HPLC method. Specific acid-base catalysis of PPD involves hydrolysis of protonated molecules of PPD catalyzed by hydrogen ions and spontaneous hydrolysis under the influence of water zwitterions, unprotonated molecules, and monoanions of PPD. The thermodynamic parameters of these reactions, energy, enthalpy, and entropy, were calculated. Also, the stability of daunorubicin and its new amidine derivatives (piperidine, morpholine, pyrrolidine, and hexahydroazepin-1-yl) in aqueous solutions was compared and discussed.

In vitro induction of apoptosis and necrosis by new derivatives of daunorubicin.

BACKGROUND/AIM: The comparative effects of daunorubicin, and its new formamidine derivatives containing either a morpholine moiety (DAUFmor) or a hexamethyleneimine moiety (DAUFhex) in the amidine group, on induction of programmed cell death were determined. MATERIALS AND METHODS: The experiments were performed on human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells. The research was conducted using the flow cytometry annexin V-fluorescein (FITC)/propidium iodide (PI) method and tetramethylrhodamine ethyl ester (TMRE) assay. RESULTS: The various patterns of temporary changes of early apoptotic cells, late apoptotic and necrotic cells, and in the frequency of the acute leukemia cells with high values of mitochondrial membrane potential (MMP) were found. Phosphatidylserine externalization, plasma membrane disruption, and changes in MMP occurring in the leukemia cells were dependent on the agent tested, its concentration, the time intervals after daunorubicin, DAUFmor, and DAUFhex application, and on the leukemia cell line used. CONCLUSION: The structural modifications of daunorubicin producing two new analogs, DAUFmor and DAUFhex, induced the different responses of MOLT-4 and ML-1 cells to triggering of programmed death.

Effects of structural modifications of daunorubicin on in vitro antileukemic activity.

BACKGROUND/AIM: In the search for new derivatives of anthracycline antibiotics, formamidinodaunorubicins containing in the amidine group either a morpholine moiety (DAUFmor) or a hexamethyleneimine moiety (DAUFhex) were synthesized. The biological effects of daunorubicin (DAU), DAUFmor and DAUFhex were compared. MATERIALS AND METHODS: The experiments were performed on human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells. The research was conducted using the spectrophotometric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and the electronic Beckman-Coulter method. RESULTS: Temporary changes in the leukemia cell viability, size and count were found. The antileukemic activities of the new DAU analogs were weaker than that of daunorubicin. MOLT-4 cells were more sensitive than ML-1 cells to the action of all agents. Among the formamidinodaunorubicins, DAUFmor appeared to be more active in ML-1 cells than DAUFhex, but there were not differences between the analyzed values in MOLT-4 cells. CONCLUSION: The structural modifications of daunorubicin were responsible for the different antileukemic potentials of the two formamidinodaunorubicins.

Oxazolinodoxorubicin - a promising new anthracycline.

Oxazolinodoxorubicin, a doxorubicin analog with a modified daunosamine moiety was synthesized. The properties of this compound and the parent doxorubicin were compared. The cytotoxicity in vitro studies against several human tumor cell lines (PC-3, MCF-7, SW707, HL-60, RPMI 8226, ACHN) showed higher antiproliferative potency for this new compound. Moreover, its ability to completely overcome the drug resistance of cancer cells in vitro was revealed (LoVo, LoVo/DX, MES-SA, MES-SA/DX5, HL-60, HL-60/Vinc, HL-60/MX2 cell lines). Cellular uptake analyzed on HL-60 and HL-60/MX2 cells, demonstrated higher penetration levels of oxazolinodoxorubicin compared to that of doxorubicin. In animal experiments, general toxicity of oxazolinodoxorubicin was lower than that observed for doxorubicin. Furthermore, similar antitumor effects was observed in NOD/SCID mice bearing resistant HL-60/Vinc leukemia tumor and in mice treated with the new or parent compounds. The presented results suggest that oxazolinodoxorubicin is a new anthracycline with an advantageous biological activity profile.