Development of a Supermolecular Radionuclide-Drug Conjugate System for Integrated Radiotheranostics for Non-small Cell Lung Cancer.

Radionuclide-drug conjugates (RDCs) designed from small molecule or nanoplatform shows complementary characteristics. We constructed a new RDC system with integrated merits of small molecule and nanoplatform-based RDCs. Erlotinib was labeled with 131I to construct the bulk of RDC (131I-ER). Floxuridine was mixed with 131I-ER to develop a hydrogen bond-driving supermolecular RDC system (131I-ER-Fu NPs). The carrier-free 131I-ER-Fu NPs supermolecule not only demonstrated integrated merits of small molecule and nanoplatform-based RDC, including clear structure definition, stable quality control, prolonged circulation lifetime, enhanced tumor specificity and retention, and rapidly nontarget clearance, but also exhibited low biological toxicity and stronger antitumor effects. In vivo imaging also revealed its application for tumor localization of nonsmall cell lung cancer (NSCLC) and screening of patients suitable for epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. We considered that 131I-ER-Fu NPs showed potentials as an integrated platform for the radiotheranostics of NSCLC.

Aptamers Entirely Built from Therapeutic Nucleoside Analogues for Targeted Cancer Therapy.

Owing to the specific and high binding affinity of aptamers to their targets, aptamer-drug conjugates (ApDCs) have emerged as a promising drug delivery system for targeted cancer therapy. However, in a conventional ApDC, the aptamer segment usually just serves as a targeting moiety, and only a limited number of drug molecules are sequentially conjugated to the oligonucleotide, giving a relatively low drug loading capacity. To address this challenge, herein we employ four clinically approved nucleoside analogues, including clofarabine (Clo), ara-guanosine (AraG), gemcitabine (Ge), and floxuridine (FdU), to replace all natural nucleosides in aptamer sequences, generating a series of whole drug-constituted DNA-like oligomers that are termed drugtamers. Similar to their parent aptamers, the obtained drugtamers maintain the targeting capability and can specifically bind to the target receptors overexpressed on the cancer cell surface. With 100% drug loading ratio, active targeting capability, and enzyme-mediated release of active therapeutics, our drugtamers can strongly induce the apoptosis of cancer cells and inhibit the tumor progression, which enables a new potential for a better targeted cancer therapy.

Construction of synergistic pH/H2O2-responsive prodrug for prolonging blood circulation and accelerating cellular internalization.

We have developed a real-time and multifunctional doxifluridine-conjugate prodrug (LYX), which involved the preliminary methylfluorescein with 5-fluorouracil linker as protecting group, the targeting biotin unit, and a model therapeutic drug (doxifluridine). The shielding group (5'-DFUR) was found to be effective in prolonging circulation at physiological pH 7.4 and improving accumulation in the acidic microenvironment of the tumor. Based on this strategy, the stability and stimulus responsive properties of prodrug could enhance drug release efficiency and exhibit fewer side effects, thereby providing a unique opportunity for diagnosis and imaging additional analytes or enzymatic activities.

Supramolecular Amphiphiles Based on Pillar[5]arene and Meroterpenoids: Synthesis, Self-Association and Interaction with Floxuridine.

In recent years, meroterpenoids have found wide biomedical application due to their synthetic availability, low toxicity, and biocompatibility. However, these compounds are not used in targeted drug delivery systems due to their high affinity for cell membranes, both healthy and in cancer cells. Using the approach of creating supramolecular amphiphiles, we have developed self-assembling systems based on water-soluble pillar[5]arene and synthetic meroterpenoids containing geraniol, myrtenol, farnesol, and phytol fragments. The resulting systems can be used as universal drug delivery systems. It was shown by turbidimetry that the obtained pillar[5]arene/synthetic meroterpenoid systems do not interact with the model cell membrane at pH = 7.4, but the associates are destroyed at pH = 4.1. In this case, the synthetic meroterpenoid is incorporated into the lipid bilayer of the model membrane. The characteristics of supramolecular self-assembly, association constants and stoichiometry of the most stable pillar[5]arene/synthetic meroterpenoid complexes were established by UV-vis spectroscopy and dynamic light scattering (DLS). It was shown that supramolecular amphiphiles based on pillar[5]arene/synthetic meroterpenoid systems form monodisperse associates in a wide range of concentrations. The inclusion of the antitumor drug 5-fluoro-2'-deoxyuridine (floxuridine) into the structure of the supramolecular associate was demonstrated by DLS, 19F, 2D DOSY NMR spectroscopy.

Evaluation of Floxuridine Oligonucleotide Conjugates Carrying Potential Enhancers of Cellular Uptake.

Conjugation of small molecules such as lipids or receptor ligands to anti-cancer drugs has been used to improve their pharmacological properties. In this work, we studied the biological effects of several small-molecule enhancers into a short oligonucleotide made of five floxuridine units. Specifically, we studied adding cholesterol, palmitic acid, polyethyleneglycol (PEG 1000), folic acid and triantennary N-acetylgalactosamine (GalNAc) as potential enhancers of cellular uptake. As expected, all these molecules increased the internalization efficiency with different degrees depending on the cell line. The conjugates showed antiproliferative activity due to their metabolic activation by nuclease degradation generating floxuridine monophosphate. The cytotoxicity and apoptosis assays showed an increase in the anti-cancer activity of the conjugates related to the floxuridine oligomer, but this effect did not correlate with the internalization results. Palmitic and folic acid conjugates provide the highest antiproliferative activity without having the highest internalization results. On the contrary, cholesterol oligomers that were the best-internalized oligomers had poor antiproliferative activity, even worse than the unmodified floxuridine oligomer. Especially relevant is the effect induced by palmitic and folic acid derivatives generating the most active drugs. These results are of special interest for delivering other therapeutic oligonucleotides.

Bifunctional and Unusual Amino Acid ß- or γ-Ester Prodrugs of Nucleoside Analogues for Improved Affinity to ATB0,+ and Enhanced Metabolic Stability: An Application to Floxuridine.

Floxuridine (FUdR, 5-fluoro-2-deoxyuridine) was widely used in patients with tumor. But the poor activity and severe side effects have been observed in the clinic, which resulted from increased degradation cleavage of FUdR to 5-FU by thymidine phosphorylase and reduced transporter-mediated entry into cells. In this study, we have synthesized a series of l-aspartic acid ß-esters and l-glutamic acid γ-esters of FUdR to improve the metabolic stability of FUdR and target FUdR to cancer cells via amino acid transporter ATB0,+ which was exclusively up-regulated in some cancerous tissue. The uptake mechanism, stability, in vitro/in vivo antiproliferation action, pharmacokinetics, and tissue distribution were studied. The combined results showed the unusual 5'-ß-l-Asp-FUdR possessed a better tumor inhibition rate and a better metabolic stability than FUdR through a ATB0,+-mediated prodrug approach. The present study provided the first proof-of-concept of exploiting ATB0,+ for tumor-selective delivery of nucleoside analogues in the form of prodrug.

Hypoxia-Activated and Indomethacin-Mediated Theranostic Prodrug Releasing Drug On-Demand for Tumor Imaging and Therapy.

A smart theranostic prodrug IMC-FDU-TZBC-NO2, releasing active drug on-demand based on hypoxia-activated and indomethacin-mediated, for solid tumor imaging and efficient therapy was designed. This prodrug was constructed by conjugating chemotherapy drug 5-fluoro-2-deoxyuridine (FDU), targeting moiety indomethacin (IMC), and the hypoxic trigger 4-nitrobenzyl group to a fluorescent dye precursor, which was mediated by IMC and activated by NTR under hypoxic conditions. The fluorescent dye IMC-TZBCM was generated and FDU was released at the same time in tumor cells. The rates and amounts of FDU release and IMC-TZBCM generation were regulated by hypoxia status, and increased with increasing degree of hypoxia. Nevertheless, it is "locked" in normal cells. It combined the advantages of tumor targeting, diagnosis, and chemotherapy functions, showed excellent targeting ability to cancer cells, excellent stability in physiological conditions, high cellular uptake efficiency, and on-demand drug release behavior. The in vitro and in vivo assays demonstrated that IMC-FDU-TZBC-NO2 exhibits enhanced anticancer potency and low side effects. The novel targeted theranostic prodrug activated by hypoxia shows a great potential in cancer therapy.

Nucleoside dimers analogs containing floxuridine and thymidine with unnatural linker groups: synthesis and cancer line studies. Part III.

Two series of novel fluorinated nucleosides dimers with an unnatural 1,2,3-triazole linkage were synthesized. The obtained molecules were prepared using "click" chemistry approach based on copper(I) catalyzed Huisgen azide-alkyne cycloaddition. It was performed between 3'- and 5'-azido-nucleosides as the azide components, and the 3'-O- and 5'-O-propargyl-nucleosides as the alkyne components. Based on analysis of the 3 JHH, 3 JH1'C2 and 3 JH1'C6 we estimated conformational preferences of sugar part and orientation around glycosidic bond. All described nucleosides dimers analogs were characterized by spectroscopic methods and evaluated for their in vitro cytotoxicity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7).

An Organic Afterglow Protheranostic Nanoassembly.

Cancer theranostics holds potential promise for precision medicine; however, most existing theranostic nanoagents are simply developed by doping both therapeutic agents and imaging agent into one particle entity, and thus have an "always-on" pharmaceutical effect and imaging signals regardless of their in vivo location. Herein, the development of an organic afterglow protheranostic nanoassembly (APtN) that specifically activates both the pharmaceutical effect and diagnostic signals in response to a tumor-associated chemical mediator (hydrogen peroxide, H2 O2 ) is reported. APtN comprises an amphiphilic macromolecule and a near-infrared (NIR) dye acting as the H2 O2 -responsive afterglow prodrug and the afterglow initiator, respectively. Such a molecular architecture allows APtN to passively target tumors in living mice, specifically release the anticancer drug in the tumor, and spontaneously generate the uncaged afterglow substrate. Upon NIR light preirradiation, the afterglow initiator generates singlet oxygen to react and subsequently transform the uncaged afterglow substrate into an active self-luminescent form. Thus, the intensity of generated afterglow luminescence is correlated with the drug release status, permitting real-time in vivo monitoring of prodrug activation. This study proposes a background-free design strategy toward activatable cancer theranostics.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells.

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

Prototyping kinase inhibitor-cytotoxin anticancer mutual prodrugs activated by tumour hypoxia: A chemical proof of concept study.

Amide- and ester-linked kinase inhibitor-cytotoxin conjugates were rationally designed and synthesised as prototype hypoxia-activated anticancer mutual prodrugs. Chemical reduction of an aryl nitro trigger moiety was shown to initiate a spontaneous cyclisation/fragmentation reaction that simultaneously released the kinase inhibitor semaxanib (SU5416) and the amine- or alcohol-linked cytotoxin from the prodrugs. Preliminary cell testing and reduction potential measurements support optimisation of the compounds towards tumour-selective mutual prodrugs.

Loading Lovastatin into Camptothecin-Floxuridine Conjugate Nanocapsules for Enhancing Anti-metastatic Efficacy of Cocktail Chemotherapy on Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a malignant and refractory disease with high morbidity and mortality. The TNBC shows no response to hormonal therapy nor targeted therapy due to the lack of known targetable biomarkers. Furthermore, the TNBC also exhibits a high degree of heterogeneity that leads to cancer evolution, drug resistance, metastatic progression, and recurrence, arising from the tumor-initiating properties of cancer stem cells (CSCs). Thus, the development of radical therapeutic regimens with high efficacy and limited side effects is crucial. In this study, we designed an innovative ternary cocktail chemotherapy by using Lovastatin (L)-loaded Janus camptothecin-floxuridine conjugate (CF) nanocapsules (NCs) with ultrahigh drug loading capacity. The obtained LCF NCs were shown to be able to suppress growth of TNBC, including inhibition of growth and metastasis of CSCs, both in vitro and in tumor-bearing mice. Moreover, in animal experiments, the LCF NCs showed sustained and synchronous drug release (half-life > 300 min), 85.2% reduction in pulmonary metastases, and no cancer recurrence during one-month observation post-treatment. Thus, this innovative LCF NC design provides a simple and synergistic strategy for the development of simultaneous triple chemotherapy and could be an efficacious, safe, and amenable choice with higher therapeutic relevance and fewer toxic complications than conventional multidrug delivery systems for TNBC treatment in the future.

Simultaneous determination of gemcitabine prodrug, gemcitabine and its major metabolite 2', 2'-difluorodeoxyuridine in rat plasma by UFLC-MS/MS.

To improve bioavailability and provide resistance to deamination, an array of gemcitabine (dFdC) prodrugs carrying the acyl modifications has been successful in the optimization of pharmacokinetic properties of dFdC, but the reports about 4-N-carbobenzoxy-dFdC (Cbz-dFdC), a dFdC prodrug bearing alkyloxycarbonyl modification, are relatively rare. Notably, in vivo enzymatic hydrolysis was an absolutely essential factor for the activation of these prodrugs, which is correlated with the anti-tumor activity. Therefore, detailed metabolism studies of Cbz-dFdC should be carried out for a more authentic pharmacodynamic evaluation. In order to detect the pharmacokinetic characteristics of Cbz-dFdC, a selective, sensitive and accurate method for the simultaneous determination of Cbz-dFdC, along with dFdC and its major metabolite dFdU in rat plasma was developed and validated using UFLC-MS/MS techniques. Column was at 40 °C for separation using an eluent with acetonitrile and 0.1% formic acid, 1 mM ammonium formate at a flow rate of 0.2 mL/min. Detection was performed using ESI source in positive ion selected reaction monitoring mode by monitoring the following ion transitions m/z 398.1 → 202.2 (Cbz-dFdC), m/z 264.1 → 112.0 (dFdC), m/z 265.3 → 113.2 (dFdU) and m/z 246.1 → 112.0 (IS). Analytes were extracted by simple precipitation with acetonitrile containing internal standards followed by liquid-liquid extraction with ethyl acetate. The calibration curves of Cbz-dFdC, dFdC and dFdU were linear in the concentration range of 2 to 500 ng/mL, 2 to 500 ng/mL and 40 to 10,000 ng/mL, respectively. The assay ranges selected for the three analytes were appropriate and minimized the need for reanalysis. All the validation data, such as intra- and inter-day precision, accuracy, selectivity and stability, were within the required limits. In conclusion, the sensitive analytical assay was selective and accurate for the determination of rat plasma concentrations of Cbz-dFdC, dFdC and dFdU from a single LC-MS/MS analysis and well-suited to support pharmacokinetic studies.

Effect of nucleoside analogue antimetabolites on the structure of PEO-PPO-PEO micelles investigated by SANS.

The effect of three nucleoside analogue antimetabolites (5-fluorouracil, floxuridine, and gemcitabine) on the structure of Pluronic L62 copolymer micelles was investigated using small-angle neutron scattering. These antimetabolites used for cancer chemotherapy have analogous molecular structures but different molecular sizes and aqueous solubilities. It was found that the addition of the three antimetabolites slightly reduced the micellar size and aggregation number, and the micellar anisotropy. The added antimetabolites also changed the internal molecular distribution of the micelles as measured by the scattering length densities, resulting in enhanced hydration of the hydrophobic core region of the micelle. The strength of the effect was found to correlate with the molecular properties of the model drugs, i.e. a larger molecular size and a higher aqueous solubility lead to enhanced hydration of the micellar core.

Doxifluridine-conjugated 2-5A analog shows strong RNase L activation ability and tumor suppressive effect.

RNase L is activated by 2',5'-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2'-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5'-modified 2-5A analogs with 8-methyladenosine at the 2'-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5'-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells.

3'-O- and 5'-O-Propargyl Derivatives of 5-Fluoro-2'-Deoxyuridine: Synthesis, Cytotoxic Evaluation and Conformational Analysis.

A series of new 3'-O- and 5'-O-propargyl derivatives of 5-fluoro-2'-deoxyuridine (1-4) was synthesized by means of propargyl reaction of properly blocked nucleosides (2,4), followed by the deprotection reaction with ammonium fluoride. The synthesized propargylated 5-fluoro-2'-deoxyuridine analogues (1-4) were evaluated for their cytotoxic activity in three human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7), using the sulforhodamine B (SRB) assay. The highest activity and the best SI coefficient in all of the investigated cancer cells were displayed by 3'-O-propargyl-5-fluoro-2'-deoxyuridine (1), and its activity was higher than that of the parent nucleoside. The other new compounds exhibited moderate activity in all of the used cell lines.

2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors.

Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-ß expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.

Synergistic Combination Chemotherapy of Camptothecin and Floxuridine through Self-Assembly of Amphiphilic Drug-Drug Conjugate.

Combination chemotherapy has been widely applied in cancer treatment; however, the cocktail administration of combination chemotherapy could cause the nonuniform biodistribution of anticancer agents, thus impairing the therapeutic efficacy. In the present study, to address this concern, we proposed a novel strategy of preparing self-assembled nanoparticles from amphiphilic drug-drug conjugate for synergistic combination chemotherapy. The conjugate was synthesized by two-step esterification of hydrophobic camptothecin (CPT) and hydrophilic floxuridine (FUDR) through a linker compound. Because of its amphiphilic nature, the CPT-FUDR conjugate self-assembled into stable nanoparticles which could simultaneously release fixed dosage of the two drugs in cancer cells. In vitro studies demonstrated synergistic anticancer efficacy of the CPT-FUDR nanoparticles including improved cell apoptosis, varied cell cycle arrest, as well as effective inhibition of cancer cell proliferation.

Preanalytical Stability of Gemcitabine and its Metabolite 2', 2'-Difluoro-2'-Deoxyuridine in Whole Blood-Assessed by Liquid Chromatography Tandem Mass Spectrometry.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) and metabolite (2',2'-difluoro-2'-deoxyuridine, dFdU) quantification is warranted for individualized treatment strategies. Analyte stability is crucial for the validity of such quantification. We therefore studied the impact of the time interval from blood sampling to separation of plasma on gemcitabine stability. Blood from gemcitabine-treated patients was drawn into tetrahydrouridine (THU)-spiked heparin and ethylenediaminetetraacetic acid tubes and kept on ice until separation. Plasma was separated sequentially up to 24 h after sampling and dFdC and dFdU were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The change in plasma concentrations over time was compared with the highest imprecision for concentrations above the lower limit of quantification of the LC-MS/MS method. Analyte concentrations decreased slightly over time, but for samples stored for 4 h on ice, the decline was smaller than the expected analytical imprecision. After 24 h, the maximum decline was 14.0%, which exceeded the expected analytical imprecision. dFdC and dFdU stabilities were acceptable for at least 4 h when THU-spiked whole blood samples were kept on ice. This is within the scope of routine sampling procedures. Further, variations in separation time intervals within this time frame are negligible when interpreting drug concentrations.

Locked 2'-Deoxy-2',4'-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity.

2'-Deoxy-2',4'-difluorouridine (2',4'-diF-rU) was readily incorporated into DNA and RNA oligonucleotides via standard solid phase synthesis protocols. NMR and thermal denaturation (Tm) data of duplexes was consistent with the 2',4'-diF-rU nucleotides adopting a rigid North (RNA-like) sugar conformation, as previously observed for the nucleoside monomer. The impact of this modification on Tm is neutral when incorporated within RNA:RNA duplexes, mildly destabilizing when located in the RNA strand of a DNA:RNA duplex, and highly destabilizing when inserted in the DNA strand of DNA:RNA and DNA:DNA duplexes. Molecular dynamics calculations suggest that the destabilization effect in DNA:DNA and DNA:RNA duplexes is the result of structural distortions created by A/B junctions within the helical structures. Quantum mechanics calculations suggest that the "neutral" effect imparted to A-form duplexes is caused by alterations in charge distribution that compensate the stabilizing effect expected for a pure North-puckered furanose sugar. 2',4'-diF-RNA modified siRNAs were able to trigger RNA interference with excellent efficiency. Of note, incorporation of a few 2',4'-diF-rU residues in the middle of the guide (antisense) strand afforded siRNAs that were more potent than the corresponding siRNAs containing LNA and 2'-F-ANA modifications, and as active as the 2'-F-RNA modified siRNAs.