Investigation into Stability of Poly(Vinyl Alcohol)-Based Opadry® II Films

Poly(vinyl alcohol) (PVA)-based formulations are used for pharmaceutical tablet coating with numerous advantages. Our objective is to study the stability of PVA-based coating films in the presence of acidic additives, alkaline additives, and various common impurities typically found in tablet formulations. Opadry® II 85F was used as the model PVA-based coating formulation. The additives and impurities were incorporated into the polymer suspension prior to film casting. Control and test films were analyzed before and after exposure to 40°C/75% relative humidity. Tests included film disintegration, size-exclusion chromatography, thermal analysis, and microscopy. Under stressed conditions, acidic additives (hydrochloric acid (HCl) and ammonium bisulfate (NH₄HSO₄)) negatively impacted Opadry® II 85F film disintegration while NaOH, formaldehyde, and peroxide did not. Absence of PVA species from the disintegration media corresponded to an increase in crystallinity of PVA for reacted films containing HCl. Films with NH₄HSO₄ exhibited slower rate of reactivity and less elevation in melting temperature with no clear change in melting enthalpy. Acidic additives posed greater risk of compromise in disintegration of PVA-based coatings than alkaline or common impurities. The mechanism of acid-induced reactivity due to the presence of acidic salts (HCl vs. NH₄HSO₄) may be different.     

Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p?=?0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.     

Molecular basis of toxicity of Clostridium perfringens epsilon toxin

Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.     

Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells

Effect of headgroup structure on catonic lipid-mediated transfection was investigated with either a (i) tertiary amine, (ii) quaternary amine with a hydroxyl, or (iii) quaternary amine with mesylate as headgroups. Liposomes were formulated using cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) as colipids, and transfection efficiencies were determined in rapidly dividing colon carcinoma (CT 26) and rat aortic smooth muscle (RASM) cells as well as in nondividing human pancreatic islets using luciferase and green fluorescent protein expression plasmids, pcDNA3-Luc and pCMS-EGFP, respectively. Liposome/pDNA complexes were evaluated for DNA conformational state by circular dichroism (CD), DNA condensation by electrophoretic mobility shift assay (EMSA), particle size and zeta potential by laser diffraction technique, and surface morphology by transmission electron microscopy (TEM). Encouraging transfection results were obtained with the mesylate headgroup based lipid in liposome formulations with DOPE as a colipid, which were higher than the commercially available Lipofectamine formulation. We hypothesize that the additional hydrogen bonding or covalent interactions of the headgroup with the plasmid DNA, leading to higher binding affinity of the cationic lipids to pDNA, results in higher transfection. This hypothesis is supported by TEM observations where elongated complexes were observed and more lipid was seen associated with the DNA.     

Analysis of chimeric RGS proteins in yeast for the functional evaluation of protein domains and their potential use in drug target validation

For the identification of regulators of G-protein signaling (RGS) modulators, previously, we developed a luciferase based yeast pheromone response (YPhR) assay to functionally investigate RGS4 (K.H. Young, Y. Wang, C. Bender, S. Ajit, F. Ramirez, A. Gilbert, B.W. Nieuwenhuijsen, in: D.P. Siderovski (Ed.), Meth. Enzymol. 389 Regulators of G_protein Signaling, Part A, 2004.). To extend the diversity of this assay, additional RGS proteins were evaluated for functional complementation in a RGS (sst2Delta) knockout yeast strain. For RGS proteins that did not function in their native form, a series of chimeric constructs were generated with the N terminus of RGS4 fused in frame with the partial or full-length RGS cDNA of interest. RGS4 N terminus fused to either full-length or the C terminus of RGS7 successfully complemented sst2Delta. On the contrary, the RGS7N/RGS4C chimera (N terminus of RGS7 in frame with RGS domain of RGS4) was not effective, showing that N terminus of RGS4 helps in targeting. RGS10 exists as two splice variants, differing only by 8 amino acids (aa) in the N terminus, being either 168 aa (RGS10S), or 174 aa (RGS10). While RGS10 was functional in yeast, RGS10S required the presence of the N terminus of RGS4 for its activity. Although the same RGS4 N terminus domain was present in chimeras generated, the GTPase accelerating protein (GAP) function observed was not similar, suggesting differences in the RGS domain function. In conclusion, the use of RGS4 N terminus chimeric constructs enabled us to develop a selectivity assay for different RGS proteins.     

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.     

Collection devices for cervicovaginal cytology: a comparison

OBJECTIVE: To compare cervicovaginal smears obtained by a cotton-tipped swab with those obtained by cervix brush and modified Ayre spatula. STUDY DESIGN: A combined cervicovaginal smear was collected from 100 women using 3 different collection devices: cotton-tipped swab, cervix brush, and modified Ayre spatula. In each patient a set of 3 smears was collected by the same cytotechnologist using all 3 devices in random order. Smears were evaluated using parameters mandatory for an optimal smear: evenly dispersed, well-preserved, adequate cells from the transformation zone. The cost and availability of the collection devices were also considered. RESULTS: The swab was the most effective device in obtaining thin, evenly spread, adequate, well-preserved smears as against the cervix brush and Ayre spatula. The pickup of abnormal cells was similar with the cotton-tipped swab and cervix brush, while the Ayre spatula failed to yield high grade squamous intraepithelial lesions, atypical glandular cells of undetermined significance and adenocarcinoma cells. The cotton-tipped swab proved to be the most cost effective. CONCLUSION: A properly prepared cotton-tipped swab is an inexpensive, readily available, nontraumatic collection device that yields smear of optimal quality.