Factor VIII was purified from cryoprecipitate by ion exchange chromatography on solid phase polyelectrolyte E-5 (PE-E5). The product was highly purified (3.5 u VIII:C/mg protein) compared to conventional concentrate (0.3 u VIII:C/mg protein) with low fibrinogen, low isoagglutinin titre, and a ratio of factor VIII coagulant activity (VIII:C) to factor VII related antigen (VIIIR:Ag) of 16:1. Trial infusions of this material (PE VIII) were given to three patients with severe haemophilia A and one patient with homozygous von Willebrand's disease. These patients also each received separate infusions of intermediate purity concentrate (IPC) for comparison. There were no adverse effects. The mean half life of VIII:C after PE VIII infusion in the haemophiliacs was 10.9 h and after IPC was 12.1 h, a statistically insignificant difference. The survival of factor VIII coagulant antigen (VIII:CAg) was similar to that of VIII:C. In contrast, the half life of VIII:C and of VIII:CAg was very short after infusion of PE VIII in the patient wih von Willebrand's disease (2.4 h). IPC when infused in this patient produced a typical secondary rise of VIII:C. Two bleeding episodes in severe haemophiliacs were satisfactorily treated with PE VIII. PE-E5 deserves further study as a means of preparing clinical concentrates of factor VIII.

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Linearized esculentin 2 EM (E2EM-lin) from the frog, Glandirana emeljanovi was highly active against Gram-positive bacteria (minimum lethal concentration ≤ 5.0 μM) and strongly α-helical in the presence of lipid mimics of their membranes (> 55.0 %). The Nterminal α-helical structure adopted by E2EM-lin showed the potential to form a membrane interactive, tilted peptide with an hydrophobicity gradient over residues 9 to 23. E2EM-lin inserted strongly into lipid mimics of membranes from Gram-positive bacteria (maximal surface pressure changes ≥ 5.5 mN m -1 ), inducing increased rigidity (Cs -1 ↑), thermodynamic instability (ΔGmix < 0 → ΔGmix > 0) and high levels of lysis (> 50.0%). These effects appeared to be driven by the high anionic lipid content of membranes from Gram-positive bacteria; namely phosphatidylglycerol (PG) and cardiolipin (CL) species. The high levels of α-helicity (60.0%), interaction (maximal surface pressure change = 6.7 mN m -1 ) and lysis (66.0%) shown by E2EM-lin with PG species was a major driver in the ability of the peptide to lyse and kill Gram-positive bacteria. E2EM-lin also showed high levels of α-helicity (62.0%) with CL species but only low levels of interaction (maximal surface pressure change = 2.9 mN m -1 ) and lysis (21.0%) with the lipid. These combined data suggest that E2EM-lin has a specificity for killing Gram-positive bacteria that involves the formation of tilted structure and appears to be primarily driven by PG-mediated membranolysis. These structure / function relationships are used to help explain the pore forming process proposed to describe the membranolytic, antibacterial action of E2EM-lin. Highlights• E2EM-lin shows specificity and potent efficacy towards Gram-positive bacteria • PG-driven membranolysis promotes E2EM-lin action against Gram-positive bacteria • PE-driven membranolysis promotes E2EM-lin action against Gram-negative bacteria • CL-mediated mechanisms contribute to E2EM-lin action against both bacterial types

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Summary. A repeated finding of national and international collaborative studies of standard factor VIII preparations has been that systematic differences exist between laboratories in their measurement of the relative activities of the same pairs of factor VIII preparations. A workshop meeting was held at the Oxford Haemophilia Centre during 23–26 November 1976 to investigate which of the possible sources of variation between laboratories were responsible. Participants from 16 British laboratories (nine using one‐stage assays and seven using two‐stage assays) performed a total of 273 valid assays using three freeze‐dried preparations of differing purity (a plasma, an intermediate and a high purity concentrate). The results of assay with each participant using their normal system established that, if the participants were a representative cross‐section, approximately one‐third of one‐stage laboratories would show a systematic difference from the overall mean of at least 16% with a corresponding figure for the two‐stage laboratories of 9%. Various features of the assay systems were then modified in a controlled series of experiments. The results showed conclusively that (i) differences between reagents accounted for most of the variation between laboratories, and (ii) the two‐stage assays were, on average, detecting relatively more activity in the more purified preparations than the one‐stage assays. The results also suggested that the use of buffer or citrate‐saline as opposed to haemophilic plasma for the initial dilution of concentrates did not affect the assay results.

Several C-β-D-glucopyranosyl azoles have recently been unravelled among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Towards further exploring their translational potential, ex-vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the MM-GBSA method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (Rp) and Spearman (Rs) correlations (both 0.98), predictive index (PI = 0.99) and AU-ROC (0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using DFT calculations. Seven 2-aryl-4(5)-(β-D-glucopyranosyl)imidazoles and 2-aryl-4-(β-D-glucopyranosyl)-thiazoles were subsequently synthesized and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki-s in the low µM range (5c = 1.97 µM; 13b = 4.58 µM). Ten C-β-D-glucopyranosyl azoles were then tested ex-vivo in mouse primary hepatocytes. Four of these (5a-c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30-60 µM). Structural and predicted physicochemical properties associated with their effectiveness was analysed, with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favoured, while within the cell there is predicted more favourable binding to GP in the protonated form.

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m−1) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with −  < µH > increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.

A fractionation method has been modified to produce a factor VIII concentrate of intermediate purity and a potency of about 12 iu/ml, suitable for injection by syringe. The solubility, ease of filtration and purity of the concentrate have been improved by including a stage in which contaminants are precipitated at controlled temperature, pH and ionic strength. A summary of the first 50 production-scale batches is presented. Plasma can be harvested from whole blood and frozen within eight hours of donation, or after overnight storage of whole blood, with no significant difference in overall yield.