ABSTRACT
Over the last decade, upconversion nanoparticles (UCNP) have been widely investigated in nanomedicine due to their high potential as imaging agents in the near-infrared (NIR) optical window of biological tissues. Here, we successfully develop active targeted UCNP as potential probes for dual NIR-NIR fluorescence and radioactive-guided surgery of prostate-specific membrane antigen (PSMA)(+) prostate cancers. We designed a one-pot thermolysis synthesis method to obtain oleic acid-coated spherical NaYF4:Yb,Tm@NaYF4 core/shell UCNP with narrow particle size distribution (30.0 ± 0.1 nm, as estimated by SAXS analysis) and efficient upconversion luminescence. Polyethylene glycol (PEG) ligands bearing different anchoring groups (phosphate, bis- and tetra-phosphonate-based) were synthesized and used to hydrophilize the UCNP. DLS studies led to the selection of a tetra-phosphonate PEG(2000) ligand affording water-dispersible UCNP with sustained colloidal stability in several aqueous media. PSMA-targeting ligands (i.e., glutamate-urea-lysine derivatives called KuEs) and fluorescent or radiolabelled prosthetic groups were grafted onto the UCNP surface by strain-promoted azide-alkyne cycloaddition (SPAAC). These UCNP, coated with 10 or 100% surface density of KuE ligands, did not induce cytotoxicity over 24 h incubation in LNCaP-Luc or PC3-Luc prostate cancer cell lines or in human fibroblasts for any of the concentrations evaluated. Competitive binding assays and flow cytometry demonstrated the excellent affinity of UCNP@KuE for PSMA-positive LNCaP-Luc cells compared with non-targeted UCNP@CO2H. Furthermore, the binding of UCNP@KuE to prostate tumour cells was positively correlated with the surface density of PSMA-targeting ligands and maintained after 125I-radiolabelling. Finally, a preliminary biodistribution study in LNCaP-Luc-bearing mice demonstrated the radiochemical stability of non-targeted [125I]UCNP paving the way for future in vivo assessments.
Subject(s)
Antigens, Surface/metabolism , Coated Materials, Biocompatible/chemistry , Glutamate Carboxypeptidase II/metabolism , Magnetite Nanoparticles/chemistry , Animals , Antigens, Surface/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Coated Materials, Biocompatible/metabolism , Coated Materials, Biocompatible/pharmacology , Coated Materials, Biocompatible/therapeutic use , Cycloaddition Reaction , Fluorides/chemistry , Glutamate Carboxypeptidase II/chemistry , Humans , Ligands , Magnetite Nanoparticles/therapeutic use , Magnetite Nanoparticles/toxicity , Male , Mice , Oleic Acids/chemistry , Optical Imaging , Particle Size , Polyethylene Glycols/chemistry , Prostatic Neoplasms/diagnostic imaging , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/surgery , Thulium/chemistry , Tissue Distribution , Ytterbium/chemistry , Yttrium/chemistryABSTRACT
Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. Surgical resection with wide margins remains the mainstay of treatment. To address the lack of therapy, our strategy aims to increase anticancer drugs targeting and delivery in the tumour, by leveraging specific chondrosarcoma hallmarks: an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia. A dual targeted therapy for chondrosarcoma was investigated by conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions which exhibit a high affinity for polyanionic sites of proteoglycans (PGs), the major components of the chondrosarcoma extracellular matrix. Based on preclinical results, an imidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study. A series of 27 QA-phosphoramide mustard conjugates, differing by the type of QA function and the length of the alkyl linker, was yielded by a common multi-step sequence involving phosphorylation of a key 2-nitroimidazole alcohol. Then, a screening was realized by surface plasmon resonance technology to assess biomolecular interactions between QA derivatives and aggrecan, the most abundant PG in chondrosarcoma. Results revealed that affinity depends more on the type of QA function, than on the linker length. Moreover, the presence of a benzyl group enhanced affinity to aggrecan. Twelve compounds were shortlisted and evaluated for antiproliferative activity (i.e., growth inhibiting concentration 50), under normoxic and hypoxic conditions using the human extraskeletal myeloid chondrosarcoma cell line (HEMC-SS). For all prodrugs, hypoxic selectivity was maintained and even increased, compared with the lead. From this study, compound 31f emerged as the most effective PG-targeted HAPs with a dissociation constant of 2.10⯵M in the SPR experiment, a hypoxia cytotoxicity ratio of 24 and an efficient reductive cleavage under chemical and enzymatic conditions.
Subject(s)
Antineoplastic Agents/pharmacology , Bone Neoplasms/drug therapy , Chondrosarcoma/drug therapy , Prodrugs/pharmacology , Proteoglycans/metabolism , Quaternary Ammonium Compounds/pharmacology , Aggrecans/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Chondrosarcoma/metabolism , Chondrosarcoma/pathology , Humans , Molecular Targeted Therapy , Oxygen/metabolism , Prodrugs/chemistry , Prodrugs/metabolism , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/metabolism , Structure-Activity Relationship , Tumor Hypoxia/drug effectsABSTRACT
The present study aimed at developing an original pre-column HPLC assay allowing rapid characterization of nitric oxide (NO) scavengers from complex plant extracts. Sodium nitroprusside (SNP) was employed as a NO donor and spiked with an aqueous extract from Aloysia triphylla leaves prior to HPLC analysis. Relying on the ability of radical scavenging constituents to be oxidized upon reaction with radicals, this assay successfully allowed direct identification of three potential NO scavengers, including verbascoside, isoverbascoside, and luteolin-7-O-diglucuronide. These three phenolics were also individually assessed for their NO scavenging activities by using a Griess colorimetric assay. With respective IC50 values of 56 ± 4, 51 ± 3, and 69 ± 5 µg/mL, verbascoside, isoverbascoside, and luteolin-7-O-diglucuronide were all reported as potent NO scavenging compounds, confirming the efficiency of the SNP spiking HPLC assay. The present method can, thus, be considered as a valuable and effective approach for speeding up the discovery of NO scavenging constituents.
Subject(s)
Chromatography, High Pressure Liquid/methods , Nitric Oxide/chemistry , Plant Leaves/chemistry , Verbenaceae/chemistry , Antioxidants/chemistry , Flavones/chemistry , Glucosides/chemistry , Phenols/chemistry , Plant Extracts/chemistryABSTRACT
Nitrogen mustards, such as chlorambucil (CLB), can cause adverse side-effects due to ubiquitous distribution in non-target organs. To minimize this toxicity, strategies of tumor-targeting drug delivery have been developed, where a cytotoxic warhead is linked to a tumor-cell-specific small ligand. Malignant cells exhibit marked glucose avidity and an accelerated metabolism by aerobic glycolysis, known as the Warburg effect, and recognized as a hallmark of cancer. A targeting approach exploiting the Warburg effect by conjugation of CLB to 2-fluoro-2-deoxyglucose (FDG) was previously reported and identified two peracetylated glucoconjugates 2 and 3 with promising antitumor activities in vivo. These results prompted us to investigate the importance of the spacer in this tumor-targeting glucose-based conjugates. Here we report the chemical synthesis and an in vitro cytotoxicity evaluation, using a 5-member panel of human tumor cell lines and human fibroblasts, of 16 new CLB glucoconjugates in which the alkylating drug is attached to the C-1 position of FDG via different linkages. We studied the structure-activity relationships in the linker, and evidenced the positive impact of an aromatic linker on in vitro cytotoxicity: compound 51 proved to be the most active FDG-CLB glucoside, characterized by a bis-aromatic spacer tethered to CLB through an amide function.
Subject(s)
Antigens, Neoplasm , Chlorambucil/chemistry , Drug Delivery Systems , Fluorodeoxyglucose F18/chemistry , Fluorodeoxyglucose F18/pharmacology , Antigens, Neoplasm/chemistry , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Agents, Alkylating/toxicity , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival , Chlorambucil/chemical synthesis , Chlorambucil/pharmacology , Fluorodeoxyglucose F18/chemical synthesis , Fluorodeoxyglucose F18/toxicity , Humans , Inhibitory Concentration 50 , Molecular Structure , Structure-Activity RelationshipABSTRACT
PURPOSE: This study was conducted during the development of innovative treatment targeting the microenvironment of chondrosarcoma. In this context, MMP inhibitors were conjugated with a quaternary ammonium (QA) function as a targeting ligand to proteoglycans of chondrosarcoma extracellular matrix. Here we report the proof of concept of this strategy applied to the MMP13 inhibitor, doxycycline (Dox). METHODS: A quaternary ammonium derivative of the MMP13 inhibitor doxycycline (QA-Dox) was synthesized, and its anticancer activity was evaluated in the Swarm rat chondrosarcoma (SRC) model compared with the parent drug doxycycline, in vitro and in vivo. In vivo, dox and QA-Dox efficiency was assessed at equimolar doses according to a q4dx4 schedule by monitoring tumour volume by MRI and PG-targeted scintigraphy. Molecular mechanism (MMP13 expression, proteoglycan level) and histology studies were performed on tumours. RESULTS: The link of QA targeting function to Dox maintained the MMP13 inhibitory activity in vitro. Interestingly, the bacteriostatic activity was lost. SRC cells incubated with both drugs were blocked in S and G2 M phases. Tumour growth inhibition (confirmed by histology) was observed for both Dox and QA-Dox. Undesirable blood effects (leukocyte decrease) were reduced when Dox was targeted to tumour tissue using the QA function. CONCLUSIONS: In the SRC model, the MMP13 inhibitor Dox and its QA derivative are promising as adjuvant therapies for chondrosarcoma management.
Subject(s)
Ammonium Compounds/therapeutic use , Chondrosarcoma/drug therapy , Doxycycline/therapeutic use , Ammonium Compounds/administration & dosage , Ammonium Compounds/pharmacology , Chondrosarcoma/pathology , Doxycycline/administration & dosage , Doxycycline/pharmacology , HumansABSTRACT
The proteasome is a promising target in cancer therapy. However, it is ubiquitous and its inhibitors cause side effects. To target melanoma cells we synthesized new peptide aldehyde and vinylsulfone inhibitors of the proteasome conjugated to the melanin-targeting ligand (MTL) derived from radiotracer [(123)I]-N-(2-diethylaminoethyl)benzamide ([(123)I]BZA) or [(125)I]-N-(4-dipropylaminobutyl)-4-iodobenzamide ([(125)I]BZ18). Influence on the cytotoxicity of the benzamide alkyl side chain length and the composition of the amino acid sequence was assessed. Among the conjugates evaluated, compound 16 and 22 presented the highest cytotoxicity (IC(50), 0.71 and 0.64 µM respectively), which persisted in the presence of an MTL derived from N-(dialkylaminoalkylenyl)benzamide residue.
Subject(s)
Aldehydes/pharmacology , Antineoplastic Agents/pharmacology , Melanoma/drug therapy , Molecular Targeted Therapy , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Sulfones/pharmacology , Aldehydes/chemistry , Aldehydes/metabolism , Aldehydes/therapeutic use , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Design , Humans , Inhibitory Concentration 50 , Ligands , Melanins/metabolism , Melanoma/pathology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/therapeutic use , Sulfones/chemistry , Sulfones/metabolism , Sulfones/therapeutic useABSTRACT
Proteasome inhibition is a new strategy in cancer therapy. We synthesized three new peptide aldehyde inhibitors linked to the benzamide derivative structure to use their cytotoxic activity against malignant melanoma cells. Of these, 10 displayed the highest cytotoxicity (0.18 +/- 0.16 microM). A radiosynthesis of the iodine aldehyde was performed. Its drug biodistribution showed that some selectivity of the benzamide group toward malignant melanoma tissue was conserved.
Subject(s)
Antineoplastic Agents/chemical synthesis , Benzamides/chemical synthesis , Oligopeptides/chemical synthesis , Phthalic Acids/chemical synthesis , Proteasome Inhibitors , Radiopharmaceuticals/chemical synthesis , Triazenes/chemical synthesis , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Benzamides/pharmacokinetics , Benzamides/pharmacology , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Iodine Radioisotopes , Leucine/analogs & derivatives , Leucine/chemical synthesis , Leucine/pharmacokinetics , Leucine/pharmacology , Melanoma, Experimental/metabolism , Mice , Neoplasm Transplantation , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Phthalic Acids/pharmacokinetics , Phthalic Acids/pharmacology , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/pharmacology , Stereoisomerism , Tissue Distribution , Transplantation, Heterologous , Triazenes/pharmacokinetics , Triazenes/pharmacologyABSTRACT
The proteasome is a multicatalytic protease that plays a critical role in the cell. The control of proteasomes could, thus, provide a weapon for the treatment of cancer. Therefore, we have synthesized six new peptide aldehyde inhibitors of the proteasome linked to the N-(2-diethylaminoethyl)benzamide (BZA-CO) structure, in order to target the cytotoxic activity to malignant melanoma cells. Biological studies demonstrated the influence of length and composition of the amino acid chain on the cytotoxicity of our compounds. Among them, compound 19 presents the highest cytotoxicity (IC50 = 0.64 +/- 0.07 micromol): this cytotoxicity was maintained in the presence of BZA-CO but decreased 8-fold compared to the control MG132. Fluorescence activated cell sorter (FACS) and cytotoxic activity analysis demonstrated the selectivity of compound 19 for melanoma cells. Finally, western blottings of ubiquitinated proteins in IPC227F cells as well as proteasome assays confirmed that the cytotoxicity was linked to an inhibition of the proteasome activity.