Process for the preparation and use of a bivalent vaccine against morphine-heroin addiction

Abstract

Provided are bivalent immunogenic composition against morphine-heroin addiction comprising a carrier protein (“CP”) and a morphinic product, wherein the CP and the morphinic product are connected by a spacer-linker arm having a total molecular size of 16-21 Å, and methods of using the same.

1 . A surrogate structural formulation of the bivalent vaccine against morphine-heroin addiction obtained from a synthetic process wherein the CP-TFCS intermediate is covalently coupled to EDC-3-O-carboxymethylmorphine, and wherein the synthetic process is characterized by the following chemical reactions or steps:
 a) reaction of the EDC-(morphine-6-hemisuccinate) intermediate product, obtained through a standard synthetic process for covalent haptenization to the tetanus toxoid-TFCS intermediate conjugate, obtained through a synthetic process so as to generate free reactive amine groups in the tetanus toxoid-TFCS conjugate, enhancing a covalent condensation between both intermediate products, which reaction is carried out in a stoichiometric ratio by reacting mol:mol, 100 μmol of EDC-(M-6-H) per each 0.07 μmol of active free amino groups of the intermediate tetanus toxoid-TFCS conjugate;   b) mix 100 mg of the tetanus toxoid-TFCS intermediate conjugate (1 μmol of tetanus toxoid-TFCS=0.07 μmol of active sites) with 30 mg (70 μmol of active covalent condensation sites) of the EDC-(M-6-H) intermediate product, in a final volume of 100 ml of 0.1 M phosphate buffer solution/0.15 M NaCl, pH 7-7.5;   c) incubation of reactants obtained in step a) during 2-3 hours at room temperature under slow and constant stirring;   d) purification of morphine-6-hemisuccinyl-tetanus toxoid vaccine from reactive formed by-products, such as urea and EDC-(M-6-H) non-haptenized molecules, using standard dialysis procedures. The morphine-6-hemisuccinyl-tetanus toxoid vaccine is dialyzed against three-6 liters replacements, every 8 hours of 0.1 M phosphate buffer solution, pH 7.2 in 10 kDa of molecular weight cut-off dialysis membranes, for up to 24 hours at 4° C.;   e) sterilization of the vaccine product through filtration procedures under positive-pressure using 0.45 μm pore-size membrane filters;   f) dry-freezing of 1 ml aliquots of the morphine-6-hemisuccinyl-tetanus toxoid vaccine in sterile glass vials vacuum sealed and storage at 4° C. to preserve vaccine preparation. In addition, several chemical reagents can be added to the therapeutic bivalent vaccine formulation against morphine-heroin addiction, to enhance the stabilization and avoid damage and deterioration, during the dry-freezing and storage procedures of the vaccine;   
       obtaining of the bivalent vaccine against morphine-heroin addiction with a percent yield of the conjugated hapten (morphine)-carrier protein (tetanus toxoid) of up to 75-85%. 
     
     
         2 . The surrogate structural formulation of the bivalent vaccine against morphine-heroin addiction obtained from the synthetic process according to  claim 1 , displaying the following structural formula: 
       
         
           
           
               
               
           
         
       
     
     
         3 . The surrogate bivalent vaccine against morphine-heroin addiction according to  claim 2 , characterized by the presence of the TFCS reagent, which is used for the synthesis of the spacer-linker arm to haptenize morphine with the tetanus toxoid. 
     
     
         4 . The surrogate bivalent vaccine against morphine-heroin addiction according to  claim 2 , characterized by the presence of a spacer-linker arm which displays a total molecular size of approximately 16.47 Å, where the 9.03 Å segment comprising an alkane chain introduced by the TFCS reagent, the 7.44 Å segment comprising a α-carbon atom and four extended carbon atoms from the lateral chain of exposed lysine residues in the tetanus toxoid, as depicted in the following formula: 
       
         
           
           
               
               
           
         
       
     
     
         5 . The surrogate bivalent vaccine against morphine-heroin addiction according to  claim 2 , which contains morphine as the haptenic drug. 
     
     
         6 . The surrogate bivalent vaccine against morphine-heroin addiction according to  claim 2 , which contains the tetanus toxoid as the carrier protein. 
     
     
         7 . A process to prepare a typical dosage of the formulation of the bivalent vaccine against morphine-heroin addiction by adding an adjuvant used for active vaccination protocols in rodents, which process is characterized by the following chemical reactions or steps:
 a) mix 1 ml of the bivalent vaccine against morphine-heroin addiction in a 1:2 (v:v) ratio; resuspended in sterile deionized H 2 O with 2 ml of 45 mg/ml aluminum hydroxide stock solution, added at room temperature through slow dripping in a 3 minute time period under moderate and constant stirring;   b) incubation of reactants for 1-2 hours, so that the final aluminum hydroxide concentration should not exceed 1.12-2.25 mg/100 μl in the mixture containing the bivalent vaccine conjugate against morphine-heroin addiction; and   c) loading the dosage formulation which comprise the anti-morphine-heroin bivalent vaccine/aluminum hydroxide adjuvant mixed composition into sterile plastic syringes under continuous stirring.   
     
     
         8 . An active immunization method applied to a rodent detoxified and abstinent from either morphine or heroin addiction, which is characterized on the basis that it may receive a subcutaneous administration of an initial dose-unit of the haptenic drug-carrier protein conjugate of about 1-2 mg/kg according to the individual's body weight. 
     
     
         9 . The method according to  claim 8 , wherein the initial “unit dose” must be followed by 3-6 re-vaccinations at 14-21 day intervals using the same initial dose. 
     
     
         10 . A method to produce specific antisera against morphine-heroin in rodents, wherein the method comprises the active vaccination of an individual with a bivalent vaccine against morphine-heroin addiction having one of the following structural formulas: 
       
         
           
           
               
               
           
         
       
     
     
         11 . The antiserum resulting from the active immunization procedure with a bivalent vaccine against morphine-heroin addiction according to  claim 10 . 
     
     
         12 . The polyclonal antibodies resulting from the active immunization procedure with the bivalent vaccine against morphine-heroin addiction according to  claim 10 . 
     
     
         13 . The method according to  claim 10 , wherein the bivalent vaccine against morphine-heroin addiction generates high and sustained titers of circulating polyclonal antibodies showing equivalent cross-recognition to morphine and/or heroin in the blood, thereby preventing its blood-brain barrier permeation into the brain. 
     
     
         14 . The method according to  claim 13 , wherein generated antibodies also display equivalent specificities for morphine/heroin metabolites such as, 6-monoacetylmorphine, morphine-3-glucuronide and morphine-6-glucuronide. 
     
     
         15 . A method to blunt the reacquisition to morphine-heroin self-administering behavior in rodents by the repeated boosting of a bivalent vaccine against morphine-heroin addiction, wherein the bivalent vaccine has one of the following structural formulas:
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