Eco-Friendly Stability-Indicating HPLC Method for Related Compounds in Pemetrexed Ditromethamine (Antineoplastic Agent) for Injection.

BACKGROUND: An eco-friendly analytical technique was developed with the intention of preserving the environment by using green chemistry principles. Pemetrexed is a folate analogue indicated for the treatment of advanced lung cancer. OBJECTIVE: Development of a green stability-indicating HPLC method for the quantification of pemetrexed ditromethamine (PDT) impurities in Active Pharmaceutical Ingredient (API) and parenteral dosage form. METHODS: Chromatographic separation was achieved using a Zorbax SB C18 column (150 mm × 4.6 mm i.d., 3.5 µ particle size) with perchlorate buffer (pH 3.0 ± 0.1, 50 mM) as mobile phase A and acetonitrile-perchlorate (90 + 10, v/v) buffer as mobile phase B at a flow rate of 0.8 mL/min with a column temperature of 40°C ± 0.5°C. All analytes were well resolved by gradient elution with a total run time of 75 min. The UV detection wavelength was 230 nm. RESULTS: The RP-HPLC method is capable of resolving all the degradation and process impurities for PDT API and parenteral dosage form. The related compounds method was validated in accordance with International conference on harmonization (ICH) Q2(R1) and United states of Pharmacopoeia (USP) <1225> guidelines, and found to be accurate, specific, precise, linear, robust and stability-indicating. The precision and intermediate results were <5% CV for all the impurities. The accuracy for all the impurities was found to be between 90 and 110%. The linearity of regression co-efficient values for all the impurities were found to be more than 0.999. CONCLUSION: The proposed related compounds method is found suitable for the determination of process and degradation impurities of commercial formulations, stability samples in QC analysis for PDT API, and drug product. HIGHLIGHTS: The developed liquid chromatographic method greenness and eco-friendliness were assessed using the green analytical procedure index (GAPI) and the analytical greenness (AGREE) tool, and found to be green. A PDT detoxification procedure was also developed to reduce environmental pollution.

Trojan-Horse Diameter-Reducible Nanotheranostics for Macroscopic/Microscopic Imaging-Monitored Chemo-Antiangiogenic Therapy.

Although nanotheranostics have displayed striking potential toward precise nanomedicine, their targeting delivery and tumor penetration capacities are still impeded by several biological barriers. Besides, the current antitumor strategies mainly focus on killing tumor cells rather than antiangiogenesis. Enlightened by the fact that the smart transformable self-targeting nanotheranostics can enhance their targeting efficiency, tumor penetration, and cellular uptake, we herein report carrier-free Trojan-horse diameter-reducible metal-organic nanotheranostics by the coordination-driven supramolecular sequential co-assembly of the chemo-drug pemetrexed (PEM), transition-metal ions (FeIII), and antiangiogenesis pseudolaric acid B. Such nanotheranostics with both a high dual-drug payload efficiency and outstanding physiological stability are responsively decomposed into numerous ultra-small-diameter nanotheranostics under stimuli of the moderate acidic tumor microenvironment and then internalized into tumor cells through tumor-receptor-mediated self-targeting, synergistically enhancing tumor penetration and cellular uptake. Besides, such nanotheranostics enable visualization of self-targeting capacity under the macroscopic monitor of computed tomography/magnetic resonance imaging, thereby realizing efficient oncotherapy. Moreover, tumor microvessels are precisely monitored by optical coherence tomography angiography/laser speckle imaging during chemo-antiangiogenic therapy in vivo, visually verifying that such nanotheranostics possess an excellent antiangiogenic effect. Our work will provide a promising strategy for further tumor diagnosis and targeted therapy.

Highly sensitive quantification of pemetrexed in human plasma using UPLC-MS/MS to support microdosing studies.

Pemetrexed is an antifolate drug approved for the treatment of non-small-cell lung cancer and mesothelioma. Assessing pemetrexed pharmacokinetics after administration of a microdose (100 µg) may facilitate drug-drug interaction and dose individualization studies with cytotoxic drugs, without causing harm to patients. Therefore, a highly sensitive bioanalytical assay is required. A reversed-phase ultra-high performance liquid chromatography method was developed to determine pemetrexed concentrations in human ethylenediaminetetraacetic acid-plasma after microdosing. [13 C5 ]-Pemetrexed was used as the internal standard. The sample preparation involved solid-phase extraction from plasma. Detection was performed using MS/MS in a total run time of 9.5 min. The assay was validated over the concentration range of 0.0250-25.0 µg/L pemetrexed. The average accuracies for the assay in plasma were 96.5 and 96.5%, and the within-day and between-day precision in coefficients of variations was <8.8%. Extraction recovery was 59 ± 1 and 55 ± 5% for pemetrexed and its internal standard. Processed plasma samples were stable for 2 days in a cooled autosampler at 10°C. The assay was successfully applied in a pharmacokinetic curve, which was obtained as a part of an ongoing clinical microdosing study.

Facile Synthetic Protocols for the Preparation of New Impurities in Pemetrexed Disodium Heptahydrate as an Anti-cancer Drug.

A facile synthetic protocol was employed to prepare process-related impurities associated with the synthesis of pemetrexed disodium heptahydrate, Alimta. The research work is described for the development of the novel synthetic methods and their structure elucidation of Pemetrexed glutamide, N-methyl pemetrexed, and N-methyl pemetrexed glutamide impurities. The listed impurities were deduced through spectral analysis, such as 1H-NMR, 13CNMR, and HRMS. The target compounds can be used as the reference substances for quality control.

Structural basis of antifolate recognition and transport by PCFT.

Folates (also known as vitamin B9) have a critical role in cellular metabolism as the starting point in the synthesis of nucleic acids, amino acids and the universal methylating agent S-adenylsmethionine1,2. Folate deficiency is associated with a number of developmental, immune and neurological disorders3-5. Mammals cannot synthesize folates de novo; several systems have therefore evolved to take up folates from the diet and distribute them within the body3,6. The proton-coupled folate transporter (PCFT) (also known as SLC46A1) mediates folate uptake across the intestinal brush border membrane and the choroid plexus4,7, and is an important route for the delivery of antifolate drugs in cancer chemotherapy8-10. How PCFT recognizes folates or antifolate agents is currently unclear. Here we present cryo-electron microscopy structures of PCFT in a substrate-free state and in complex with a new-generation antifolate drug (pemetrexed). Our results provide a structural basis for understanding antifolate recognition and provide insights into the pH-regulated mechanism of folate transport mediated by PCFT.

Heterocyclic Substitutions Greatly Improve Affinity and Stability of Folic Acid towards FRα. an In Silico Insight.

Folate receptor alpha (FRα) is known as a biological marker for many cancers due to its overexpression in cancerous epithelial tissue. The folic acid (FA) binding affinity to the FRα active site provides a basis for designing more specific targets for FRα. Heterocyclic rings have been shown to interact with many receptors and are important to the metabolism and biological processes within the body. Nineteen FA analogs with substitution with various heterocyclic rings were designed to have higher affinity toward FRα. Molecular docking was used to study the binding affinity of designed analogs compared to FA, methotrexate (MTX), and pemetrexed (PTX). Out of 19 FA analogs, analogs with a tetrazole ring (FOL03) and benzothiophene ring (FOL08) showed the most negative binding energy and were able to interact with ASP81 and SER174 through hydrogen bonds and hydrophobic interactions with amino acids of the active site. Hence, 100 ns molecular dynamics (MD) simulations were carried out for FOL03, FOL08 compared to FA, MTX, and PTX. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of FOL03 and FOL08 showed an apparent convergence similar to that of FA, and both of them entered the binding pocket (active site) from the pteridine part, while the glutamic part was stuck at the FRα pocket entrance during the MD simulations. Molecular mechanics Poisson-Boltzmann surface accessible (MM-PBSA) and H-bond analysis revealed that FOL03 and FOL08 created more negative free binding and electrostatic energy compared to FA and PTX, and both formed stronger H-bond interactions with ASP81 than FA with excellent H-bond profiles that led them to become bound tightly in the pocket. In addition, pocket volume calculations showed that the volumes of active site for FOL03 and FOL08 inside the FRα pocket were smaller than the FA-FRα system, indicating strong interactions between the protein active site residues with these new FA analogs compared to FA during the MD simulations.

Cytotoxicity study and influence of SBA-15 surface polarity and pH on adsorption and release properties of anticancer agent pemetrexed.

Mesoporous material SBA-15 was functionalized with different polar and nonpolar groups: 3-aminopropyl, (SBA-15-NH2), 3-isocyanatopropyl (SBA-15-NCO), 3-mercaptopropyl (SBA-15-SH), methyl (SBA-15-CH3) and phenyl (SBA-15-Ph). The resulting surface grafted materials were investigated as matrices for controlled drug delivery. Anticancer agent, pemetrexed (disodium pemetrexed heptahydrate) was selected as a model drug and loaded in the unmodified and functionalized SBA-15 materials. Materials were characterized by elemental analysis, infrared spectroscopy, transmission electron microscopy, nitrogen adsorption/desorption analysis, small angle X-ray scattering, powder X-ray diffraction, solid state NMR spectroscopy and thermogravimetry. It was shown that surface modification has an impact on both encapsulated drug amount and release properties. Release experiments were performed into two media with different pH: simulated body fluid (pH = 7.4) and simulated gastric fluid (pH = 2). In general, the effect of pH was reflected by the lower release of pemetrexed under acidic conditions (pH = 2) compared to slightly alkaline saline environment (pH = 7.4). The release rate of pemetrexed from propylamine-, propylisocyanate- and phenyl-modified SBA-15 was found to be effectively controlled by intermolecular interactions as compared to that from pure SBA-15, SBA-15-SH, and SBA-15-CH3, that evidenced a steady and similar release. The highest release was observed for methyl-functionalized material whose hydrophobic surface accelerates the pemetrexed release. The data obtained from release studies were fitted using various kinetic models to determine the pemetrexed release mechanism and its release rate. The best correlations were found for Korsmeyer-Peppas and Higuchi models. Moreover, the theoretical three-parameter model for drug release kinetic was applied to calculate the strength of drug-support interactions. The in vitro cell study was performed on SKBR3 cancer cells and obtained results demonstrated that the modification of the mesoporous silica material by grafted polar/nonpolar groups may significantly affect the compatibility of this material with cells, drug release from this material and subsequent biological activity of PEM.

Synthesis of lactoferrin mesoporous silica nanoparticles for pemetrexed/ellagic acid synergistic breast cancer therapy.

Despite the clinical approval of few nanomedicines for cancer therapy, some drawbacks still impede their improved efficiency including low drug loading, off-target toxicity and development of multi-drug resistance. Herein, lactoferrin (Lf)-coupled mesoporous silica nanoparticles (MSNPs) were developed for combined delivery of the cytotoxic drug pemetrexed (PMT) and the phytomedicine ellagic acid (EA) for synergistic breast cancer therapy. While the hydrophobic EA was physically encapsulated within the pores of MSNPs via the adsorptive properties of MSNPs and the electrostatic interactions between the negatively charged EA and positively charged amino modified MSNs, the highly water soluble PMT was chemically anchored to the Lf shell through chemical conjugation to the surface of lactoferrin coated MSNPs by carbodiimide reaction to avoid pre-mature drug release and systemic toxicity. The dual drug-loaded Lf-MSNPs (284 nm) demonstrated a sequential faster release of EA followed by a sustained release of PMT. The dual drug-loaded Lf-MSNPs exhibited highest cytotoxicity against MCF-7 (Michigan Cancer Foundation-7) breast cancer cells as revealed by the lowest combination index (CI = 0.885) compared to free drugs. The combination index value (< 1) revealed synergy between both loaded drugs. Furthermore, high cellular uptake of the nanocarriers into MCF-7 breast cancer cells was observed via Lf-receptor mediated endocytosis. Altogether, the dual drug-loaded Lf-targeted MSNPs showed to be a promising carrier for breast cancer therapy through triggering different signaling pathways, and hence overcoming the multi-drug resistance and minimizing the systemic toxicity.

The Development of Pemetrexed Diacid-Loaded Gelatin-Cloisite 30B (MMT) Nanocomposite for Improved Oral Efficacy Against Cancer: Characterization, In-Vitro and Ex-Vivo Assessment.

AIM: The intention of this investigation was to develop Pemetrexed Diacid (PTX)-loaded gelatine-cloisite 30B (MMT) nanocomposite for the potential oral delivery of PTX and the in vitro, and ex vivo assessment. BACKGROUND: Gelatin/Cloisite 30 B (MMT) nanocomposites were prepared by blending gelatin with MMT in aqueous solution. METHODS: PTX was incorporated into the nanocomposite preparation. The nanocomposites were investigated by Fourier Transmission Infra Red Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscope (SEM) X-Ray Diffraction (XRD) and Confocal Laser Microscopy (CLSM). FT-IR of nanocomposite showed the disappearance of all major peaks which corroborated the formation of nanocomposites. The nanocomposites were found to have a particle size of 121.9 ± 1.85 nm and zeta potential -12.1 ± 0.63 mV. DSC thermogram of drug loaded nanocomposites indicated peak at 117.165 oC and 205.816 oC, which clearly revealed that the drug has been incorporated into the nanocomposite because of cross-linking of cloisite 30 B and gelatin in the presence of glutaraldehyde. RESULTS: SEM images of gelatin show a network like structure which disappears in the nanocomposite. The kinetics of the drug release was studied in order to ascertain the type of release mechanism. The drug release from nanocomposites was in a controlled manner, followed by first-order kinetics and the drug release mechanism was found to be of Fickian type. CONCLUSION: Ex vivo gut permeation studies revealed 4 times enhancement in the permeation of drug present in the nanocomposite as compared to plain drug solution and were further affirmed by CLSM. Thus, gelatin/(MMT) nanocomposite could be promising for the oral delivery of PTX in cancer therapy and future prospects for the industrial pharmacy.

Delivery of pemetrexed by magnetic nanoparticles: design, characterization, in vitro and in vivo assessment.

Drug-loaded magnetic nanoparticles have been developed because of the advantages of specific drug targeting in cancer treatment. Pemetrexed (PEM) is a multi-targeting antifolate agent that is effective for the treatment of many cancers, for example, non-small cell lung cancer. Here, PEM loaded magnetic O-carboxymethyl chitosan (O-CMC) nanoparticles were prepared to deliver PEM on tumor tissue with an external magnetic field. The modification of chitosan to O-CMC was confirmed by FTIR analysis. Nanoparticle synthesis was performed via ionic gelation method. The diameter of magnetic O-CMC nanoparticles (MCMC) was found to be 130.1 ± 22.96 nm. After PEM loading, diameter was found to be 123.9 ± 11.42 nm. The drug release of PEM loaded MCMC (PMCMC) was slower in physiological medium than in acidic medium. A549-luc-C8 and CRL5807 cell lines were used for MTT test which showed that IC50 values of nanoparticles were lower than PEM. The antitumor efficiency of PMCMC in xenograft tumor model was examined with in vivo imaging system (IVIS) and caliper and with hematological analyses. In vivo studies revealed that PMCMC had targeted antitumor activity in A549-luc-C8-tumor-bearing mice compared to PEM. As a result, it was suggested that PMCMC have great potential for the treatment of non-small cell lung cancer.

Diselenide-Pemetrexed Assemblies for Combined Cancer Immuno-, Radio-, and Chemotherapies.

Immunotherapy has emerged as a promising new approach for cancer treatment. However, clinically available drugs have been limited until recently, and the antitumor efficacy of most cancer immunotherapies still needs to be improved. Herein, we develop diselenide-pemetrexed assemblies that combine natural killer (NK) cell-based cancer immunotherapy with radiotherapy and chemotherapy in a single system. The assemblies are prepared by co-assembly between pemetrexed and cytosine-containing diselenide through hydrogen bonds. Under γ-radiation, the hydrogen bonds are cleaved, resulting in the release of pemetrexed. At the same time, diselenide can be oxidized to seleninic acid, which suppresses the expression of human leukocyte antigen E (HLA-E) in cancer cells, thus activating the immune response of NK cells. In this way, cancer immunotherapy is combined with radiotherapy and chemotherapy, providing a new strategy for cancer treatment.

Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models.

Serine hydroxymethyltransferases (SHMTs) reversibly transform serine into glycine in a reaction accompanied with conversion of tetrahydrofolate (THF) into 5,10-methylene-THF (5,10-meTHF). In vivo, 5,10-meTHF is the main carrier of one-carbon (1C) units, which are utilized for nucleotide biosynthesis and other processes crucial for every living cell, but hyperactivated in overproliferating cells (e.g. cancer tissues). SHMTs are emerging as a promising target for development of new drugs because it appears possible to inhibit growth of cancer cells by cutting off the supply of 5,10-meTHF. Methotrexate (MTX) and pemetrexed (PTX) are two examples of antifolates that have cured many patients over the years but target different enzymes from the folate cycle (mainly dihydrofolate reductase and thymidylate synthase, respectively). Here we show crystal structures of MTX and PTX bound to plant SHMT isozymes from cytosol and mitochondria-human isozymes exist in the same subcellular compartments. We verify inhibition of the studied isozymes by a thorough kinetic analysis. We propose to further exploit antifolate scaffold in development of SHMT inhibitors because it seems likely that especially polyglutamylated PTX inhibits SHMTs in vivo. Structure-based optimization is expected to yield novel antifolates that could potentially be used as chemotherapeutics.

Tumor Microenvironment Responsive Shape-Reversal Self-Targeting Virus-Inspired Nanodrug for Imaging-Guided Near-Infrared-II Photothermal Chemotherapy.

Tumor microenvironment responsive multimodal synergistic theranostic strategies can significantly improve the therapeutic efficacy while avoiding severe side effects. Inspired by the fact that special morphology could enhance photothermal conversion efficiency (PCE) and cellular delivery, we developed an acidic tumor microenvironment responsive shape-reversal metal-organic virus-inspired nanodrug for enhancing near-infrared (NIR)-II PCE, increasing cell adhesion, and activating tumor targeting. First, a NIR-I fluorescence probe (IR825), a chemo-drug (pemetrexed, PEM), and a rare-earth metal ion (Nd(III)) were chosen to synthesize a virus-like nanodrug via coordination-driven assembly. Then, the spike-like surface of the nanodrug was further camouflaged by an acidity-sensitive poly(ethylene glycol) "shell" to create virus-core and sphere-shell hierarchical nanoassemblies, which could efficiently prevent immune clearance and prolong systemic circulation. Interestingly, the acidic tumor microenvironment could trigger the shell detachment of nanoassemblies for shape reversal to produce a virus-like surface followed by re-exposure of PEM to synergistically amplify the cellular internalization while enhancing NIR-II PCE. By utilizing the shell-detached virus-like nanodrug core, the tumor microenvironment specific enhanced NIR-II photothermal chemotherapy can be realized under the precise guidance of fluorescence/photoacoustic imaging, thereby achieving complete tumor elimination without recurrence in a single treatment cycle. We envision that integrating the tumor microenvironment responsive ability with  "sphere-to-virus" shape reversal will provide a promising strategy for biomimetic targeted cancer therapy.

Supramolecular Amphiphilic Assembly Formed by the Complexation of Calixpyridinium with Alimta.

In this work, the host-guest interaction between calixpyridinium and anionic anticancer drug Alimta was studied in aqueous media. Spherical supramolecular amphiphilic assembly rather than simple complex was accidentally fabricated by the complexation of calixpyridinium with Alimta. It is the third kind of anionic guest to be discovered to form the higher-order assembly by the complexation of calixpyridinium besides polyanionic guest and anionic gemini surfactant guest. The finding of this assembly approach supplies a new idea to construct various self-assembly architectures in water via the complexation of calixpyridinium with anionic drugs. The resulting calixpyridinium-drug assemblies may also have the potential to adjust the effects of drugs.

In vivo imaging characterization and anticancer efficacy of a novel HER2 affibody and pemetrexed conjugate in lung cancer model.

INTRODUCTION: In this study, a new agent consisting of HER2-specific affibody ZHER2:V2 and chemotherapy drug pemetrexed was synthesized to develop a new targeted drug. Its biological characteristics and anticancer efficacy were assessed in cells level and xenografts models by radiolabeling with technetium-99m. METHODS: After the ZHER2:V2-pemetrexed conjugate was synthesized, radiolabeling of the conjugate was performed using its C-terminal 4 amino acids (Gly-Gly-Gly-Cys) as the chelating moiety. The radiochemical yield of the [99mTc]Tc-ZHER2:V2-pemetrexed was identified by instant thin-layer chromatography (ITLC). Stability of the radiolabeled conjugate was investigated both in vitro and in vivo. In vitro binding affinity and cell internalization study of the probe were performed in A549 cells (HER2-positive). Tumor uptake was evaluated by in vitro uptake assay in A549 cells and H23 cells (HER2-negative), and by in vivo biodistribution and SPECT imaging in A549 and H23 tumor-bearing mice. The antitumor efficacy of the ZHER2:V2-pemetrexed conjugate was evaluated in cells and xenograft models. RESULTS: The ZHER2:V2-pemetrexed was successfully synthesized and conjugated with technetium-99 m, and acquired the radiochemical yield of 97.0 ±â€¯0.3%. The stability of [99mTc]Tc-ZHER2:V2-pemetrexed was good in both physiological saline and human serum. The radiolabeled agent displayed excellent HER2-binding specificity and affinity in vitro, and was gradually internalized into the cells. Biodistribution study revealed obvious tumor uptake in A549 xenografts (percentage injected dose per gram, 2.6 ±â€¯1.0%ID/g at 4 h postinjection), while the uptake in HER2-negative H23 tumors was much lower (0.2 ±â€¯0.1%ID/g at 4 h postinjection, P < 0.01). SPECT imaging exhibited an intensity in the A549 xenograft which could be blocked by excess ZHER2:V2-pemetrexed. Treatment with ZHER2:V2-pemetrexed significantly impaired the tumor growth (P < 0.05), with less weight loss than pemetrexed. CONCLUSION: [99mTc]Tc-ZHER2:V2-pemetrexed showed desirable property and HER2-specificity. The ZHER2:V2-pemetrexed conjugate could inhibit tumor growth of HER2-positive lung adenocarcinoma and may have the potential to become a targeted drug for lung cancer. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The compound described herein performs HER2-targeting with favorable anticancer efficacy and offers the potential of novel targeting strategies for further tumor therapy.

Combining Albumin-Binding Properties and Interaction with Pemetrexed to Improve the Tissue Distribution of Radiofolates.

Folic-acid-based radioconjugates have been developed for nuclear imaging of folate receptor (FR)-positive tumors; however, high renal uptake was unfavorable in view of a therapeutic application. Previously, it was shown that pre-injection of pemetrexed (PMX) increased the tumor-to-kidney ratio of radiofolates several-fold. In this study, PMX was combined with the currently best performing radiofolate ([177Lu]cm13), which is outfitted with an albumin-binding entity. Biodistribution studies were carried out in mice bearing KB or IGROV-1 tumor xenografts, both FR-positive tumor types. SPECT/CT was performed with control mice injected with [177Lu]folate only and with mice that received PMX in addition. Control mice showed high uptake of radioactivity in KB and IGROV-1 tumor xenografts, but retention in the kidneys was also high, resulting in tumor-to-kidney ratios of ~0.85 (4 h p.i.) and ~0.60 (24 h p.i.) or ~1.17 (4 h p.i.) and ~1.11 (24 h p.i.) respectively. Pre-injection of PMX improved the tumor-to-kidney ratio to values of ~1.13 (4 h p.i.) and ~0.92 (24 h p.i.) or ~1.79 (4 h p.i.) and ~1.59 (24 h p.i.), respectively, due to reduced uptake in the kidneys. It was found that a second injection of PMX­3 h or 7 h after administration of the radiofolate­improved the tumor-to-kidney ratio further to ~1.03 and ~0.99 or ~1.78 and ~1.62 at 24 h p.i. in KB and IGROV-1 tumor-bearing mice, respectively. SPECT/CT scans readily visualized the tumor xenografts, whereas accumulation of radioactivity in the kidneys was reduced in mice that received PMX. In this study, it was shown that PMX had a positive impact in terms of reducing the kidney uptake of albumin-binding radiofolates; hence, the administration of PMX resulted in ~1.3⁻1.7-fold higher tumor-to-kidney ratios. This is, however, a rather moderate effect in comparison to the previously shown effect of PMX on conventional radiofolates (without albumin binder), which led to 5⁻6-fold increased tumor-to-kidney ratios. An explanation for this result may be the different pharmacokinetic profiles of PMX and long-circulating radiofolates, respectively. Despite the promising potential of this concept, it is believed that a clinical translation would be challenging, particularly when PMX had to be injected more than once.

Lung cancer specific and reduction-responsive chimaeric polymersomes for highly efficient loading of pemetrexed and targeted suppression of lung tumor in vivo.

Lung cancer is one of the worldwide leading and fast-growing malignancies. Pemetrexed disodium (PEM, Alimta®), a small hydrophilic drug, is currently used for treating lung cancer patients. However, PEM suffers from issues like fast elimination, low bioavailability, poor tumor cell selectivity and penetration. Here, we report on lung cancer specific CSNIDARAC (CC9) peptide-functionalized reduction-responsive chimaeric polymersomes (CC9-RCPs) for efficient encapsulation and targeted delivery of PEM to H460 human lung cancer cells in vitro and in vivo. PEM-loaded CC9-RCPs (PEM-CC9-RCPs) was obtained from co-self-assembly of poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate)-b-polyethylenimine (PEG-P(TMC-DTC)-PEI) and CC9-functionalized PEG-P(TMC-DTC) in the presence of PEM followed by self-crosslinking. PEM-CC9-RCPs displayed an optimal CC9 density of 9.0% in targeting H460 cells, a high PEM loading content of 14.2 wt%, a small hydrodynamic size of ca. 60 nm and glutathione-triggered PEM release. MTT assays showed that PEM-CC9-RCPs was 2.6- and 10- fold more potent to H460 cells than the non-targeting PEM-RCPs and free PEM controls, respectively. Interestingly, PEM-CC9-RCPs exhibited 22-fold longer circulation time and 9.1-fold higher accumulation in H460 tumor than clinical formulation Alimta®. Moreover, CC9-RCPs showed obviously better tumor penetration than RCPs. Remarkably, PEM-CC9-RCPs at 12.5 mg PEM equiv./kg effectively suppressed growth of H460 xenografts and significantly prolonged mouse survival time as compared to PEM-RCPs and Alimta® controls. These lung cancer specific and reduction-responsive chimaeric polymersomes provide a unique pemetrexed nanoformulation for targeted lung cancer therapy. STATEMENT OF SIGNIFICANCE: Multitargeted antifolate agent pemetrexed (PEM, Alimta®) is currently used for treating lung cancer patients and has low side-effects. However, PEM suffers from issues like fast elimination, low bioavailability, poor tumor cell selectivity and penetration. Scarce work on targeted delivery of PEM has been reported, partly because most conventional nanocarriers show a low and instable loading for hydrophilic, negatively charged drugs like PEM. Herewith, we report on lung cancer specific CSNIDARAC (CC9) peptide-functionalized reduction-responsive chimaeric polymersomes (CC9-RCPs) which showed efficient PEM encapsulation (14.2 wt%, 60 nm) and targeted delivery of PEM to H460 human lung cancer cells, leading to effective suppression of H460 tumor xenografts and significantly prolonged survival rates of mice than Alimta®. To the best of our knowledge, this represents a first report on targeted nanosystems that are capable of efficient loading and targeted delivery of PEM to lung tumors.

Reduced interleukin-6 immunoexpression and birefringent collagen formation indicate that MTA Plus and MTA Fillapex are biocompatible.

Considering that endodontic sealers release some components which may promote delay in the repair process, our purpose was to evaluate the tissue reaction promoted by MTA Plus and MTA Fillapex in comparison with AH Plus (standard control) and Endofill, which has a long clinical track record. One hundred rats were distributed into five groups: MTA Plus (Avalon Biom Inc., Bradenton, FL, USA), MTA Fillapex (Angelus, Londrina, PR, Brazil), AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany), Endofill (Dentsply, Petrópolis, RJ, Brazil) and CG (control group, empty polyethylene tubes). The polyethylene tubes filled with sealers or empty (CG) were implanted into subcutaneous. After 7, 15, 30 and 60 days, the tubes surrounded by capsules were paraffin-embedded. In HE-stained sections, the volume density of inflammatory cells (VvIC) was estimated in the capsules. The number of interleukin-6-immunolabelled cells (IL-6), a pro-inflammatory cytokine, was also computed in the capsules. The birefringent collagen content was quantified in picrosirius-stained sections. Data were analysed by ANOVA and Tukey tests (p ≤ 0.05). At 7 days, the capsules showed moderate inflammatory reaction. In all groups, VvIC and IL-6-immunostained cells reduced significantly from 7 to 60 days. At 60 days, IL-6 immunoexpression was reduced significantly in MTA Plus and MTA Fillapex in comparison with AH Plus; no difference was found in the VvIC among MTA Plus, MTA Fillapex, AH Plus and CG whereas Endofill exhibited the highest VvIC. The reduction in VvIC was parallel to an increase in the collagen in all the groups, except Endofill. MTA Plus, MTA Fillapex and AH Plus induce a response that culminates in the regression of inflammation and formation of a fibrous capsule over time. The lower IL-6 immunoexpression in the capsules of MTA Plus and MTA Fillapex than AH Plus suggests that the immune response is suppressed more rapidly in the MTA-based sealers.

Evaluation of METase-pemetrexed-loaded PEG-PLGA nanoparticles modified with anti-CD133-scFV for treatment of gastric carcinoma.

PEG-PLGA nanoparticles (NPs) modified with anti-CD133 and tumor-targeting single-chain antibody fragment (scFV-NPs) for systemic delivery of methioninase (METase) and pemetrexed for gastric carcinoma were successfully formulated. The structure characterization and biological functions of METase-pemetrexed-loaded scFV-PEG-PLGA NPs (scFV-METase/pemetrexed-NPs) in vitro were investigated. Functional scFV-PEG-PLGA NPs or PEG-PLGA NPs present low cell cytoxicity in CD133+ SGC7901 cells. scFV-METase/pemetrexed-NPs (scFv-M/P-NP) was more effective in inhibiting tumor growth (including cell growth and migration ability) in CD133 positive expressed gastric cancer cells than METase/pemetrexed-NPs (M/P-NP). Moreover, METase enhanced the inhibitory effect of pemetrexed on thymidylate synthase (TS) synthesis and cell apoptosis. We have demonstrated the application of scFV-targeted PEG-PLGA NPs as a new potential strategy to enhance treatment benefits for gastric carcinoma.

Development of a Prodrug of Levofloxacin to Avoid Chelation with Al3+ and of Pemetrexed Dimedoxomil Esters for Oral Administration.

An ethoxycarbonyl 1-ethyl hemiacetal ester of levofloxacin (LVFX-EHE) avoids insoluble chelate formation with metal-containing drugs in the intestinal tract and is rapidly hydrolyzed to the parent drug. Furthermore, the minimum inhibitory concentration confirms that LVFX-EHE is less likely to cause pseudomembranous colitis because of less susceptibility to normal intestinal bacteria flora. Pemetrexed dimedoxomil, the prodrug of pemetrexed, was synthesized via reaction with medoxomil bromide after modification of L-glutamate with the tert-butyloxycarbonyl protecting group (BOC), followed by hydrolysis of the BOC moiety with trifluoroacetic acid (TFA) in CH2Cl2 at a temperature of 0°C for 2 h. A serum pemetrexed concentration of >2 µg/mL was observed after oral administration of pemetrexed dimedoxomil at a dose of 60 mg/kg to rats.