Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.     

Mineral deficiency and the use of the FETAX bioassay to study environmental teratogens

The Frog Embryo Teratogenesis Assay: Xenopus (FETAX) bioassay has been employed extensively to screen compounds for teratogenic activity. Recent laboratory studies have indicated that low potassium concentrations retard Xenopus laevis development. The effects of varying concentrations of minerals on Xenopus laevis embryo length and development were examined to determine the utility of the FETAX bioassay in the study of environmental teratogens. Water samples collected from 18 wetlands in Minnesota and North Dakota correlated with low mineral levels, causing delayed embryonic development in the FETAX bioassay. When the concentration of sodium or potassium was <5 ppm, Xenopus laevis development was delayed. As a result, it was difficult to assess teratogenic activity after 96 h of incubation. Furthermore, the length of the embryos-an indication of development-paralleled changes in mineral composition. Comparisons between different wetlands based on changes in one specific mineral were not possible due to a synergism between various minerals. If the concentration of sodium and/or potassium was <5 ppm but > or =2 ppm, extension of the FETAX bioassay to 120 h allowed organogenesis to proceed through stage 46, as required for scoring in accordance with ASTM guidelines for the FETAX bioassay. In those cases in which the concentration of sodium and/or potassium were <2 ppm, the embryos could not develop to stage 46 within 120 h and the FETAX bioassay was not suitable for detecting teratogenic activity. Published in 2002 by John Wiley & Sons, Ltd.     

黄柏及中成药中小檗碱和巴马亭的高效液相色谱法测定

本文以正相高效液相色谱法,用窗口图解技术对色谱条件进行了优化。对黄柏及其中成药中的有效成分——小檗碱、巴马亭的提取、测定条件、标准曲线进行了研究。并对两种含黄柏的中成药样品进行了分析。其中小檗碱的回收率均在97%以上,巴马亭的回收率均在96%以上。     

Eyelid reconstruction using temporary tissue expanders and cartilage grafts

Temporary tissue expanders have been used in general plastic surgery to expand skin for breast reconstruction and for scar revision. A specifically designed eyelid tissue expander was used to reconstruct both upper and lower eyelids in a patient suffering a severe lye burn. The implant consists of an expander and a reservoir, connected by silicone tubing. The implants are inserted into the remnant of both eyelids and expanded slowly with multiple injections of saline into the reservoir. After full expansion is achieved the expanders are removed and ear cartilage grafts inserted into the eyelids. The newly created eyelids resemble normal eyelids. The technique of tissue expansion has had very limited use in oculoplastic surgery but new applications are waiting to be discovered.