Patent EA-002127-B1 (EA)
Title: In vivo stain composition, process of manufacture, and methods of use to identify displastic tissue
Inventor: BURKETT DOUGLAS D
IPC: A61K6/00
Filed: 1998-11-11
Abstract: 1. A composition of toluidine blue O ("TBO"), in which the ratio of the combined areas of the 254 nm HPLC peaks representing conformational isomers of toluidine blue O to the combined areas of the peaks representing N-demethylation derivatives being at least 6:1. 2. A composition  of toluidine blue O ("TBO"), in which the ratio of the combined areas of the 254 nm HPLC peaks representing a first group of components, comprising the conformational isomer of toluidine blue O having the ring methyl group in the -2 position, the N-demethylation derivative thereof and the N,N-demethylation derivative thereof; and the combined areas of the HPLC peaks representing a second group of components, comprising the conformational isomer of toluidine blue O having the ring methyl group in the -4 position, the N-demethylation derivative thereof and the N,N-demethylation derivative thereof being at least 2.5:1 3. A composition of toluidine blue O ("TBO"), in which the isomer having the ring methyl group in -2 position comprises at least 58% of the total organic dye content of said composition. 4. In a method for identification of dysplastic tissue, the step of applying to human oral tissue the composition of claim 1 in a liquid carrier. 5. In a process for manufacturing toluidine blue O ("TBO") of claims 1 to 3, in which the process includes the steps of  (A) oxidizing N,N-dimethyl-p-phenylene diamine in a first reaction mixture, to form a first intermediate, 2-amino-5-dimethylaminophenyl thiosulfonic acid,  (B) oxidizing said first intermediate and condensing the oxidizate in a second reaction mixture with o-toluidine, forming a second intermediate, indamine-thio-sulfonic acid, (C) oxidizing said second intermediate to close the indamine ring thereof, forming a TBO-containing reaction product dissolved in a third reaction mixture, (D) introducing a complexing reagent into said third reaction mixture, to form a TBO-complex product dissolved in said third reaction mixture, (E) precipitating said TBO-complex product from said third reaction mixture, and (F) separating said TBO-complex product, containing the conformational isomers of TBO and N-demethylation and N,N-demethylation derivatives thereof, from said third reaction mixture, the improved process comprising introducing said complexing reagent to a reaction mixture before the formation of said third reaction mixture. 6. The process of claim 5 in which the temperature of the reaction mixtures is maintained at not higher than 10 degree C. 7. The process of claim 5 in which the pH of the reaction mixtures is maintained in the range of 2.8 - 3.8 in the first reaction mixture, 3.1 - 4.1 in the second reaction mixture and 3.0 in the third reaction mixture. 8. The process of claim 5 which includes the steps of introducing a source of thiosulfate ions into said first reaction mixture, while maintaining the temperature of said first reaction mixture at a temperature not higher than 10 degree C. 9. In an HPLC method for analysis of a toluidine blue O ("TBO") dye product, said method including forming a mobile phase, forming a TBO sample solution, equilibrating an HPLC column with the mobile phase flow, and injecting the sample solution into the HPLC column, in which said mobile phase is formed as a composition comprising a water-soluble salt of an organic acid. 10. The process for synthesizing 2-amino-5-dimethylaminophenyl thiosulfonic acid, comprising the step of oxidizing N,N'-dimethyl-p-phenylene-diamine in the presence of a source of thiosulfate ions, while maintaining the temperature of the reaction mixture not higher than 10 degree C.