A human-derived, mammary epithelial cell culture model would allow drug transport in the mammary epithelium to be studied in greater detail while minimizing risks to mothers and nursing infants. MCF10A and primary human mammary epithelial cells (HMECs) were investigated for their utility as human, cell-based model systems for drug transport studies. Polarized monolayers are essential for transcellular flux studies of drug transporter function, and their formation was measured by transepithelial electrical resistance, immunofluorescence microscopy and vectoral flux studies. Both cell types failed to form adequately polarized monolayers despite various modifications to the cells or culture conditions. Transporter-mediated drug uptake and efflux in MCF10A cells was measured using flow cytometry, a technique which enables the measurement of intracellular drug concentrations. The fluorescent drug, mitoxantrone, was used to assess active efflux transport by the ABC transporters ABCG2 (BCRP) and ABCB1 (MDR1). After accounting for the inter-day variability with a linear mixed effects model, inhibitor effects on intracellular drug concentrations were evident. Specific inhibition of MDR1 using verapamil increased mitoxantrone accumulation as expected; however, BCRP-specific inhibition with fumitremorgin C decreased accumulation. Flow cytometry studies on mitoxantrone uptake suggested that it is a substrate for an unidentified active uptake transporter. PEPT1 and PEPT2 transporter functionality in MCF10A cells was evaluated using a fluorescently labeled dipeptide (A-K-AMCA). A-K-AMCA uptake showed an active component which was inhibited by a general metabolic inhibitor, the dipeptide Gly-Gln, and the peptidomimetic cefadroxil, indicating the involvement of a peptide transporter in A-K-AMCA uptake. Drug transporter expression levels in MCF10A cells and HMECs were measured using RT-PCR. Transporter expression levels, which were similar in the MCF10A cells and the HMECs, were compared with expression levels in lactating and non-lactating mammary epithelial cells. Low expression of BCRP, MDR1 and PEPT1 was seen in MCF10A cells, yet the effects of these transporters could still be observed in functional flow cytometry transport assays. Flow cytometry studies MCF10A cells may useful as a mammary drug transport model for transporters which have similar expression levels to lactating mammary epithelial cells.

A series of lacunary (XM$\sb{11}$O$\sb{39}\sp{\rm n-}$ and P$\sb2$M$\sb{11}$O$\sb{61}\sp{10-}$) and plenary (SiW$\sb{12}$O$\sb{40}\sp{4-}$ and P$\sb2$M$\sb{18}$O$\sb{62}\sp{6-}$) polyoxometalates (POM) were synthesized, analyzed and their solution chemistry with lanthanide cations was studied by a variety of physical methods. These lanthanide complexes are evaluated with regard to potential applications as magnetic resonance imaging agents. / For elemental analysis of POM, quantitative methods were developed using ICP-AES for the determination of B, Si, Ge, P and Mo and atomic absorption spectroscopy for Na. The two-step procedure minimizes interelement interferences using matrix matching of sample and standard solutions, offering comparable precision to classical methods. The FTIR spectra of POM in D$\sb2$O and in KBr demonstrated their aqueous stability, and provided evidence of similarities between the solution and solid state structures. / The thermodynamics parameters of complexation ($\Delta$G, $\Delta$H, and $\Delta$S) of Eu(III) with POM were studied at 1 M ionic strength. pK$\sb{\rm a}$ values for the lacunary POM indicate weak basicity with two protonations between pcH 7 and 2. $\sp7$F$\sb0 \to \ \sp5$D$\sb0$ Selective excitation spectra coupled with the computer program, SQUAD, were used to determine 1:1 and 1:2 stability constants and to calculate $\Delta$G$\sb{\rm 10n}$ (n = 1,2). The stability constants are discussed in terms of statistical, electrostatic and geometrical effects. The enthalpies of complexation, $\Delta$H$\sb{\rm 10n}$ (n = 1,2), were determined for the formation of the 1:1 and 1:2 complexes of Eu(III) by titration calorimetry. The entropies of complexation, $\Delta$S$\sb{\rm 10n}$ (n = 1,2), were calculated from $\Delta$G$\sb{\rm 10n}$ (n = 1,2) and $\Delta$H$\sb{\rm 10n}$ (n = 1,2) values. / In addition, the $\sp7$F$\sb0 \to \sp5\rm D\sb0$ selective excitation spectra of Eu(III)-polyoxometalate complexes were evaluated qualitatively. Based on the number of peaks, the number of inner sphere H$\sb2$O molecules and the known structures, binding sites were proposed for 1:1 and 1:2 complexes. / The Gd(POM)$\sb2$ complexes were kinetically unstable in Sprague-Dawley rat serum. In vitro challenges to Gd(POM)$\sb2$ by metal cations, DTPA, and DTPA-doped serum suggest instability in serum is due primarily to a transmetalation mechanism. / Source: Dissertation Abstracts International, Volume: 56-04, Section: B, page: 2008. / Major Professor: Gregory R. Choppin. / Thesis (Ph.D.)--The Florida State University, 1995.

A re-examination of the reactions of (Z)-2,3:5,6-bis-O-(1-methylethylidene)-N-(phenylmethylidene)-$\alpha$- scD-mannofuranosylamine N-oxide and (Z)-2,3:5,6-bis-O-(1-methylethylidene)-N-(phenylmethylidene)-$\beta$- scD-gulofuranosylamine N-oxide with methylmagnesium bromide was undertaken to determine why the former gave the addition product in low yield and only moderate stereoselectivity, while the latter proceeded in high yield and with high stereoselectivity. A mechanism was proposed to explain the differences and three additional benzaldehyde nitrones bearing chiral auxiliaries derived from scD-lyxose were examined. The key interaction was indicated to be the formation of a chelated structure between the oxygen at C(5), the furanosyl oxygen and a magnesium atom. N-Glycosylnitrones that could form the chelate gave the corresponding N-hydroxy-$\alpha$-methylbenzenemethanamine after hydrolysis of the chiral auxiliary in high yield with high stereospecificity. / Pretreatment of the N-glycosylnitrones with TMSCl (2-5 equiv; 0$\sp\circ$C) prior to the addition of methylmagnesium bromide ($-$78$\sp\circ$C) afforded after hydrolysis, N-hydroxy-$\alpha$-methylbenzenemethanamine with the opposite configuration from the one in the absence of TMSCl. / In addition to benzaldehyde nitrones, N-glycosylnitrones derived from aliphatic aldehydes were shown to undergo diastereospecific addition of methylmagnesium bromide to give the corresponding N-hydroxy-$\alpha$-methylalkylamines in good yield with high ee. The absolute configuration of the product was also reversed upon pretreatment with TMSCl. / Intramolecular versions of this process were attempted. The N-methylnitrone of 3-(2-iodophenyl)propanal reacts smoothly with $\sp t$BuLi at $-$78$\sp\circ$C to give N-hydroxy-N-methyl-1-aminoindane in 92% yield. However, analogous reaction of the corresponding N-glycosylnitrone afforded the cyclic hydroxylamine in only 8.7% ee. Finally, initial attempts to achieve stereospecific carbon-carbon bond formation by radical addition to N-glycosylnitrones failed. / Source: Dissertation Abstracts International, Volume: 56-01, Section: B, page: 0247. / Major Professor: Martin A. Schwartz. / Thesis (Ph.D.)--The Florida State University, 1994.

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<p>This study analyses the communication possibilities for pharmaceutical companies in Belgium. What communication tools can they use,which tools are the most effective, useful for the companies.</p>

This study analyses the communication possibilities for pharmaceutical companies in Belgium. What communication tools can they use,which tools are the most effective, useful for the companies.

This thesis describes the structure elucidation of drug metabolites in biological samples by the use of high performance liquid chromatography (HPLC) atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). Due to their different advantages, various mass analyzers have been used in the different experiments. The metabolism of clemastine, flutamide, and meloxicam were studied in vitro and/or in vivo in different species such as humans, dogs, and horses. Accurate mass measurements with the quadrupole-time of flight mass spectrometer and MSn data supplied by the ion trap instrument were useful in the structural investigation of the product ions of the drugs and their metabolites. Different scan modes of the triple quadrupole mass spectrometer resulted in great flexibility, selectivity, and sensitivity in the qualitative and semi-quantitative studies. Additionally, hydrogen/deuterium exchange and experiments with atmospheric pressure chemical ionization were conducted, and the fungus Cunninghamella elegans was utilized to produce amounts of drug metabolites sufficient for structural investigation. Six isomers of oxidized clemastine were detected and characterized in C. elegans incubations and their retention times and mass spectral data were compared to the metabolites detected in urine samples. Two of the metabolites were concluded to be diastereomeric N-oxides. In urine from horses treated with meloxicam, the peak of 5'-hydroxymethylmeloxicam resulted in much higher intensity than the parent drug or the other metabolites, and it was detectable for at least 14 days after the last dose in some of the horses. That is useful information in the development of analytical methods for the detection of prohibited use of meloxicam. A mercapturic acid conjugate of hydroxyflutamide was detected in urine from cancer patients, which indicated that a reactive metabolite was formed. This metabolite could be responsible for the adverse events reported for flutamide. The results from the four papers included in the thesis clearly demonstrate the usefulness and the flexibility of the HPLC-API-MS/MS technique.

Tuberculosis (TB) is a major infectious disease, killing about 2 million people annually throughout the world. Today's TB treatment is a lengthy procedure involving a combination of antibiotics. No new TB drug has been introduced onto the market in the past 40 years, and the emergence of multi- and extensively drug-resistant TB calls for new drugs. Finding new drug targets is important and one such target is the Mycobacterium tuberculosis enzyme glutamine synthetase (GS), which catalyses the formation of glutamine from glutamic acid. In this work, novel GS inhibitors and new Pd(0)-catalyzed methods have been developed. A microwave-enhanced Pd(0)-catalyzed α-arylation reaction was developed using water as solvent, and a phenylglycine scaffold was identified using structure-based design. A series of α-arylated phenylglycine derivates was produced at moderate to good yields. Some of these were biologically evaluated against GS. A novel scaffold, 3-amino-imidazo[1,2-a]pyridine, was identified by high-throughput screening directed towards GS. This type of compound could be easily produced via a Ugi-type, microwave-promoted multi-component reaction in 20 min. The scaffold was investigated by changing one substituent at a time, and in an experimental design where 8 factors were varied in the same design. Several potent inhibitors were identified; amongst them the most potent inhibitor to date (IC50 = 0.38 µM). Two discrete structure-activity relationships were established, and one of the inhibitors was co-crystallized. The first general aminocarbonylation of aryl chlorides and the first aminocarbonylation of alkenyl phosphates were developed. Alkenyl chlorides, bromides and triflates were investigated in the same transformation utilizing Mo(CO)6 as a solid carbon monoxide source. Two different Pd(0)-based catalytic systems were developed. A wide variety of aryl chlorides and amines could be transformed into the corresponding amides with good yields. The alkenyl substrates produced low to good yields.

Tuberculosis kills approximately 1.7 million people each year around the world making it one of the most lethal infectious diseases. This thesis concerns the development of two computational tools that can support the early stages of drug discovery, and their use in an anti-tubercular drug discovery program. One of the tools developed is a statistical molecular design (SMD) approach that generates information-rich libraries biased towards a lead structure. The other metod is a post-filtering technique to increase the success of virtual screening, has also been developed. Both methods have been validated using literature data. Ribonucleotide reductase (RNR) has been identified as a potential anti-tubercular target, and our focus has been to develop small-molecule inhibitors of this target. The enzyme consists of two subunits (a large R1 and a small R2 subunit) that have to associate in order to generate a bioactive complex. It had previously been shown that a heptapeptide corresponding to the small R2 subunits C-terminal inhibited the enzyme. In order to investigate the requirements for inhibitory effect of the peptide a library was designed using the developed SMD approach. The designed library was synthesized and evaluated for biological activity and an OPLS-DA model was derived to understand which positions were most important for activity. In order to identify small-molecule inhibitors of RNR a combined shape- and structure-based virtual screen was performed, employing ROCS, GlideXP and the developed post-filtering technique. Starting from a library of 1.5 million compounds 24 was acquired and evaluated for enzymatic activity. The best compounds were almost as potent as the starting peptide, but considerably more drug-like.