Role of formulation composition in folate receptor-targeted liposomal doxorubicin delivery to acute myelogenous leukemia cells

Targeted drug delivery has the potential to improve the efficacy of a therapeutic agent while reducing its side effects. The folate receptor type beta (FR-beta) is a cell surface marker selectively expressed in the leukemic cells of approximately 70% of acute myeloid leukemia (AML) patients. Upregulation of FR-beta may also be selectively induced in AML cells by treatment with all-trans-retinoic acid (ATRA). In this study, the role of formulation composition in FR-targeted liposomal doxorubicin (DOX) delivery to AML cells was investigated. Liposomal formulations with a variable percentage of folate-polyethylene glycol distearoyl phosphatidylethanolamine (f-PEG-DSPE) were synthesized and evaluated for FR-beta-targeted DOX delivery in MV4-11 AML cells in vitro and for their pharmacokinetic properties in vivo. The formulation containing 0.5 mol % f-PEG-DSPE exhibited the highest efficiency of cellular uptake and in vitro cytotoxicity, as well as a long systemic circulation time in mice. In MV4-11 cells, the binding and cytotoxicity of FR-targeted liposomal DOX based on this formulation was also enhanced by ATRA-induced FR-beta upregulation.     

Albumin-conjugated PEG liposome enhances tumor distribution of liposomal doxorubicin in rats

To evaluate the effect of coupling of recombinant human serum albumin (rHSA) onto the surface of poly(ethylene glycol)-modified liposome (PEG liposome) on the in vivo disposition characteristics of liposomal doxorubicin (DXR), the pharmacokinetics and tissue distribution of DXR were evaluated after intravenous administration of rHSA-modified PEG (rHSA/PEG) liposomal DXR into tumor-bearing rats. rHSA/PEG liposome prepared using a hetero-bifunctional cross-linker, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), efficiently encapsulated DXR (over 95%). rHSA/PEG liposomal DXR showed longer blood-circulating property than PEG liposomal DXR and the hepatic and splenic clearances of rHSA/PEG liposomal DXR were significantly smaller than those of PEG liposomal DXR. It was also demonstrated that the disposition of DXR to the heart, one of the organs for DXR-related side-effects, was significantly smaller than free DXR. Furthermore, the tumor accumulation of rHSA/PEG liposomal DXR was significantly larger than that of PEG liposomal DXR. The "therapeutic index", a criterion for therapeutic outcome, for rHSA/PEG liposomal DXR was significantly higher than PEG liposomal DXR. These results clearly indicate that rHSA-conjugation onto the surface of PEG liposome would be a useful approach to increase the effectiveness and safety of PEG liposomal DXR.     

In vitro cellular accumulation and cytotoxicity of liposomal and conventional formulations of daunorubicin and doxorubicin in resistant K562 cells

Previous investigations have indicated the possibility to circumvent multidrug resistance (MDR) by incorporation of an anthracycline into liposomes. We examined the in vitro cytotoxicity and cellular drug accumulation of the anthracyclines daunorubicin and doxorubicin compared with the commercially available liposomal formulations DaunoXome and Caelyx in human myelogenous leukemia K562 cells. The drug-sensitive parental K562/K line was compared with the P-glykoprotein (P-gp)-expressing cell lines K562/Dnr and K562/Vcr. Two cell lines with reduced levels of topoisomerase II (K562/Nov and K562/Ida) were also included. The cytotoxicity was determined by fluorometric microculture cytotoxicity assay and the cellular drug levels were determined by high performance liquid chromatograghy. There was a strong inverse correlation between P-gp levels and cellular drug accumulation (rho = -0.83, p = 0.04) and cytotoxicity (rho = -0.95, p = 0.01) of daunorubicin. Also the cytotoxicity of DaunoXome and doxorubicin was related to P-gp levels (rho = -0.96, p = 0.01 and rho = -0.90, p = 0.07, respectively). Caelyx did not show any cytotoxic effect due to impaired cellular uptake of the pegylated liposome. Regardless of the P-gp levels of the treated cells, DaunoXome showed the same cytotoxic effect despite lower intracellular accumulation (range 22-47%), compared with conventional daunorubicin.     

乳酸-羟基乙酸共聚物微球的研究进展

特殊注射剂是一类复杂的载药系统,如脂质体、微球和注射混悬剂等,尽管上市的产品不多,但经济价值巨大,部分产品的市场表现甚至超过了新分子实体。分别对全球范围内已上市的注射脂质体、注射微球和注射混悬剂作简要介绍,从技术特征、市场效应和临床需求3个方面,介绍这3种特殊注射剂的发展现状和发展潜力。

     

Application of human pancreatic carcinoid BON cells for receptor-targeted drug development

In our previous study, we found that several tumor cell lines displayed high receptor-specific binding affinity, one of which, the human pancreatic carcinoid BON cell line, demonstrates high affinity binding of the bombesin (BN) and somatostatin (SST) receptor-specific ligands. In the present study, BON cells, as a representative model, were further applied to evaluate various peptide analogs and cytotoxic receptor-targeted peptide conjugates. We observed quick ligand–receptor internalization in BON cells as well as high binding affinity. Furthermore, BON cells have high expression of multidrug resistance-associated genes (MDR1) and show camptothecin (CPT) resistance. Various receptor-specific cytotoxic conjugates were synthesized and evaluated in the BON cell model via in vitro and in vivo studies. We found that all the tested conjugates displayed potent antitumor ability in xenografts. Especially, the CPT conjugates, CPT-SST, and CPT-BN, are most likely to increase sensitivity to CPT-resistant BON cells. Our findings suggest that appropriately defined tumor cell lines may provide physiologically relevant cell-based evaluations of novel peptide analogs and receptor-targeted chemotherapeutics.     

Mechanism of resistance to oxidative stress in doxorubicin resistant cells

Doxorubicin (DOX) is an anthracycline drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3-1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.     

Feasibility study of cavitation-induced liposomal doxorubicin release in an AT2 Dunning rat tumor model

Background: Targeted and triggered release of liposomal drug using heat or ultrasound represents a promising treatment modality able to increase the therapeutic-totoxicity ratio of encapsulated drugs.

Purpose: To study the ability for high-intensity focused ultrasound to induce liposomal drug release mainly by focused inertial cavitation in vitro and in an animal model.

Methods: A 1 MHz ultrasound setup has been developed for in vitro and in vivo drug release from a specific liposomal doxorubicin formulation at a target cavitation dose.

Results: Controlled cavitation at 1 MHz was applied within the tumors 48 hours after liposome injection according to preliminary pharmacokinetic study. A small non-significant therapeutic effect of US-liposomal treatment was observed compared to liposomes alone suggesting no beneficial effect of ultrasound in the current setup.

Conclusion: The in vitro study provided a suitable ultrasound setup for delivering a cavitation dose appropriate for safe liposomal drug release. However, when converting to an in vivo model, no therapeutic benefit was observed. This may be due to a number of reasons, one of which may be the difficulty in converting in vitro findings to an in vivo model. In light of these findings, we discuss important design features for future studies.     

Feasibility study of cavitation-induced liposomal doxorubicin release in an AT2 Dunning rat tumor model

Background: Targeted and triggered release of liposomal drug using heat or ultrasound represents a promising treatment modality able to increase the therapeutic-totoxicity ratio of encapsulated drugs. Purpose: To study the ability for high-intensity focused ultrasound to induce liposomal drug release mainly by focused inertial cavitation in vitro and in an animal model. Methods: A 1 MHz ultrasound setup has been developed for in vitro and in vivo drug release from a specific liposomal doxorubicin formulation at a target cavitation dose. Results: Controlled cavitation at 1 MHz was applied within the tumors 48 hours after liposome injection according to preliminary pharmacokinetic study. A small non-significant therapeutic effect of US-liposomal treatment was observed compared to liposomes alone suggesting no beneficial effect of ultrasound in the current setup. Conclusion: The in vitro study provided a suitable ultrasound setup for delivering a cavitation dose appropriate for safe liposomal drug release. However, when converting to an in vivo model, no therapeutic benefit was observed. This may be due to a number of reasons, one of which may be the difficulty in converting in vitro findings to an in vivo model. In light of these findings, we discuss important design features for future studies.     

Bombesin peptide antagonist for target-selective delivery of liposomal doxorubicin on cancer cells

Purpose: This study addresses novel peptide modified liposomal doxorubicin to specifically target tissues overexpressing bombesin (BN) receptors.

Methods: DOTA-(AEEA)₂-peptides containing the [7–14]bombesin and the new BN-AA1 sequence have been synthesized to compare their binding properties and in serum stabilities. The amphiphilic peptide derivative (MonY-BN-AA1) containing BN-AA1, a hydrophobic moiety, polyethylenglycole (PEG), and diethylenetriaminepentaacetate (DTPA), has been synthesized. Liposomes have been obtained by mixing of MonY-BN-AA1 with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

Results: Both ¹¹¹In labeled peptide derivatives present nanomolar Kd to PC-3 cells. ¹⁷⁷Lu labeled peptide DOTA-(AEEA)₂-BN-AA1 is very stable (half-life 414.1?h), while DOTA-(AEEA)₂-BN, shows a half-life of 15.5?h. In vivo studies on the therapeutic efficacy of DSPC/MonY-BN-AA1/Dox in comparison to DSPC/MonY-BN/Dox, were performed in PC-3 xenograft bearing mice. Both formulations showed similar tumor growth inhibition (TGI) compared to control animals treated with non-targeted DSPC/Dox liposomes or saline solution. For DSPC/MonY-BN-AA1/Dox the maximum effect was observed 19 days after treatment.

Conclusions: DSPC/MonY-BN-AA1/Dox nanovectors confirm the ability to selectively target and provide therapeutic efficacy in mice. The lack of receptor activation and possible acute biological side effects provided by using the AA1 antagonist bombesin sequence should provide safe working conditions for further development of this class of drug delivery vehicles.     

Recent advances in tumor vasculature targeting using liposomal drug delivery systems

Tumor vessels possess unique physiological features that might be exploited for improved drug delivery. The targeting of liposomal anticancer drugs to tumor vasculature is increasingly recognized as an effective strategy to obtain superior therapeutic efficacy with limited host toxicity compared with conventional treatments. This review introduces recent advances in the field of liposomal targeting of tumor vasculature, along with new approaches that can be used in the design and optimization of liposomal delivery systems. In addition, cationic liposome is focused on as a promising carrier for achieving efficient vascular targeting. The clinical implications are discussed of several approaches using a single liposomal anticancer drug formulation: dual targeting, vascular targeting (targeting tumor endothelial cells) and tumor targeting (targeting tumor cells).     

肿瘤转移靶向肽修饰的阿霉素脂质体对高转移性乳腺癌细胞的靶向特异性研究

肿瘤转移日渐成为肿瘤治疗的重要靶标。本研究采用肿瘤转移靶向肽（TMT）与脂质材料（PEG-DSPE）偶联获得靶向化合物（TMT-PEG-DSPE）,用以构建靶向阿霉素脂质体（TMT-LS-DOX）。结果表明,TMT-LS-DOX呈现出良好的药剂学性质。选用高转移性乳腺癌细胞（MDA-MB-435S和MDA-MB-231）对该转移特异性递送系统进行评价,采用非转移性乳腺癌细胞（MCF-7）作为对照。游离TMT多肽浓度达100μg/mL时仍未显示出细胞毒性。与MCF-7相比,MDA-MB-435S及MDA-MB-231细胞对TMT-LS-DOX摄取增加,并经受体竞争性实验证明该促进作用由TMT介导。因此,TMT修饰的纳米载体可能成为增加化疗药物对高转移性乳腺癌特异性的一种新策略。     

Novel tetrapeptide, RGDF, mediated tumor specific liposomal doxorubicin (DOX) preparations

Arginine-glycine-aspartate (RGD) has been shown to possess a strong affinity for the integrins overexpressed in tumor cells, especially during tumor invasion, angiogenesis and metasis. Based on work from others, a novel tetrapeptide, arginine-glycine-aspartate-phenylanaline (RGDF), has been designed and studied as a homing device to direct liposomal doxorubicin (DOX) to tumor cells in this work. In order to incorporate RGDF into liposomal DOX preparations, RGDF was conjugated with three different fatty alcohols to achieve RGDF-fatty alcohol conjugates. Glycine-glycine-aspartate-phenylanaline (GGDF)-lauryl alcohol conjugate was synthesized as a negative control. RGDF-fatty alcohol conjugates (RGDFO(CH(2))(n)CH(3)) and GGDF-lauryl alcohol conjugate (L-GGDFC12-DOX) incorporated liposomal preparations were obtained by first preparing liposomes using the film dispersion method followed by loading DOX using a transmembrane pH gradient method. Because of their amphipathic nature, RGDF- or GGDF-fatty alcohol conjugates are expected to be readily incorporated into liposomes with their fatty alkanyl chains being intercalated between fatty acyl chains of liposomal bilayers and the hydrophilic peptide moiety (RGDF or GGDF) being anchored on the surface of liposomes. The particle size and zeta potential of liposomal DOX preparations containing RGDF-fatty alcohol conjugate (L-RGDF-DOXs) or L-GGDFC12-DOX were measured, and their morphology was studied using transmission electron microscopy. In vitro DOX release profile from RGDF incorporated liposomal DOX was measured. The antitumor activities of RGDF incorporated liposomal DOX preparations were evaluated in ICR mice inoculated with sarcoma S(180), which is known to express α(v)β(3) integrin. Both conventional liposomal DOX preparation (L-DOX) without RGDFO(CH(2))(n)CH(3) and L-GGDFC12-DOX were used as negative controls. Our results showed improved tumor growth inhibition with L-RGDF-DOXs over doxorubicin hydrochloride solution, L-DOX and L-GGDFC12-DOX. Pathological examination of tumor biopsy demonstrated that L-RGDF-DOXs induced enhanced tumor cell death in comparison to negative controls. Pharmacokinetic studies showed that the concentrations of DOX found in tumor sites were increased by 1.7-4.5-fold when liposomal DOX preparation containing RGDF-lauryl alcohol conjugate (L-RGDFC12-DOX) was administered in comparison to when L-GGDFC12-DOX or doxorubicin hydrochloride solution was administered. The concentrations of DOX found in the heart, which is the main site of toxic effects of DOX, were significantly reduced when L-RGDFC12-DOX was administered in comparison to when L-GGDFC12-DOX or doxorubicin hydrochloride solution was administered.     

MEN15658: a new promising anti-tumoral drug active on resistant tumor cells

We describe the identification of MEN15658, a molecule characterized by a promising cytotoxic effect against human tumor cell lines, including platinum- and anthracycline-resistant ovarian carcinoma. MEN15658 induces p53 accumulation, and activation of gadd-45, p21, c-fos and bcl-2 family genes in human ovarian carcinoma A2780 cell line. The compound causes a block in S phase of the cell cycle, inducing apoptotic cell death, thus suggesting an involvement of DNA damage in the MEN15658 effect on tumor cells. The anti-tumoral activity observed against the human ovarian carcinoma A2780 cell line xenotransplanted in nude mice makes this compound a new promising anti-tumoral drug.     

A novel peptide probe for imaging and targeted delivery of liposomal doxorubicin to lung tumor

Targeted delivery of imaging agents and therapeutics to tumors would provide early detection and increased therapeutic efficacy against cancer. Here we have screened a phage-displayed peptide library to identify peptides that selectively bind to lung tumor cells. Evaluation of individual phage clones after screening revealed that a phage clone displaying the CSNIDARAC peptide bound to H460 lung tumor cells at higher extent than other phage clones. The synthetic CSNIDARAC peptide strongly bound to H460 cells and was efficiently internalized into the cells, while little binding of a control peptide was seen. It also preferentially bound to other lung tumor cell lines as compared to cells of different tumor types. In vivo imaging of lung tumor was achieved by homing of fluorescence dye-labeled CSNIDARAC peptide to the tumor after intravenous injection into mice. Ex vivo imaging and microscopic analysis of isolated organs further demonstrated the targeting of CSNIDARAC peptide to tumor. The CSNIDARAC peptide-targeted and doxorubicin-loaded liposomes inhibited the tumor growth more efficiently than untargeted liposomes or free doxorubicin. In vivo imaging of fluorescence dye-labeled liposomes demonstrated selective homing of the CSNIDARAC-liposomes to tumor. In the same context, higher levels of doxorubicin and apoptosis in tumor tissue were observed when treated with the targeted liposomes than untargeted liposomes or free doxorubicin. These results suggest that the CSNIDARAC peptide is a promising targeting probe that is able to direct imaging agents and therapeutics to lung tumor.     

Transferrin as a drug carrier: Cytotoxicity,cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells

Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin–transferrin conjugate (DOX–TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX–TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX–TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX–TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX–TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX–TRF conjugate and free drug.     

盐酸多柔比星脂质体注射液犬体内药物动力学研究

目的研究Beagle犬静脉滴注多柔比星脂质体注射液后,犬体内脂质体包合多柔比星(L-DOX)、游离多柔比星(F-DOX)和多柔比星总量(T-DOX)药动学特点。方法采用甲醇沉淀蛋白处理血浆样品以纯化血浆中T-DOX;采用固相萃取分离血浆中L-DOX和F-DOX。采用LC-MS/MS方法测定处理后样品中T-DOX、L-DOX、F-DOX浓度。结果 Beagle犬静脉滴注多柔比星脂质体注射液0.3、1.0和2.0 mg·kg~(-1)后,犬体内T-DOX的ρ_(max)分别为4.35、18.84和29.92 mg·L~(-1);AUC_(0-t)分别为144.0、546.7和1204 mg·h·L~(-1);L-DOX的ρ_(max)分别为4.77、19.93和32.17 mg·L~(-1);AUC_(0-t)分别为153.9、574.7和1 294 mg·h·L~(-1);F-DOX的ρ_(max)分别为13.00、26.53和40.70μg·L~(-1);AUC_(0-t)分别为158.2、445.2和1171μg·h·L~(-1)。结论犬给予盐酸多柔比星脂质体注射液后,血浆中主要存在形体为L-DOX。在0.3～2.0 mg·kg~(-1)剂量范围内,犬体内N-DOX和F-DOX均呈线性药动学特征。     

Ultrasound enhanced antitumor activity of liposomal doxorubicin in mice

Liposomal encapsulation of doxorubicin (DXR) improves tumor accumulation and reduces adverse effects. One possible strategy for further optimization of this delivery technology would be to design the liposome carrier to release its content within the tumor tissue in response to specific stimuli such as ultrasound (US). In this study, the tumor uptake properties and therapeutic efficacy of 1,2 distearoyl-sn-glycero-3-phosphatidylethanolamine-based liposomes containing DXR were investigated in nude mice bearing tumor xenografts. The liposomal DXR formulation alone showed no inhibitory effect on tumor growth. However, upon exposure to low frequency US in situ inhibition of tumor growth was demonstrated.