The mobility of N-alkyl nicotinamides and their solubilization equilibria in surfactant micellar solution were investigated using an NMR paramagnetic relaxation method. The spin-lattice relaxation times (Tl) . for protons of these compounds were measured in pure D20 and in cationic surfactant solution in the presence and absence of · a low concentration of paramagnetic Ma2+ ions. The rotational motion of these molecules in aqueous phase became slower when the alkyl group changed from methyl to octyl. The increase of the 1H spinlattice relaxation rate (Rl) of the molecules, when surfactants are added, implies the penetration of these solubilizates into micellar phase. The micelle to water phase distribution coefficient, 1-p, was determined by monitoring the change of Rl of the solubilizates upon addition of paramagnetic ions to the aqueous phase. The mole fraction based distribution coefficient, Kx, as well as the free energy of transfer of N-alkyl nicotinamides from the aqueous phase to the micellar phase were calculated. A model was postulated for the interaction of N -alkyl nicotinamide with micelles. Hydrophobic force between the alkyl chains of the solubilizates and the surfactants accounts for the solubilization of N -alkyl nicotinamides and can force the binding of cationic compounds to cationic micelles · despite charge repulsion. With increasing of the alkyl chain length, the hydrophobic force increases, and the interaction between them becomes stronger, and more N -alkyl nicotinamides are solubilizated into the micellar phase.

The F420-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F420 as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum of 7.5. The NADP reductase had an apparent Km of 42 uM for reduced F420, and an apparent Km of 4l uM for NADP. The enzyme was stable in 0.05 M sodium phosphate buffer (plus 10 mM cysteine) at pH 7.0, when gassed with nitrogen or hydrogen and stored at 4°C.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24516号 / 医博第4958号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 岩田 想, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The crystalline solid state is invaluable to both the pharmaceutical and fine chemical sectors. The advantages primarily relate to reducibility criteria required during processing of stable solid state materials and delivering purification, which is inherently performed by the crystal growth process. A major challenge is achieving control through crystallising solids with the desired physico-chemical properties. If this can be achieved the crystalline solid is of great financial and practical benefit. One emerging methodology for manipulating the solid crystalline form is the application of co-crystals. This work relates to key steps in the understanding of rational design of co-crystals utilizing crystal engineering concepts to determine systems before then applying screening criteria to the selected sub-set. Co-crystal screening is routinely undertaken using high-throughput solution growth. We report a low- to medium-throughput approach, encompassing both a melt and solution crystallization step as a route to the identification of co-crystals. Prior to solution studies, a melt growth step was included utilizing the Kofler mixed fusion method. This method allowed elucidation of the thermodynamic landscape within the binary phase diagram and was found to increase overall screening efficiency. This led to the discovery of a number of co-crystal systems with the co-former nicotinamide, with the single crystal structures determined for the following systems; R/S ibuprofen: nicotinamide, S ibuprofen: nicotinamide, R/S flurbiprofen: nicotinamide and salicylic acid: nicotinamide. To assess the crystallization and phase behaviours of determined co-crystals the R/S ibuprofennicotinamide system was selected and successful studies were undertaken determining the aqueous ternary phase behavior and the pre-nucleation speciation in methanol. There have, as yet, been a limited number of published examples which are concerned with pharmaceutical property enhancement by co-crystals, as vast proportion of the literature concerns the growth and isolation of these novel phases. To elucidate further the pharmaceutical relevance of co-crystals the properties of the R/S ibuprofen- nicotinamide system were then assessed showing a positive profile for this material. / AstraZeneca and the University of Bradford / The accompanying "Experimental raw data files and cifs" are not available online.

Dispersing co-crystals in a polymeric carrier may improve their physicochemical properties such as dissolution rate and solubility. Additionally co-crystal stability may be enhanced. However, such dispersions have been little investigated to date. This study focuses on the feasibility of dispersing co-crystals in a polymeric carrier and theoretical calculations to predict their stability. Acetone/chloroform, ethanol/water, and acetonitrile were used to load and grow co-crystals in a HPMCAS film. Caffeine-malonic acid and ibuprofennicotinamide co-crystals were prepared using solvent evaporation method. The interactions between each of the co-crystals components and their mixtures with the polymer were studied. A solvent evaporation approach was used to incorporate each compound, a mixture, and co-crystals into HPMCAS films. Differential scanning calorimetry data revealed a higher affinity of the polymer to acidic compounds than their basic counterparts as noticed by the depression of the glass transition temperature (Tg). Moreover, the same drug loading produced films with different Tgs when different solvents were used. Solubility parameter values (SP) of the solvents were employed to predict that effect on the depression of polymer Tg with relative success. SP values were more successful in predicting the preferential affinity of two acidic compounds to interact with the polymer. This was confirmed using binary mixtures of naproxen, flurbiprofen, malonic acid, and ibuprofen. On the other hand, dispersing basic compounds such as caffeine or nicotinamide with malonic acid in HPMCAS film revealed the growth of co-crystals. A dissolution study showed that the average release of caffeine from films containing caffeine-malonic acid was not significantly different to that of films containing similar caffeine concentration. The stability of the caffeine-malonic acid co-crystals in HPMC-AS was prolonged to 8 weeks at 95% relative humidity and 45°C. The theory developed in this project, that an acidic drug with a SP value closer to the polymer will dominate the interaction process and prevent the majority of the other material from interacting with the polymer, may have utility in designing co-crystal systems in polymeric vehicles

Strawberry plants (Fragaria x ananassa) and Scots pine (Pinus sylvestris) represent species, withinagriculture and forestry respectively, that are traditionally protected by utilization of pesticidesincluding neurotoxic insecticides. More environmentally friendly protection strategies are thereforehighly desirable. Treating plants with specific metabolites naturally occurring in their tissues might alterepigenetic mechanisms, which in turn may strengthen plants self-defense against diseases and weevilattacks. F. x ananassa and P. sylvestris seeds were treated with 2,5 mM nicotinamide and 2,5 mMnicotinic acid in order to investigate possible epigenetical effects by analyzing changes in the level ofthe DNA methylation. The epigenetic changes, for both plants, were analyzed on the global DNA level.Reduction in the DNA methylation level in strawberry leaves as well as the DNA methylation increase inpine needles were observed by means of LUMA-analysis when HpaII restriction enzyme was used in theanalysis. Further investigation is required in order to understand if NIC and NIA may have a significantimpact on pathogen attack in strawberry plants and Scots pine. More research may also unveil ifnicotinamide and nicotinic acid can play a potential role in more sustainable defense strategies ofplants. / Jordgubbsplantor (Fragaria x ananassa) och tallar (Pinus sylvestris) representerar växter inom jord- ochskogsbruk som traditionellt skyddas genom användning av bekämpningsmedel, detta inklusiveneurotoxiska insekticider. Mer miljövänliga skyddsstrategier är därför mycket önskvärda. Behandling avväxter med specifika naturligt förekommande metaboliter genererade av växterna kan påverkaepigenetiska mekanismer. Förändringar på den epigenetiska nivån kan, i sin tur, bidra till förstärkningav växternas eget självförsvar mot sjukdomar och insektsangrepp. Frön av både F. x ananassa och P.sylvestris behandlades med 2,5 mM nikotinamid och 2,5 mM nikotinsyra i syfte att undersökaeventuella epigenetiska effekter. Detta genom att analysera förändringar i graden av DNA metylering ide behandlade plantorna. De epigenetiska förändringarna för jordgubbsplantor och tallar analyseradespå den globala DNA-nivån. Minskad DNA-metylering i jordgubbsblad samt ökad DNA-metylering itallbarr observerades med hjälp av restriktionsenzymet Hpall och LUMA-analys. Ytterligareundersökningar behövs för att kunna förstå om NIC och NIA kan ha en inverkan på patogenangrepp ijordgubbsplantor och tall. Mer forskning kan också avslöja om nikotinamid och nikotinsyra kan ha enbetydande roll inom hållbara försvarsstrategier för växter.

Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. While there is much correlative evidence to suggest a connection between poly(ADP-ribose) (PAR) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 — the enzyme responsible for synthesis of 90% of cellular PAR — in mechanisms of senescence. Furthermore, many techniques currently used for analysis of protein poly(ADP-ribosyl)ation are fraught with imprecision. We therefore sought to address these issues both by developing methods for the unambiguous analysis of poly(ADP-ribosyl)ation by mass spectrometry, and by exploring the role of PARP-1 in nicotinamide-mediated cellular lifespan extension. Due to the challenges introduced by PAR's biochemical characteristics, successful mass spectrometric analysis of poly(ADP-ribosylation) will require the use of techniques to reduce the mass, charge, and heterogeneity of the polymer, as well as methods to enrich for poly(ADP- ribosyl)ated protein. To this end, we evaluated the effectiveness of several approaches, including ammonium sulfate fractionation, boronate affinity chromatography, snake venom phosphodiesterase digestion, manipulation of PARP-1 reaction conditions, and immobilized metal affinity chromatography (IMAC) for the preparation of poly(ADP-ribosyl)ated protein samples prior to MS analysis using both MALDI-TOF and Q-TRAP LC-MS. Based on this work, we developed a three-tiered scheme that may provide the first ever identification of poly(ADP- ribosyl)ated peptides from full-length wild-type PARP-1 by mass spectrometry. Past work in our laboratory has demonstrated that nicotinamide (NAM), a component of vitamin B3, significantly extends the replicative lifespan of human fibroblasts. In order to help elucidate the role of PARP-1 in cellular senescence, we then analyzed the poly(ADP-ribosyl)ation response of aging cells undergoing NAM-mediated lifespan extension. While NAM is a known PARP-1 inhibitor, we found that oxidative stress-induced poly(ADP- ribosyl)ation is increased, not decreased, in NAM-treated cells. We propose that supplemented NAM is taken up by the NAD salvage pathway, ultimately leading to increased cellular NAD and extending replicative lifespan by both preventing PARP-mediated NAD depletion and upregulating SIRT1. We further propose that the demonstrated protective effects of NAM treatment in a number of disease models are due not to PARP-1 inhibition as is commonly assumed, but to upregulation of NAD salvage. / Ph. D.

The objective of this study was to develop a new method for co-crystal preparation which adhered to green chemistry principles, and provided advantages over conventional methods. A novel, solvent-free, high-power ultrasound (US) technique, for preparing co-crystals from binary systems, was chosen as the technology which could fulfil these aims. The application of this technology for solid state co-crystal preparation was explored for ibuprofen-nicotinamide (IBU-NIC), carbamazepine-nicotinamide (CBZ-NIC) and carbamazepine-saccharin (CBZ-SAC) co-crystals. The effect of different additives and processing parameters such as power level, temperature and sonication time on co-crystallisation was investigated. Characterisation was carried out using DSC, PXRD, FTIR, Raman and HPLC. In addition, an NIR prediction model was developed and combined with multivariate analysis (PLS) and chemometric pre-treatments. It was found to be a robust, reliable and rapid method for the determination of co-crystal purity for the IBU-NIC and CBZ-NIC pairs. Co-crystal quantification of US samples helped to optimise the US method. Finally, a model formulation of paracetamol containing 5% and 10% PEG 8000 was ultrasonicated at maximum power with different exposure times. A comparison of technological and physicochemical properties of the resulting tablets with those of the tablets obtained using the pressing method evidenced significant differences. This suggested that US energy dissipation (mechanical and thermal effects) was the main mechanism which caused the PAR form I tabletability to improve. It was found that the ultrasound–compacted tablets released the drug at a slower rate compared to pure PAR. This technique was shown to be useful for improving tabletability for low-compressible drugs without the need to use a conventional tabletting machine.

The nicotinamide riboside (NR) form of vitamin B3and the ketone body ß-hydroxybutyrate (BHB) are two of the most promising natural compounds yet identified for the treatment of aging and aging-related diseases. Forms of vitamin B3are precursors for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD(H)) and nicotinamide adenine dinucleotide phosphate (NADP(H)). In aged cells levels of NAD+decline, decreasing metabolism and decreasing activity of protective sirtuin protein deacetylases. In aged cells NR, but not more common forms of vitamin B3, boost NAD+levels. BHB is naturally produced by the body when individuals fast or consume a ketogenic (KD) or calorically restricted (CR) diet. These diets have been shown to extend lifespan in mice, while they are also protective in many disease models. Caenorhabditis elegans, a roundworm with a short mean lifespan of roughly 2 to 3 weeks depending upon the temperature, is used as a model system to study aging. BHB has been previously shown to increase lifespan by roughly 20% when administered to C. elegans.We administered NR and BHB individually and together to C. elegans starting at two different developmental stages (larval stages 1 and 4) and measured lifespan. We found that administration of 20 mM DL-BHB decreased lifespan when first given at the L1 stage, while it robustly increased lifespan when first given at the L4 stage. Administration of 0.5 mM NR increased lifespan when first given at L1, with only a very slight increase when first given at L4. When initiating administration at L1, NR greatly mitigated the BHB-mediated decline in longevity, however, NR did not increase BHB-mediated lifespan extension when first administered at L4.

The objective of this study was to develop a new method for co-crystal preparation which adhered to green chemistry principles, and provided advantages over conventional methods. A novel, solvent-free, high-power ultrasound (US) technique, for preparing co-crystals from binary systems, was chosen as the technology which could fulfil these aims. The application of this technology for solid state co-crystal preparation was explored for ibuprofen-nicotinamide (IBU-NIC), carbamazepine-nicotinamide (CBZ-NIC) and carbamazepine-saccharin (CBZ-SAC) co-crystals. The effect of different additives and processing parameters such as power level, temperature and sonication time on co-crystallisation was investigated. Characterisation was carried out using DSC, PXRD, FTIR, Raman and HPLC. In addition, an NIR prediction model was developed and combined with multivariate analysis (PLS) and chemometric pre-treatments. It was found to be a robust, reliable and rapid method for the determination of co-crystal purity for the IBU-NIC and CBZ-NIC pairs. Co-crystal quantification of US samples helped to optimise the US method. Finally, a model formulation of paracetamol containing 5% and 10% PEG 8000 was ultrasonicated at maximum power with different exposure times. A comparison of technological and physicochemical properties of the resulting tablets with those of the tablets obtained using the pressing method evidenced significant differences. This suggested that US energy dissipation (mechanical and thermal effects) was the main mechanism which caused the PAR form I tabletability to improve. It was found that the ultrasound–compacted tablets released the drug at a slower rate compared to pure PAR. This technique was shown to be useful for improving tabletability for low-compressible drugs without the need to use a conventional tabletting machine.