| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 11.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 02.11.99 |
| PATENT TITLE |
Catalytic antibodies against cocaine |
| PATENT ABSTRACT |
This invention provides compounds which are analogs to the hydrolysis transition-state of a cocaine benzoyl ester group. This invention also provides such analogs linked to carrier proteins, and antibodies thereto. This invention further provides pharmaceutical composition for decreasing concentration in a subject using the antibodies produced. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 07.06.95 |
| PATENT REFERENCES CITED |
Ambre, J., et al., (1984), Urinary excretion of ecgonine methyl ester, a major metabolite of cocaine in humans, J. Anal. Toxicol., 8:23-25 (Exhibit 17). Ambre, J., (1985), The urinary excretion of cocaine and metabolites in humans: a kinetic analysis of published data, J. Anal. Toxicol., 9:241-245 (Exhibit 18). Bach, J.F., et al., (1993), Safety and efficacy of therapeutic monoclonal antibodies in clinical therapy, Immunol. Today, 14:421-425 (Exhibit 19). Baldwin, E., and Schultz, P.G., (1989), Generation of a catalytic antibody by site-directed metagenesis, Science, 245:1104-1107 (Exhibit 20). Benkovic, S.J., (1988), Catalysis of a stereospecific bimolecular amide synthesis by an antibody, Proc. Natl. Acad. Sci. USA, 85:5355-5358. Benkovic, S.J., et al., (1990), The enzymic nature of antibody catalysis: development of multistep kinetic processing, Science, 250:1135-1139 (Exhibit 22). Benuck, M., et al., (1987), Pharmacokinetics of systemically administered cocaine and locomotor stimulation in mice, J. Pharmacol. Exp. Therap., 243:144-149 (Exhibit 23). Bonese, K.F., et al., (1974), Changes in heroin self-administration by a rhesus monkey after morphine immunization, Nature, 252:708-710 (Exhibit 24). Borrebaeck, C.A.K., (1989), Strategy for the production of human monoclonal antibodies using in vitro activated B cells, J. Immunol. Methods, 23:157-165 (Exhibit 25). Carpenter, C.B., (1990), Immunosuppression in organ transplantation, N. Engl. J. Med., 332:1224-1226 (Exhibit 26). Chandrakumar, N.S., et al., (1993), Phenylphosphonate monoester analogs of cocaine, Bioorg. & Medic. Chem. Let., 3:309-312 (Exhibit 27). Chow, M.J., (1985), Kinetics of cocaine distribution, elimination, and chronotropic effects, Clin. Pharmacol. Ther., 38:318-324 (Exhibit 28). Cochran, A.G., et al., (1991), Antibody-catalyzed biomolecular imine formation, J. Am. Chem. Soc., 113:6670-6672 (Exhibit 29). Dean, R.A., et al., (1991), Human liver cocaine esterases: ethanol-mediated formation of ethylcocaine, FASEB J., 5:2753-2739 (Exhibit 30). Fischman, M.W., et al., (1990), Effects of desipramine maintenance on cocaine self-administration by humans, J. Pharmacol. & Exp. Ther., 253:760-770 (Exhibit 31). Fujii, I., et al., (1991), Enantiofacial protonation by catalytic antibodies, J. Am. Chem. Soc., 113:8528-8529 (Exhibit 32). Gatley, S.J., et al., (1990) Rapid stereoselective hydrolysis of (+)-cocaine in baboon plasma prevents its uptake in the brain: implications for behavioral studies, J. Neurochem., 54:720-723 (Exhibit 33). Gawin, F.H., et al., (1988), Cocaine and other stimulants: actions, abuse and treatment, New Eng. J. Med., 318:1173-1182 (Exhibit 34). Gawin, F.H., et al., (1989), Desipramine facilitation of initial cocaine abstinence, Arch. Gen. Psychiatry, 46:117-121 (Exhibit 35). Goeders, N.E., (1983), Cortical dopaminergic involvement in cocaine reinforcement, Science, 221:773-775 (Exhibit 37). Harris, W.J., et al., (1993), Therapeutic antibodies--the coming of age, TIBTECH, 11:42-44 (Exhibit 38). Ikeda, S., et al., (1991), Asymmetric induction via a catalytic antibody, J. Am. Chem. Soc., 113:7763-7764 (Exhibit 39). Iverson, B.L. and Lerner, R.A., (1989), Sequence-specific peptide cleavage catalyzed by an antibody, Science, 243:1184-1188 (Exhibit 40). Jackson, D.Y., et al., (1988), An antibody-catalyzed claisen rearrangement, J. Am. Chem. Soc., 110:4841-4842 (Exhibit 41). Janda, K.D., et al., (1991), Catlytic antibodies with acyl-transfer capabilities: mechanistic and kinetic investigations, J. Am. Chem. Soc., 113:291-297 (Exhibit 42). Janda, K.D., et al., (1988), Induction of an antibody that catalyzes the hydrolysis of an amide bond, Science, 241:1188-1191 (Exhibit 43). Janda, K.D., et al., (1989), Catalytic antibodies with lipase activity and R or S substrate selectivity, Science, 244:437-440 (Exhibit 44). Janda, K.D., et al., (1991), Antibody bait and switch catalysis: a survey of antigens capable of inducing abzymes, J. Am. Chem. Soc., 113:5427-5434 (Exhibit 45). Janda, K.D., et al., (1991), Substrate attenuation: an approach to improve antibody catalysis, Tetrahedron, 47:2503-2506 (Exhibit 46). Kitazume, T., et al., (1991), Antibody-catalyzed double stereoselection in fluorinated materials, J. Am. Chem. Soc., 113:8573-8575 (Exhibit 47). Lesko, L.M., et al., (1982), Introgenous cocaine psychosis, New Eng. J. Med., 307:1153 (Exhibit 49). Mayforth, R.D., et al., (1990), Designer and catalytic antibodies, N. Engl. J. Med., 323:173-178 (Exhibit 50). Osband, M.E., et al., (1990), Problems in the investigational study and clinical use of cancer immunotherapy, Immunol. Today, 11:193-9195 (Exhibit 51). Pollack, S.J., (1986), Selective chemical analysis by an antibody, Science, 234:1570-1573 (Exhibit 52). Queen, C., et al., (1989), A humanized antibody that binds to the interleukin 2 receptor, Proc. Natl. Acad. Sci. USA, 86:10029-10033 (Exhibit 53). Reichmann, L.M., et al., (1988), Reshaping human antibodies for therapy, Nature, 332:323-327 (Exhibit 54). Robins, R.J., (1996), The measurement of low-molecular-weight, non-immunogenic compounds by immunoassay. In: H. Linskens and J.F. Jackson (Eds.), Immunology in Plant Sciences, Springer-Verlag, Berlin Heidelberg, pp. 86-141, (Exhibit 55). Schultz, P.G., (1988), The interplay between chemistry and biology in the design of enzymatic catalysts, Science, 240:426-433 (Exhibit 56). Seaver, S.S., (1994), Monoclonal antibodies in industry: more difficult than originally thought, Genetic Engineering News, 14:10, 21 (Exhibit 57). Shokat, K.M., et al., (1989), A new strategy for the generation of catalytic antibodies, Nature, 338:269-271 (Exhibit 58). Tramontano, et al., (1986), Catalytic antibodies, Science, 234:1566-1570 (Exhibit 59). Tramontano, A., et al., (1986), Chemical reactivity at an antibody binding site elicited by mechanistic design of a synthetic antigen, Proc. Natl. Acad. Sci. USA, 83:6736-6740 (Exhibit 60). Tramontano, A., et al., (1988), Antibody catalysis approaching the activity of enzymes, J. Am. Chem. Soc., 110:2282-2286 (Exhibit 61). Waldman, T.A., (1991), Monoclonal antibodies in diagnosis and therapy, Science, 252:1657-1662 (Exhibit 62). Ziegler, E.J., et al., (1991), Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin, N. Engl. J. Med., 324:429-436 (Exhibit 63). National Institute On Drug Abuse "Development of immunological and molecular biological approaches to effect reduction of cocaine use." NIH Guide vol. 21, No. 34, Sep. 25, 1991. National Institute On Drug Abuse "Development of innovative methods to identify medications for treating cocaine abuse." NIH Guide vol. 21, No. 31, Aug. 28, 1992. Erlanger, B.F. (1980) "The Preparation of Antigenic Hapten-Carrier Conjugates: A Survey." Methods in Enzymology 70: 85-105. Colburn, W.A. (1980) "Specific Antibodies and Fab Fragments to Alter the Pharmaco-kinetics and Reverse the Pharmacologic/Toxicoligic Effects of Drugs." Drug Metabolism Reviews 11: 223-262. McConnell, et al. (1981) The Immune System, Blackwell Scientific Publications, Boston, MA, pp. 157-159. Hames, et al. (1981) Gel Electrophoresis of Proteins, (IRL Press, Washington, D.C.) p. 44. Scopes, R.K. (1982) Protein Purification, (Springer-Verlag, NY), p. 254. Giannini, et al. (1989) "Bromocriptine and Amantadine in Cocaine Detoxification." Psychiatry Research 29: 11-16. Gawin, et al. (1989) "Cocaine Dependence." Ann. Rev. Med. 40: 149-161. Pentel, et al. (1991) "Pretreatment with Drug-Specific Antibody Reduces Desipramine Cardiotoxicity in Rats." Life Sciences 48: 675-683. Bagasra, et al. (1992) "A Potential Vaccine for Cocain Abuse Prophylaxis." Immunopharmacology 23: 173-179. Yu, et al. (1992) "Synthesis of Carbon-11 Labeled Iodinated Cocaine Derivatives and Their distribution in Baboon Brain Measured Using Positron Emission Tomography.". Chandrakumar, et al. (1993) "Phenylphosphonate Monoester Analogs of Cocaine. Potential Haptens for the Generation of Catalytic Antibodies." Bioorganic & Medicinal Chemistry Letters 3: 309-312. Landry, et al. (1993) "Antibody-Catalyzed Degradation of Cocaine." Science 259: 1899-1901. Abraham, et al. (1992) "N-Modified Analogues of Cocaine: Synthesis and Inhibition of Binding to the Cocaine Receptor." J. Med. Chem. 35: 141-144. Lewin, A.H., et al. (1992) "2 beta-substituted Analogues of Cocaine. Synthesis and Inhibition of Binding to the Cocaine Receptor." J. Med. Chem. 35: 135-140. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An antibody capable of catalyzing the hydrolysis of cocaine at benzoyl ester group and against the compound having the structure: ##STR60## wherein R' is O or CH2 and R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. 2. An antibody capable of catalyzing the hydrolysis of cocaine at benzoyl ester group and against the compound having the structure: ##STR61## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. 3. An antibody capable of catalyzing the hydrolysis of cocaine at benzoyl ester group and against the compound having the structure: ##STR62## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. 4. An antibody capable of catalyzing the hydrolysis of cocaine at benzoyl ester group and against the compound having the structure: ##STR63## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. 5. An antibody capable of catalyzing the hydrolysis of cocaine at benzoyl ester group and against the compound having structure: ##STR64## wherein each of R.sub.1, R.sub.2, R.sub.3, or R.sub.4 is independently hydrogen, or a lower alkyl; or wherein one but only one of R.sub.1, R.sub.2, or R.sub.3 is a lower alkyl substituted on the terminal carbon atom by an azido or amino group, a group comprising a lower alkyl group linked to a lower alkyl carboxylic acid or derivative, with each of the remaining two of R1, R2, or R3 is independently hydrogen or a lower alkyl and R4 is hydrogen, a lower alkyl or a negative charge. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Within this application, publications are referenced by arabic numerals within par-theses. Full citations for these publications may be found at the end of each series of experiments. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to witch this invention pertains. Cocaine has been used by over 30,000,000 Americans since 1980 and frank addiction afflicts at least 1,700,000(1). The medical and social consequences of this stimulant abuse are well known and range from acute psychoses to cardiac failure and from violent behavior to crack-addicted newborns(2-4). Cocaine-induced disinhibition and an increased propensity for high risk behavior now pose a special peril with the advent of the acquired immunodeficiency syndrome (AIDS). The highly reinforcing nature of stimulants makes this form of substance abuse especially pernicious and despite a variety of pharmacologic and non-pharmacologic approaches to treatment, no modality is adequately successful(5,6). The reinforcing potential is clearly related to peak serum concentration(7-9). Also, the rapidity with which the Peak is achieved appears critical and may relate to the observation that tolerance to the psychopharmacologic and physiologic effects of cocaine manifests during the course of a single administration(10). The rampaging abuse of crack, a smokeable form of cocaine, likely corresponds in part to its rapid delivery across the lung with an efficiency approaching that of an intravenous injection(1,5). Pharmacokinetics may also explain the propensity for binge use associated with crack smoking(1). An agent that reduced the velocity to and magnitude of peak serum levels would permit this hypothesis to be tested as well as have major therapeutic potential. The neuropharmacologic approach to treatment has focused on receptor systems such as the dopaminergic pathways that mediate the effects of cocaine(11). A direct antagonist to cocaine is not available but agents such as desipramine show some promise for maintaining abstinence(12,13). However, there is a lag of several weeks in the onset of desipramine's effect and during this induction period a marked potential for recidivism remains(5,14). An agent effective even for just this period could have important clinical applications but at present no such agent exists. An alternative to receptor based approaches would be to interfere with the delivery of cocaine to the central nervous system (CNS) so that a dose of cocaine no longer had a reinforcing behavioral effect. Since there is no prospect for excluding cocaine from the circulation, this approach would require binding of cocaine by a circulating agent. In the 1970's Schuster and colleagues investigated an immunologic approach to substance abuse based on the possibility of interference with CNS delivery(15). A rhesus monkey was allowed to self-administer heroin to dependence, and then was immunized to an opiate. Despite access to the heroin, the animal no longer self-administered it. The serum anti-opiate antibody titer greatly exceeded the cerebrospinal fluid titer and this localized the antibody effect to the serum. Thus, the association of heroin and circulating heroin antibody must have been sufficiently rapid to block the heroin's effect. However, the limitation of the approach was identified in that continued administration of very high doses of heroin exhausted the pool of circulating antibody and the animal resumed heroin self-administration. Thus, the approach worked in that the antibody effectively bound the drug and did modify behavior but the approach was limited in that the antibody supply was exhaustible. An antibody would need the characteristics of an enzyme to avoid being "depleted" itself as it depleted its target. Recently, the exciting development of catalytic antibodies has been reported(16,17). Catalytic antibodies not only bind but also act as artificial enzymes which metabolize their target thus freeing the antibody for further binding(18-25). The principles of this startling advance are illustrated by considering the hydrolysis of a carboxylic acid ester by an enzyme. As seen in FIG. 1, hydrolysis of the planar ester commonly proceeds through a tetrahedral intermediate which decomposes to yield alcohol and planar carboxylic acid. The rate of the reaction varies with the magnitude of the activation barrier (.DELTA.G) between the starting ester and the peak or transition state structure. An enzyme's active site contains a pocket that complements the structure of the hydrolysis transition-state and through various binding interactions, the enzyme stabilizes the transition-state relative to the starting material. This differential stabilization decreases .DELTA.G and contributes to catalysis. The transition state corresponds to a particular configuration of atoms and is thought to resemble the definable species closest to it in energy, i.e. the tetrahedral intermediate in the case of ester hydrolysis. The transition state is unstable and evanescent but phosphonate monoesters are stable compounds which resemble this species in geometry and distribution of charge and on this basis, may serve as transition state analogs. An antibody elicited to such an analog will manifest binding interactions complementary to the hydrolysis transition state being modeled. This antibody, by binding to the modeled substrate, will stabilize the transition state relative to the starting states, lower the activation barrier and catalyze the hydrolysis. By binding and destroying its target, the catalytic antibody is then freed to bind an additional target. Ample literature precedent supports the use of catalytic antibodies as artificial enzymes for the hydrolysis of esters(16,17,26-33). Analogs based on An-oxide structure, rather than the phosphonate structure, can also be used to yield catalytic antibodies. Of all the commonly abused substances, cocaine is the best candidate for this approach (Scheme I). Attached to the ecgonine nucleus of cocaine is a benzoyl ester group which when hydrolyzed results in a virtually inactive product(35,36)--this is one of the pathways of deactivating metabolism in humans(35,36). The transition state of that reaction resembles the tetrahedral intermediate of hydrolysis and can be mimicked by a suitably designed phosphonate ester analog of the hydrolysis transition state of the cocaine benzoyl ester. A subpopulation of the antibodies elicited by this cocaine analog will function as esterases highly specific for cocaine. Thus, the principal impediment to the immunologic approach suggested two decades earlier--the exhaustibility of the circulating antibody--could be overcome. The anti-cocaine catalytic antibody generated ##STR1## in this fashion would destroy cocaine and be itself available for continued function. The application of such a reagent antibody to the problem of chronic cocaine abuse would be to deprive the abuser of the reinforcing effect of the drug, thereby providing a window for appropriate psychosocial and relapse prevention interventions, and promoting extinction of the addiction. Only a subpopulation of anti-analog antibodies will possess catalytic activity, so the production of a monoclonal antibody and passive immunization of subjects is required(37,38). Monoclonal antibodies have become established pharmaceutical agents for the treatment of organ transplant rejection(39) and Gram negative septicemia(40). Passive immunization with an anti-cocaine catalytic monoclonal antibody appears to be practical in clinical terms. Second is the duration of effectiveness. The currently available monoclonal pharmaceuticals are administered daily since, as these antibodies bind, the antibody-antigen complexes are removed from the circulation. In contrast, a monoclonal antibody functioning as an artificial enzyme could be designed for longevity(41)--the Fc portion of the antibody genetically engineered for a low clearance rate and portions of the antibody "humanized" by substitution of human in place of mouse epitopes to reduce antigenicity and clearance by a host immune response(42,43). Ideally an administration of an artificial enzyme against cocaine would last for several weeks and provide the extended coverage important for populations with a record of poor compliance. Third, the efficacy of an artificial enzyme against cocaine relies on a kinetic argument that the rate of cocaine destruction will be able to match the rate of delivery to the CNS. In order to specify the kinetic requirements for the anti-cocaine catalytic antibody, a kinetic model of cocaine delivery is needed. If a dose of smoked crack is absorbed across the lung over the 90-120 second period of one circulation of the intravascular volume (the volume of distribution for the antibody) then an even mixing of cocaine and antibody pools may be assumed. The volume of distribution of cocaine is over twice the total body water(44), but we may neglect this since partitioning of cocaine into other compartments would only decrease on antibody activity. From the moment of cocaine and antibody mixing in the lung, approximately fifteen to twenty seconds elapse before cocaine reaches the CNS capillaries and the most stringent criterion would require complete destruction of cocaine by that time. For a large 100 mg dose (0.36 mmoles) and complete hydrolysis in 15 seconds, the required rate is 0.023 mmol/sec. Thus, the product of the quantity of enzymatic antibody and the antibody's intrinsic enzymatic turnover rate must exceed this value. The assumptions in this model are uniformly conservative and if liberalized would decrease the demand on enzyme performance accordingly. Thus at a monoclonal dose of 200 mg (the monoclonal HA-1A(40) is dosed at 100 mg) the required turnover-rate would be on the order of 2 sec.sup.-1 to 20 sec.sup.-1. Catalytic antibodies have been reported with esterase turnover rates from 20-40 sec.sup.1 and although these estarases were directed toward particularly susceptible target esters, activity of this order of magnitude is possible(20,29). Also, the K.sub.m values for artificial esterases are as low as 2-15 .mu.M(20,31) less than likely pulmonary venous concentrations of cocaine from crack inhalation. We conclude that the kinetic requirements for a clinically useful anti-cocaine catalytic antibody are attainable. An added advantage is that an antibody suitable for the treatment of addiction by the above criteria could be suitable for the treatment of acute overdose. A final concern is the possibility of saturating the enzyme with massive dosages of cocaine. However, the reinforcing effect of cocaine may not be as significant if peak serum levels are reached more gradually and the large dose of crack may be blunted in effect to a weak dose of nasal cocaine hydrochloride. Thus, the protection afforded by an anti-cocaine enzymatic antibody may not need to be complete in order to be useful. SUMMARY OF THE INVENTION The present invention provides a compound having the structure: ##STR2## wherein each of R.sub.1, R.sub.2, R.sub.3, or R.sub.4 is independently hydrogen, or a lower alkyl; or wherein one but only one of R.sub.1, R.sub.2, or R.sub.3 is an azide lower alkyl group, a lower alkyl amine, a group comprising a lower alkyl group linked to a lower alkyl carboxylic acid or derivative, with each of the remaining two of R.sub.1, R.sub.2, or R.sub.3 is independently hydrogen or a lower alkyl and R.sub.4 is hydrogen, a lower alkyl or a negative charge. The present invention provides a compound having the structure: ##STR3## The present invention provides the compound having the structure: ##STR4## This invention also provides the compound having the structure: ##STR5## This invention also provides a compound having the structure: ##STR6## wherein X is a primary amine of a carrier protein. This invention further provides the compound having the structure: ##STR7## wherein X is a primary amine of a carrier protein. This invention also provides the compound having the structure: ##STR8## wherein X is a primary amine of a carrier protein. This invention also provides the compound having the structure: ##STR9## wherein R' is O or CH2 and R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention also provide the compound having the structure: ##STR10## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention also provides the compound having the structure: ##STR11## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention also provides a compound having structure: ##STR12## wherein each of R.sub.1, R.sub.2, R.sub.3 or R.sub.4 is independently hydrogen, or a lower alkyl; or wherein one but only one of R.sub.1, R.sub.2 or R.sub.3 is a lower alkyl azide group, a lower alkyl amine, a group comprising a lower alkyl group linked to a lower alkyl carboxylic acid or derivative, with each of the remaining two of R.sub.1, R.sub.2 or R.sub.3 is independently hydrogen or a lower alkyl and R.sub.4 is hydrogen, a lower alkyl or a negative charge. This invention further provides the above-described compounds linked to a carrier protein. This invention also provides an antibody against the above-described compounds. This invention further provides the genes which are coding for the antibodies against the above-described compounds. This invention also provides a human chimeric antibody and human monoclonal antibody against the above-described compounds. This invention further provides a pharmaceutical composition for decreasing the concentration of cocaine in a subject's blood which comprises an amount of the antibody against the above-described compounds effective to decrease the concentration of cocaine in the subject and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 The kinetic model of the hydrolysis of a caroxylic acid ester. Hydrolysis of the planar ester proceeds through an evanescent tetrahedral intermediate which decomposes to yield alcohol and planar carboxylic acid. The rate of this reaction varies with the magnitude of the difference of the energy states (.DELTA.G) of the starting ester and the peak or transition state structure. The effective catalyst reduces the .DELTA.G of a reaction and thereby increases the rate of reaction. FIG. 2A. Hydrolysis of the benzoyl ester of cocaine. Presumed tetrahedral intermediate formed along the reaction pathway is shown. 2B. General structure of a phosphonate monoester analog of the benzoyl ester. The R substituent for 1a corresponds to the tether depicted in FIG. 3. FIG. 3 Synthesis of transition-state analog 1a. Reagents and conditions: a, I-(CH.sub.2).sub.4 N.sub.3, (CH.sub.3).sub.4 NOH, dimethylformamide (DMF), CH.sub.3 OH 50.degree. C. (92% yield); b, PhP(O)Cl.sub.2, tetrazole (0.1 eq), benzene, diisopropylethylamine room temperature (rt) then MeOH (80% yield); c, P(CH.sub.3).sub.3, tetrahydrofuran (THF)/CH.sub.3 OH/H.sub.2 O (9:9:2) rt (62% yield); d, .sup.14 C-succinic anhydride (2.2 mci/mmol), THF, rt (purified as benzylester, regenerated with H.sub.2 /Pd on C, yield--50%); e, dicyclohexylcarbodiimide, N-hydroxyphthalimide, DMF, rt (85% yield); f, (CH.sub.3).sub.3 SiBr, CDCl.sub.3, rt (unstable,--90% yield); g, bovine serum album (coupling ratio 1:6) or ovalbumin (coupling ratio 1:15). No epimerization was observed at C-2 of the tropane nucleus by 300 MHz .sup.1 H-nmr. FIG. 4 Lineweaver-Burke plot of (1/V) as a function of (1/[S]) for hydrolysis of .sup.3 H.sub.phenyl -cocaine by mAb 3B9 (closed circles) and mAb 6A12 (open circles). Artificial enzyme (2 .mu.M) in phosphate buffered saline was incubated with .sup.3 H-cocaine at five concentrations between 100 .mu.M and 2000 .mu.M. At 10 min intervals, aliquots were acidified with cold HCL (aq) to final pH2, partitioned with CH.sub.2 Cl.sub.2 and the organic phase was assayed by scintillation counting The optimal pH was determined and employed for each enzyme: 3B9 pH7.7 and 6A12 pH7.0. Background hydrolysis was determined in otherwise identical reactions without antibody and observed rates were corrected. Uncatalyzed hydrolysis rates were determined under similar conditions. Assays were performed in triplicate and standard error limits are indicated by brackets. (3B9 r=0.99; 6A12 r=0.98). DETAILED DESCRIPTION OF THE INVENTION The present invention provides a compound having the structure: ##STR13## wherein each of R.sub.1, R.sub.2, R.sub.3, or R.sub.4 is independently hydrogen, or a lower alkyl; or wherein one but only one of R.sub.1, R.sub.2, or R.sub.3 is a lower alkyl azide group, a lower alkyl amine, a group comprising a lower alkyl group linked to a lower alkyl carboxylic acid or derivative, with each of the remaining two of R.sub.1, R.sub.2, or R.sub.3 is independently hydrogen or a lower alkyl and R.sub.4 is hydrogen, a lower alkyl or a negative charge. The invention further provides examples of this compound which include but are not limited to the following: ##STR14## The invention further provides that one but only one of R.sub.1, R.sub.2, or R.sub.3 has the structure: ##STR15## wherein A is a lower alkyl group and X is a primary amine of a carrier protein. The invention provides for a compound having the structure: ##STR16## wherein X is a primary mine of a carrier protein. The invention further provides a method of synthesizing this compound comprising selectively alkylating: ##STR17## with 4-iodo-n-butyl azide, in the presence of tetraethyl ammonium hydroxide, to yield: ##STR18## to which was added sequentially an equivalent of phenylphosphonic dicloride and methanol, in the presence of 1H-tetrazole, to obtain: ##STR19## which was subsequently reduced with trimethyl phosphine in benzene to obtain: ##STR20## which was acylated with succinic anhydride to obtain: ##STR21## which was converted by acylation with N-hydroxyphthalimide in combination with dicyclohexylcarbodiimide to: ##STR22## which was selectively dealkylated to: ##STR23## which was coupled to the primary amine of a carrier protein. The invention provides for a compound having the structure: ##STR24## wherein X is a primary amine of a carrier protein. The invention provides a method of synthesizing this compound comprising starting with the structure: ##STR25## with this acid esterified with acidic methanol and reduced with Dibal to the corresponding alcohol. The alcohol was protected with t-butyldimethylsilyl chloride under imidazole catalysis to yield A: ##STR26## from the starting alcohol. This was transmetalated with n-butyl lithium to the following Lithium B: ##STR27## and this structure was phosphorylated with diethylchlorophosphate to yield C: ##STR28## the silyl group of (C) was removed with tetra-n-butylammonium fluoride to yield the corresponding alcohol in 62% yield from (B). This alcohol was transformed to the bromide via tosylate; the phosphonate ester was converted from ethyl to methyl via bromotrimethylsilane followed by methanol; the bromide was displaced by azide; and finally the phosphonate ester was transformed to the phosphorylchloride with the following structure D: ##STR29## This was in an approximately 30% yield. Using the tetrazole catalysis method, this structure D was coupled with methyl ecgonine: ##STR30## followed by methanol to yield the mixed diester E in 30% yield after column chromatography, having the following structure: ##STR31## The azide of E was reduced to the corresponding amine with triphenylphosphine and coupled to .sup.14 C labeled succinic anhydride. The resulting acid was converted to its benzyl ester to facilitate column chromatography in 65% yield from E. The benzoyl ester was removed by catalytic hydrogenation, activated by DCC esterification with N-hydroxyphthalimide. Finally, the phosphonate was demethylated with bromotrimethylsilane and the product used directly for coupling to carrier proteins including bovine serum albumin or ovalbumin. The invention provides for a compound having the structure: ##STR32## wherein X is a primary amine of a carrier protein. This invention also provides a compound having the structure: ##STR33## wherein R is a ##STR34## or other aromatic substitute. This invention further provides a compound having the structure: ##STR35## wherein R' is O or CH2 and R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention also provides a compound having the structure: ##STR36## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention provides a compound having the structure: ##STR37## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention provides a compound having the structure: ##STR38## wherein R is a hydrocarbon chain or a series of hydrocarbon linked by amide, ester or other functional group, capable of linking to a carrier protein. This invention provides a compound having structure: ##STR39## wherein each of R.sub.1, R.sub.2, R.sub.3 or R.sub.4 is independently hydrogen, or a lower alkyl; or wherein one but only one of R.sub.1, R.sub.2 or R.sub.3 is a lower alkyl azide group, a lower alkyl amine, a group comprising a lower alkyl group linked to a lower alkyl carboxylic acid or derivative, with each of the remaining two of R.sub.1, R.sub.2 or R.sub.3 is independently hydrogen or a lower alkyl and R.sub.4 is hydrogen, a lower alkyl or a negative charge. This invention also provides the above-described compounds linked to a carrier protein wherein the carrier protein is bovine serum albumin, bovine serum ovalbumin, keyhole limpet hemocyanin or thyroglobulin. As stated herein, carrier proteins are well-known to an ordinary skilled artisan. Any protein which may help to facilitate to induce an immune response are meant to be covered by this invention. Typical carrier proteins are stated in the above such as bovine serum albumin. This invention provides antibodies against the above-described compound. One utility of these antibodies is to detect the intermediates of cocaine formed in a subject. The other utility of these antibodies is to serve as starting materials for generation of high affinity antibodies for pharmaceutical uses. This invention further provides antibodies against the above-described compounds, which upon binding to an intermediate of the hydrolysis transition-site of a cocaine benzoyl ester group decreases the .DELTA.G of the hydrolysis reaction. Generally, an antibody comprises two molecules, each molecule having two different polypeptides, the shorter of which is the light chain and the longer is the heavy chain. A fragment of a naturally occuring or recombinant antibody molecule is encompassed within the scope of this invention. A Fab protein or a F(ab)' protein which exhibits innunoreactive activity is part of this invention. Methods to generate antibodies against chemical compounds are well-known to a person of ordinary skill in the art. One method is to link the compound to a carrier protein and immunize animal with such a linked compound. Sera from the animals may then be tested for the antibody produced against the compound. This invention further provides monoclonal antibody against the above-described compounds. Methods to generate monoclonal antibodies are well-known to an ordinary skilled artisan. In an embodiment, the monoclonal antibody is produced by a hybridoma, 3B9, having ATCC Accession No. HB 11313. This hybridoma call line, 3B9 was deposited on Mar. 31, 1993 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. The hybridoma call line 3B9 was accorded ATCC Accession number HB 11313. In another embodiment, the monoclonal antibody is produced by a hybridoma, 6A12, having ATCC Accession No. HB 11314. This hybridoma cell line, 6A12 was deposited on Mar. 31, 1993 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent Procedure. The hybridoma call line 6A12 was accorded ATCC Accession number HB 11314. As an alternative method to generate the desirable antibody, genes which code for the heavy chain and light chain of the antibody may be isolated. Both genes may be co-expressed in an host vector system to produce the desirable antibody. Standard methods are available in the art to obtain gene coding for the heavy and light chain of a monoclonal antibody. This invention further provides an isolated nucleic acid molecule encoding the light chain protein of the monoclonal antibody against the above-described compounds. In an embodiment, the isolated nucleic acid molecule is DNA. In another embodiment, the isolated nucleic acid molecule is cDNA. This invention further provides an isolated nucleic acid molecule encoding the heavy chain protein of the monoclonal antibody against the above-described compounds. In an embodiment of this isolated nucleic acid molecule encoding the heavy chain protein of the monoclonal antibody, the molecule is DNA. In a further embodiment, it is a cDNA. This invention further provides a vector comprising the isolated nucleic acid molecule encoding the light chain protein of the monoclonal antibody operably linked to a promoter of RNA transcription. This invention also provides a vector comprising the nucleic acid molecule encoding the heavy chain protein of the monoclonal antibody operably linked to a promoter of RNA transcription. This invention also provides a host vector system comprising the above-described vectors in a suitable host cell. The suitable host of this host vector system may be a bacterial call, insect cell, or animal cell. There are other suitable host cell known in the art such as bacterial cells (such as E.coli), yeast cells, fungal cells, insect cells and animal cells. Suitable animal cells include, but are not limited to Vero cells, HeLa cells, Cos cells, CV1 cells and various primary mammalian cells. The affinity of a specific antibody may be improved by changing the amino acid residue of the antibody molecule. Site-directed mutagenesis may be performed to achieve that. In addition, improvement of the affinity of the antibody may be achieved by generating composite antibody consisting of the heavy chain of the anti-analog antibody and a metal-binding light chain. The affinity of new monoclonal antibody generated may be examined and the monoclonal antibody with batter affinity will be selected. This invention further provides a human chimeric antibody against the above-described compound. There are known standard methods to produce such human chimeric antibody. One approach is to link the variable region of the monoclonal antibody with the human Fc region. This invention further provides human monoclonal antibodies. Methods to make human monoclonal antibodies are known in the art. This invention also provides a pharmaceutical composition for decreasing the concentration of cocaine in a subject's blood which comprises an amount of the above-described antibody effective to decrease the concentration of cocaine in the subject and a pharmaceutically acceptable carrier. In an embodiment of the pharmaceutical composition, the antibody is a human chimeric antibody. For the purposes of this invention "pharmaceutically acceptable carrier" means any of the standard pharmaceutical carrier. Examples of suitable carriers are well known in the art and may include, but not limited to, any of the standard pharmaceutical vehicles such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents. The invention further provides that the carrier protein is bovine serum albumin, bovine serum ovalbumin, keyhole limpet hemocyanin or thyroglobulin. The invention further provides antibodies which upon binding to an intermediate of the hydrolysis transition-site of a cocaine benzoyl ester group decreases the .DELTA.G of the hydrolysis reaction. Preferably the antibody is a monoclonal antibody. The invention further provides a method of decreasing the concentration of cocaine in a subject's blood which comprises administering to the subject an amount of an antibody effective to catalyze hydrolysis of cocaine and thereby reduce the concentration of cocaine in the subject's blood. Preferably the antibody is administered intravenously, yet it is speculated that it can be administered intramuscularly. This invention provides a pharmaceutical composition for treating cocaine overdose in a subject which comprises an amount of at least one of the above-described antibodies effective to decrease the concentration of cocaine in the subject and a pharmaceutically acceptable carrier. This invention further provides a method for treating cocaine overdose in a subject which comprises administering to the subject an amount of at least one of the above-described antibodies effective to catalyze hydrolysis of cocaine and thereby reduce cocaine overdose in the subject. This invention provides a pharmaceutical composition for treating cocaine addiction in a subject by diminishing an achievable concentration of cocaine which comprises an amount of at least one of the above-described antibodies effective to diminish the achievable concentration of cocaine in the subject. This invention provides a method for treating cocaine addiction in a subject by diminishing the achievable concentration of cocaine which comprises administering to the subject an amount of at least one of the above-described antibodies effective to catalyze the hydrolysis of cocaine and thereby diminish the achievable concentration of cocaine in the subject. This invention further provides a method of identifying an antibody with hydrolytic activity against the benzoyl ester linkage of cocaine which comprises (a) contacting the antibody with radioactive cocaine labelled at the benzoyl group in a reaction mixture under conditions permitting the release of the radioactively labelled benzoyl group; (b) separating the released radioactively labelled benzoyl group from the radioactive cocaine; (c) determining the radioactivity of the released benzoly group; and (d) comparing the radioactivity determined at step (c) with the radioactivity released in a reaction mixture where no antibody is added, the higher radioactivity at step c indicating the hydrolytic activity of the antibody against the benzoyl ester linkage of cocaine. In an embodiment, step (b) comprises acidifying the reaction mixture to an extend that the released radioactively labelled benzoly group from the cocaine will be extracted into the organic phase and the cocaine will be in the aqueous phase and extracting the mixture with an organic solvent, thereby separating the released radioactively labelled benzoly group into the organic solvent. Finally, this invention provides a method of determining the specificity of an antibody with hydrolytic activity against the benzoyl ester linkage of cocaine to an analog which comprises: (a) contacting an antibody with the analog to the hydrolysis transition-state of the cocaine benzoyl ester group in a reaction mixture under conditions permitting the binding of antibody and the analog; (b) adding cocaine radioactively labelled at the benzoly group into the reaction mixture and modifying the conditions to permit the release of the radioactively labelled benzoly groupseparating the released radioactively labelled benzoly group from the cocaine, if the conditions of step (a) do not permit the release; (c) determining the radioactivity of the released benzoly group; and (d) comparing the radioactivity determined at step (c) with the radioactivity released in a reaction mixture where no antibody is added, a similar radioactivity indicating that the antibody is specific to the analog. |
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