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1.
Int J Parasitol Drugs Drug Resist ; 23: 120-129, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38043188

ABSTRACT

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.


Subject(s)
Parasites , Toxoplasma , Humans , Animals , Toxoplasma/genetics , Topotecan/pharmacology , Topotecan/metabolism , DNA Repair , DNA Damage
2.
Biomolecules ; 12(9)2022 09 02.
Article in English | MEDLINE | ID: mdl-36139065

ABSTRACT

Immune dysfunction and pro-oncogenic inflammation play critical roles in malignant progression and non-response to immunotherapy for hepatocellular carcinoma (HCC). In particular, PD-1/PD-L1 blockade therapy could induce durable tumor remissions and improve the prognosis of patients to a certain extent. However, PD-L1, as a promising biomarker, has limited knowledge about its relevance to tumor microenvironment (TME) characterization and endogenous inflammatory immune responses. In this study, we systematically investigated and characterized the important intercommunication of PD-L1 with immunosuppressive TME and inflammatory response activity in HCC and predicted promising therapeutic drugs to improve the current therapeutic strategy for specific patients. We identified aberrant expression patterns of PD-L1 in HCC and completely different clinical and molecular characteristics among the PD-L1 subgroups. PD-L1 positively associated with immunosuppressive macrophages and macrophage-derived cytokines, which may contribute to the polarization of macrophages. Moreover, inflammatory response activity exhibited significant differences between high and low PD-L1 expression groups and had robust positive correlativity of the infiltration level of tumor-associated macrophages. Notably, given the immunosuppressive and inflammatory microenvironment in HCC, we screened four candidate drugs, including dasatinib, vemurafenib, topotecan and AZD6482, and corroborated in two pharmacogenomics databases, which might have potential therapeutic implications in specific HCC patients. Our results enhanced the understanding of linkage in PD-L1 expression patterns with macrophages and inflammation, which may provide new insight into the pathogenic mechanisms and potential therapeutic strategy for HCC.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , B7-H1 Antigen/metabolism , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cytokines/metabolism , Dasatinib/therapeutic use , Humans , Immunosuppression Therapy , Inflammation/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Macrophages/metabolism , Programmed Cell Death 1 Receptor/metabolism , Topotecan/metabolism , Topotecan/therapeutic use , Tumor Microenvironment , Vemurafenib/metabolism , Vemurafenib/therapeutic use
3.
Protoplasma ; 259(6): 1455-1466, 2022 Nov.
Article in English | MEDLINE | ID: mdl-35195768

ABSTRACT

The drug development process is one of the important aspects of medical biology. The classical lead identification strategy in the way of drug development based on animal cell is time-consuming, expensive and involving ethical issues. The following study aims to develop a novel plant-based screening of drugs. Study shows the efficacy of certain anti-cancerous drugs (Pemetrexed, 5-Fluorouracil, Methotrexate, Topotecan and Etoposide) on a plant-based (Lathyrus sativus L.) system. Two important characteristics of cancer cells were observed in the colchicine-treated polyploid cell and the callus, where the chromosome numbers were unusual and the division of cells were uncontrolled respectively. With increasing concentration, the drugs significantly reduced the mitotic index, ploidy level and callus growth. Increasing Pemetrexed concentration decreased the plant DHFR activity. A decrease in total RNA content was observed in 5-FU and Methotrexate with increasing concentrations of the drugs. Etoposide and Topotecan inhibited plant topoisomerase II and topoisomerase I activities, which was justified through plasmid nicking and comet assay, respectively. Molecular and biochemical study revealed similar results to the animal system. The in silico study had been done, and the structural similarity of drug binding domains of L. sativus and human beings had also been established. The binding site of the selected drugs to the domains of plant target proteins was also determined. Experimental results are significant in terms of the efficacy of known anti-cancerous drugs on the plant-based system. The proposed assay system is a cost-effective, convenient and less time-consuming process for primary screening of anti-cancerous lead molecules.


Subject(s)
Lathyrus , Colchicine/metabolism , DNA Topoisomerases, Type I/metabolism , DNA Topoisomerases, Type II/metabolism , Etoposide/pharmacology , Fluorouracil/metabolism , Humans , Lathyrus/chemistry , Lathyrus/genetics , Lathyrus/metabolism , Methotrexate/metabolism , Methotrexate/pharmacology , Pemetrexed/metabolism , Plant Proteins/metabolism , RNA/metabolism , Topotecan/metabolism
4.
Ophthalmic Genet ; 41(4): 397-400, 2020 08.
Article in English | MEDLINE | ID: mdl-32490703

ABSTRACT

BACKGROUND: . Intravitreal administration of topotecan shows activity against tumor vitreous seeding in the conservative treatment of retinoblastoma, a malignant tumor originated in the retina of small children. Adequate storage of the intravitreal topotecan solution would allow immediate availability for patients at health care institutions. The goal of the work was to address the stability of the intravitreal topotecan formulation upon reconstitution. MATERIALS AND METHODS: . Intravitreal topotecan solutions were reconstituted (at a concentration of 0.2 mg topotecan in 1 mL saline solution vehicle, aliquoted in 1 mL plastic syringes) and stored either frozen or at room temperature for different times. Topotecan content was analyzed at time zero and at different conditions using a high performance liquid chromatography method to quantify topotecan lactone (active) and to detect its pH-dependent hydrolysis product, the open carboxylate. RESULTS: . We found that intravitreal topotecan syringes remained stable at room temperature at least for 24 h, at least for 167 days upon stored frozen at -20°C, and up to 8 h after thawing at day 6. The degradation carboxylate product did not appear in the analyzed thawed samples during the whole study. CONCLUSIONS: . This study confirms the stability of frozen intravitreal topotecan syringes and will help optimize the use of this chemotherapy modality at institutions with low resources. Storage of aliquots will also help reduce personnel exposure to chemotherapy at hospital pharmacies.


Subject(s)
Drug Stability , Drug Storage/standards , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/metabolism , Topotecan/chemistry , Topotecan/metabolism , Humans , Intravitreal Injections , Topoisomerase I Inhibitors/analysis , Topotecan/analysis
5.
Bioorg Med Chem ; 28(1): 115234, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31831297

ABSTRACT

Spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1) is a debilitating neurological disease that is caused by the mutation the Tyrosyl-DNA phosphodiesterase 1 (TDP1) DNA repair enzyme. The crucial His493 in TDP1's binding site is replaced with an arginine amino acid residue rendering the enzyme dysfunctional. A virtual screen was performed against the homology model of SCAN1 and seventeen compounds were identified and tested in a novel SCAN1 specific biochemical assay. Six compounds showed activity with IC50 values between 3.5 and 25.1 µM. The most active ligand 5 (3.5 µM) is a dicoumarin followed by a close structural analogue 6 at 6.0 µM. A less potent series of ß-carbolines (14 and 15) was found with potency in the mid-teens. According to molecular modelling an excellent fit for the active ligands into the binding pocket is predicted. To the best of our knowledge, data on inhibitors of the mutant form of TDP1 has not been reported previously. The virtual hits were also tested for wild type TDP1 activity and all six SCAN1 inhibitors are potent for the former, e.g., ligand 5 has a measured IC50 at 99 nM. In the last decade, TDP1 is considered as a promising target for adjuvant therapy against cancer in combination with Topoisomerase 1 poisons. The active ligands are mostly non-toxic to cancer cell lines A-549, T98G and MCF-7 as well as the immortalized WI-38 human fetal lung cells. Furthermore, ligands 5 and 7, show promising synergy in conjunction with topotecan, a clinically used topoisomerase 1 anticancer drug. The active ligands 5, 7, 14 and 15 have a good balance of the physicochemical properties required for oral bioavailability making the excellent candidates for further development.


Subject(s)
Enzyme Inhibitors/chemistry , Molecular Docking Simulation , Phosphoric Diester Hydrolases/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line , Cell Survival/drug effects , Coumarins/chemistry , Coumarins/metabolism , Coumarins/pharmacology , Drug Design , Drug Synergism , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Ligands , Mutation , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/genetics , Protein Structure, Tertiary , Topotecan/chemistry , Topotecan/metabolism , Topotecan/pharmacology
6.
Mater Sci Eng C Mater Biol Appl ; 103: 109799, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31349439

ABSTRACT

Retinoblastoma (Rb) is the most common primary malignant intraocular tumor in children which develops from the retinal stem cells. Systemic chemotherapy is the typical therapeutic treatment and though most children survive Rb, they often lose their vision, or the eye needs to be enucleated. Regarding to the pure availability of the target tumor by systemic chemotherapy, the local anticancer drug administration would be advantageous to increase the local drug concentration and minimize adverse side effects of chemotherapy. The present paper describes a new hydrogel implant enabled to deliver therapeutically active doses of low molecular weight hydrophilic antitumor drugs topotecan and vincristine. The hydrogel implant is proposed as bi-layered with an inner hydrophilic layer from 2-hydroxyethyl methacrylate (HEMA) serving as a reservoir of the chemotherapeutic agent and an outer hydrophobic layer from 2-ethoxyethyl methacrylate (EOEMA) acting as a barrier to protect the surrounding vascularized tissue against cytotoxicity of the delivered chemotherapeutics. The experiments with enucleated pig eyes demonstrated the ability of tested drugs to diffuse through sclera and reach the vitreous humor. HEMA-based hydrogels were examined in terms of sorption, release and transport properties, showing the possibility of adjusting the loading capacity and diffusion of the drugs by the degree of crosslinking. The EOEMA-based gels proved to be an inert for drug sorption and diffusion. A chorioallantoic membrane assay demonstrated excellent biocompatibility of unloaded hydrogels, and in vitro experiments confirmed significant cytotoxicity of drug-loaded hydrogels against a Rb cell line; 2 days for those topotecan-loaded and a minimum of 6 days for vincristine-loaded hydrogels. The bi-layered hydrogel implant can be considered promising for local administration of active agents to eye-globe for the treatment of Rb and also other ocular disorders.


Subject(s)
Drug Carriers/chemistry , Hydrogels/chemistry , Animals , Cell Line, Tumor , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Drug Stability , Eye/drug effects , Eye/metabolism , Humans , Kinetics , Methacrylates/chemistry , Prostheses and Implants , Retinoblastoma/metabolism , Retinoblastoma/pathology , Swine , Topotecan/chemistry , Topotecan/metabolism , Topotecan/pharmacology , Vincristine/chemistry , Vincristine/metabolism , Vincristine/pharmacology
8.
Eur J Pharm Sci ; 123: 515-523, 2018 Oct 15.
Article in English | MEDLINE | ID: mdl-30086354

ABSTRACT

Topotecan is a relatively large, planar, asymmetric and polar molecule with a lactone moiety. In neutral or basic aqueous solutions, this ring opens forming the carboxylate form of Topotecan that is biologically inactive and uncapable of passively cross membranes. Nevertheless, despite this inability to cross membranes at this form, Topotecan may still be able to interact with phospholipid bilayers, disturbing them. In this context, phospholipid models, mimicking normal (DMPC at pH 7.4) and cancer cell lipid membranes (DMPC:DMPS (5:1) at pH 6.5), were used to assess structural modifications upon interaction with Topotecan. Langmuir isotherms of monolayers coupled with Brewster angle microscopy, differential scanning calorimetry of liposomes and X-ray scattering of small and wide angle of stacked multilayers were used as complementary techniques. The overall results show that the interaction of Topotecan with lipid membranes is deeply conditioned by their composition and that Topotecan seems to have a preferential interaction with the glycerol backbone of phosphatidylcholine phospholipids.


Subject(s)
Cell Membrane/drug effects , Dimyristoylphosphatidylcholine/chemistry , Membranes, Artificial , Neoplasms/drug therapy , Topoisomerase I Inhibitors/pharmacology , Topotecan/pharmacology , Animals , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Membrane Permeability , Dimyristoylphosphatidylcholine/metabolism , Humans , Models, Biological , Molecular Structure , Neoplasms/chemistry , Neoplasms/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/metabolism , Topotecan/chemistry , Topotecan/metabolism
9.
J Pharm Pharmacol ; 69(10): 1318-1326, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28703281

ABSTRACT

OBJECTIVES: This work aimed to evaluate semisolid formulations containing topotecan (TPT) loaded nanostructured lipid carriers (NLC) for topical treatment of skin cancers, as TPT is effective against a variety of tumours. A formulation which increases TPT skin permeation would be extremely desirable. METHODS: TPT-NLC were prepared and incorporated in hydrogels with hydroxyethyl cellulose and chitosan (TPT-NLC-HEC and TPT-NLC-Ch, respectively). Control formulations were obtained by dispersing TPT in HEC and Ch hydrogels (TPT-HEC and TPT-Ch). KEY FINDINGS: TPT-NLC-HEC and TPT-NLC-Ch showed to maintain the drug and nanoparticle dispersions stable for up to 30 days. When nanoparticles were incorporated into gels, TPT release was significantly decreased (P < 0.05). Still, TPT-NLC-HEC increased 2.37 times permeation compared with TPT-HEC (11.9 and 5.0 µg/cm2 , respectively). Cell culture experiments with B16F10 melanoma demonstrated that nanoencapsulation significantly increased TPT cytotoxicity (P < 0.05). TPT-NLC was more toxic than free TPT, with IC50 value of 5.74 µg/ml, whereas free TPT had an IC50 > 20 µg/ml. As skin permeated values of TPT from developed formulation (TPT-NLC) were superior to melanoma IC50, it can be extrapolated that chemotherapeutic permeated amounts may be sufficient for a therapeutic effect. CONCLUSIONS: TPT-NLC-HEC may be a valuable tool for the topical treatment of skin cancers.


Subject(s)
Drug Carriers/administration & dosage , Melanoma, Experimental/drug therapy , Nanoparticles/administration & dosage , Skin Absorption/physiology , Skin Neoplasms/drug therapy , Topotecan/administration & dosage , Administration, Topical , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/metabolism , Cell Survival/drug effects , Cell Survival/physiology , Dose-Response Relationship, Drug , Drug Carriers/metabolism , Hydrogels/administration & dosage , Hydrogels/metabolism , Lipids/administration & dosage , Melanoma, Experimental/metabolism , Mice , Nanoparticles/metabolism , Organ Culture Techniques , Skin Absorption/drug effects , Skin Neoplasms/metabolism , Swine , Topoisomerase I Inhibitors/administration & dosage , Topoisomerase I Inhibitors/metabolism , Topotecan/metabolism , Treatment Outcome
10.
J Pharm Sci ; 106(4): 1149-1161, 2017 04.
Article in English | MEDLINE | ID: mdl-28007561

ABSTRACT

Actively loaded liposomal formulations of anticancer agents have been widely explored due to their high drug encapsulation efficiencies and prolonged drug retention. Mathematical models to predict and optimize drug loading and release kinetics from these nanoparticle formulations would be useful in their development and may allow researchers to tune release profiles. Such models must account for the driving forces as influenced by the physicochemical properties of the drug and the microenvironment, and the liposomal barrier properties. This study employed mechanistic modeling to describe the active liposomal loading and release kinetics of the anticancer agent topotecan (TPT). The model incorporates ammonia transport resulting in generation of a pH gradient, TPT dimerization, TPT lactone ring-opening and -closing interconversion kinetics, chloride transport, and transport of TPT-chloride ion-pairs to describe the active loading and release kinetics of TPT in the presence of varying chloride concentrations. Model-based predictions of the kinetics of active loading at varying loading concentrations of TPT and release under dynamic dialysis conditions were in reasonable agreement with experiments. These findings identify key attributes to consider in optimizing and predicting loading and release of liposomal TPT that may also be applicable to liposomal formulations of other weakly basic pharmaceuticals.


Subject(s)
Models, Chemical , Topotecan/metabolism , Biological Transport/physiology , Liposomes , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/metabolism , Topotecan/chemistry
11.
J Photochem Photobiol B ; 163: 185-93, 2016 Oct.
Article in English | MEDLINE | ID: mdl-27585365

ABSTRACT

Protein - ligand interactions play pivotal role in almost all the biological processes occurring in living organisms, and therefore such studies hold immense importance from the standpoint of rational drug design and development. In this study the binding of the topoisomerase I inhibitor drug, topotecan to hemoglobin was probed using various biophysical and microcalorimetry techniques. Spectrofluorimetric data confirmed the static nature of the quenching mechanism of the protein induced by the drug. Significant conformational changes in the protein were ascertained from circular dichroism and three dimensional fluorescence results. Synchronous fluorescence study revealed an increase in the polarity around the Trp residues of the protein while atomic force microscopy study enabled to obtain images of the bound molecules. Isothermal titration calorimetry studies indicated an exothermic binding with a negative Gibbs energy change; ionic strength variation suggested a greater contribution from non-polyelectrolytic forces in the binding process. Differential scanning calorimetry studies indicated an increased thermal stabilization of the protein upon topotecan binding which is also in close agreement with the results obtained from absorbance and circular dichroism melting studies. Overall this manuscript presents results on the molecular interaction from structural and energetic perspectives providing an in depth insight into drug-protein interaction.


Subject(s)
Antineoplastic Agents/metabolism , Hemoglobins/metabolism , Topotecan/metabolism , Binding Sites , Hemoglobins/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Protein Binding , Protein Conformation , Thermodynamics , Transition Temperature
12.
Cancer Res ; 75(18): 3879-89, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26199091

ABSTRACT

While a small number of plasma membrane ABC transporters can export chemotherapeutic drugs and confer drug resistance, it is unknown whether these transporters are expressed or functional in less therapeutically tractable cancers such as Group 3 (G3) medulloblastoma. Herein we show that among this class of drug transporters, only ABCG2 was expressed at highly increased levels in human G3 medulloblastoma and a mouse model of this disease. In the mouse model, Abcg2 protein was expressed at the plasma membrane where it functioned as expected on the basis of export of prototypical substrates. By screening ABC substrates against mouse G3 medulloblastoma tumorspheres in vitro, we found that Abcg2 inhibition could potentiate responses to the clinically used drug topotecan, producing a more than 9-fold suppression of cell proliferation. Extended studies in vivo in this model confirmed that Abcg2 inhibition was sufficient to enhance antiproliferative responses to topotecan, producing a significant survival advantage compared with subjects treated with topotecan alone. Our findings offer a preclinical proof of concept for blockade of ABCG2 transporter activity as a strategy to empower chemotherapeutic responses in G3 medulloblastoma.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Cerebellar Neoplasms/drug therapy , Drug Resistance, Neoplasm/physiology , Medulloblastoma/drug therapy , Neoplasm Proteins/physiology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/analysis , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/biosynthesis , Adenine/analogs & derivatives , Adenine/metabolism , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Biological Transport, Active , Cell Division/drug effects , Cell Line, Tumor , Cell Membrane/chemistry , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/pathology , High-Throughput Screening Assays , Humans , Medulloblastoma/classification , Medulloblastoma/metabolism , Medulloblastoma/pathology , Mice , Mice, Knockout , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Propionates/pharmacology , Protoporphyrins/biosynthesis , Quinolines/pharmacology , Topoisomerase I Inhibitors/metabolism , Topoisomerase I Inhibitors/pharmacology , Topotecan/metabolism , Topotecan/pharmacology
13.
Biochem Pharmacol ; 91(2): 144-56, 2014 Sep 15.
Article in English | MEDLINE | ID: mdl-25058526

ABSTRACT

The overexpression of ATP-binding cassette (ABC) transporters often leads to the development of multidrug resistance (MDR), which is the major factor contributing to the failure of chemotherapy. The objective of this study was to investigate the enhancement of CEP-33779, a small-molecule inhibitor of Janus kinase 2 (JAK2), on the efficacy of conventional chemotherapeutic agents in MDR cells with overexpression of P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Our results showed that CEP-33779, at nontoxic concentrations, significantly sensitized ABCB1 overexpressing MDR cells to its anticancer substrates. CEP-33779 significantly increased intracellular accumulation and decreased the efflux of doxorubicin by inhibiting the ABCB1 transport function. Furthermore, CEP-33779 did not alter the expression of ABCB1 both at protein and mRNA levels but did stimulate the activity of ABCB1 ATPase. CEP-33779 was predicted to bind within the large hydrophobic cavity of homology modeled ABCB1. In addition, the down-regulation of JAK2 by shRNA altered neither the expression of ABCB1 nor the cytotoxic effect of chemotherapeutic agents in ABCB1-overexpressing cells. Significantly, CEP-33779 enhanced the efficacy of vincristine against the ABCB1-overexpressing and drug resistant KBv200 cell xenograft in nude mice. In conclusion, we conclude that CEP-33779 enhances the efficacy of substrate drugs in ABCB1-overexpressing cells by directly inhibiting ABCB1 transport function. The findings encouraged to further study on the combination therapy of CEP-33779 with conventional chemotherapeutic agents in ABCB1 mediated-MDR cancer patients.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/drug effects , Pyridines/pharmacology , Triazoles/pharmacology , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Doxorubicin/metabolism , Heterografts , Humans , Mice , Mice, Nude , Molecular Structure , Neoplasms, Experimental , Real-Time Polymerase Chain Reaction , Rhodamine 123/metabolism , Topotecan/administration & dosage , Topotecan/metabolism
14.
Cancer Chemother Pharmacol ; 72(1): 127-38, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23680920

ABSTRACT

PURPOSE: Pharmacokinetic analyses estimate the mean concentration of drug within a given tissue as a function of time, but do not give information about the spatial distribution of drugs within that tissue. Here, we compare the time-dependent spatial distribution of three anticancer drugs within tumors, heart, kidney, liver and brain. METHODS: Mice bearing various xenografts were treated with doxorubicin, mitoxantrone or topotecan. At various times after injection, tumors and samples of heart, kidney, liver and brain were excised. RESULTS: Within solid tumors, the distribution of doxorubicin, mitoxantrone and topotecan was limited to perivascular regions at 10 min after administration and the distance from blood vessels at which drug intensity fell to half was ~25-75 µm. Although drug distribution improved after 3 and 24 h, there remained a significant decrease in drug fluorescence with increasing distance from tumor blood vessels. Drug distribution was relatively uniform in the heart, kidney and liver with substantially greater perivascular drug uptake than in tumors. There was significantly higher total drug fluorescence in the liver than in tumors after 10 min, 3 and 24 h. Little to no drug fluorescence was observed in the brain. CONCLUSIONS: There are marked differences in the spatial distributions of three anticancer drugs within tumor tissue and normal tissues over time, with greater exposure to most normal tissues and limited drug distribution to many cells in tumors. Studies of the spatial distribution of drugs are required to complement pharmacokinetic data in order to better understand and predict drug effects and toxicities.


Subject(s)
Antineoplastic Agents/pharmacokinetics , Doxorubicin/pharmacokinetics , Mitoxantrone/pharmacokinetics , Neoplasms/blood supply , Neoplasms/metabolism , Neovascularization, Pathologic/metabolism , Topotecan/pharmacokinetics , Animals , Antibiotics, Antineoplastic/blood , Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/pharmacokinetics , Antineoplastic Agents/blood , Antineoplastic Agents/metabolism , Cell Line, Tumor , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Coronary Vessels/pathology , Doxorubicin/blood , Doxorubicin/metabolism , Female , Humans , Kidney/blood supply , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Mice , Mice, Nude , Mitoxantrone/blood , Mitoxantrone/metabolism , Myocardium/metabolism , Myocardium/pathology , Neoplasms/drug therapy , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Tissue Distribution , Topoisomerase I Inhibitors/blood , Topoisomerase I Inhibitors/metabolism , Topoisomerase I Inhibitors/pharmacokinetics , Topotecan/blood , Topotecan/metabolism , Xenograft Model Antitumor Assays
15.
PLoS One ; 7(4): e35073, 2012.
Article in English | MEDLINE | ID: mdl-22558117

ABSTRACT

BACKGROUND: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays. PRINCIPAL FINDINGS: LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis. CONCLUSION: LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.


Subject(s)
Antineoplastic Agents/metabolism , Cell Death/drug effects , Oligopeptides/pharmacology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Analysis of Variance , Annexin A5 , Blotting, Western , Cell Line, Tumor , Drug Synergism , Female , Flow Cytometry , Humans , In Vitro Techniques , Oligopeptides/metabolism , Topotecan/metabolism , Transduction, Genetic
16.
PLoS One ; 7(4): e35487, 2012.
Article in English | MEDLINE | ID: mdl-22530032

ABSTRACT

Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing lysosomal photodestruction of normal breast epithelial cells. Thus, MDR modalities including ABCG2-dependent drug sequestration within EVs can be rationally converted to a pharmacologically lethal Trojan horse to selectively eradicate MDR cancer cells.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/metabolism , Drug Resistance, Neoplasm , Neoplasm Proteins/metabolism , Photosensitizing Agents/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Aminoacridines/metabolism , Aminoacridines/pharmacology , Antineoplastic Agents/pharmacology , Biological Transport/drug effects , Breast/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Macrolides/pharmacology , Neoplasm Proteins/genetics , Photosensitizing Agents/pharmacology , Protein Transport , Topotecan/metabolism , Transcription Factors/metabolism
17.
Chem Commun (Camb) ; 48(35): 4205-7, 2012 May 04.
Article in English | MEDLINE | ID: mdl-22441353

ABSTRACT

The intracellular glutathione-triggered release of the anticancer drug topotecan from gold nanoparticles in serum-containing media was directly monitored in real time using a label-free fluorescence live-cell imaging technique.


Subject(s)
Antineoplastic Agents/metabolism , Glutathione/metabolism , Gold/chemistry , Metal Nanoparticles/chemistry , Topotecan/metabolism , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Culture Media , Glutathione/chemistry , Humans , Mice , Microscopy, Confocal , Neoplasms/drug therapy , Topotecan/therapeutic use
18.
Drug Metab Dispos ; 40(3): 602-9, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22190695

ABSTRACT

It has been reported that in vivo biliary clearance can be predicted using sandwich-cultured rat and human hepatocytes. The predicted apparent biliary clearance (CL(bile, app)) from sandwich- cultured rat hepatocytes (SCRH) based on medium concentrations correlates to in vivo CL(bile, app) based on plasma concentrations of angiotensin II receptor blockers (ARBs), HMG-CoA reductase inhibitors (statins), ß-lactam antibiotics, and topotecan. However, the predicted biliary clearance from SCRH was 7- to 300-fold lower than in vivo biliary clearance. We speculated that the process of biliary excretion might not have been evaluated using sandwich-cultured hepatocytes. To evaluate this issue, intrinsic biliary clearance (CL(bile, int)) based on intracellular compound concentrations was evaluated to investigate the in vitro-in vivo correlation of this process among ARBs, statins, ß-lactam antibiotics, and topotecan. Intrinsic biliary clearance in SCRH correlated to in vivo values obtained by constant intravenous infusion of six compounds, but not rosuvastatin and cefmetazole, to rats. Moreover, differences between SCRH and in vivo CL(bile, int) (0.7-6-fold) were much smaller than those of CL(bile, app) (7-300-fold). Therefore, in vivo CL(bile, int) is more accurately reflected using SCRH than CL(bile, app). In conclusion, to predict in vivo biliary clearance more accurately, CL(bile, int) should be evaluated instead of CL(bile, app) between SCRH and in vivo.


Subject(s)
Biliary Tract/metabolism , Hepatocytes/metabolism , Pharmaceutical Preparations/metabolism , Angiotensin Receptor Antagonists/metabolism , Angiotensin Receptor Antagonists/pharmacokinetics , Animals , Biological Transport , Cells, Cultured , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacokinetics , Male , Rats , Rats, Sprague-Dawley , Topotecan/metabolism , beta-Lactams/metabolism
19.
J Oncol Pharm Pract ; 18(2): 222-8, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22075006

ABSTRACT

PURPOSE: DC Bead™ is successfully used for chemoembolization of various liver cancers. The purpose of this study was to determine the loading capacity of the semi-synthetic topoisomerase-1 inhibitor topotecan into the DC Bead™ microspheres under static or agitated conditions and to assess the physicochemical stability over a period of 7 days. METHODS: Commercially available topotecan hydrochloride powder (Hycamtin®) was reconstituted with water for injection to yield a nominal concentration of 1 mg/mL topotecan. Polyvinyl alcohol (PVA)-based microspheres (DC Bead™, 300-500 µm, 2 mL/vial) were mixed with 4 mL of the reconstituted topotecan solution. Vials were stored light protected at room temperature under static or agitated conditions for 7 days (n = 3, for each loading condition). At different time intervals, samples were taken from the excess solution and assayed via a stability-indicating HPLC assay. Drug-loading profiles were determined by measuring the remaining topotecan concentration in the excess solution. RESULTS: Under agitated conditions, topotecan was loaded into the microspheres rapidly after mixing. After 5 min 86.4 ± 0.1% of topotecan was loaded. Under static conditions, drug uptake was slower. Only 65.0 ± 0% were loaded after 5 min; 86.6 ± 0.1% drug uptake was achieved not until 1 h. Over a storage period of 7 days, topotecan remained loaded in the DC Bead™ microspheres at a level of >90%. CONCLUSION: Drug uptake of 4 mg topotecan (1 mg/mL solution) into DC Beads™ was faster under agitated loading conditions. Nevertheless, after 1 h, ∼90% of topotecan was loaded into the DC Bead™ microspheres independent from the type of loading condition. The loading rate remained >90% over the observation period of 7 days and light-protected storage at room temperature. Loading and stability of topotecan-loaded DC Beads™ is suitable and convenient for preparation in a pharmacy-based cytotoxic preparation unit.


Subject(s)
Chemistry, Pharmaceutical/methods , Drug Carriers/metabolism , Microspheres , Polyvinyl Alcohol/metabolism , Topotecan/metabolism , Chromatography, High Pressure Liquid/methods , Drug Carriers/chemistry , Polyvinyl Alcohol/chemistry , Time Factors , Topotecan/chemistry
20.
Nucl Med Biol ; 38(5): 707-14, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21718946

ABSTRACT

INTRODUCTION: Topotecan (TPT) is a camptothecin derivative and is an anticancer drug working as a topoisomerase-I-specific inhibitor. But TPT cannot penetrate through the blood-brain barrier. In this study, we synthesized a new positron emission tomography (PET) probe, [(11)C]TPT, to evaluate the P-glycoprotein (Pgp)- and breast cancer resistance protein (BCRP)-mediated brain penetration of [(11)C]TPT using small-animal PET. METHODS: [(11)C]TPT was synthesized by the reaction of a desmethyl precursor with [(11)C]CH(3)I. In vitro study using [(11)C]TPT was carried out in MES-SA and doxorubicin-resistant MES-SA/Dx5 cells in the presence or absence of elacridar, a specific inhibitor for Pgp and BCRP. The biodistribution of [(11)C]TPT was determined using small-animal PET and the dissection method in mice. RESULTS: The transport of [(11)C]TPT to the extracellular side was determined in MES-SA/Dx5 cells exhibiting the expressions of Pgp and BCRP at high levels. This transport was inhibited by coincubation with elacridar. In Mdr1a/b(-/-)Bcrp1(-/-) mice, PET results indicated that the brain uptake of [(11)C]TPT was about two times higher than that in wild-type mice. Similarly, the brain penetration of [(11)C]TPT in wild-type mice was increased by treatment with elacridar. The radioactivity in the brain of elacridar-treated mice was maintained at a certain level after the injection of [(11)C]TPT, although the radioactivity in the blood decreased with time. CONCLUSIONS: We demonstrated the increase of brain penetration of [(11)C]TPT by deficiency and inhibition of Pgp and BCRP functions using small-animal PET in mice.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Antineoplastic Agents/metabolism , Brain/diagnostic imaging , Brain/metabolism , Neoplasm Proteins/metabolism , Positron-Emission Tomography , Topotecan/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Acridines/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Brain/drug effects , Carbon Radioisotopes , Cell Line, Tumor , Gene Knockout Techniques , Humans , Male , Mice , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Tetrahydroisoquinolines/pharmacology , Topotecan/pharmacokinetics , Topotecan/pharmacology
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