Phenelfamycins A, B, C, E, F and unphenelfamycin make up a recently isolated group of elfamycin-type antibiotics. All of the phenelfamycins were active against Gram-positive anaerobes, including Clostridium difficile. Phenelfamycin A was also active in vitro against Neisseria gonorrhoeae and Streptococci. Phenelfamycin A was found to be effective in prolonging the survival of hamsters in an animal model of C. difficile enterocolitis. After oral administration of phenelfamycin A to hamsters, antibiotic was detected in the caecal contents but not in the blood.

Methods were developed for the isolation of gm. quantities of human antithrombin-III (AT-III) from Cohn Fraction IV-1 of human plasma using heparin covalently attached to agarose. Attachment of heparin carboxyl groups to alkylamino-agarose yielded a support with no affinity for AT-III. Linkage via the heparin hydroxyl groups yielded a support with approximately 1 mg of heparin/ml agarose and with a low capacity for binding AT-III. Linkage of heparin to agarose thru its amino groups yielded a heparin-agarose with the highest capacity for AT-III. Reaction of heparin containing a free α-amino group with cyanogen bromide activated agarose resulted in agarose substituted with 5 mg of heparin/ml. The conditions of buffer, pH, ionic strength and temperature which maximized AT-III binding were 0.05M sodium phosphate, 0.02M sodium citrate, 0.15M NaCl, pH 8.3 and 4°. The heparin-agarose bound 0.1-0.2 mg of AT-IIl/ml. The AT-III isolated by affinity chromatography was further purified by gel permeation and yielded a homogeneous product as judged by Polyacrylamide disc gel electrophoresis of native and reduced protein and by sedimentation velocity (S20, w = 4.1). This material had an activity of 1700 units/A280 as measured by inhibition of human thrombin. The AT-III is stable to heating at 60° for 10 hours in a buffer of 0.5M sodium citrate at pH 7-8. Injection of bovine thrombin (3000 units/Kg) into heparinized dogs (150 units/Kg) decreased circulating AT-III levels to 50%. (Supported by NHLI, Contract NOl-HB-4-2946).

Since improvements in the isolation procedure have resulted in heparin of high biological activity, the question of species difference has been reexamined. We have isolated heparin from dog, beef, hog, sheep and human tissue by selective fractionation with quaternary ammonium compounds. Based on our results we would suggest that heparins isolated from various mammalian species are biologically, chemically and physically similar. The biological variations previously observed may well be a result of different degree of purity in the heparin isolated.

A wide range in the content of the linkage-region sugar units and of 2-acetamido-2-deoxy-d-glucose was found in heparin samples from different sources. All heparin preparations examined contained 2-acetamido-2-deoxy-d-glucose, and this constituent was not altered by bleaching reagents which significantly lowered the neutral sugar and l-serine content. This indicated that the variability of the 2-acetamido-2-deoxy-d-glucose content was not due to the methods of purification. Most of the purified heparin preparations showed single compact spots of varying mobilities on electrophoresis, although unbleached beef-lung fractions showed distinct double spots.

1. Previously reported light scattering molecular weights of hexadimethrine bromide are in error due to the presence of fluorescent material. The true molecular weights are much lower and may in fact be nearly identical for all commercial samples. 2. Fractionation by gel filtration followed by pharmacological and toxicological studies indicate that a polymer of 4 monomer units would be the preferred one for clinical use.