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To enhance the physical stability of Clostridium difficile toxoids A and B, screening for stabilizing compounds was performed. The screening of 30 GRAS compounds at various concentrations and in several combinations was performed in two parts. First, a high-throughput aggregation assay was used to screen for compounds which delayed or prevented aggregation of toxoids under stress conditions (toxoids at pH 5-5.5 were incubated at 55 degrees C for 55 or 75 min). Compounds which stabilized both proteins were further studied for their ability to delay unfolding under conditions leading to a presumably native-like folded state (pH 6.5). The thermal stability of the toxoids on the surface of Alhydrogel was monitored with DSC and also showed significant improvement in the presence of certain excipients. This study has generated information concerning the free and adjuvant bound toxoids behavior under a range of conditions (temperature, solutes) that can be used to design pharmaceutical formulations of enhanced physical stability.

The synthesis and biological evaluation of several enantioenriched schweinfurthin B analogs were undertaken to develop structure-activity relationships and guide design of probes for their putative molecular target. The desired stilbenes contain a common left-half hexahydroxanthene ring system and an aromatic right-half with varied substituents. The synthesis involves penultimate Horner-Wadsworth-Emmons coupling of one of several right-half phosphonates with the aldehyde comprising the left-half of 3-deoxyschweinfurthin B. Preparation of the requisite phosphonates, and the respective stilbenes, as well as the cytotoxicity profiles of these new compounds in the National Cancer Institute's 60 cell-line anticancer screen is described. Several of these analogs displayed cytotoxicity patterns well-correlated with the natural product and differences in activity of approximately 10(3) across the various cell lines. Together, these assay results indicate the importance of at least one free phenol group on the aromatic D-ring of this system for differential cytotoxicity.

The total synthesis of the opioid modulator pawhuskin C has been accomplished in eight steps from methyl 3,5-dihydroxybenzoate. The key step in this sequence is a directed ortho metalation reaction conducted without protection of a benzylic alcohol and thus presumed to involve a formal dianion intermediate.

Nosocomial diarrhea and pseudomembranous colitis causing toxins A and B from Clostridium difficile were studied at pH 5-8 and over the temperature range of 10-85 degrees C. The proteins were crosslinked with formaldehyde to inactivate them to toxoid forms and permit their use as vaccines. Structural changes and aggregation behavior were monitored with circular dichroism, intrinsic and extrinsic (ANS) fluorescence spectroscopy, turbidity measurements, high-resolution UV absorbance spectroscopy and dynamic light scattering. The combined results were summarized in empirical phase diagrams. Toxins A and B had similar secondary structure with a combination of helical, beta-sheet and unordered character and were partially unfolded at pH 5-5.5. Upon heating, toxin A at all pH values partially unfolded at approximately 45 degrees C and formed insoluble aggregates at approximately 50 degrees C. Toxin B partially unfolded at approximately 40 degrees C, and upon heating to approximately 50 degrees C precipitated at pH 5.0-6.0 and formed soluble aggregates at pH 6.5-7.5. The thermal stability of the toxins was pH-dependent with the proteins more thermally stable at higher pH. Similar studies of formaldehyde crosslinked toxoids A and B revealed enhanced thermal stability, in which secondary and tertiary structure changes as well aggregation were delayed by about 10 degrees C. These studies reveal both similarities and differences between C. difficile toxins A and B and demonstrate the stabilizing effect of formaldehyde crosslinking on the thermal stability of their corresponding toxoids.

To evaluate relationships between the extent of protein-excipient interactions, structural relaxation of an amorphous matrix, and the physico-chemical stability of a protein, human growth hormone (hGH) was lyophilized with sucrose and trehalose in a 1:2 weight ratio. The protein-excipient interactions were analyzed immediately after lyophilization with isoperibol solution calorimetry (ISC), water sorption analysis (WSA), differential scanning calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR). The physical and chemical stability of hGH during storage at 50 degrees C was monitored by reverse phase (RP)-HPLC, SEC-HPLC and UV absorption spectroscopy. The hGH formulation containing sucrose demonstrated greater protein-excipient interactions and faster initial relaxation times compared to the trehalose formulation. Although both formulations had similar chemical stability (rate of deamidation), physical stabilities (e.g. degree of aggregation) were different. The hGH/sucrose formulation manifested a higher rate and lower extent of insoluble aggregate formation. The decreased amount of aggregation in the sucrose formulation could be correlated with a greater extent of protein-excipient interactions and the presence of a more homogeneous mixture. In contrast, the higher rate of aggregation in the sucrose formulation could be directly correlated with the higher molecular mobility of the matrix.

The total synthesis of the opioid modulator pawhuskin C has been accomplished in eight steps from methyl 3,5-dihydroxybenzoate. The key step in this sequence is a directed ortho metalation reaction conducted without protection of a benzylic alcohol and thus presumed to involve a formal dianion intermediate.