Content measurement of doxorubicin hydrochloride and lonidamine by HPLC / 药学实践与服务

Objective To establish a method for the simultaneous determination of DOX·HCl and LND. Methods HPLC was performed on Agilent 5 HC-C18（2） （4.6 mm × 250 mm, 5 µm） column. The mobile phase was methanol-0.1% TFA aqueous solution, and the gradient elution procedure were: 0 to 3 min, 65% methanol; 3 to 7 min, 65%→90% methanol; 7 to 13 min, 90% methanol; 13 to 15 min, 90%→65% methanol; 15 to 20 min, 65% methanol. The collection time was 20 min, the balance time was 3 min, the UV detection wavelengths were 205 nm and 253 nm. The flow rate was 1.0 ml/min and the column temperature was 35℃. The amount of inlet was 10 µl. Results The method was highly specific, and both DOX·HCl and LND exhibited good linearity in the concentration range of 1-40 µg/ml and 6-240 µg/ml, respectively. The two compounds’ precision, stability, and recovery satisfied the requirements of the method. Conclusion This study established a HPLC method that was suitable for the simultaneous detection of DOX·HCl and LND. This method’s high level of specificity, accuracy, and reliability .

Lysophosphatidic Acid Down-regulated SKOV3 Cells of Ovarian Cancer in Doxorubicin Hydrochloride-induced Apoptosis / 中国生物化学与分子生物学报

Lysophosphatidic acid (LPA), a bioactive lipid medium, plays an important role in the development and progression of ovarian cancer. Doxorubicin hydrochloride (DOX) is a first-line drug in the ovarian cancer clinical therapy, while the effect and molecular mechanism of LPA in the ovarian cancer with DOX treatment is still unclear. This study intended to explore the effect and molecular mechanism of LPA in ovarian cancer treated with DOX. SKOV3 and OVCAR-3 cells of human ovarian cancer and Chinese hamster ovary cells were treated with control, LPA (lOp-mol/L), DOX (2jjLmol/L) and LPA (10jJLmol/L) + DOX (2p,mol/L) respectively for 24 hours. The morphological changes of SKOV3 cells were observed under optical microscope and transmission electron microscope. Results showed that LPA reduced cell death and the degree of chromatin aggregation in SKOV3 cells treated with DOX; RT-qPCR showed that LPA treatment could down-regulate the mRNA levels of caspase-3 in DOX-treated SKOV3 cells (P<0. 05); Western blot showed that LPA treatment could reduce caspase-3 and cleaved caspase-3 levels treated with DOX in SKOV3, OVCAR-3 and CHO cells (P<0. 05); Flow cytometry using Annexin V/PI double staining showed that LPA could down-regulate apoptosis in SKOV3 cells treated with DOX (P<0. 05); DCFH-DA method was used to detect intracellular levels of reactive oxygen species (ROS) in SKOV3 cells. It was found that LPA reduced the intracellular ROS level treated with DOX (P<0. 05). Our preliminarily study showed the effect of LPA in the apoptosis of ovarian cancer treated with DOX, which may provide a reference for the drug therapy of ovarian cancer targeting LPA.

Decrease of the DXR-induced genotoxicity and nongenotoxic effects of Theobroma cacao revealed by micronucleus assay / Diminuição da genotoxicidade induzida por DXR e efeitos não genotóxicos de Theobroma cacao revelados pelo ensaio de micronúcleo

This study evaluated the genotoxicity of lyophilized glycolic extract of Theobroma cacao Linné seeds (TCL), using the micronucleus assay in bone marrow of mice. The interaction between TCL and doxorubicin (DXR) was also analyzed. Experimental groups were evaluated 24-48 h after treatment with N-Nitroso-N-ethylurea (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0.5-2 g/kg), and TCL (2 g/kg) in combination with DXR (antigenotoxic assays). Analysis of micronucleated polychromatic erythrocytes (MNPCEs) showed no significant differences between all the treatment doses of TCL and NaCl control. Mice experimentally treated with DXR and NEU significantly induced MNPCEs. However, a significant reduction of MNPCEs was also observed when TCL was administered in combination with the chemotherapeutic agent DXR. The analysis of the PCE/NCE ratio revealed no significant differences between the NaCl control, all doses of TCL, and DXR. However, there were significant differences in the PCE/NCE ratio between positive NEU control and all other treatments. The PCE/NCE ratio observed after treatment with TCL and DXR showed significant differences and intermediate values to controls (NaCl and NEU). This study suggests absence of genotoxicity and cytotoxicity of TCL, regardless of dose, sex, and time. TCL reduced genotoxic effects induced by DXR, suggesting potential antigenotoxic effects.

Este estudo avaliou a genotoxicidade do extrato glicólico liofilizado de sementes de Theobroma cacao Linné (TCL), usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre TCL e doxorrubicina (DXR) foi também analisada. Grupos experimentais foram avaliados 24-48 h após tratamento com N-Nitroso-N-etilureia (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0,5-2 g/kg), e TCL (2 g/kg) em combinação com DXR (ensaio antigenotóxico). As análises de eritrócitos policromáticos micronucleados (EPCMNs) não mostraram diferenças significantes entre todas as doses de tratamento do TCL e o controle NaCl. Camundongos experimentalmente tratados com DXR e NEU induziram significativamente EPCMNs. Contudo, uma redução significante de EPCMNs foi também observada quando TCL foi administrada em combinação com o agente quimioterapêutico DXR. As análises da relação EPC/ENC (eritrócito policromático/eritrócito normocromático) revelaram ausência de diferenças significantes entre o controle NaCl, todas as doses de TCL e DXR. Contudo, houve diferenças significantes na relação EPC/ENC entre o controle positivo NEU e todos os outros tratamento. A relação ECP/ENC observada após o tratamento com TCL e DXR mostrou diferenças significantes e valores intermediários aos controles (NaCl e NEU). Este estudo sugere ausência de genotoxicidade e citotoxicidade de TCL, independentemente da dose, sexo e tempo. TCL reduziu os efeitos genotóxicos induzidos por DXR, sugerindo potencial efeitos antigenotóxicos.

Optimization of Preparation Technology of Near-infrared Light Response-based Doxorubicin Hydrochloride Nano-liposomes / 中国药房

OBJECTIVE： To establish a methaod for content determination of doxorubicin hydrochloride nano-liposomes， and to optimize its preparation technology. METHODS： The contents of doxorubicin hydrochloride nano-liposomes was determined by UV spectrophotometry. The membrane dispersion method was used to prepare doxorubicin hydrochloride nano-liposomes. Using particle size， encapsulation efficiency and drug-loading amount as indexes， the weight ratio of phospholipid to drug （mg/mg）， the weight ratio of phospholipid to cholesterol （mg/mg） and ultrasonic time （min） as factors， central composite design-response surface methodology was used to optimize the preparation technology. The photothermal conversion effect of doxorubicin hydrochloride nano-liposomes was investigated by near infrared irradiation. RESULTS： The linear range of doxorubicin hydrochloride were 1.01-16.16 μg/mL（r＝0.999 7）； precision， stability and reproducibility tests were all in line with the requirments of Chinese Pharmacopoeia. The optimal preparation technology included that the weight ratio of phospholipid to drug was 13.30 ∶ 1（mg/mg）； the weight ratio of phospholipid to cholesterol was 4.09 ∶ 1 （mg/mg）； the ultrasonic time was 10 min. Under this technology， the particle size and drug-loading amount of doxorubicin hydrochloride nano-liposomes were （200.5±25.1） nm and （11.02±0.20）％， relative errors of which to predicted value （196.3 nm， 10.68％） were 1.82％ and 1.63％. The consistency between measured value and predicted value was good. Doxorubicin hydrochloride nano-liposomes exhibited concentration- dependent and time-dependent photothermal conversion characteristics under near infrared irradiation at 808 nm. CONCLUSIONS： Established method is simple and good accuracy. The optimized preparation technology is simple and feasible.

Preparation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes and determination of encapsulation efficiency / 中草药

Objective To develop a method to determine the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. Methods In this paper, the thin-film rehydration method was used to prepare doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. Liposomes and free drugs were separated by dialysis, gel microcolumn centrifugation, and ultra-high speed centrifugation. The content of free drugs and drugs in liposomes was determined by HPLC, and the entrapment efficiency of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes was calculated. Results The optimal formulation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes was DPPC-DSPE-PEG2000-TAIII-DOX with a molar ratio of 5:1:1:1. The liposomes prepared using thin-film rehydration method had a well-defined spherical shape with a size of (55.4 ± 0.40) nm, a PDI of (0.20 ± 0.02), and a weakly negative zeta potential of (-17.4 ± 0.6) mV. The excipients in the liposomal formulation can be well separated from doxorubicin hydrochloride and timosaponin AIII in the selected chromatographic conditions. The calibrated linear curve of doxorubicin hydrochloride was within 24.9-498.0 μg/mL (r = 0.999 9) and that of timosaponin AIII was within 50.55-1 011.0 μg/mL (r = 0.999 6). Free doxorubicin hydrochloride and timosaponin AIII were well separated from liposome by gel microcolumn centrifugation, and the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AIII was (85.12 ± 1.27)% and (76.51 ± 0.46)% respectively. Conclusion The thin-film dispersion- method can be used for the preparation of doxorubicin hydrochloride and timosaponin AIII co-loaded liposomes. The method of gel microcolumn centrifugation is accurate, reproducible, simple, and suitable for determination of the encapsulation efficiency of co-loaded liposomes.

Effect of Ciwujia Injection on decrease of cardiac contractile function induced by doxorubicin hydrochloride / 中草药

Objective To investigate the protective effect of Ciwujia Injection (CI) on the decrease of cardiac contractile function induced by doxorubicin hydrochloride, and provide experimental basis for its clinical application. Methods The cardiotoxicity model in rats was established by ip injection of doxorubicin hydrochloride with a dose of 2.5 mg/kg every week for 6 weeks, and the total cumulative dose was 15 mg/kg. Rats were equally divided into five groups with 10 rats in each group (e.g. model group, three test groups of CI, positive group). In addition, a normal control group of 10 rats was injected intraperitoneally with normal saline (NS) once a week for 6 weeks. Normal control and model group were given NS intravenously. The test groups were given CI and the positive group was given Shenqi Fuzheng Injection. The CI and Shenqi Fuzheng Injection were infused by tail vein for 1 h when doxorubicin hydrochloride administrated. The parameters of left ventricular systolic function including ejection fraction (EF), shortening fraction (FS), the maximal rate of rise of left ventricular pressure (+LVdp/dtmax), and heart geometric shape were measured. Cardiomyocyte apoptosis was measured with immunohistochemistry, apoptosis-related protein and oxidative stress factors such as SOD, MDA, and LPO were measured with enzyme-linked immune sorbent assay, or chemical method. The ultrastructural changes of myocardium under electron microscope were observed. Results Compared with model group, after CI treatment (50, 100, and 200 mg/kg), the cardiac systolic function was improved; the declined EF, FS, and + LVdp/dtmax increased (P < 0.05, 0.01). The heart geometric shape was improved significantly with CI treatment, the expanded LVIDs and LVVs decreased (P < 0.05, 0.01). CI can inhibit myocardial cell apoptosis (P < 0.01), the myocardial apoptosis rate decreased (P < 0.01), and the Bax/Bcl-2 decreased (P < 0.01). In addition, CI (100 and 200 mg/kg) can decline the generation of LPO, MDA and increase the activity of SOD. Conclusion CI can effectively relieve cardiotoxicity induced by doxorubicin hydrochloride in rats, increase cardiac function, improve the cardiac configuration, and reduce the damage of myocardial ultrastructure. The mechanism may be related to decreasing oxidative stress and inhibition of apoptosis in impaired myocardial cells.

Variety methods study on hemolytic effect of colored injection doxorubicin hydrochloride in vitro / 药物评价研究

Objective The visual inspection method were not appropriate to perform a hemolysis evaluation for colored injection like doxorubicin hydrochloride,this article adopted three methods to evaluate the hemolysis test of doxorubicin hydrochloride in vitro and provide reference for clinical drug safety.Methods Using rabbit erythrocytes as experimental object,the durg concentration 4.0 and 2.0 mg/mL was chosen which range of clinical concentration and preclinical safety evaluation concentration,to evaluate the hemolysis test of doxorubicin hydrochloride injection with blood analyzer test,direct colorimetric assay,and indirect colorimetric assay.Results The evaluation results of three different methods were very consistent.The tube's hemolysis rate of 4.0 mg/mL dose was far greater than 5％,which means serious hemolysis;Only 0.1 mL tube of 2.0 mg/mL dose (according to the drug concentration equal to 0.5 mL tube of 0.4 mg/mL drug concentration) without hemolysis occurring,the other tubes' hemolysis rates were far greater than 5％,which means serious hemolysis.Conclusion The hemolysis phenomenon may occur when 2.0 mg/mL dose of doxorubicin hydrochloride solution for iv injection is used in clinic and dilution (final concentration not more than 0.4 mg/mL) is recommended.

Decrease of the DXR-induced genotoxicity and nongenotoxic effects of Theobroma cacao revealed by micronucleus assay

Abstract This study evaluated the genotoxicity of lyophilized glycolic extract of Theobroma cacao Linné seeds (TCL), using the micronucleus assay in bone marrow of mice. The interaction between TCL and doxorubicin (DXR) was also analyzed. Experimental groups were evaluated 24-48 h after treatment with N-Nitroso-N-ethylurea (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0.5-2 g/kg), and TCL (2 g/kg) in combination with DXR (antigenotoxic assays). Analysis of micronucleated polychromatic erythrocytes (MNPCEs) showed no significant differences between all the treatment doses of TCL and NaCl control. Mice experimentally treated with DXR and NEU significantly induced MNPCEs. However, a significant reduction of MNPCEs was also observed when TCL was administered in combination with the chemotherapeutic agent DXR. The analysis of the PCE/NCE ratio revealed no significant differences between the NaCl control, all doses of TCL, and DXR. However, there were significant differences in the PCE/NCE ratio between positive NEU control and all other treatments. The PCE/NCE ratio observed after treatment with TCL and DXR showed significant differences and intermediate values to controls (NaCl and NEU). This study suggests absence of genotoxicity and cytotoxicity of TCL, regardless of dose, sex, and time. TCL reduced genotoxic effects induced by DXR, suggesting potential antigenotoxic effects.

Resumo Este estudo avaliou a genotoxicidade do extrato glicólico liofilizado de sementes de Theobroma cacao Linné (TCL), usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre TCL e doxorrubicina (DXR) foi também analisada. Grupos experimentais foram avaliados 24-48 h após tratamento com N-Nitroso-N-etilureia (NEU: 50 mg/kg), DXR (5 mg/kg), NaCl (145 mM), TCL (0,5-2 g/kg), e TCL (2 g/kg) em combinação com DXR (ensaio antigenotóxico). As análises de eritrócitos policromáticos micronucleados (EPCMNs) não mostraram diferenças significantes entre todas as doses de tratamento do TCL e o controle NaCl. Camundongos experimentalmente tratados com DXR e NEU induziram significativamente EPCMNs. Contudo, uma redução significante de EPCMNs foi também observada quando TCL foi administrada em combinação com o agente quimioterapêutico DXR. As análises da relação EPC/ENC (eritrócito policromático/eritrócito normocromático) revelaram ausência de diferenças significantes entre o controle NaCl, todas as doses de TCL e DXR. Contudo, houve diferenças significantes na relação EPC/ENC entre o controle positivo NEU e todos os outros tratamento. A relação ECP/ENC observada após o tratamento com TCL e DXR mostrou diferenças significantes e valores intermediários aos controles (NaCl e NEU). Este estudo sugere ausência de genotoxicidade e citotoxicidade de TCL, independentemente da dose, sexo e tempo. TCL reduziu os efeitos genotóxicos induzidos por DXR, sugerindo potencial efeitos antigenotóxicos.

Graphene quantum dot modified glassy carbon electrode for the determination of doxorubicin hydrochloride in human plasma$ / 药物分析学报

Low toxic graphene quantum dot (GQD) was synthesized by pyrolyzing citric acid in alkaline solution and characterized by ultraviolet–visible (UV–vis) spectroscopy, X-ray diffraction (XRD), atomic force micro-scopy (AFM), spectrofluorimetery and dynamic light scattering (DLS) techniques. GQD was used for electrode modification and electro-oxidation of doxorubicin (DOX) at low potential. A substantial de-crease in the overvoltage ( ? 0.56 V) of the DOX oxidation reaction (compared to ordinary electrodes) was observed using GQD as coating of glassy carbon electrode (GCE). Differential pulse voltammetry was used to evaluate the analytical performance of DOX in the presence of phosphate buffer solution (pH 4.0) and good limit of detection was obtained by the proposed sensor. Such ability of GQD to promote the DOX electron-transfer reaction suggests great promise for its application as an electrochemical sensor.

Redox-Responsive Vesicles Prepared from Supramolecular 2-Hydroxypropyl-β-Cyclodextrin and Ferrocene Derivative for Drug Delivery / 分析化学

A novel hydrophobic ferrocene polymer ( P-Fc) was synthesized successfully. Taken the advantage of 2-hydroxypropyl-β-cyclodextrin ( HP-β-CD ) with hydrophilic outside and hydrophobic inside, P-Fc was successfully enveloped in the cavity of HP-β-CD to form macromolecular vesicle by a self-assembly process. The structure and morphology of the macromolecular vesicle was characterized by FTIR, 1HNMR, SEM and CV. Rhodamine 6G (R6G) and doxorubicin hydrochloride (DOX) was used as target drugs and loaded in the vesicles successfully. After added the oxidant, ferrocene was oxidized to dicyclopentadienyl iron, which destroyed the vesicles successfully. The rapid directional release of the drugs was realized. The maximum loading values of R6G and DOX were 6. 89 and 39. 06 μg/mg, respectively. The release rates were 73. 7%and 88. 2%, respectively. It would increase the utilization level of the drugs and enhance their efficiency.

Determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection by HPLC-ELSD / 中国药学杂志

OBJECTIVE: To develop an HPLC-ELSD method for determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection. METHODS: The column was Waters Symmetry 300 C18(4.6 mm × 150 mm, 5 μm; pore size; 300 Å). The mobile phase was methonal-tetrahydrofuran-0.17 mol · L-1 ammonium acetate(93: 6:1). The flow rate was 1.0 mL · min-1. The column temperature was 25℃ and the injection volume was 10 μL. The ELSD conditions were as follows: Alltech 2000ES ELSD detector; drift temperature; 110℃; rate; 2.6 L · min-1. RESULTS: This method had good specificity. The linear ranges of the calibration curves for MPEG-DSPE and HSPC were 0.03-0.48 mg · mL-1 (r=0.999 8) and 0.1-1.0 mg · mL-1 (r=0.9998), respectively. The average recovery rates of MPEG-DSPE and HSPC were 100.0% (n=3 × 3) and 101.0% (n=3 × 3), respectively. The LODs of MPEG-DSPE and HSPC were 13 and 52 ng, respectively. The repeatability and intermediate precision of MPEG-DSPE were 0.9% (n=5) and ≤1.9% (n=3), respectively. The repeatability and intermediate precision of HSPC were 1.1% (n=5) and ≤1.3%(n=3), respectively. CONCLUSION: The established method is accurate, reliable, repeatable and suitable for the determination of MPEG-DSPE and HSPC in doxorubicin hydrochloride liposome injection.

Study on influencing factors of the in vitro release rate of doxorubicin hydrochloride sustained release implants for glioma / 中国药学杂志

OBJECTIVE: To study the factors affecting the in vitro release rate of doxorubicin hydrochloride sustained release implants for glioma to provide information for pharmacodynamic evaluation in animals. METHODS: The implants were prepared by incorporating doxorubicin hydrochloride into poly(lactide-co-glycolide) (PLGA) matrix. The in vitro release rate of the implants was determined by UV. The copolymer ratio and molecular weight (Mw) of PLGA, and the drug loading were examined to study their effects on the in vitro release rate of the implants. RESULTS: The release rate of 10% drug-loaded implants was greater than that of 5% drug-loaded ones, and it was enhanced by an increase in the proportion of copolymerized glycollic acid in PLGA. The cumulative release rate of the implants with LA-GA ratio of 50: 50 was 90% at 35 d, while that of the implants with LA-GA ratio of 75: 25 was only about 60%. The molecular weight of PLGA was inversely proportional to the release rate of the implants. The release rate of the implants with Mw of 20748 was greater than that with Mw of 30834. CONCLUSION: The factors affecting the in vitro release rate of the implants include the copolymer ratio of PLGA, the molecular weight of PLGA and the drug loading, among which the copolymer ratio and molecular weight of PLGA are the major factors. The drug release rate of the implants can be well controlled by regulating the copolymer ratio and molecular weight of PLGA matrix. Copyright 2012 by the Chinese Pharmaceutical Association.