Development of a topically active imiquimod formulation

The purpose of this work was to develop a topical formulation of imiquimod, a novel immune response modifier, to induce local cytokine production for the treatment of external genital and perianal warts. A pH-solubility profile and titration data were used to calculate a pKa of 7.3, indicative of a weak base. Solubility experiments were conducted to identify a solvent that dissolves imiquimod to achieve a 5% formulation concentration. Studies to select surfactants, preservatives, and viscosity-enhancing excipients to formulate an oil-in-water cream indicated that fatty acids were the preferred solvent for topical imiquimod formulations, and isostearic acid (ISA) was selected. A relationship existed between the fatty acid composition of four commercially available ISA sources and the solubility of imiquimod. A combination of polysorbate 60, sorbitan monostearate, and xanthan gum was used to produce a physically stable cream. The preservative system included parabens and benzyl alcohol to meet the USP criteria for preservative activity. An in vitro method was developed to demonstrate that imiquimod was released from the formulation. Topical application of the formulation induced local cytokine activity in mice.     

Estimation of Surface Polarity of Silicas by Absorption Spectroscopy of Glycerin Suspensions of Adsorbed 6-Nitrobenzoindolinopyran

The title compound (6-NO₂-BIPS) is adsorptiochromic, becoming colored upon adsorption to a polar surface. Powders of 6-NO₂-BIPS adsorbed to silica gel or silicic acid are suspended in glycerin, and the absorption spectrum of the adsorbate is recorded by conventional absorption spectroscopy. The wave number of maximum absorption is related to the effective surface polarity by v*/cm^–1 = 90.85 Z + 11,571, where Z is the Kosower polarity measure. Silica surface polarity corresponds to Z = 86–89.     

Studies on adsorptiochromism. II: Diffuse reflectance spectroscopy of adsorptiochromic spiropyrans adsorbed to some pharmaceutically useful solids

The colored powders produced by the adsorption of four adsorptiochromic spiropyrans to many solids (silica gel, silicic acid, fumed silica, alumina, microcrystalline cellulose, talc, titanium dioxide) were examined by diffuse reflectance spectroscopy. The reflectance spectra were dominated by two bands, one at 550 nm and the other in the range 400-500 nm, often at 472 nm. Plots of the Kubelka-Munk function [F(R' infinity)] against g, the coverage expressed in nmol/m2, were linear at very low g and approached a limiting value independent of g at high coverage. The color formation upon adsorption terminates at coverages much lower than the maximum binding capacity of the solid. The slope of the plot of F(R' infinity) against g, at low g (denoted f0), appears to be sensitive to the scattering properties of the solid. For a single solid (silica gel), comparison of f0 for adsorptiochromic adsorbates with f0 for permanent dyes allowed estimates to be made of the fraction of adsorbed spiropyran in the colored form on the surface.     

Studies on adsorptiochromism. I: Binding of adsorptiochromic spiropyrans to some pharmaceutically useful solids

The adsorption of four adsorptiochromic spiropyrans to many solids (silica gel, silicic acid, fumed silica, alumina, microcrystalline cellulose, talc, titanium dioxide) was studied at 25 degrees C from cyclohexane solution. Nineteen adsorption isotherms were determined; and the binding data were fitted to the Langmuir equation. A model for binding of adsorptiochromic substances is described, and the parameters of the model are related to the experimental binding constant K.     

Archaeal replicative primases can perform translesion DNA synthesis

DNA replicases routinely stall at lesions encountered on the template strand, and translesion DNA synthesis (TLS) is used to rescue progression of stalled replisomes. This process requires specialized polymerases that perform translesion DNA synthesis. Although prokaryotes and eukaryotes possess canonical TLS polymerases (Y-family Pols) capable of traversing blocking DNA lesions, most archaea lack these enzymes. Here, we report that archaeal replicative primases (Pri S, primase small subunit) can also perform TLS. Archaeal Pri S can bypass common oxidative DNA lesions, such as 8-Oxo-2''-deoxyguanosines and UV light-induced DNA damage, faithfully bypassing cyclobutane pyrimidine dimers. Although it is well documented that archaeal replicases specifically arrest at deoxyuracils (dUs) due to recognition and binding to the lesions, a replication restart mechanism has not been identified. Here, we report that Pri S efficiently replicates past dUs, even in the presence of stalled replicase complexes, thus providing a mechanism for maintaining replication bypass of these DNA lesions. Together, these findings establish that some replicative primases, previously considered to be solely involved in priming replication, are also TLS proficient and therefore may play important roles in damage tolerance at replication forks.The DNA replication machinery rapidly and accurately copies genomes but is prone to stalling at lesions and physical barriers (1). A variety of cellular pathways have evolved to restart stalled replication forks. These include translesion DNA synthesis (TLS) that is performed by specialized polymerases that synthesize short tracts of DNA opposite lesions, thus enabling reinitiation of replication (2). Error-free bypass mechanisms, mediated by homologous recombination, use an alternative undamaged template to rescue stalled replication forks (3). Stalled replisomes can also be rescued by repriming downstream of the blockage, leaving a gap opposite the lesion (4, 5).Eukaryotes and prokaryotes encode distinct TLS polymerases required for DNA damage tolerance (e.g., Y-family Pols). Although much of our understanding of TLS mechanisms has come from studies of archaeal Y-family DNA polymerases, the majority of archaeal species lack canonical TLS enzymes (Fig. 1A) (6), surprising given the otherwise high degree of conservation between eukaryotic and archaeal replisomes. Many archaea do not appear to encode nucleotide excision repair or photolyase pathways that remove UV light-induced damage (6). These anomalies pose the question as to how archaea, lacking canonical TLS or lesion repair pathways, tolerate the presence of lesions that stall replication. This is particularly pertinent to archaea because of the harsh environmental conditions under which many species reside, including extreme temperatures, which promote increased levels of DNA damage.Open in a separate windowFig. 1.A. fulgidus replisomal enzymes displaying DNA polymerase activity. Analysis of 173 archaeal genomes revealed that only 79 archaea encode canonical TLS DNA polymerases. DNA polymerization activities of A. fulgidus replisomal enzymes. (A) The absence of genes encoding Y-family DNA polymerases in most archaea is shown. (B) Structural elements present in A. fulgidus replisomal enzymes. Abbreviations: AEP, archaeo-eukaryotic primase; CTD, carboxy terminal domain; Exo, exonuclease; NTD, amino terminal domain; Pol, polymerase; and Zn, zinc binding site. (C) Polymerization on nondamaged templates. (D) Single nucleotide incorporation on nondamaged templates. The letter C denotes no enzyme control. The triangles above gel panels indicate time course of the polymerization (30 s, 1'', 5'', and 10'').Archaeal replicases (B- and D-family Pols) specifically arrest at deoxyuracil (dU) (7, 8). This unique feature is limited to replicases from archaea (9). Two important questions regarding dU-induced stalling of archaeal replisomes remain unanswered. First, why do archaea stall replication in response to the template strand dU? Second, how are archaeal genomes containing dU copied? This stalling mechanism may have evolved to prevent promutagenic bypass of the template strand dU, resulting in C–T transition (7, 9). The mechanism used by archaea to resume replication after dU-induced replisome stalling has not been identified.In this study, we report that archaeal replicative primases (primase small subunit, Pri S) can perform translesion DNA synthesis on damaged DNA templates. Pri S can bypass common DNA lesions, such as oxidative and UV damages, faithfully bypassing cyclobutane pyrimidine dimers (CPDs). Additionally, we report that Pri S can replicate past template strand dUs, even in the presence of stalled replicative polymerase B and proliferating cell nuclear antigen (Pol B/PCNA) complexes, thus providing a specific mechanism for maintaining timely replication of DNA containing dU lesions. Together, these findings establish that the archaeal primase is not only required for de novo primer synthesis during initiation of DNA replication but also actively participates during the elongation step by assisting the major DNA replicases in traversing DNA lesions.     

Characterization of a Hot-Melt Fluid Bed Coating Process for Fine Granules

The equipment modifications and process changes necessary to perform hot-melt particle coating in a fluid bed granulator are reviewed. A specific case is presented in which partially hydrogenated cottonseed oil is coated onto fine granules (mean particle size, 77 µm; range, 10–150 µm; one standard deviation is 10 µm) composed of a hydrophobic drug and sucrose. The major variables were product bed temperature, temperature of the wax, spray rate, and atomization air pressure. The product bed temperature was selected to give the optimum congealing rate, and the latter three variables were varied in a statistically designed experiment. The physical properties of wax-coated granules fabricated using combinations of process variables were examined. Response surface analysis was used to determine the optimum process settings in terms of dissolution, particle size, and density of the coated product. This system proved quite adequate for the production of uniformly coated granules, with the best product being obtained at the optimized conditions using 120°C atomization air and molten coating temperature, 30 g/min as the spray rate, and an atomization air pressure of 5 bar.