Reproducibility warning: The curious case of polyethylene glycol 6000 and spheroid cell culture.

Reproducibility of results is essential for a well-designed and conducted experiment. Several reasons may originate failure in reproducing data, such as selective reporting, low statistical power, or poor analysis. In this study, we used PEG6000 samples from different distributors and tested their capability inducing spheroid formation upon surface coating. MALDI-MS, NMR, FTIR, and Triple SEC analysis of the different PEG60000s showed nearly identical physicochemical properties different, with only minor differences in mass and hydrodynamic radius, and AFM analysis showed no significant differences in the surface coatings obtained with the available PEG6000s. Despite these similarities, just one showed a highly reproducible formation of spheroids with different cell lines, such as HT-29, HeLa, Caco2, and PANC-1. Using the peculiar PEG6000 sample and a reference PEG6000 chosen amongst the others as control, we tested the effect of the cell/PEG interaction by incubating cells in the PEG solution prior to cell plating. These experiments indicate that the spheroid formation is due to direct interaction of the polymer with the cells rather than by interaction of cells with the coated surfaces. The experiments point out that for biological entities, such as cells or tissues, even very small differences in impurities or minimal variations in the starting product can have a very strong impact on the reproducibility of data.

Stability data of extemporaneous suspensions of hydroxychloroquine sulphate in oral liquid bases after tablet manipulation.

Hydroxychloroquine is a well-known anti-malarial and anti-rheumatic drug that has garnered recently unprecedented attention as potential therapeutic agent against virus infections. Hydroxychloroquine sulphate is authorized in the EU as film-coated tablets (i.e. Plaquenil®), which cannot be administered to non-cooperative patients, such as those in intensive care units or, more in general, unable to swallow solid dosage forms. Therefore, the hospital pharmacist must manipulate the solid dosage form for the preparation of suspension, even if it can strongly affect the product quality. In this scenario, it is crucial to offer useful information and advice to assist hospital pharmacists in their activity. The data presented in this article suggest that extemporaneous suspensions of hydroxychloroquine sulphate in oral liquid bases after tablet manipulation are stable for at least 30 days.

S-preactivated thiolated glycol chitosan useful to combine mucoadhesion and drug delivery.

This work describes S-preactivated N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential mucoadhesive platform. Preactivated thiomers (GC-NAC-MNA, GC-GSH-MNA) were synthesized by bond formation between GC-NAC or GC-GSH and 2-mercaptonicotinic acid (MNA) used as ligand. The presence of protected thiol moieties on this new class of thiolated GC made them not subject to oxidation. The structural modifications of the resulting derivatives were confirmed by proton Nuclear Magnetic Resonance (1H NMR) and Size Exclusion Chromatography (SEC). The conjugates displayed 91.2% and 90.1% of S-preactivation for GC-NAC-MNA and GC-GSH-MNA, respectively. The polymers were tested in ex-vivo and in vitro for their mucoadhesive properties and toxicity. The results showed that the preactivation of GC-NAC and GC-GSH increased their mucoadhesive abilities compared to their thiolated precursors by 1.4-, 4.4-fold in time of adhesion evaluated using rotating cylinder method, 1.6-, 1.5-fold in total work of adhesion (TWA) and 2.0-, 1.3-fold in maximum detachment force (MDA) determined using tensile studies, respectively. Moreover, water-uptake studies showed an improved in weight indicating water-uptake strongly dependent on derivations, before erosion occurred, whereas disintegration took place for the thiolated polymers within the first hour. The S-preactivated modification did not affect the cell viability of Caco2 cells exposed to the polymers. The release of the model drug sodium naproxen from tablets prepared with a lyophilized mixture of drug and polymer was studied via dissolution apparatus revealing that the preactivation on GC-GSH and GC-NAC involves a slowdown in the drug release rate. The results shown that the novel preactivated thiolated GC-derivatives can be considered promising excipients for the development of mucoadhesive drug delivery systems.

Delivery of Proapoptotic Agents in Glioma Cell Lines by TSPO Ligand-Dextran Nanogels.

Translocator protein 18-kDa (TSPO) is a versatile mitochondrial target for molecular imaging and therapy. Moreover, selective TSPO ligands have been widely investigated for diagnostic purposes and explored to target drug delivery systems directed to cancer cells overexpressing TSPO. Indeed, poly(d,l-lactic-co-glycolic acid (PLGA) polymers and nanocarriers decorated with TSPO ligands are capable of transporting TSPO ligands inside cancer cells, inducing survival inhibition in cancer cells and producing mitochondrial morphology modification. The aim of this work was to prepare nanogels (NGs) made with TSPO ligand dextran conjugates (TSPO-Dex) that are useful as potential delivery systems of two TSPO ligands as apoptotic agents. Synthesis and complete characterization of TSPO-dextran conjugates, an average molecular weights analysis, TSPO ligand release profiles, thermal behaviour and swelling studies were achieved. NG preparation, characterization and in vitro biological studies were also performed. The release of TSPO ligands released from dextran conjugates at 37 °C occurred in human serum at a faster rate than that detected in phosphate buffer. Cytotoxicity studies demonstrated that NGs produced from TSPO ligand-dextran conjugates induce survival inhibition in rat C6 glioma cell lines. Cellular uptake was also proven by fluorescence microscopy.

Effect of diazoxide on Friedreich ataxia models.

Friedreich ataxia (FRDA) is an inherited recessive disorder caused by a deficiency in the mitochondrial protein frataxin. There is currently no effective treatment for FRDA available, especially for neurological deficits. In this study, we tested diazoxide, a drug commonly used as vasodilator in the treatment of acute hypertension, on cellular and animal models of FRDA. We first showed that diazoxide increases frataxin protein levels in FRDA lymphoblastoid cell lines, via the mammalian target of rapamycin (mTOR) pathway. We then explored the potential therapeutic effect of diazoxide in frataxin-deficient transgenic YG8sR mice and we found that prolonged oral administration of 3 mpk/d diazoxide was found to be safe, but produced variable effects concerning efficacy. YG8sR mice showed improved beam walk coordination abilities and footprint stride patterns, but a generally reduced locomotor activity. Moreover, they showed significantly increased frataxin expression, improved aconitase activity, and decreased protein oxidation in cerebellum and brain mitochondrial tissue extracts. Further studies are needed before this drug should be considered for FRDA clinical trials.

Oxazepam-Dopamine Conjugates Increase Dopamine Delivery into Striatum of Intact Rats.

The neurotransmitter dopamine (DA) was covalently linked to oxazepam (OXA), a well-known positive allosteric modulator of γ-aminobutyric acid type-A (GABAA) receptor, through a carbamate linkage (4) or a succinic spacer (6). These conjugates were synthesized with the aim of improving the delivery of DA into the brain and enhancing GABAergic transmission, which may be useful for the long-term treatment of Parkinson disease (PD). Structure-based permeability properties, in vitro stability, and blood-brain barrier (BBB) permeability studies led to identify the OXA-DA carbamate conjugate 4a as the compound better combining sufficient stability and ability to cross BBB. Finally, in vivo microdialysis experiments in freely moving rats demonstrated that 4a (20 mg/kg, i.p.) significantly increases extracellular DA levels into striatum, with a peak (more than 15-fold increase over the baseline) at about 80 min after a single administration. The stability and delivery data proved that 4a may be a promising candidate for further pharmacological studies in animal models of PD.

Preactivated thiolated glycogen as mucoadhesive polymer for drug delivery.

The purpose of this study was to synthesize and characterize a novel thiolated glycogen, so-named S-preactivated thiolated glycogen, as a mucosal drug delivery systems and the assessment of its mucoadhesive properties. In this regard, glycogen-cysteine and glycogen-cysteine-2-mercaptonicotinic acid conjugates were synthesized. Glycogen was activated by an oxidative ring opening with sodium periodate resulting in reactive aldehyde groups to which cysteine was bound via reductive amination. The obtained thiolated polymer displayed 2203.09±200µmol thiol groups per gram polymer. In a second step, the thiol moieties of thiolated glycogen were protected by disulfide bond formation with the thiolated aromatic residue 2-mercaptonicotinic acid (2MNA). In vitro screening of mucoadhesive properties was performed on porcine intestinal mucosa using different methods. In particular, in terms of rheology investigations of mucus/polymer mixtures, the S-preactivated thiolated glycogen showed a 4.7-fold increase in dynamic viscosity over a time period of 5h, in comparison to mucus/Simulated Intestinal Fluid control. The S-preactivated polymer remained attached on freshly excised porcine mucosa for 45h. Analogous results were obtained with tensile studies demonstrating a 2.7-fold increase in maximum detachment force and 3.1- fold increase in total work of adhesion for the S-preactivated polymer compared to unmodified glycogen. Moreover, water-uptake studies showed an over 4h continuing weight gain for the S-preactivated polymer, whereas disintegration took place for the unmodified polymer within the first hour. Furthermore, even in the highest tested concentration of 2mg/ml the new conjugates did not show any cytotoxicity on Caco-2 cell monolayer using an MTT assay. According to these results, S-preactivated glycogen represents a promising type of mucoadhesive polymers useful for the development of various mucosal drug delivery systems.

Natural dendrimers: Synthesis and in vitro characterization of glycogen-cysteamine conjugates.

The aim of this study was to synthesize, characterize and evaluate the mucoadhesive properties of the first thiolated hyperbranched natural polysaccharide with biodegradability and biocompatibility features. In detail, glycogen-cysteamine conjugates were synthesized through a first step of oxidative ring opening applying increasing concentrations of sodium periodate, to obtain polymers with different degrees of oxidation, and a second step of reductive amination with a constant amount of cysteamine. The obtained glycogen-cysteamine conjugates were characterized regarding their content of free and total thiol groups by Ellman's assay, biocompatibility, swelling/erosion behavior, rheological synergism and mucoadhesive properties in comparison to the unmodified glycogen. The higher the concentration of periodate was, the higher was the content of total thiol groups being in the range of 255.7±12-1194.5±82µmol/g, biocompatibility remained unaffected by these structural changes. On the contrary, the mucoadhesive properties, evaluated by tensile, rheological synergism and rotating cylinder studies, appear to be influenced by the thiol groups concentration on the glycogen. In particular the glycogen-cysteamine conjugate exhibiting the highest degree of thiolation showed a 79-fold increase in viscosity over a time period of 8h, as well as, remained attached on freshly excised porcine mucosa 32-fold longer than the unmodified polymer. The higher was the amount of conjugated thiol groups, the higher was the water absorption capacity of glycogen-cysteamine tablets in Simulated Intestinal Fluid pH 6.8 (SIF). The introduction of thiol moieties on polymer changed the characteristics of the polysaccharide by improving mucoadhesion properties. Therefore, this work represents the first study describing thiolated natural dendrimers as potential platform useful to realize appropriate mucoadhesive nanocarrier systems suitable to prolong mucosal residence time.

Spray-dried mucoadhesives for intravesical drug delivery using N-acetylcysteine- and glutathione-glycol chitosan conjugates.

UNLABELLED: This work describes N-acetylcysteine (NAC)- and glutathione (GSH)-glycol chitosan (GC) polymer conjugates engineered as potential platform useful to formulate micro-(MP) and nano-(NP) particles via spray-drying techniques. These conjugates are mucoadhesive over the range of urine pH, 5.0-7.0, which makes them advantageous for intravesical drug delivery and treatment of local bladder diseases. NAC- and GSH-GC conjugates were generated with a synthetic approach optimizing reaction times and purification in order to minimize the oxidation of thiol groups. In this way, the resulting amount of free thiol groups immobilized per gram of NAC- and GSH-GC conjugates was 6.3 and 3.6mmol, respectively. These polymers were completely characterized by molecular weight, surface sulfur content, solubility at different pH values, substitution and swelling degree. Mucoadhesion properties were evaluated in artificial urine by turbidimetric and zeta (ζ)-potential measurements demonstrating good mucoadhesion properties, in particular for NAC-GC at pH 5.0. Starting from the thiolated polymers, MP and NP were prepared using both the Büchi B-191 and Nano Büchi B-90 spray dryers, respectively. The resulting two formulations were evaluated for yield, size, oxidation of thiol groups and ex-vivo mucoadhesion. The new spray drying technique provided NP of suitable size (<1µm) for catheter administration, low degree of oxidation, and sufficient mucoadhesion property with 9% and 18% of GSH- and NAC-GC based NP retained on pig mucosa bladder after 3h of exposure, respectively. STATEMENT OF SIGNIFICANCE: The aim of the present study was first to optimize the synthesis of NAC-GC and GSH-GC, and preserve the oxidation state of the thiol moieties by introducing several optimizations of the already reported synthetic procedures that increase the mucoadhesive properties and avoid pH-dependent aggregation. Second, starting from these optimized thiomers, we studied the feasibility of manufacturing MP and NP by spray-drying techniques. The aim of this second step was to produce mucoadhesive drug delivery systems of adequate size for vesical administration by catheter, and comparable mucoadhesive properties with respect to the processed polymers, avoiding thiolic oxidation during the formulation. MP with acceptable size produced by spray-dryer Büchi B-191 were compared with NP made with the apparatus Nano Büchi B-90.

Synthesis and Evaluation of Tricarbonyl (99m)Tc-Labeled 2-(4-Chloro)phenyl-imidazo[1,2-a]pyridine Analogs as Novel SPECT Imaging Radiotracer for TSPO-Rich Cancer.

The 18-kDa translocator protein (TSPO) levels are associated with brain, breast, and prostate cancer progression and have emerged as viable targets for cancer therapy and imaging. In order to develop highly selective and active ligands with a high affinity for TSPO, imidazopyridine-based TSPO ligand (CB256, 3) was prepared as the precursor. (99m)Tc- and Re-CB256 (1 and 2, respectively) were synthesized in high radiochemical yield (74.5% ± 6.4%, decay-corrected, n = 5) and chemical yield (65.6%) by the incorporation of the [(99m)Tc(CO)3(H2O)3]⁺ and (NEt4)2[Re(CO)3Br3] followed by HPLC separation. Radio-ligand 1 was shown to be stable (>99%) when incubated in human serum for 4 h at 37 °C with a relatively low lipophilicity (logD = 2.15 ± 0.02). The rhenium-185 and -187 complex 2 exhibited a moderate affinity (Ki = 159.3 ± 8.7 nM) for TSPO, whereas its cytotoxicity evaluated on TSPO-rich tumor cell lines was lower than that observed for the precursor. In vitro uptake studies of 1 in C6 and U87-MG cells for 60 min was found to be 9.84% ± 0.17% and 7.87% ± 0.23% ID, respectively. Our results indicated that (99m)Tc-CB256 can be considered as a potential new TSPO-rich cancer SPECT imaging agent and provides the foundation for further in vivo evaluation.

Spray Dried Chitosan Microparticles for Intravesical Delivery of Celecoxib: Preparation and Characterization.

PURPOSE: Chitosan microparticles containing celecoxib (CB), were developed as chemoprevention of bladder cancer. Furthermore two inclusion complexes of CB with methyl-ß-cyclodextrin (C1 and C2) were prepared to improve the solubility of the drug. METHODS: C1 and C2 were obtained by freeze-drying and characterized in the solid state and in solution. Microparticles loaded with CB or C1 or C2 were prepared by spray drying and fully characterized. RESULTS: The yield and encapsulation efficiencies of microparticles depended by both the viscosity and the presence of the inclusion complex in the feed medium nebulised. Generally, the microparticles exhibited a spherical shape with mean diameter of approximately 2 µm which was compatible with local intravesical administration using a catheter. The CB release studies from the microparticles allowed us to identify both immediate release systems (microparticles including the complexes) and prolonged release systems (microparticles including CB alone). The latter exhibited good adhesion to the bladder mucosa, as highlighted by a mucoadhesion study. Histological studies revealed a desquamation of the superficial cells when the bladder mucosa was treated with microparticles loaded with CB, while the morphology of the urothelium did not change when it was treated with microparticles loaded with the inclusion complex. CONCLUSION: A new CB intravesical formulation than can easily be administered with a catheter and is able to release the drug at the target site for several hours was realized. This new delivery system could be a good alternative to classic oral CB administration.

Thiolated graphene oxide as promising mucoadhesive carrier for hydrophobic drugs.

The aim of this study was to improve the mucoadhesive properties of graphene by conjugating thiol ligands, in order to formulate an oral delivery system for hydrophobic drugs showing long mucus residence time. Graphene oxide was obtained by oxidation of graphite and then was thiolated following two synthetic paths. On the one hand, the hydroxyl groups were conjugated with thiourea passing through the formation of a brominated intermediate. On the other hand, the carboxylic acid groups were conjugated with cysteamine via carbodiimide chemistry. The mucoadhesive properties of thiolated graphene were evaluated by rheological measurements and by residence time assay. Then, valsartan was loaded on thiolated graphene and the release profile was evaluated in simulated intestinal fluid. Following both synthetic paths it was possible to obtain thiolated graphene bearing 215-302µmol SH/g product. Both products induced after 1h incubation an increase of mucus viscosity of about 22-33-fold compared to unmodified graphite. The residence time assay confirmed that 60% of thiolated graphene could be retained on intestinal mucosa after 4h incubation, whereas just 20% of unmodified graphite could be retained. Valsartan could be loaded with a drug loading of about 31±0.3% and a sustained release profile was observed for both formulations. According to the presented data, the thiolation of graphene could improve its mucoadhesive properties. Therefore, thiolated graphene represents a promising platform for oral delivery of hydrophobic drugs, possessing a long residence time on intestinal mucosa which allows the release of the loaded drug close to the adsorptive epithelium.

TSPO Ligand-Methotrexate Prodrug Conjugates: Design, Synthesis, and Biological Evaluation.

The 18-kDa translocator protein (TSPO) is a potential mitochondrial target for drug delivery to tumors overexpressing TSPO, including brain cancers, and selective TSPO ligands have been successfully used to selectively deliver drugs into the target. Methotrexate (MTX) is an anticancer drug of choice for the treatment of several cancers, but its permeability through the blood brain barrier (BBB) is poor, making it unsuitable for the treatment of brain tumors. Therefore, in this study, MTX was selected to achieve two TSPO ligand-MTX conjugates (TSPO ligand α-MTX and TSPO ligand γ-MTX), potentially useful for the treatment of TSPO-rich cancers, including brain tumors. In this work, we have presented the synthesis, the physicochemical characterizations, as well as the in vitro stabilities of the new TSPO ligand-MTX conjugates. The binding affinity for TSPO and the selectivity versus central-type benzodiazepine receptor (CBR) was also investigated. The cytotoxicity of prepared conjugates was evaluated on MTX-sensitive human and rat glioma cell lines overexpressing TSPO. The estimated coefficients of lipophilicity and the stability studies of the conjugates confirm that the synthesized molecules are stable enough in buffer solution at pH 7.4, as well in physiological medium, and show an increased lipophilicity compared to the MTX, compatible with a likely ability to cross the blood brain barrier. The latter feature of two TSPO ligand-MTX conjugates was also confirmed by in vitro permeability studies conducted on Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) monolayers. TSPO ligand-MTX conjugates have shown to possess a high binding affinity for TSPO, with IC50 values ranging from 7.2 to 40.3 nM, and exhibited marked toxicity against glioma cells overexpressing TSPO, in comparison with the parent drug MTX.

A Novel PET Imaging Probe for the Detection and Monitoring of Translocator Protein 18 kDa Expression in Pathological Disorders.

A new fluorine-substituted ligand, compound 1 (CB251), with a very high affinity (Ki = 0.27 ± 0.09 nM) and selectivity for the 18-kDa translocator protein (TSPO), is presented as an attractive biomarker for the diagnosis of neuroinflammation, neurodegeneration and tumour progression. To test compound 1 as a TSPO PET imaging agent in vivo, 2-(2-(4-(2-[(18)F]fluoroethoxy)phenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide ([(18)F]1; [(18)F]CB251) was synthesized by nucleophilic aliphatic substitution in a single-step radiolabelling procedure with a 11.1 ± 3.5% (n = 14, decay corrected) radiochemical yield and over 99% radiochemical purity. In animal PET imaging studies, [(18)F]CB251 provided a clearly visible image of the inflammatory lesion with the binding potential of the specifically bound radioligand relative to the non-displaceable radioligand in tissue (BPND 1.83 ± 0.18), in a neuroinflammation rat model based on the unilateral stereotaxic injection of lipopolysaccharide (LPS), comparable to that of [(11)C]PBR28 (BPND 1.55 ± 0.41). [(18)F]CB251 showed moderate tumour uptake (1.96 ± 0.11%ID/g at 1 h post injection) in human glioblastoma U87-MG xenografts. These results suggest that [(18)F]CB251 is a promising TSPO PET imaging agent for neuroinflammation and TSPO-rich cancers.

A new complex of curcumin with sulfobutylether-ß-cyclodextrin: characterization studies and in vitro evaluation of cytotoxic and antioxidant activity on HepG-2 cells.

Curcumin (CR) is a natural polyphenol with antioxidative, anti-inflammatory, and anticancer properties but its therapeutic potential is substantially hindered by the rather low-water solubility and bioavailability. Thus, in this work, a new soluble inclusion complex of CR with sulfobutylether-ß-cyclodextrin (SBE-ß-CD) was prepared in solution and at the solid state using different preparation techniques and characterized by Fourier transform infrared, nuclear magnetic resonance, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and Job's plot method. Results clearly indicate that CR reacts with SBE-ß-CD to form a host-guest complex with an apparent formation constant of 1455 M(-1) . Moreover, SBE-ß-CD strongly increases water solubility of CR (from 0.56 to 102.78 µg/mL, at 25°C), and lyophilization method seems to be the best preparation technique to obtain the complex at the solid state. Finally, an in vitro test on a human hepatic cancer cell line (HepG-2) shows that complexation positively influences CR anticancer and antioxidant activity.