Patent GB-771068-A (GB)
Title: Process for the preparation of biologically active preparations
IPC: A61K31/00
Filed: 1954-08-23
Abstract: The invention comprises a highly effective preparation of a vitamin of the B12 group in dosage unit form, comprising about 2 mg. of a preparation containing about 7 m g. of said vitamin bound to a protein fraction containing intrinsic factor activity and having, in the free state, binding power for vitamins of the B12 group, and the process of preparing biologically active preparations with a component having, in the free state, a binding power for Vitamin B12 or intrinsic factor activity, characterized in that an aqueous solution of a combination of a protein substance with intrinsic factor activity and a vitamin of the B12 group is subjected to fractional precipitation with an alkanol of no more than 2 C atoms, e.g. ethanol, the precipitate formed at alcohol concentrations of about 40 per cent by volume-about 60 per cent by volume being separated, an aqueous solution of this precipitate is then subjected to a fractional salting-out with ammonium sulphate, the fractions precipitated at a saturation of the solution of about 0.7-1.0 being separated after which this precipitate is separated into more or less strongly coloured fractions in aqueous medium by means of electrophonesis and eventually the cobalamin-protein is isolated from the most strongly red-coloured fraction according to well-known methods.  Preferably the various process steps are performed at a temperature not exceeding 3 DEG  C. and preferably at - 5 DEG  C. Zone electrophonesis may be applied using as vehicle cellulose powder, starch, glass powder or other inert vehicles. In an example a physiologically active cobalamin-protein compound is obtained, said compound being of strongly red colour, its Vitamin B12 content corresponding to a ratio of 1 mol. of Vitamin B12 to 1 mol. of said cobalamin-protein compound, said compound containing 1 atom of cobalt per molecule, its Vitamin B12 content being about 13 m g. per mg., said compound being a muco protein compound, having a molecular weight of the order of 100,000, not being dialysable, being soluble in water and physiological sodium chloride solution, being soluble in and not precipitated from its aqueous solutions at an alcohol concentration of 43.7 per cent by volume and precipitating at an alcohol concentration of 56 per cent by volume at a temperature of -5 DEG  C. to a pH of about 7.0, its mobility measured in the electrophoresis apparatus of Tiselius (moving boundary method) in phosphate buffer of pH 7.7 and ionic strength 0.1 being at the descending limb - 3.0 x 10-5 cm.2 sec.-1 volt-1, the absorption spectrum of its aqueous solution showing peaks at 550, 518, 413, 363, 324 and 278 mm ., the ratio between the optical densities D413/D550 being about 0.5, that between D363/D550 being about 8.0. Specification 733,875 is referred to.ALSO:The invention comprises a pharmaceutical preparation for treating pernicious anaemia comprising, in dosage unit form, cobalamin-protein (see Group IV(b)), and organs or purified preparations thereof having intrinsic factor activity prepared from animal material such as hog stomach, pylorus or duodenum. Mention is made of a tablet form comprising 0.375 mg of cobalamin-protein with about 1 grm of hog pylorus or stomach or duodenum mucosa, or the corresponding quantity of aqueous extracts, dry powders and like preparations prepared from such mucosa.