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Hepatocytes isolated from adult rat livers were hypothermically preserved for 24 or 48 hr before being plated under conventional culture conditions. They were stored either in the Leibovitz medium, a cell culture medium with and without polyethylene glycol (PEG), a compound known to suppress ischemia-induced cell swelling, or in the University of Wisconsin solution, the most effective solution for cold organ preservation. After 24 or 48 hr of storage at 4.5 degrees C in Leibovitz medium, cell viability and adherence efficiency to plastic dish, were only slightly reduced, whereas University of Wisconsin hepatocytes had a decreased viability and (especially after 48-hr storage) lost their adhesion ability; they did not survive in vitro. The metabolic competence of hepatocytes maintained in Leibovitz medium was retained over the 3 days of culture, as shown by low extracellular levels of the membrane-bound and cytosolic hepatic enzymes, as well as by intracellular glutathione content, albumin secretion rate and several phase I and phase II drug metabolic reactions very close to those found with fresh hepatocytes maintained under similar culture conditions. Addition of polyethylene glycol to the Leibovitz medium resulted in slightly higher viability and function of hepatocytes after cold storage. These results clearly demonstrate that viability of a transplanted liver does not correlate with long-term in vitro viability of isolated hepatocytes after hypothermic preservation in University of Wisconsin solution. They also suggest that nutritional and energy substrates as found in the Leibovitz medium are probably required to define a suitable solution for cold preservation of isolated parenchymal cells. The findings with Leibovitz medium favor the conclusion that hypothermically preserved hepatocytes could be used for various metabolic studies and for the treatment of liver insufficiency.

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BackgroundArtemisinin-based combination therapy is currently recommended worldwide for the treatment of uncomplicated malaria. Fixed-dose combinations are preferred as they favour compliance. This paper reports on the initial phases of the pharmaceutical development of an artesunate-amodiaquine (ASAQ) bilayer co-formulation tablet, undertaken following pre-formulation studies by a network of scientists and industrials from institutions of both industrialized and low income countries.MethodsPharmaceutical development was performed by a research laboratory at the University Bordeaux Segalen, School of Pharmacy, for feasibility and early stability studies of various drug formulations, further transferred to a company specialized in pharmaceutical development, and then provided to another company for clinical batch manufacturing. The work was conducted by a regional public-private not-for-profit network (TropiVal) within a larger Public Private partnership (the FACT project), set up by WHO/TDR, Médecins Sans Frontières and the Drugs for Neglected Disease initiative (DNDi).ResultsThe main pharmaceutical goal was to combine in a solid oral form two incompatible active principles while preventing artesunate degradation under tropical conditions. Several options were attempted and failed to provide satisfactory stability results: incorporating artesunate in the external phase of the tablets, adding a pH regulator, alcoholic wet granulation, dry granulation, addition of an hydrophobic agent, tablet manufacturing in controlled conditions. However, long-term stability could be achieved, in experimental batches under GMP conditions, by physical separation of artesunate and amodiaquine in a bilayer co-formulation tablet in alu-alu blisters. Conduction of the workplan was monitored by DNDi.ConclusionsCollaborations between research and industrial groups greatly accelerated the process of development of the bi-layered ASAQ tablet. Lack of public funding was the main obstacle hampering the development process, and no intellectual property right was claimed. This approach resulted in a rapid technology transfer to the drug company Sanofi-Aventis, finalizing the process of development, registration and WHO pre-qualification of the fixed-dose co-formulation together with DNDi. The bi-layered tablet is made available under the names of Coarsucam® and Artesunate amodiaquine Winthrop®, Sanofi-Aventis. The issue related to the difficulty of public institutions to valorise their participation in such initiative by lack of priority and funding of applied research is discussed.

The structure integrity of sensitive molecule due to the small energy involved by HHP and the development of industrial plants (intended for the decontamination of food products) confer to this technology the potential of a new method for sterilization of fragile drugs and an original alternative to aseptic processes and sterilizing filtration.

After oral administration of amineptine (7-[(10-11)-dihydro-5H-dibenzo(a,d)cycloheptane-5yl] amino heptanoic acid), an original tricyclic antidepressant, seven metabolites were isolated from urine and plasma of rat, dog and man. The metabolic pathways were similar for the three species studied. The two major pathways consisted of the beta-oxidation of the heptanoic side chain leading to pentanoic (first step) and propanoic (second step) side chain metabolites and the hydroxylation of the dibenzocycloheptyl ring on carbon atom 10 (C10) causing the formation of two diastereoisomers. Lactamization by internal dehydration of beta-oxidized metabolites appeared to be a minor route of biotransformation. Conjugation reactions were of minor importance in the rat, in contrast to findings for dog and man. Urinary elimination was the major route of excretion in man while in dog and in rat faecal excretion was predominant.

The aim of this work was to develop a high-pressure decontamination and sterilization process for pharmaceutical treatments as was developed in food processing in the late eighties. The lack of normalized biological indicators able to validate sterilizing treatments under high pressure led us to select representative pathogenic strains from flora and the European Pharmacopoeia. We selected the following four bacterial strains: Candida albicans (ATCC 10231), Psuedomonas aeruginosa (ATCC 9027), spores of Aspergillus niger (ATCC 16404) and Staphylococcus aureus (ATCC 6538).This present study is focussed on S. aureus. Successive pressurization and depressurization cycles appeared to be more efficient than a continuous high-pressure treatment. Importantly, these pressure conditions, temperature and process duration are perfectly compatible with current industrial plants. These results show that HHP technology is a new alternative to inactivate pathogenic strains in accordance with pharmaceutical requirements.