By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed DielsAlderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.biocatalysis | computational enzyme design | Diels-Alder reaction | laboratory evolution | enzyme mechanism

Onchocerciasis, lymphatic filariasis (LF), schistosomiasis and soil transmitted, helminthiasis (STH) are all coendemic in Nigeria. Annual mass drug administration (MDA) with ivermectin (for onchocerciasis), albendazole (for STH and with ivermectin for LF) and praziquantel (for schistosomiasis) is the WHO-recommended treatment strategy for preventive chemotherapy. Separate delivery rounds for distribution of these drugs have been the usual approach to MDA. All three drugs, however, have now been shown to be clinically and programmatically safe for co-administration with what has come to be known as triple drug administration (TDA). We examined the cost savings of converting from separate delivery rounds to TDA in two states in Nigeria. In 2008, eight local government areas received a single round of ivermectin with albendazole followed at least 1 week later by a single round of praziquantel to school-aged children. The following year, a single round was administered with TDA. The number of treated individuals was essentially unchanged during both years (1 301 864 in 2008 and 1 297 509 in 2009) and no change in adverse events was reported. The total programmatic costs for the MDA, not including drug and overhead costs, reduced by 41% from $123, 624 to $72, 870. Cost savings were limited in larger populations due to economies of scale. TDA is recommended for mature MDA.

Many potent drugs are difficult to administer intravenously due to poor aqueous solubility. One validated approach for addressing this issue is to process them into colloidal dispersions known as "nanocrystals" (NCs). However, NCs possess high-energy surfaces that must be stabilized with surfactants to prevent aggregation. In addition, the stabilizer provides a means of anchoring targeting moieties to the NCs for achieving deposition or uptake at specified locations. Nevertheless, a critical challenge is that the surfactant (and consequently the targeting agents) can be shed upon high dilution. This work demonstrates successful cross-linking by click chemistry of stabilizers around paclitaxel NCs to form polymeric "nanocages". Cross-linking does not cause aggregation, as evidenced by transmission electron microscopy, and the nanocages retained the particulate drug through a combination of physical entrapment and physisorption. Size measurements by dynamic light scattering showed that nanocages act as sterically stabilizing barriers to particle-particle interactions and aggregation. The nanocages were shown to be less shed from the NCs than comparable non-cross-linked stabilizers. This contribution provides crucial general tools for preparing poorly sheddable stabilizing coatings to NCs and potentially other classes of nanoparticles for which covalent attachment of the stabilizer to the particle is undesirable (e.g., a drug) or impossible (chemically inert). The presented approach also offers the possibility of more stably attaching targeting moieties to the latter by use of heterotelechelic PEG derivatives, which may favor active targeting and internalization by cells.

Objectives Elafin, an endogenous serine protease inhibitor, modulates colonic inflammation. We investigated the role of elafin in celiac disease (CD) using human small intestinal tissues and in vitro assays of gliadin deamidation. We also investigated potential beneficial effects of elafin in a mouse model of gluten sensitivity. Methods Epithelial elafin expression in the small intestine of patients with active CD, treated CD and controls without CD was determined by immunofluorescence. Interaction of elafin with human tissue transglutaminase-2 (TG-2) was investigated in vitro. The 33-mer peptide, a highly immunogenic gliadin peptide, was incubated with TG-2 and elafin at different concentrations. The degree of deamidation of the 33-mer peptide was analyzed by liquid chromatography-mass spectrometry. Elafin was delivered to the intestine of gluten-sensitive mice using a recombinant Lactococcus lactis vector. Small intestinal barrier function, inflammation, proteolytic activity, and zonula occludens-1 (ZO-1) expression were assessed. Results Elafin expression in the small intestinal epithelium was lower in patients with active CD compared to control patients. In vitro, elafin significantly slowed the kinetics of the deamidation of the 33-mer peptide to its more immunogenic form. Treatment of gluten-sensitive mice with elafin delivered by the L. lactis vector normalized inflammation, improved permeability and maintained ZO-1 expression. Conclusions The decreased elafin expression in small intestine of patients with active CD, the reduction of 33-mer peptide deamidation by elafin, coupled to the barrier enhancing and anti-inflammatory effects observed in gluten sensitive mice, suggest this molecule may have pathophysiological and therapeutic importance in gluten-related disorders.

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Background Yearly, millions of children are treated globally with ivermectin mainly for neglected tropical diseases. Anatomical, physiological and biochemical differences between children and adults may result in changes in pharmacokinetics. However, paediatric pharmacokinetic data of ivermectin are lacking. Methods In the framework of a randomized controlled dose-finding trial in rural Côte d’Ivoire, Trichuris trichiura -infected pre-school-aged children (PSAC, 2–5 years) and school-aged children (SAC, 6–12 years) were assigned to 100 or 200 μg/kg and 200, 400 or 600 μg/kg ivermectin, respectively (ISRCTN registry no. ISRCTN15871729). Capillary blood was collected on dried blood spot cards until 72 h post-treatment. Ivermectin was quantified by LC-MS/MS, and pharmacokinetic parameters were evaluated by non-compartmental analysis. Results C max and AUC increased in PSAC and SAC with ascending doses and were similar in both age groups when the current standard dose (200 μg/kg) was administered (∼23 ng/mL and ∼350 ng×h/mL, respectively). PSAC with lower BMI were associated with significantly higher AUCs. AUC and C max were ∼2-fold lower in children compared with parameters previously studied in adults, whereas body weight-adjusted CL/F (∼0.35 L/h/kg) was significantly higher in children. T max (∼6 h), t 1/2 (∼18 h), mean residence time (MRT INF ) (∼28 h) and V/F (∼8 L/kg) were similar in all paediatric treatment arms. Conclusions A positive association of AUC or C max with dose was observed in both age groups. Undernutrition might influence the AUC of ivermectin in PSAC. Ivermectin shows a lower exposure profile in children compared with adults, highlighting the need to establish dosing recommendations for different age groups.

ISRCTN15871729 (www.isrctn.com).

The site-specific conjugation of polymers to multiple engineered cysteine residues of a prolyl endopeptidase leads to its stabilization in the gastrointestinal tract of rats, without compromising the activity relative to the native enzyme. The importance of polymer attachment sites is investigated, as well as the significance of polymer structure.