Simultaneous high-performance liquid chromatographic determination of a glucuronyl prodrug of doxorubicin, doxorubicin and its metabolites in human lung tissue

A rapid and sensitive method was developed for the simultaneous determination of the new doxorubicin glucuronide prodrug HMR 1826, the parent drug doxorubicin and its metabolites in human lung tissue samples. Homogenization of frozen tissue samples with the micro-dismembrator was followed by a silver nitrate precipitation step. By removing the exceeding silver ions with sodium chloride further purification steps could be omitted. Compounds were separated by isocratic high-performance liquid chromatography on a LiChrospher 100 RP18 column and a mobile phase consisting of citric acid buffer–acetonitrile–methanol–tetrahydrofuran within 30 min and quantified with fluorescence detection. The method showed good recoveries for all compounds (86–99%) and a linear calibration range of 20 ng/g–80 μg/g for doxorubicin and 1–600 μg/g for HMR 1826.     

Synthesis and antitumor activity of new D-galactose-containing derivatives of doxorubicin

A general scheme of synthesis of antibiotic doxorubicin derivatives is based on the 13-dimethyl ketal of 14-bromodaunorubicin (4). The interaction of 4 with melibiose (5), lactose (6), 3-methoxy-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-4-oxybenzaldehyde (12) or 4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-4-oxybenzaldehyde (13) by reductive alkylation followed by hydrolysis of the corresponding intermediate bromoketals produced 3'-N-[alpha-D-galactopyranosyl-(1-->6)-O-1-deoxy-D-glucit-1-yl]doxorubicin (7), 3'-N-[beta-D-galactopyranosyl-(1-->4)-O-1-deoxy-D-glucit-1-yl]doxorubicin (8), 3'-N-[3"-methoxy-4"-O-(beta-D-galactopyranosyl)-4"-oxybenzyl]doxorubicin (16), and 3'-N-[4"-O-(beta-D-galactopyranosyl)-4"-oxybenzyl]doxorubicin (17). Cytotoxic and antitumor activity of the synthesized drug candidates compared to the parent doxorubicin was studied using various experimental models, in particular, on mice bearing lymphocyte leukemia P-388 at single and multiple i.v. injection regimens.     

Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).     

叶酸介导的普鲁兰多糖- 阿霉素聚合物前药的制备及生物学性能初探

目的:制备叶酸介导的普兰多糖-阿霉素聚合物前药(FA-MP-DOX),实现阿霉素药物的靶向控制释放。方法:将普鲁兰多糖用马来酸酐进行修饰后,通过酰胺键键合阿霉素制备得到普鲁兰多糖-阿霉素(MP-DOX),继而酯键键合叶酸制备得到叶酸介导的普鲁兰多糖-阿霉素聚合物前药(FA-MP-DOX)。红外光谱、核磁共振光谱表征聚合物药物的结构,动态透析法模拟体外释药特性,监测不同pH值聚合物药物中阿霉素的释药特性,同时采用人口腔表皮样癌细胞(KB细胞)测定聚合物药物体系的细胞毒性。结果:①经核磁共振表征FA-MP-DOX聚合物合成完成。②在pH2.5、pH5.0及pH7.4的PBS缓冲体系16h中,阿霉素药物累积释放率分别为49.1%,30.3%和15.3%,证实FA-MP-DOX中阿霉素的释放具有pH依赖性。③细胞实验证实FA-MP-DOX的细胞毒性高于阿霉素和MP-DOX。结论:FA-MP-DOX聚合物药物有望成为阿霉素智能型控释和靶向性药物载体。     

High-performance liquid chromatographic validated assay of doxorubicin in rat plasma and tissues

A specific and selective high-performance liquid chromatography (HPLC) technique that requires few manipulations, and is readily adaptable to analysis for a large series of samples, has been developed for the determination of the concentration of the anticancer drug doxorubicin (DXR) in rat serum and tissues. The biological samples were efficiently deproteinised and resolved from a reversed-phase nucleosil C₁₈ column with fluorescence detection. The validation study of the proposed method was successfully carried out in an assay range of between 5 and 5000 ng/ml and was subsequently implemented in a pharmacokinetic study of DXR in Wistar rats that were treated by intravenous administration of the drug.     

Synthesis and anti-cancer activity of bis-amino-phosphine ligand and its ruthenium(II) complexes

The development of both chemotherapeutic drug resistance as well as adverse side effects suggest that the current chemotherapeutic drugs remain ineffective in treating the various types of cancers. The development of new metallodrugs presenting anti-cancer activity is therefore needed. Ruthenium complexes have gained a great deal of interest due to their promising anti-tumour properties and reduced toxicity in vivo. This study highlighted the effective induction of cell death in a malignant melanoma cell by two novel bis-amino-phosphine ruthenium(II) complexes referred to as GA105 and GA113. The IC₅₀ concentrations were determined for both the complexes, the ligand and cisplatin, for comparison. Both complexes GA105 and GA113 displayed a high anti-cancer selectivity profile as they exhibited low IC₅₀ values of 6.72 µM and 8.76 µM respectively, with low toxicity towards a non-malignant human cell line. The IC₅₀ values obtained for both complexes were lower than that of cisplatin. The new complexes were more effective compared to the free ligand, GA103 (IC₅₀ = >20 µM). Morphological studies on treated cells induced apoptotic features, which with further studies could indicate an intrinsic cell death pathway. Additionally, flow cytometric analysis revealed that the mode of cell death of complex GA113 was apoptosis. The outcomes herein give further insight into the potential use of selected Ru(II) complexes as alternative chemotherapeutic drugs in the future.     

Synthesis and anti-cancer activity of chalcone linked imidazolones

A series of novel chalcone linked imidazolones were prepared and evaluated for their anti-cancer activity against a panel of 53 human tumour cell lines derived from nine different cancer types: leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate and breast. Some of these hybrids (6, 7 and 8) showed good anti-cancer activity with GI₅₀ values ranging from 1.26 to 13.9 μM. When breast carcinoma cells (MCF-7) were treated with 10 μM concentration of compounds TMAC, CA-4, 6 and 8 cell cycle arrest was observed in G2/M phase. Surprisingly, the increased concentration of the same compound to 30 μM caused accumulation of cells in G0/G1 phase of the cell cycle.     

Synthesis and biological evaluation of novel doxorubicin-containing ASGP-R-targeted drug-conjugates

Asialoglycoprotein receptor (ASGP-R) belongs to a wide family of C-type lectins and it is currently regarded as an attractive protein in the field of targeted drug delivery (TDD). It is abundantly expressed in hepatocytes and can be found predominantly on the sinusoidal surface especially of HepG2 cells. Therefore, ASGP-R can be used for the TDD of anticancer therapeutics against HCC and molecular diagnostic tools. To date, a variety of mono- and multivalent selective ASGP-R ligands have been discovered. Although many of these compounds have demonstrated a relatively high binding affinity towards the target, the reported synthetic schemes are not handled, complicated and include many non-trivial steps. In the current study, we describe a convenient and versatile synthetic approach to novel monovalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose fragment as an ASGP-R-recognition “core-head” and well-known nonselective cytostatic – Doxorubicin (Dox). This is the first example of the direct conjugation of a drug molecule to the ASGP-targeted warhead by a really convenient manner via a simple linker sequence. The performed MTS-based biological evaluation in HepG2 cells revealed the novel conjugates as having anticancer activity. Confocal microscopy showed that the molecules readily penetrated HepG2 membrane and were mainly localized within the cytoplasm instead of the nucleus. Per contra, Dox under the same conditions demonstrated good anticancer activity and was predominantly concentrated in the nucleus. Therefore, we speculate that the amide “trigger” that we have used in this study for linker attachment is a sufficiently stable inside the cells to be enzymatically or spontaneously degraded. As a consequence, we did not observe the release of the drug. Ligands containing triggers that are more liable towards endogenous hydrolysis within the tissue of targeting are strongly required.     

Synthesis and delivery activity of new cationic cholesteryl glucosides

Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-methylmorpholinium, and N-methylpiperidinium) linked via β-glucosyl spacer were prepared by alkylation of the corresponding bases with 6-О-mesyl-β-d-cholesteryl glucopyranoside. IC₅₀ values were in the range 20-35 μM for the series of compounds and liposomal formulations with DOPE (1:1) were significantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleotide in cells, and the efficiency of this process was comparable to that of Lipofectamine® 2000. Cationic liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum (up to 30%).     

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C₁₈ solid-phase extraction cartridge, elution from a Spherisorb C₈ reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8±3.5% and 89.1±6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 μg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.     

Detection of doxorubicin and metabolites in cell extracts and in single cells by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 microM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635 +/- 27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61 +/- 13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.     

Synthesis and biological evaluation of Doxorubicin-containing conjugate targeting PSMA

Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), has recently emerged as a prominent biomarker of prostate cancer (PC) and as an attractive protein trap for drug targeting. At the present time, several drugs and molecular diagnostic tools conjugated with selective PSMA ligands are actively evaluated in different preclinical and clinical trials. In the current work, we discuss design, synthesis and a preliminary biological evaluation of PSMA-specific small-molecule carrier equipped by Doxorubicin (Dox). We have introduced an unstable azo-linker between Dox and the carrier hence the designed compound does release the active substance inside cancer cells thereby providing a relatively high Dox concentration in nuclei and a relevant cytotoxic effect. In contrast, we have also synthesized a similar conjugate with a stable amide linker and it did not release the drug at all. This compound was predominantly accumulated in cytoplasm and did not cause cell death. Preliminary in vivo evaluation has showed good efficiency for the degradable conjugate against PC3-PIP(PSMA⁺)-containing xenograft mine. Thus, we have demonstrated that the conjugate can be used as a template to design novel analogues with improved targeting, anticancer activity and lower rate of potential side effects. 3D molecular docking study has also been performed to elucidate the underlying mechanism of binding and to further optimization of the linker area for improving the target affinity.     

Synthesis and anti-cancer activity evaluation of new aurone derivatives

Abstract

In this study, we have synthesized 2-[3- or 4-(2-aryl-2-oxoethoxy)arylidene]benzofuran-3-one derivatives (D1–D38) and evaluated their anti-cancer activities. The final compounds were obtained in multistep synthesis reactions using benzofuranon-3-one derivatives (A1–A4, B) as starting materials which were gained in various synthetic ways. Aurone derivatives (C1–C10) were acquired with the condensation reaction of these starting materials and 3-/4-hydroxybenzaldehyde which were then reacted with α-bromoacetophenones to get final compounds. The anti-cancer activity of the selected compounds was performed by National Cancer Institute (NCI), USA against 60 human tumor cell lines derived from nine neoplastic diseases. Compounds exhibited anti-cancer activity in varying ratios.     

Controlling the thickness of polymeric shell on magnetic nanoparticles loaded with doxorubicin for targeted delivery and MRI contrast agent

The polymeric functionalization of superparamagnetic iron oxides nanoparticles is developed for cancer targeting capability and magnetic resonance imaging. Here the nanoparticles (NP) are decorated through the adsorption of a polymeric layer around the particle surface for the formation of core-shell. The synthesized magnetic nanoparticles (MNPs) are conjugated with fluorescent dye, targeting ligand, and drug molecules for improvement of target specific diagnostic and possible therapeutics applications. In this investigation doxorubicin was loaded into the shell of the MNPs and release study was carried out at different pH. The core-shell structure of magnetic NP coated chitosan matrix was visualized by TEM observation. The cytotoxicity of these magnetic NPs is investigated using MTT assay and receptor mediated internalization by HeLa and NIH3T3 cells are studied by fluorescence microscopy. Moreover, compared with T₂-weighted magnetic resonance imaging (MRI) in the above cells, the synthesized nanoparticles are showed stronger contrast enhancements towards cancer cells.     

Synthesis and characterization of a photoresponsive doxorubicin/combretastatin A4 hybrid prodrug

To explore the application of photoremovable protecting groups (PPGs) in the field of combination chemotherapy, we designed and synthesized a photoresponsive hybrid prodrug 4 that bearing both doxorubicin (DOX) and combretastatin A4 (CA4). Light triggered drug release investigation found that DOX release was mainly accomplished by 405?nm light while CA4 release was mainly triggered by 365?nm light, i.e., prodrug 4 exhibited a quasi-sequential release behavior when a sequential light irradiation strategy was applied. Cell viability evaluation confirmed the increased cytotoxicity of prodrug 4 compared with individual drugs towards MDA-MB-231cells, indicating that a synergistic effect was achieved.     

Glucose-aspirin: Synthesis and in vitro anti-cancer activity studies

Glucose-aspirin (GA) was synthesized by conjugating aspirin (ASA) to glucose. The water solubility and biological activity of GA was studied in comparison to aspirin. The human serum protease activity on the ester showed a slower hydrolysis rate, compared to ASA. Glucose-aspirin was sevenfold more water soluble than aspirin and it was about 8- to 9-fold more active in inhibiting cell growth than aspirin in their anti-cancer cell culture activity on breast (SKBR3), pancreatic (PANC-1), and prostate (PC3) cell lines, whereas the activity was similar on a benign non-cancerous cell line (WI 38). In conclusion, GA is a highly water soluble derivative of aspirin. Although the serum hydrolysis for GA was slower, there was significant anti-cancer activity at the doses studied under the experimental conditions.     

Internalization of an intact doxorubicin immunoconjugate

An immunoconjugate between doxorubicin and anti-(carcinoembryonic antigen) (CEA) was prepared by using aminodextran (M _r=40 000) as the intermediate carrier, and the carbohydrate moiety of the antibody as the linking site. The resulting immunoconjugate was subjected to an in vitro evaluation for the internalization on the target cells (LoVo), and compared to that of unconjugated antibody, as well as the cellular uptake of unconjugated doxorubicin. The internalization was evaluated microscopically by following the translocation of the red fluorescence of doxorubicin and the green fluorescence of the fluorescein-isothiocyanate-labeled goat anti-(mouse Ig) antibody, which visualizes the location of the primary mouse antibody. Anti-CEA monoclonal antibody (NP-4) was found to internalize into LoVo cells. The immunoconjugate made with this antibody was similarly internalized, and the doxorubicin was found to distribute with the primary antibody. The cell surface and cytoplasm were the major compartments of their distribution. These results indicate that the drug molecules were indeed delivered into the cells by the antibody as an intact conjugate. Unconjugated doxorubicin, on the contrary, was quickly absorbed by the cells and concentrated in the nucleus within 30 min, and never showed a distribution in the cytoplasm or cell membrane as in the nucleus by this procedure. The intermediate drug conjugate, doxorubicin-dextran, did not show internalization. The internalization of NP-4 antibody (or the doxorubicin conjugate) was also confirmed by studying the intracellular catabolism of the cell-bound antibody (or conjugate). The release of the degraded antibody by the cells, as differentiated by trichloroacetic acid precipitation techniques, was considered an indication of internalization. Lysosomes were involved in the degradation, since the process was markedly inhibited in the presence of the lysosomal enzyme inhibitor, ammonium chloride.Supported in part by USPHS grant CA 39841 from the NIH, grant EDT-16 from the American Cancer Society, and grant 89-240360-6 from the New Jersey Commission on Science and Technology.     

Synthesis and anti-cancer activity of some novel 5-azacytosine nucleosides

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-D-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-L-threo-pentofuranose were coupled with 5-azacytosine to obtain alpha and beta anomers of nucleosides. All four nucleosides were reduced to the corresponding dihydro derivatives and deblocked to give target compounds. All eight target compounds were evaluated in a series of human cancer cell lines in culture. Only 2'-deoxy-4'-thio-5-azacytidine (3beta) was found to be cytotoxic in all the cell lines and was further evaluated in vivo. Details of the synthesis and biological activity are reported.     

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and ¹H NMR spectra. The polymer–DOX conjugate could self-assemble into spherical nanoparticles (∼150 nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.