Patent GB-744988-A (GB)
Title: Improvements in preparations of insulin
Inventor: ROBINSON FRANK ARNOLD
IPC: C07K14/62
Filed: 1952-01-31
Abstract: The general methods of preparing polyamino acids described by Bergmann et al J. Biol. Chem. Soc. 111 (1935), 245; Katchalski et al JAmer Chem. Soc. 70 (1948) 2094 and Katchalski et al Nature 164 (1949), 1092 are employed in the preparation of polyamino acids having more than 8 amino acid residues per molecule, said polyamino acid being polylysine, poly-ornithine or polyarginine, or a copolymer of lysine or ornithine with each other or with other amino acids selected from valine, phenylalanine, leucine, isoleucine and glycine or guanidization products of said copolymers.  The removal of carbobenzoxy groups from the poly-carbobenzoxyamino acid stage of the process is as disclosed in these publications, in which a solution of the polycarbobenzoxypeptide in a suitable anhydrous solvent is treated with anhydrous hydrogen chloride. Examples relate to the preparation of the following: (a) poly-L-lipine hydrochloride; (b) polyornithine hydrochloride having a chain length of at least 10 amino acid residues; (c) polyarginine sulphate having a chain length of at least 10 amino acid residues.  (a) L-lipine monohydrochloride is reacted with benzyl chloroformate in aqueous sodium hydroxide at 0 DEG C. to form a :e  dicarbobenzyloxyL-lipine (formula II), which is reacted with phosphorus pentachloride in ether to form first a :e -dicarbobenzoxylysyl chloride (III) and then e -carbobenzoxy a -N-carboxy-lysine anhydride (IV).  The anhydride is dissolved in benzene and refluxed with a small quantity of water and dioxan.  The polymer (V) so produced is dissolved in glacial acetic acid and dry hydrochloric acid is passed for 6 hours at 45-50 DEG  C. Poly-L-lysine hydrochloride (VI) is thereby formed.  In the polymerization step:pyridine, dioxane, ethylacetate or nitrobenzene may be used in place of the benzene; and ammonia or glycine dimethylamide may be used in place of the water, which initiates the polymerization.  In the decarbobenzoxylation step, nitromethane may be used in place of the glacial acetic acid.  (b) DL-ornithine hydrobromide is reacted with carbobenzoxy chloride in aqueous sodium hydroxide to form dicarbobenzoxyornithine.  The anhydride is formed by treating a benzene solution with phosphorus pentachloride; the polymer by refluxing a benzene solution with water as initiator; and decarbobenzoxylation of the polymer by treating a dioxan solution with dry hydrogen chloride.  (c) Polyarginine sulphate is prepared by adding 5-methyl isothiourea sulphate to a solution of polyornithine hydrochloride in ammonia.  Co-polymers are produced by polymerizing mixed anhydrides, e.g. e -carbobenzoxy-L-lysine-a -N-carboxy anhydride and d -carbobenzoxy-DL-ornithine-a -N-carboxy anhydride to give poly-(L-lysine:DL-ornithine)-hydrochloride.  The polyamines are employed in the preparation of an insulin complex (see Group VI). Specification 518,624 is referred to.ALSO:An insulin product of prolonged effect consists of a complex of insulin with a polyamino acid having more than 8 amino acid residues per molecule, said polyamino acid being poly-lysine, polyornithine or poly-arginine, or a copolymer of lysine or ornithine with each other or with other aminoacids selected from valine, phenylalanine, leucine, isoleucine and glycine, or guanidisation products of said copolymers.  The complex is prepared by reacting the insulin and polyaminoacid in solution, e.g. aqueous solution, preferably at the isophane point of said polyamino acid, and preferably at a pH of 5 to 8.  The solution may also contain a zinc salt e.g. zinc chloride.  The resultant aqueous suspension may be used as such for a pharmaceutical injection, in which case, there may be added (a) isotonics e.g. sodium chloride or glycerol (b) antiseptics e.g. phenol or cresol including tricresol, (c) buffers such as sodium phosphate buffer to any desired pH e.g. 7,1.  Alternatively, the complex may be separated from the reaction liquors and then put up in suitable pharmaceutical form.  The polyamino acid is produced by a process which includes a one step polymerisation reaction performed on an amino acid derivative, or mixture of amino acid derivatives (see Group IV(b)). Preferably the polyamino acid contains 10 to 15 amino acid residues per molecule.  Copolymers containing 25 moles per cent of lysine, ornithine or arginine are preferred.  The poly-amino acids may be formed from L-, D- or DL-amino acids. Specification 518,624 is referred to.