In view of their native medicinal uses, a systematic investigation of the phytochemistry and Hippocratic screening of three members of the Saururaceac namely, Anemopsis californica, Saururus cernuus and Houttuynia cordata was undertaken.

Improving the therapeutic efficacy and quality of life of patients by reducing the side effects caused by non-specificity of cytotoxic drugs has been a challenge in cancer treatment. A hypothesis was developed where integrin binding induced conformational change in a drug conjugated to hairpin peptide with an integrin binding ligand can lead to preferential accumulation of drug and reduced collateral damage by decreased premature prodrug activation. A model drug, MTX and a tripeptide ligand, RGD, known to specifically bind tumor overexpressing α v β 3 integrin receptors, were selected to test the hypothesis. A twelve amino acid sequence that has been previously shown to preferentially adopt an anti-parallel beta hairpin conformation in aqueous environment was flanked with MTX and RGD on N and C termini respectively by solid phase peptide synthesis to form a labile link between Arg-Glu specifically cleaved by SGPE, a Streptomyces griseus derived endopeptidase. Adenoviral vector was developed using AdEasy system for β 3 cDNA transfection to overexpress integrin α v β 3 receptor. MTX-α-RGD and MTX-β-hairpin-RGD were characterized using MALDI-TOF (MTX-α-RGD, 782.6(M+H + ); MTX-β-hairpin-RGD, 2272.1(M+H + )). Cell adhesion assay using HUVEC and A549 cells that overexpress α v β 3 showed that RGD conjugated prodrugs recognize and preferentially bind to integrin α v β 3 in RGD dependent manner. In rabbit plasma, MTX-β-hairpin-RGD was found to be 3 times more stable than MTX-α-RGD. In the absence of α v β 3 binding, SGPE mediated hydrolysis rate of MTX-β-hairpin-RGD was 0.7±0.1 ng/hr, that was significantly (P<0.025) lower than that of MTX-α-RGD (1.0±0.1ng/hr), a prodrug without hairpin structure. In presence of α v β 3 over-expressing cells, significant increase (P<0.025) in hydrolysis rate of MTX-β-hairpin-RGD to 1.0±0.1 ng/hr was observed, not significantly (P=0.6) different from that of MTX-α-RGD (1.1±0.1ng/hr). In addition, there was 400% increase in the fluorescence when FRET based quenching was abolished by the binding induced unfolding. These experiments along with docking studies using molecular modeling support the binding induced unfolding. Results from this investigation suggest that drugs conjugated to peptide ligands such as RGD may reduce the dose needed to achieve therapeutic concentrations by preferential recognition and binding to overexpressed integrin markers. Secondly, reduction of premature activation of prodrugs and thus reduced collateral damage may be achieved by making the the drug release to occurs preferentially upon binding to cells expressing specific integrin markers.

Novel buccal mucoadhesive copolymers of acrylic acid (AA) and poly (ethylene glycol) monomethylether monomethacrylate (PEGMM), (P(AA-co-PEG)), were designed, synthesized, and characterized for systemic delivery of acyclovir across the buccal mucosa. To achieve spontaneous intimate contact between polymer and buccal mucosa, the surface properties of P(AA-co-PEG) were optimized for buccal adhesion by varying the composition of the copolymers. It was found that the mole ratio of the repeat units of PEG, ethylene glycol, to AA is of great importance for mucoadhesion. ATR-FTIR studies revealed that the PEG moiety enhanced intra- and intermolecular H-bond formation. The copolymer films containing 16 mole% PEGMM (PEG MW 400) and 1.3 wt% ethylene glycol dimethacrylate (EGDMA) yielded the most favorable mucoadhesive properties within the investigated range. Factors influencing drug loading included equilibrium hydration, crosslinking density, loading solution medium, and concentration of drug in loading solution. In vitro release studies, performed in isotonic McIlvaine buffer pH 6.8 (IMB), showed a Non-Fickian release behavior. The release of acyclovir from the buccal mucoadhesive films was controlled by a combination of diffusion and macromolecular chain relaxation mechanism. In vitro permeation studies were conducted to determine the buccal transport pathway of acyclovir and to investigate the feasibility of buccal delivery of the drug. The paracellular route was found as the dominant buccal transport route of acyclovir. Permeation enhancement of acyclovir across the buccal mucosa was investigated using 2-100 mM sodium glycocholate (SGC). In the presence of SGC, the drug permeability was enhanced 2 to 9 times. The mechanism of this enhancement was attributed to SGC facilitating the paracellular route. The feasibility of buccal delivery of acyclovir was demonstrated and controlled release was achieved for up to 12 hours from P(AA-co-PEG) devices with a unidirectional design. The rate-limiting barrier to drug delivery from the mucoadhesive device was found to be the buccal mucosa. The target flux for systemic delivery of acyclovir was achieved with the incorporation of SGC into the tailor-made mucoadhesive copolymers of acrylic acid and PEGMM.

The local structures of the iron atoms for a series of strongly coupled Fe 2 (TIED)L 4 complexes (TIED = tetraiminethylenedimacrocycle, L = axail ligand) have been investigated by K-edge X-ray Absorption Spectroscopy (XAS). These complexes include not only the well characterized iso-valence CH 3 CN complex, mixed-valence CH 3 CN and Cl − complexes, and previously reported iso-valence CO complex but also the new isolated solids of iso-valence Fe 2 TIED complexes with Cl − , Br − , imidazole, pyridine, histidine, N,N-dimethyformamide (DMF), SCN − , and CN − as axial ligands and mixed-valence complexes with Br − and imidazole as axial ligands. The average Fe-N distances for the first coordination sphere of the iron atom obtained by EXAFS analysis are 1.94, 1.94, 1.95, 1.96, 1.94, 1.93, 1.96, 1.96, 1.96, and 1.96 Å for the iso- and mixed-valence CH 3 CN and imidazole complexes and iso-valence complexes with SCN − , CN − , CO, pyridine, histidine, and DMF as axial ligands, respectively. Two-shell fitting analyses of the complexes gave average iron to the four planar coordinated nitrogen distance of 1.90, 1.91, 1.91, 1.92, and 1.92 Å for the Fe 2 (TIED)L 4 with L = DMF, pyridine, Cl − , Br − , imidazole, and histidine complexes, respectively. The average distances from the center iron to: N(DMF), 2.05; N(pyridine), 2.05; Cl − , 2.33; Br − , 2.45; N(imidazole), 2.08; and N(histidine), 2.07 Å. They are all comparable to related bond distances in the literature. The above data indicate that there is no significant difference in the average Fe-N distances between each of the iso- and mixed-valence pairs. Also different axial ligands do not cause significant impact on the average Fe-N distances from the iron atom to the four coordinated N in the TIED ligand. The threshold edge positions shift about +1 eV from the iso-valence CH 3 CN, Cl − , and Br − complexes to their corresponding mixed-valence complexes. The relatively small shift compared with the normal +2 [special characters omitted] +3 eV edge shift from Fe 2+ to Fe 3+ reflects the oxidation state change of iron from Fe 2+ to Fe 2.5+ . The edge energy of the isovalence diiron complexes with different axial ligands increases in the order of the spectrochemical series of the axial Iigands from strong to weak field Iigands as follows [58, 59]: [special characters omitted] All the complexes studied here have a weak dipole-forbidden 1s → 3d pre-edge transition. The low intensity indicates only a small distortion of the octahedral coordination geometry of the central iron atom.

In view of current interest in several oligopeptide drugs currently being investigated for controlled implantable delivery, glycine, diglycine, triglycine, tetraglycine and pentaglycine were chosen as model compounds for encapsulation in biodegradable microspheres of DL-polylactide (DL-PLA) by a technique based on oil-in-oil emulsion and the solvent evaporation principle. A DL-polylactide concentration of 10.3% w/w and emulsifier (sorbitan sesquioleate) concentration of 0.3% v/v produced good yields of microspheres with excellent entrapment when processed under following conditions: emulsion time, 1 min; solvent evaporation time, 2 min; internal phase-external phase ratio, 1:7; stirring speed, 1100 rpm; emulsion temperature, 5$\sp\circ$C; and maximum processing temperature, 35$\sp\circ$C. Microspheres prepared as above at four different loadings (2.5, 5.0, 7.5, and 10.0% w/w) were analyzed for morphological and in vitro release characteristics. Analysis of the release data and scanning electron photographs suggested that the release of glycine and its homopeptides from DL-PLA microspheres was most likely by diffusion through the matrix. However, for models having low aqueous solubility, e.g., tetra- and pentaglycine, dissolution played a rate-limiting role. Microspheres of glycine prepared with DL-PLA plasticized with 10% triacetin demonstrated the slowest release, with the first 50% entrapped glycine released over four days and next 25% released at a constant rate over 17 days. This was in sharp contrast to unplasticized microspheres from which glycine was completely leached out in 24 h. Interestingly, while the plasticizer decreased the rate of release of glycine, it appeared to promote the degradation of the polymer DL-PLA.

The effect of environmental condition on the mechanism and kinetics of fibril formation for bovine insulin were investigated. Results showed environmental conditions played a significant role in determining the mechanism and kinetics of fibril formation. Increased protein concentration, elevated temperature, and higher ionic strengths induced insulin to form fibrils through oligomeric intermediates that were consistent with the nucleated conformational conversion (NCC) mechanism. Bovine insulin was also shown to generate fibrils without formation of oligomeric intermediates at study conditions of lower protein concentration, lower temperature, and lower ionic strength. Fibril formation without oligomeric intermediate can be described by the nucleated polymerization (NP) mechanism. Different relative amounts of oligomeric intermediate were generated at the various combinations of protein concentration, temperature, and ionic strength. The kinetic parameters, lag time, and rate of fibril formation, correlated with the relative amount of oligomeric intermediates detected. Longer lag times and slower rates of fibril formation were observed with greater amounts of oligomeric intermediate present. The effects of excipients, trifluoroethanol, ethanol, glycerol, and urea on the apparent rate constants of oligomeric intermediate and fibril formation were also investigated. At the concentrations studied, all the excipients tested were observed to decrease the rate and relative amount of oligomeric intermediate formation in an excipient concentration-dependent manner. The excipients were less effective at preventing fibril formation. In conclusion, bovine insulin can form fibrils with and without oligomeric intermediates. Protein concentration and environmental conditions, such as temperature, ionic strength, and excipients played a significant role in determining the relative amount of oligomeric intermediates, which in turn, determined the mechanism and kinetics of bovine insulin fibril formation under the conditions studied.

The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N -terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris , a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N -terminal partner to several C -terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C -terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C -terminal of the MBP domain, including the spacer and cargo regions, but the MEP domain could still act to enhance the secretion of certain cargo proteins. The attempt to identify the unknown protease was unsuccessful. However, in contrast to other fusion partners, MBP was secreted with the cargo when it was fused as a C -terminal peptide to an N -terminal cargo protein. These studies provide insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris , suggesting new strategies to optimize this expression system.

This investigation focused on four aspects of hexamethylene bisacetamide's ( HMBA ) involvement in induction of differentiation in murine erythroleukemia (MEL) cells: (a) Effects of APAH , a N 8 -acetylspermidine deacetylase inhibitor, on differentiation induced by HMBA and its two deacetylated metabolites, NADAH and DAH , (b) influence of APAH on intracellular levels of HMBA and its deacetylated metabolites in HMBA treated MEL cells, (c) Ca 2+ mobilizing effects of HMBA, NADAH and DAH and (d) effect of APAH on HMBA induced changes in c- myc gene expression during differentiation. HMBA (5 mM) and DAH (2 mM) were equally effective in inducing MEL cell differentiation as measured by the amount of hemoglobin (Hb) produced, while NADAH (5 mM) was least effective. APAH (10–500 0μM) inhibited HMBA and NADAH induced differentiation without affecting DAH induced differentiation. APAH (500 μM) was shown to affect the deacetylation pathway for HMBA. There was a significant increase in intracellular NADAH levels and a decrease in DAH levels in MEL cells treated with both HMBA and APAH compared to HMBA alone (measured by LC/MS). This indicated that APAH inhibited the second deacetylation step, the conversion of NADAH to DAH but not the first, the conversion of HMBA to NADAH. Ca 2+ influx is necessary for HMBA induced MEL cell differentiation. BAPTA-AM (10 μM), a calcium chelator, inhibited HMBA induced Hb production while Tg (0.5 nM), the SERCA pump blocker, potentiated Hb production. 2-APB, a store operated channel (SOC) regulator, at higher concentrations (50,75 μM) prevented HMBA induced differentiation while at lower concentrations (5,10 μM) potentiated induced differentiation. DAH (0.5 mM), caused an immediate increase in [Ca 2+ ] i in MEL cells, while a slower response was seen with NADAH (3 mM). HMBA (5 mM) had the longest lag period (∼6 min) before it elevated [Ca 2+ ] i . APAH effectively prevented [Ca 2+ ] i increase caused by HMBA and NADAH but failed to alter DAH induced increase suggesting that DAH was the metabolite that raised [Ca 2+ ] i levels. Permeabilized cell assays demonstrated that DAH mobilized Ca 2+ from intracellular IP 3 sensitive stores in the ER. The identity of SOC for DAH induced Ca 2+ influx was inconclusive since 2-APB was not able to alter DAH induced Ca 2+ mobilizing responses. In addition to preventing HMBA induced MEL cell differentiation, APAH also inhibited the second phase of repression of c- myc gene expression, a hallmark of induced differentiation. In summary, the present study suggests the mechanism of action of HMBA requires the active involvement of a metabolite, DAH, in differentiation of hematopoietic cells.

The objective of this research was to investigate two important aspects of buccal drug delivery, transport and mucoadhesion. Buspirone was chosen as a model drug for the in vitro buccal transport studies, polyvinyl alcohol and sodium alginate polymer blends were prepared to investigate the mucoadhesive properties through a Lewis acid-base approach and finally, the effect of formulation factors on the force of mucoadhesion, surface energy parameters, release rate and flux was studied. In vitro permeation studies were conducted to investigate the buccal transport pathway of buspirone. Mathematical models were developed to quantify the process of permeation. Permeation enhancement of buspirone across the buccal mucosa was investigated using bile salts (sodium glycocholate and taurodeoxycholate), propylene glycol, propylene. Effect of formulation factors like drug, enhancer, and plasticizer was studied through statistically designed experiments. These experiments aided in characterizing the buccal delivery system. Mathematical models were developed for surface energy parameters, force of mucoadhesion, release rate, and flux. Research conducted in this dissertation focused on two important aspects of transbuccal delivery, drug transport and mucoadhesion by studying a model drug and polymer blends. The results obtained in these investigations can be utilized in the development of other bioadhesive delivery systems with respect to drug transport and mucoadhesion. Polymer blends of polyvinyl alcohol (PVA) and sodium alginate (Alg) were prepared to evaluate their mucoadhesive properties and investigate mucoadhesive mechanism by a Lewis acid-base approach. (Abstract shortened by UMI.)

Trichomonas vaginalis carries out unique mode of carbohydrate decarboxylation by an intracellular compartment, hydrogenosome. It was suggested that enzymes exclusively associated with hydrogenosome were responsible for activating metronidazole. This provoked researchers to target hydrogensosomal enzymes such as pyruvate: ferredoxin oxioreductase and ferredoxin, to treat trichomoniasis caused by T. vaginalis. Recent studies have shown alternative pathways responsible for activating metronidazole without the involvement of hydrogenosomal enzymes. This pathway requires the participation of thioredoxin and thioredoxin reductase. These enzymes are essential for T.vaginalis' survival as it is responsible for maintaining cell's redox system, as well as many other cellular processes. Targeting thioredoxin and thioredoxin reductase could be a novel drug target to treat metronidazole-resistant T. vaginalis. In this study, recombinant T. vaginalis TrxR and T. vaginalis Trx were produced to test its activity against known TrxR inhibitor, auranofin, using DTNB assay. Cell lysates of metronidazole susceptible and metronidazole resistant lines were also tested to see its activity against auranofin. Auranofin derivatives that were effective on E. histolytica recombinant TrxR were also tested to compare their effects on T. vaginalis recombinant TrxR. The results showed that auranofin effectively inhibits recombinant TvTrxR. Aurnofin derivatives were also shown to have different effects on inhibiting the activity of recombinant TvTrxR. It is known that auranofin also targets mammalian TrxR. With the degree of different inhibitions observed between auranofin derivatives, it opened the possibility of developing an auranofin derivative that can specifically targets thioredoxin reductase of parasitic protozoan, T. vaginalis.