Identification of aggregation breakers for bevacizumab (Avastin®) self-association through similarity searching and interaction studies.

Aggregation is a common challenge in the optimization of therapeutic antibody formulations. Since initial self-association of two monomers is typically a reversible process, the aim of this study is to identify different excipients that are able to shift this equilibrium to the monomeric state. The hypothesis is that a specific interaction between excipient and antibody may hinder two monomers from approaching each other, based on previous work in which dexamethasone phosphate showed the ability to partially reverse formed aggregates of the monoclonal IgG1 antibody bevacizumab back into monomers. The current study focuses on the selection of therapeutically inactive compounds with similar properties. Adenosine monophosphate, adenosine triphosphate, sucrose-6-phosphate and guanosine monophosphate were selected in silico through similarity searching and docking. All four compounds were predicted to bind to a protein-protein interaction hotspot on the Fc region of bevacizumab and thereby breaking dimer formation. The predictions were supported in vitro: An interaction between AMP and bevacizumab with a dissociation constant of 9.59±0.15 mM was observed by microscale thermophoresis. The stability of the antibody at elevated temperature (40 °C) in a 51 mM phosphate buffer pH 7 was investigated in presence and absence of the excipients. Quantification of the different aggregation species by asymmetrical flow field-flow fractionation and size exclusion chromatography demonstrates that all four excipients are able to partially overcome the initial self-association of bevacizumab monomers.

Glycoprofiling with micro-arrays of glycoconjugates and lectins.

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.

Calcium supplementation of chocolate: effect on cocoa butter digestibility and blood lipids in humans.

BACKGROUND: The digestibility of cocoa butter was reported in animal but not human studies to be low (60-70% and 89-94%, respectively). These differences could be due to the much higher ratio of calcium to fat (by wt) in the diet of rats (0.04-0.18) than in that of humans (0.01). OBJECTIVE: We investigated whether supplementation of chocolate with 0.9% calcium (by wt), as an integral part of a Western diet, reduces absorption of cocoa butter and hence the digestible energy value of chocolate. We also assessed the effect of calcium supplementation on the blood lipid profile. DESIGN: Ten men were fed control diets containing 98-101 g chocolate/d with or without a 0.9%-Ca supplement (0.9 g Ca/d) for 2 periods of 2 wk each. The study was conducted with use of a randomized, double-blind crossover design under free-living conditions but with strict control of food intake. RESULTS: Calcium supplementation of chocolate increased fecal fat 2-fold (from 4.4 to 8.4 g/d; P < 0.0001) and reduced the absorption of cocoa butter by 13.0%. This was due mainly to an increase in the excretion of palmitic and stearic acids (3.4 g/d), which reduced the absorbable energy value of the chocolate by approximately 9%. This supplementation also reduced plasma LDL cholesterol by 15% (P < 0.02); HDL cholesterol was unchanged. CONCLUSIONS: Calcium supplementation can be used as a means of reducing the absorbable energy value of chocolate. Supplementation with 2.25% CaCO3 had no effect on the taste of chocolate, was well tolerated by the subjects, and reduced LDL cholesterol in a short-term study.

Effect of dietary phytic acid on zinc absorption in the healthy elderly, as assessed by serum concentration curve tests.

Zn absorption was investigated in healthy elderly subjects aged 71-78 years and in young subjects aged 23-43 years using serum concentration curve (SCC) tests. Both groups had similar Zn and protein status. The increase in serum Zn was monitored for 180 min after ingestion of 200 ml of soya milk enriched with 50 mg of Zn. Three levels of phytic acid were used: 0 g/200 ml (totally dephytinized soya milk), 0.13 g/200 ml (half dephytinized), and 0.26 g/200 ml (natural phytic acid content). In a first study the effect of 0 v. 0.26 g/200 ml phytic acid was compared in 10 elderly and 10 young subjects, each subject receiving both treatments. In a second study soya milks with 0 and 0.13 g/200 ml were tested in nine elderly and ten young subjects, again receiving both treatments. Mean areas under the curve of the SCC tests conducted with the 0 g/200 ml soya milk were found to be the same in both studies. Phytic acid strongly depressed Zn absorption in both studies (P < or = 0.05), but to a greater extent at the 0.26 g/200 ml level. No difference was found between the groups of young and elderly subjects. Therefore, no significant effect of aging on Zn absorption, as evaluated by the SCC test, or on the inhibitory effect of phytic acid was detected.

Influence of large intakes of trace elements on recovery after major burns.

Because Cu, Se, and Zn are involved in immune and antioxidative defense mechanisms and tissue repair, deficiencies might aggravate complications classically observed with burns. After measuring massive cutaneous trace element losses in 10 burn patients, our aim in this study was to determine whether large intravenous intakes of Cu, Zn, and Se can modify serum trace element levels and recovery after major burns. Ten patients, aged 34 +/- 6 yr (mean +/- SD), admitted to the burns center of a Swiss university hospital with thermal burns on 41 +/- 9% of their body surface were studied prospectively, with trace element balance studies from day 1 (D1) to D7 postinjury. Urine and blood samples were also collected on D10, D15, D20, and D25. The patients were divided into two groups of five and received either standard (group 1, control) or greatly increased (group 2, treatment: 4.5 mg Cu, 190 micrograms Se, and 40 mg Zn/day) trace element intakes. Energy and protein intake and wound treatment were similar in both groups. The treatment group was characterized by improved Cu, Se, and Zn status (increase in serum levels and various protein indicators), a much larger leukocyte increase between D4 and D14 (mainly neutrophils), and shorter hospital stay (45 days) compared with the untreated group (57 days). Grafting requirements were more extensive in group 1. Although severity of injury and wound treatment were similar in the groups, the duration of hospitalization was lower in the treated group. Further studies are required to determine whether this is related to trace element supplementation.

Zinc absorption in healthy elderly humans and the effect of diet.

Absorption of a zinc stable isotope was measured on two consecutive occasions in nine young and eight elderly healthy men aged 24-40 and 70-83 y, respectively. A zinc stable-isotope label (0.8 mg 70Zn) was added to a test meal of either high or low zinc bioavailability, depending mainly on phytic acid content. Zinc absorption from the high-bioavailability test meal was not significantly different (P > 0.05) in the young (38.9 +/- 9.8%, mean +/- SD) and elderly (35.0 +/- 10.9%) subjects. Zinc absorption from the low-bioavailability test meal was 40% and 43% lower, at 23.4 +/- 10.2% and 19.8 +/- 6.1% in these young and elderly men, respectively. Again, no significant effect of age was found. These results show that aging does not lead to nutritionally relevant changes in zinc absorption and in the effect of dietary inhibitors on zinc absorption. Thus, zinc absorption ability seems to be preserved in healthy elderly people, at least until the age of 80 y.

Cutaneous copper and zinc losses in burns.

To measure the exudative cutaneous copper (Cu) and zinc (Zn) losses in burns, 10 patients, aged 36 +/- 9 years (mean +/- s.d.) with burns covering 33 +/- 10 per cent of the total body surface area, were studied from the first postburn day (D1) until D7. All intakes and losses were analysed for Cu, Zn and nitrogen (N) content. Cutaneous losses were extracted from textiles surrounding the patients. Urinary excretions were 0.12 +/- 0.06mg/24h for Cu, 0.9 +/- 0.6mg/24h for Zn, and 14.1 +/- 4.4g/24h for N. Mean daily exudative losses through wound seepage from D1 to D7 were 4.7 +/- 2.1mg/24h for Cu, 27.1 +/- 14.4mg/24h for Zn, and 8.7 +/- 3.8g/24h for N. The cumulated mean losses over 7 days were 37mg for Cu, and 212mg for Zn, representing respectively 20-40 per cent and 5-10 per cent of normal body content. Serum Cu and Zn levels were strongly depressed. The urinary Cu/N ratios correlated with clinical improvement. We conclude that the exudative Cu and Zn losses during the first week postburn contribute significantly to the increased nutrient requirements in burns.