The synthesis of the COC-TT vaccine begins with the production of benzoylecgonine (BE). Briefly, cocaine hydrochloride is dissolved at PH 9.5 in 0.1 M phosphate buffered saline (PBS) at a temperature of 70°C. The benzoylecgonine was activated for coupling with water-soluble 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide (EDC, Pierce, Rockford, IL, USA).
To prepare the TT-TFCS conjugate (tetanus toxoid+N-( -trifluoroacetyl caproyloxy) succinimide ester), the TFCS (Pierce, Rockford, IL, USA) was dissolved in a freshly prepared solution of 10–20% DMSO (Sigma – Aldrich, St. Louis, MO, USA)/80% distilled H2O. This solution was mixed with the tetanus toxoid (TT) at a volume of 4 ml of PBS, pH 7.2, and incubated at room temperature overnight. To remove the TFCS trifluoroacetyl protecting group, which is required to couple the TFCS to the -amino groups of the side chain of lysine residues of the TT, it was incubated at pH 8.1 in PBS at room temperature for up to 3 hours. The final purification of the TT-TFCS derivative was carried out by exhaustive dialysis against PBS, pH 7.2.
To prepare the TT-COC conjugate, the activated EDC-COC was added to TT-TFCS in a volume of 100 ml of PBS, pH 7.5, and the reaction mixture was incubated under gentle stirring at room temperature overnight. After exhaustive dialysis against PBS, pH 7.4, the purified conjugate was concentrated by pressure dialysis, aliquoted (unit dose = 1 mg TT/ml), and stored in sealed sterile glass vials. The COC BSA conjugate was synthesized using the same method as for TT.
To prepare the TT-TFCS conjugate (tetanus toxoid+N-( -trifluoroacetyl caproyloxy) succinimide ester), the TFCS (Pierce, Rockford, IL, USA) was dissolved in a freshly prepared solution of 10–20% DMSO (Sigma – Aldrich, St. Louis, MO, USA)/80% distilled H2O. This solution was mixed with the tetanus toxoid (TT) at a volume of 4 ml of PBS, pH 7.2, and incubated at room temperature overnight. To remove the TFCS trifluoroacetyl protecting group, which is required to couple the TFCS to the -amino groups of the side chain of lysine residues of the TT, it was incubated at pH 8.1 in PBS at room temperature for up to 3 hours. The final purification of the TT-TFCS derivative was carried out by exhaustive dialysis against PBS, pH 7.2.
To prepare the TT-COC conjugate, the activated EDC-COC was added to TT-TFCS in a volume of 100 ml of PBS, pH 7.5, and the reaction mixture was incubated under gentle stirring at room temperature overnight. After exhaustive dialysis against PBS, pH 7.4, the purified conjugate was concentrated by pressure dialysis, aliquoted (unit dose = 1 mg TT/ml), and stored in sealed sterile glass vials. The COC BSA conjugate was synthesized using the same method as for TT.
