Class of 2005 Abstract / Objective: The goal of this study was to determine the presence of erythro-methylphenidate (erythro-MPH) isomers contained within methylphenidate HCl (Metadate CD®) capsules. Methods: This experiment was conducted at a pharmaceutical manufacturing facility located in Tucson, Arizona. Methylphenidate HCl (MPH) capsules by Celltech Pharmaceuticals, Inc. were analyzed and compared to a reference standard. Thin-layer chromatography (TLC) was the technique used to qualitate the samples. The main outcome measure was the Rf values which were used to determine whether or not the MPH capsules contained erythro-MPH. Results: The study included ten, 20 mg MPH capsules and a reference standard (50 mg strength USP MPH related compound). The Rf value of the reference standard was 0.073 while the Rf value of the MPH samples ranged from 0.42 - 0.85. Conclusion: In this qualitative analysis of MPH capsules, there was no erythro-MPH isomers present in the MPH capsules.

The goal of this PhD research was directed to the isolation and structural elucidation of the secondary metabolites from four species of Durio plants, especially from those growing in the island of Borneo; This island contains most Durio species. The structures of isolated compounds were deduced using state-of-the-art approaches, including 1D and 2D nuclear magnetic resonance (NMR), mass spectrometric (MS) and circular dichroism (CD) analyses. From the bark of the woody species Durio zibethinus Murr. (Bombacaceae), that was collected in Pontianak, a chemically diverse group of oxygenated compounds was obtained. These included caffeoyl triterpenes, lignans and phenylpropanoid derivatives. During this research, one new compound 27-O-cis-caffeoylcylicodiscate was obtained which resulted from a trans to cis isomerization; this process might be caused by acid and light. Two presumed fungal metabolites were also isolated during this study, these possibly arising from fungal (unknown species) contamination of the bark. The relative stereochemistry of 4-hydroxymellein were also established by conversion to it diacetate and measurement of the coupling constant between H-3 and H-4. The absolute configuration was determined by comparison of []D values with those from the literature. Assignment of the 13C NMR spectrum for (+)-(R)-de-O-methyllasiodiplodin was completed using HSQC-TOCSY data. The methanol extract of the bark of Durio kutejensis (Hassk.) Becc. was fractionated and purified by C18-HPLC. Two new caffeoyl pentacyclic triterpenes 3-O-trans-caffeoyl-2-hydroxyolean-12-en-28-oic acid and 3-O-trans-caffeoyl-2-hydroxyurs-12-en-28-oic acid, together with five known compounds were isolated. The known compound fraxidin had previously been obtained from D. zibethinus Murr. The bark of Durio carinatus Mast., a plant species which has non edible fruit was collected in Sambas district, West Kalimantan. Three new lignans, boehmenan X, (-)-(7S,8S)-threo-carolignan X and (-)-(7R,8S)-erythro-carolignan X, and a new caffeoyl triterpene 3-O-cis-caffeoyl betulinic acid, together with three known lignans and one known caffeoyl triterpene, were obtained from the crude MeOH extract. The relative configurations and conformational models of the diastereomers threo- and erythro-carolignan X were determined by the coupling constant data for H-7′ and H-8′ and supported by 2D NMR spectroscopic analysis including NOESY and HSQC-HECADE in CDCl3. However, the magnitude of the coupling constants for both compounds in MeOH-d4 was similar. The stability of the individual conformers was solvent dependent and must be considered for their contribution to the overall conformational equilibria. This stability was determined by steric interactions, dipole repulsion effects and intramolecular hydrogen bonding. The absolute configuration of the two new diastereomers was established by application of circular dichroism (CD) and optical rotation []D measurements. Another Durio plant studied and collected in Bengkayang region, West Kalimantan, was Durio oxleyanus Griff. From the MeOH extract of its bark, two new lignans, (-)-(7S,8S)-threo-carolignan Y and (-)-(7R,8S)-erythro-carolignan Y were isolated using C18-HPLC. The relative stereochemistry of the carolignan Y diastereomers could not be deduced from the coupling constants for H-7′ and H-8′ either in CDCl3 and MeOH-d4, because of the presence of multiple conformers. The conformational model for the diastereomers of carolignan Y was similar to that of threo- and erythro-carolignan X in MeOH-d4. Using 2D NMR spectroscopic methods, including NOESY and HSQC-HECADE, the relative stereochemistry of the threo and erythro isomers of carolignan Y was confirmed. Further, the absolute configuration of threo- and erythro-carolignan Y was explored via analysis of CD spectra and by comparison of experimentally obtained []D values with literature values.

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that targets joints, resulting in in permanent disability. Synovial macrophages have been implicated in the pathogenesis of RA; however, their exact origins and functions remains unclear. In this study, we show evidence that synovial macrophages are mostly derived from embryonic origin during normal development. Macrophages are derived from either hematopoietic stem cells (HSC) or erythro-myeloid progenitors (EMP), and it is postulated that different subpopulations of synovial macrophages may have distinct functions contributing to either homeostasis or inflammation. To investigate the phenotypes of synovial macrophage populations and characterize their lineage-specific functions in arthritic joints, we utilized both cell lineage-tracing and K/BxN serum-transfer arthritis mouse models. Utilizing Flt3Cre;Rosa26LSL-YFP mice to label HSC-derived cells, we demonstrated that there is minimal HSC contribution to synovial macrophage populations during homeostasis. Use of RankCre;Rosa26LSL-YFP and Cx3cr1CreERT2;Rosa26LSL-tdTomato mice to label EMP-derived cells corroborated the finding that the EMP compartment maintains the largest contribution to synovial macrophage populations during normal development. Analysis of macrophages in Csf1rMericreMer;Rosa26-LSLtdTomato mice provided definitive prove that synovial macrophages derived from yolk-sac EMP precursors in adult mice. Use of serum transfer arthritis (STA) mice demonstrated that while most macrophages in the inflamed synovium were EMP-derived, there was a marked increase in HSC-derived cells compared to those present in homeostasis. Although this study has contributed to eluding that the heterogeneity of synovial macrophages in both homeostasis and inflammatory arthritis (IA) is complex and lineage-specific, further studies are needed to clearly define lineage-specific functions of macrophages in synovial tissues and in IA.

Indiana University-Purdue University Indianapolis (IUPUI) / A version of this thesis was published as: Masters AR, McCoy M, Jones DR, and Desta Z. Stereoselective method to quantify bupropion and its three major metabolites, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion using HPLC-MS/MS. J Chromatography B Analyt Technol Biomed Life Sci 1015-1016:201-208, 2016. / Bupropion is a dual dopamine-norepinephrine uptake inhibitor and a nicotine receptor antagonist. Clinically, bupropion is given as a racemate for the management of depression, smoking cessation aid, and for the management of weight. Bupropion has also been targeted as a phenotypic probe of CYP2B6 activity. Bupropion metabolites are formed via oxidation (4-hydroxybupropion) through CYP2B6, and reduction (erythro- and threo-dihydrobupropion) through carbonyl reductases. These metabolites exhibit pharmacological activity, but little is known regarding their stereoselective disposition due to the lack of a chiral assay. A novel reversed phase chiral-HPLC-MS/MS method involving a simple liquid-liquid extraction procedure and a small plasma sample volume (50µL) was developed that allowed simultaneous separation and quantification of enantiomers of bupropion, 4-hydroxybupropion, and those of threo- and erythro-dihydrobupropion in human plasma. This method was successfully implemented to determine the unique stereoselective disposition of bupropion and its metabolites in 15 human volunteers administered a single 100 mg oral dose of racemic bupropion. Significant differences (p<0.05) in the stereoselective metabolism were observed for all of the enantiomers. The highest plasma exposure (AUC0-∞) was (2R, 3R)-4-hydoxybupropion, almost 65 fold higher, than (2S, 3S)-4-hydoxybupropion, and over 32 fold greater than the parent R-bupropion. The second highest plasma exposure was threo-dihydrobupropion A, which was almost 5 fold higher than threo-dihydrobupropion B. (Nomenclature of the enantiomers for erythro- and threo-dihydrobupropion was based on the chromatography of the first eluting peak as “A” and the second eluting peak as “B”.) Threo-dihydrobupropion A and B showed the most significant difference between the racemic and enantiomer profiles. Although the AUC was greater for threo-dihydrobupropion B, threo-dihydrobupropion A had a significantly (p<0.05) higher Cmax. The half-life for threo-dihydrobupropion A and erythro-dihydrobupropion A were the longest for all analytes, which could indicate accumulation in multiple dosing. The importance of this study was, for the first time, to be able to characterize the stereoselective metabolism of bupropion and its three major metabolites. This new method and subsequent pharmacokinetic data should enhance further research into bupropion stereoselective metabolism, drug interactions, and effect. / A version of this thesis was published as: Masters AR, McCoy M, Jones DR, and Desta Z. Stereoselective method to quantify bupropion and its three major metabolites, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion using HPLC-MS/MS. J Chromatography B Analyt Technol Biomed Life Sci 1015-1016:201-208, 2016.

La thrombopoïétine (TPO) favorise successivement la prolifération et la maturation des progéniteurs mégacaryocytaires, soulevant la question du mécanisme expliquant cette dualité d'action. La signalisation SCF/ c-Kit est essentielle pour la prolifération de tous les progéniteurs hématopoïétiques, alors que l'extinction de l'expression du récepteur c-Kit est requise pour l'engagement en différenciation terminale. Réciproquement, l'équipe a montré que la stimulation de la voie Notch affecte une sous-population de progéniteurs bipotents érythro-mégacaryocytaires exprimant fortement CD9 (tétraspanine induite durant la maturation mégacaryocytaire) et favorise la reprise de leurs divisions au détriment de leur différenciation mégacaryocytaire terminale. Cet effet de la voie Notch s'accompagne d'une augmentation de l'expression de c-Kit. Ces observations m'ont conduite à m'intéresser aux mécanismes de régulation de c-Kit par la TPO en m'appuyant sur un modèle de progéniteurs bipotents immortalisés et dont la prolifération est strictement dépendante de la TPO (cellules G1ME). Les travaux réalisés durant ma thèse m'ont permis d'établir que (i) La stimulation des cellules G1ME par le ligand de Notch DLL1 favorise l'expression de c-Kit et réprime celle de CD9 (ii) L'activation inattendue de c-Kit par la TPO contribue à la prolifération (iii) c-Kit contribue activement à restreindre la polyploïdisation des cellules G1ME en présence de TPO (iv) La tétraspanine CD9 elle-même réprime l'expression de c-Kit à la membrane. Sur la base de ces résultats, nous proposons le modèle selon lequel, la TPO participerait à la fois à la prolifération des progéniteurs du fait de sa capacité à activer c-Kit, mais contribue aussi à l'augmentation de l'expression de CD9 qui en atteignant un seuil suffisant conduit à l'extinction de l'expression de c-Kit à la surface, entrainant alors l'arrêt des divisions et la différenciation mégacaryocytaire terminale / The Thrombopoietin (TPO) favors both the proliferation and the maturation of megakaryocytic progenitors, raising the question of the molecular mechanism explaining its dual function. SCF/ c-Kit signaling is essential for all hematopoietic progenitors amplification, whereas terminal differentiation requires the extinction of c-Kit receptor expression. Reciprocally, we evidenced in our team that Notch stimulation enables the induction of c-Kit expression and act on a particular subpopulation of bipotent erythro-megakaryocytic progenitors highly expressing the tetraspanin CD9 (induced during megakaryocytic maturation) and favors their re-entry in a cycling state by blocking their megakaryocytic maturation. These observations lead to the investigation of the molecular mechanism of c-Kit regulation by TPO in a cellular model of bipotent progenitors immortalized and dependent on TPO, the G1ME cells. During my thesis, I evidenced that: i) Notch stimulation induces the expression of c-Kit while repressing CD9 expression; ii) Surprisingly TPO is able to activate c-Kit allowing its contribution to cell proliferation; iii) c-Kit also represses megakaryocytic polyploidization (endomitosis characterizing megakaryocytic maturation) of G1ME cells; iv) The tetraspanin CD9 represses the expression of c-Kit. The ensemble of these data allows us to propose the following model wherein TPO activates c-Kit allowing the proliferation of megakaryocytic progenitors, while concomitantly induces the expression of the tetraspanin CD9 that will reach a sufficient level to provoke the extinction of c-Kit expression at the cell surface, thus enabling the arrest of cell cycling progress and the engagement into terminal megakaryocytic maturation

La transcétolase (TK, EC 2.2.1.1) est une enzyme catalysant la formation de cétoses de configuration D-thréo à partir d’aldéhydes α-hydroxylés (2R), par formation stéréospécifique d’une liaison C-C. L’objectif de ces travaux est d’inverser l’énantiosélectivité de cette enzyme par ingénierie afin d’obtenir des cétoses L-érytho (recherchés pour leurs applications potentielles dans les domaines pharmaceutique et/ou nutritionnel) à partir d’aldéhydes α-hydroxylés (2S). Dans ce but, une TK thermostable (mTKgst) issue de la bactérie thermophile Geobacillus stearothermophillus a d’abord été identifiée et produite. L’étude de sa structure tridimensionnelle a permis d’identifier deux résidus du site actif ayant un rôle potentiel dans l’inversion de son énantiosélectivité : Leu382 et Asp470. Des banques demTKgst mutées ont alors été créées, selon deux stratégies : rationnelle et semi-rationnelle. La première a consisté à muter les deux résidus sélectionnés par mutagenèse par saturation de site, tandis que la seconde a consisté à modifier deux séquences de cinq résidus contigus à aux positions clés, selon la mutagenèse par cassette. Afin d’identifier les mTKgst mutées d’intérêt, un test de criblage à haut-débit a été mis au point, basé sur le suivi pH-métrique de la réaction en présence de rouge de phénol. A l’issue du criblage, le variant mTKgst-L382D/D470S a été mis en évidence. Son activité vis-à-vis d’un aldéhyde modèle de configuration (2S) a été augmentée d’un facteur 5 par rapport à l’enzyme sauvage et la perte de l’énantiosélectivité vis-à-vis desaldéhydes (2R) a été confirmée. / Transketolase (TK, EC 2.2.1.1) catalyzes the formation of D-threo ketoses from (2R)-α-hydroxyaldehydes by the stereospecific formation of a C-C bond. Our aim was to invert the enantioselectivity of TK by protein engineering in order to obtain L-erytho ketoses (sought after for their potential pharmaceutical and/or nutritional applications) from (2S)-α-hydroxyaldehydes. For that purpose, a thermostable TK from thermophilic bacterium Geobacillus stearothermophilus (mTKgst) has been identified and overexpressed. After the study of the 3D-structure of mTKgst, two residues located in its active site (Leu382 and Asp470) were selected as mutation targets for the inversion of the enzyme’s enantioselectivity. Both rational and semi-rational approaches were considered for the construction of the mutant mTKgst libraries. In the former, the two residues were modified by site-saturation mutagenesis. In the latter, short sequences of five amino acids, neighboring target ones, were modified using a cassette mutagenesis technique. A novel continuous pH-based assay has been developed for the high-throughput screening of the mTKgst libraries, using phenol red as pH indicator. The screening revealed mTKgst-L382D/D470S as the top mutant, showing a 5-fold activity improvement towards a model (2S)-hydroxyaldehyde and the loss of enantioselectivity towards the (2R)-aldehyde.