Preclinical characterization of alginate-poly-L-lysine encapsulated HepaRG for extracorporeal liver supply.

We recently demonstrated that HepaRG cells encapsulated into 1.5% alginate beads are capable of self-assembling into spheroids. They adequately differentiate into hepatocyte-like cells, with hepatic features observed at Day 14 post-encapsulation required for external bioartificial liver applications. Preliminary investigations performed within a bioreactor under shear stress conditions and using a culture medium mimicking acute liver failure (ALF) highlighted the need to reinforce beads with a polymer coating. We demonstrated in a first step that a poly-l-lysine coating improved the mechanical stability, without altering the metabolic activities necessary for bioartificial liver applications (such as ammonia and lactate elimination). In a second step, we tested the optimized biomass in a newly designed perfused dynamic bioreactor, in the presence of the medium model for pathological plasma for 6 h. Performances of the biomass were enhanced as compared to the steady configuration, demonstrating its efficacy in decreasing the typical toxins of ALF. This type of bioreactor is easy to scale up as it relies on the number of micro-encapsulated cells, and could provide an adequate hepatic biomass for liver supply. Its design allows it to be integrated into a hybrid artificial/bioartificial liver setup for further clinical studies regarding its impact on ALF animal models.

Complementary mass spectrometric approaches and scanning electron microscopy to study the structural stability of polyurethane tunneled dialysis catheters after exposure to ethanol solutions.

RATIONALE: Ethanol lock is an emerging therapeutic option for preventing and/or controlling catheter-associated infection. A previous study of silicone catheters showed they underwent no polymer degradation when kept in 60% ethanol for 15 days at 37 °C. The stability of the more widely used polyurethane catheters was studied here in the same way. METHODS: A qualitative and quantitative study of the stability of Carbothane® catheters was performed following their immersion at 37 °C in different solvents (0.9% sodium chloride as control medium and 40%, 60%, 95% ethanol solutions) for different periods of time (from 5 min to 15 days) using scanning electron microscopy and complementary mass spectrometry techniques. RESULTS: Electron ionization analysis of the 95% ethanol storage solutions revealed the release of about 45 products (8 of which were major) subdivided into two groups according to their fragmentation patterns. Combining all the mass spectrometric data made it possible to propose structures. Group I (major) originated from the polycarbonate diol component (soft segment) and group II (minor) from the dicyclohexylmethane-4,4'-diisocyanate component (rigid segment). Semi-quantitative gas chromatography/mass spectrometry (GC/MS) analysis showed that no significantly higher release was observed after immersion for 30 min at 37 °C in 40% ethanol (mean ratio = 0.677 ± 0.068) than after immersion in reference 0.9% sodium chloride solution for 15 days (0.837 ± 0.127). CONCLUSIONS: A 30 min-40% (v/v) ethanol solution can be considered as safe for preventing the infectious complications of Carbothane® dialysis catheters, and a 30 min-60% (v/v) ethanol treatment can be occasionally used to eradicate established biofilm.

HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver.

In liver tissue engineering, cell culture in spheroids is now well recognized to promote the maintenance of hepatic functions. However, the process leading to spheroids formation is time consuming, costly, and not easy to scale-up for further use in human bioartificial liver (BAL) applications. In this study, we encapsulated HepaRG cells (precursors of hepatocyte-like cells) in 1.5% alginate beads without preforming spheroids. Starting from a given hepatic biomass, we analyzed cell differentiation and metabolic performance for further use in a fluidized-bed BAL. We observed that cells self-rearranged as aggregates within the beads and adequately differentiated over time, in the absence of any differentiating factors classically used. On day 14 postencapsulation, cells displayed a wide range of hepatic features necessary for the treatment of a patient in acute liver failure. These activities include albumin synthesis, ammonia and lactate detoxification, and the efficacy of the enzymes involved in the xenobiotic metabolism (such as CYP1A1/2). Impact statement It has been recognized that culturing cells in spheroids (SPHs) is advantageous as they better reproduce the three-dimensional physiological microenvironment. This approach can be exploited in bioartificial liver applications, where obtaining a functional hepatic biomass is the major challenge. Our study describes an original method for culturing hepatic cells in alginate beads that makes possible the autonomous formation of SPHs after 3 days of culture. In turn, the cells differentiate adequately and display a wide range of hepatic features. They are also capable of treating a pathological plasma model. Finally, this setup can easily be scaled-up to treat acute liver failure.

Evaluation of radiolabeled (hetero)aromatic analogues of N-(2-diethylaminoethyl)-4-iodobenzamide for imaging and targeted radionuclide therapy of melanoma.

Targeted radionuclide therapy using radioiodinated compounds with a specific affinity for melanoma tissue is a promising treatment for disseminated melanoma, but the candidate with the ideal kinetic profile remains to be discovered. Targeted radionuclide therapy concentrates the effects on tumor cells, thereby increasing the efficacy and decreasing the morbidity of radiotherapy. In this context, analogues of N-(2-diethylaminoethyl)-4-iodobenzamide (BZA) are of interest. Various (hetero)aromatic analogues 5 of BZA were synthesized and radioiodinated with (125)I, and their biodistribution in melanoma-bearing mice was studied after i.v. administration. Most [ (125)I] 5-labeled compounds appeared to bind specifically and with moderate-to-high affinity to melanoma tumor. Two compounds, 5h and 5k, stood out with high specific and long-lasting uptake in the tumor, with a 7- and 16-fold higher value than BZA at 72 h, respectively, and kinetic profiles that makes them promising agents for internal targeted radionuclide therapy of melanoma.

New opportunities with the Duff reaction.

The Duff reaction (HMTA, AcOH or TFA) was studied on substituted [6 + 5] heterocyclic compounds. This reaction provides a useful route to aldehydes for compounds bearing sensitive amide functions. It gives also access to tricyclic lactams of potential biological interest. The formation of an aminomethyl intermediate in the Duff reaction mechanism is unequivocally demonstrated.

Design, synthesis and preliminary biological evaluation of acridine compounds as potential agents for a combined targeted chemo-radionuclide therapy approach to melanoma.

Various iodo-acridone and acridine carboxamides have been prepared and evaluated as agents for targeted radionuclide and/or chemotherapy for melanoma, due to their structural similarity to benzamides which are known to possess specific affinity for melanin. Three of these carboxamides selected for their in vitro cytotoxic properties were radioiodinated with [(125)I]NaI at high specific activity. Biodistribution studies carried out in B16F0 murine melanoma tumour-bearing mice highlighted that acridone 8f and acridine 9d, presented high, long-lasting tumour concentrations together with an in vivo kinetic profile favourable to application in targeted radionuclide therapy.

Compatibility of Injectable Anticoagulant Agents in Ethanol; In Vitro Antibiofilm Activity and Impact on Polyurethane Catheters of Enoxaparin 400 U/mL in 40% v/v Ethanol.

BACKGROUND AND OBJECTIVES: Interdialytic lock solutions should maintain catheter patency and prevent catheter infections. We aimed to determine in which conditions injectable anticoagulant agents (IAAs) combined with ethanol are compatible and to assess the antibiofilm activity of the selected combination and its effects on dialysis catheters (DC). METHODS: The solubility and compatibility of unfractionated heparin (UFH), low molecular weight heparins (LMWHs), heparinoids and fondaparinux (50 to 2,500 U/mL) in 30 to 70% ethanol were determined by visual observation. The stability of enoxaparin in ethanol and the ethanol content were assessed by high performance liquid chromatography (HPLC) and titrimetric control, respectively. The bactericidal effect was determined on 24h-old biofilms embedded in silicone-DC. The integrity of polyurethane-DC immersed in anticoagulant-ethanol was assessed by gas chromatography-mass spectrometry (GC-MS) and compared with previously published results. RESULTS: The compatibility of IAAs and ethanol varied according to IAA type and concentration, and ethanol content. UFH in 40% ethanol was not compatible, whatever the UFH concentration used. Established limits of compatibility of enoxaparin, nadroparin, dalteparin and tinzaparin in 40% ethanol were 1350, 575, 307 and 207 U/ml, respectively, and up to 300 U/ml for danaparoid and 1 mg/mL for fondaparinux. Enoxaparin 400 U/mL in 40% ethanol (Enox/Eth) eradicated biofilm after 4 hours of exposure for Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans and after 24 hours for Klebsiella pneumoniae and S. aureus. Aliphatic carbonate and alcohol compounds were released by polyurethane-DC after Enox/Eth exposure, as after 40% ethanol or saline exposure. There was no significant difference between the amounts released after 30 minutes of exposure to Enox/Eth and 15 days to saline. CONCLUSIONS: A 40% ethanol solution can be combined with all IAAs but UFH. Enox/Eth was effective as an anti-biofilm agent with minor impacts on DC integrity and could be a useful interdialytic lock solution.

Identification of degradation products of diclofenac by electrospray ion trap mass spectrometry.

The degradation products of diclofenac in aqueous dosage form in accelerated storage conditions were characterized by electrospray ionization-ion trap mass spectrometry (ESI-MS). Liquid chromatography (LC)-MS analyses revealed the presence of three degradation products. ESI-MS(n) spectra were used to study diclofenac fragmentation in detail and to characterize the structures of degradation products. A previously described degradation product, formed by a cyclization reaction of diclofenac producing the indolinone derivative, was found. As any hydroxylated product was found, no oxidation seems to occur in the dosage form used. On the contrary, two degradates have been detected and identified, leading to a primary alcohol structure or an aldehyde function in place of the acetate group of diclofenac.

Anticoagulant properties of enoxaparin 400 IU/mL-40 % ethanol catheter lock solution.

Unfractionated heparin (UFH) is the most widely used interdialytic lock solution but has no anti-infectious properties. Ethanol at a content ≥40 %v/v eradicates experimental biofilm but has no anticoagulant properties. In contrast to UFH, enoxaparin (Enox) can be combined with 40 % ethanol without precipitation. Enoxaparin 400 UI/mL-40 % ethanol (Enox/Eth) has antibiofilm properties and therefore has promise as an alternative lock solution. This study assessed the anticoagulant properties of Enox/Eth. Enox and Enox/Eth were diluted in whole blood at a final Enox concentration of 0.5, 1 (N = 6 samples), 1.5 (N = 4) and 2 (N = 6) IU/mL. Anti-Xa activity was determined by chromogenic assay and the inhibition of endogenous thrombin potential (ETP) by thrombinography. Quantitative data were compared by the Mann-Withney U test. For Enox concentrations of 0.5, 1, 1.5 and 2 UI/mL in whole blood samples, the mean ± SD values of the anti-Xa activity were 0.68 ± 0.09, 1.26 ± 0.14, 1.73 ± 0.30, 2.35 ± 0.32 UI/mL for Enox/Eth and 0.94 ± 0.15, 1.80 ± 0.22, 2.74 ± 0.23, 3.54 ± 0.44 UI/mL for Enox (P = 0.03, P = 0.03, P = 0.13, P = 0.03); and of the percentage of ETP inhibition was 17.36 ± 9.65, 30.27 ± 17.06, 36.5 ± 17.06, 57.82 ± 15.42 for Enox/Eth, and 42.96 ± 15.68, 68.93 ± 10.01, 83.5 ± 8.81, 91.19 ± 4.67 for Enox (P = 0.03, P = 0.03, P = 0.13, P = 0.03), respectively. The median and IQR values of Enox concentration inhibiting 50 % of ETP (IC50 ETP) were 1.8 [1.1-2.4] IU/mL for Enox/Eth and 0.7 [0.3-0.9] IU/mL for Enox, P = 0.03. Enox/Eth has strong anticoagulant activity, albeit lower than that of Enox, but with an extremely low IC50 ETP compared to the Enox concentration of non-diluted Enox/Eth.

Synthesis and antiviral activity of an imidazo[1,2-a]pyrrolo[2,3-c]pyridine series against the bovine viral diarrhea virus.

A series of imidazo[1,2-a]pyrrolo[2,3-c]pyridines has been prepared and evaluated for their anti-BVDV activities in MDBK cells. From the synthesized analogues bearing modifications of the substituents at positions 2, 3, 7 and 8, compounds 10a, b, 16, 24, 25 and 26 exhibited significant anti-BVDV activities.

Mass spectrometry and scanning electron microscopy study of silicone tunneled dialysis catheter integrity after an exposure of 15 days to 60% ethanol solution.

Anti-infectious lock is an emerging therapeutic option for preventing and/or controlling catheter-associated infection. Ethanol has widespread bactericidal activity, limited side effects, and low risk of inducing antimicrobial resistance. However, concerns have been raised about ethanol-induced catheter structural degradation. In this study, silicone catheters were immersed at 37 degrees C in three different solvents: 0.9% sodium chloride, 60% ethanol, and 95% ethanol for 4 h, 15 days and 15 days after a first storage of 4 h. Scanning electron microscopy (magnification 1000-20 000 times) of the inner surface of the catheter revealed no damage to the lumen surfaces of catheters immersed in 95% ethanol for 15 days compared with the reference catheter. Gas chromatography/mass spectrometry (GC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis of the storage solutions revealed a significant release of polydimethylsiloxanes having a number of dimethylsiloxane units lower than 30 in the 95% ethanol solution and a structure highly consistent with a cyclic structure. Most release occurred within the first 4 h of exposure. In contrast, there was no difference in the small amounts of silicone released in 0.9% sodium chloride as reference and 60% ethanol solution, whatever the exposure time. These results should allow the development of clinical trials to assess the efficacy of the 60% ethanol lock technique in preventing or controlling the infectious complications of silicone dialysis catheters.

New 3- and 4-hydroxyfuranones as anti-oxidants and anti-inflammatory agents.

Two series of new furanones substituted by methylsulfonylphenyl or methylsulfamidophenyl moieties were found to protect against oxidation damage by inhibiting or quenching free radicals and reactive oxygen species in in vitro experiments. The effect on lipid peroxidation was also examined. In addition, we investigated the activity of products in two models of inflammation: phorbol ester-induced ear edema in mice and carrageenan-induced paw edema in rat. The most powerful compounds and with reducing activity against DPPH (IC50=1779 and 57 microM, respectively), superoxide anion quenching capacity (IC50=511 and 49 microM, respectively), lipid peroxidation inhibitory effect and anti-inflammatory properties (about 50-65% inhibition of edema at 200 mg/kg ip in both tests used) were selected for further pharmacological and toxicological tests because of their attractive profile for the treatment of inflammatory diseases.