COCAINE HAPTENS
Description
Background:
Despite intensive efforts, the development of effective therapies for ***e craving and addiction remain elusive. An improved pharmacotherapy would increase the effectiveness of rehabilitative programs. One approach to the development of therapies for *** addiction has relied on immunological reagents and the immune system. It has been shown that the antibody-mediated binding of ***e impeded passage of the drug into the central nervous system that resulted in a suppression of its characteristic actions (Carrera, M. R. A.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6202; Carrera, M.; et al. Nature 1995, 378, 727; Fox, B. S.; et al. Nat. Med. 1996, 2, 1129). Administration of a monoclonal antibody (mAb) endowed with not only binding, but also catalytic activity to metabolize ***e, would have enhanced therapeutic effects if the kinetic properties of the mAb were sufficient.
Catalytic antibodies have emerged as a powerful tool at the interface of chemistry and biology (Wentworth, Jr., P.; Janda, K. D. Curr. Opin. Chem. Biol. 1998, 2, 138; Schultz, P. G.; Lerner, R. A. Science 1995, 269, 1835). In this regard, the hallmark reaction catalyzed by antibodies is ester hydrolysis. Since cleavage of the benzoate ester of ***e 1 produces the nonpsychoactive metabolite ecgonine methyl ester 2(Misra, A. L; et al. J. Pharm. Pharmacol. 1975, 27, 784), it is an excellent target for an immunopharmacological strategy (Figure 1 ).
Landry and co-workers used a transition-state (TS) analog approach for hapten design and reported several ***e-hydrolyzing mAbs (Yang, G. X.-Q.; et al. J. Am. Chem. Soc. 1996, 118, 5881 ; Landry, D. W.; et al. Science 1993, 259, 1899). In this model, the benzoyl ester of the ***e framework is replaced by a phenylphosphonate that approximates the TS for ester hydrolysis (Figure 2).
Subsequently, other workers also used a phosphonate analog to obtain hybridomas which were subjected to high-throughput screening using ***e benzoyl thioester (Cashman, J. R.; et al. J. Pharmacol. Exp. Ther. 2000, 293, 952).
More than 10 years ago, the TS design was applied by the present investigators to the preparation of phosphonate based ***e haptens. The first two structures founded on this principle were 3 (code named GNP) and 4 (code named GNN) in which the site of a linker attachment for coupling to carrier proteins was different. Yet, despite screening nearly 1000 clones, no mAbs with catalytic activity above the background rate were discovered. In an effort to elicit a ***e esterase that will have utility for human use, the present investigators continue to examine TS-analog designs, as well as other approaches. Herein, it is reported that a specific change in the linker composition of 3 and 4 is critical for obtaining ***e catalytic mAbs, which provides a foundation for further advances.
Summary of Invention:
One aspect of the invention is directed to a ***e hapten represented by the following structure:
In the above structure, m and n are integers such that 0<m≤5 and 1 <n≤3. A preferred ***e hapten is represented by the following structure:
Another aspect of the invention is directed to a process for eliciting a catalytic antibody from an immune responsive animal, the catalytic antibody having a catalytic activity for converting ***e to ecgonine methyl ester by
hydrolysis. The process comprises the step of immunizing the immune responsive animal with a sufficient quantity of an immunogen for eliciting an immune response, the immunogen having a hapten represented by the following radical:
In the above structure, m and n are integers such that 0≤m≤5 and 1 <n<3. In a preferred mode, the hapten is represented by the following radical:
Another aspect of the invention is directed to a catalytic antibody having a catalytic activity for converting ***e to ecgonine methyl ester by hydrolysis. The catalytic antibody is of a type that is isolated from an immune responsive animal that has been immunized with a quantity of an immunogen sufficient to elicit an immune response. More particularly, the immunogen has a hapten represented by the following radical:
In the above structure, m and n are integers such that 0<m≤5 and 1≤n≤3. A preferred hapten is represented by the following radical:
Another aspect of the invention is directed to a process for obtaining a catalytic monoclonal antibody having a catalytic activity for converting ***e to
ecgonine methyl ester by hydrolysis. The process comprises the following steps: Firstly, an immune responsive animal is immunized with an immunogen having a hapten represented by the following radical:
In the above structure, m and n are integers such that 0≤m<5 and 1 ≤n≤3. In a preferred mode, the hapten is represented by the following radical:
Secondly, antibody producing cells that express catalytic antibody having a catalytic activity for converting ***e to ecgonine methyl ester by hydrolysis are isolated from the immune responsive animal. Thirdly, the antibody producing cells are cloned and the catalytic monoclonal antibody is isolated therefrom.
Another aspect of the invention is directed to a catalytic monoclonal antibody having a catalytic activity for converting ***e to ecgonine methyl ester by hydrolysis. The catalytic monoclonal antibody is of a type that is isolated from an antibody producing cell obtained by the following process. Firstly, an immune responsive animal is immunized with an immunogen having a hapten represented by the following radical:
In the above structure, m and n are integers such that 0<m<5 and 1 ≤n<3. In a preferred embodiment, the hapten is represented by the following radical:
Secondly, antibody producing cells that express catalytic antibody having a catalytic activity for converting ***e to ecgonine methyl ester by hydrolysis are isolated from the immune responsive animal. Thirdly, the antibody producing cells are cloned and the catalytic monoclonal antibody are isolated therefrom.
Another aspect of the invention is directed to a process for converting ***e to ecgonine methyl ester by hydrolysis. The process comprises the following steps. Firstly, an immune responsive animal is immunized with a quantity of an immunogen sufficient to elicit an immune response, the immunogen having a hapten represented by the following radical:
In the above structure, m and n are integers such that 0≤m≤5 and 1 ≤n≤3. In a preferred mode, the hapten is represented by the following radical:
Secondly, catalytic antibody is isolated from the immune responsive animal, the catalytic antibody having a catalytic activity for converting ***e to ecgonine methyl ester by hydrolysis. Thirdly, the ***e is contacted twith a concentration of the catalytic antibody sufficient to catalyze conversion of the ***e by hydrolysis to ecgonine methyl ester.
In an alternative mode of this aspect of the invention, a process for converting ***e to ecgonine methyl ester by hydrolysis comprises the following
steps. Firstly, an immune responsive animal is immunized with an immunogen having a hapten represented by the following radical:
In the above structure, m and n are integers such that 0<m<5 and 1 ≤n≤3. In a preferred mode of this aspect of the invention, the hapten is represented by the following radical:
Secondly, antibody producing cells are isolated from the immune responsive animal that express catalytic antibody having a catalytic activity for converting ***e to ecgonine methyl ester by hydrolysis. Thirdly, the antibody producing cells are cloned and catalytic monoclonal antibody is isolated therefrom. Fourthly, ***e is contacted with a concentration of the catalytic monoclonal antibody sufficient to catalyze a conversion of the ***e to ecgonine methyl ester by hydrolysis.
Brief Description of Drawings:
Figure 1 illustrates the antibody-catalyzed hydrolysis of ***e.
Figure 2 illustrates the principle of TS stabilization and haptens based on this concept. Figure 3 illustrates a scheme for the synthesis of hapten 9 GNL.
Figure 4 is a scheme illustrating the synthetic steps used in the synthesis of hapten 13 GNK.
Figure 5 is a scheme illustrating the synthetic steps used in the synthesis of hapten 17 GNJ. Figure 6 is a table illustrating the mAbs screened for the hydrolysis of ***e by following the release of benzoic acid using HPLC. Measurements were determined in 100 mM phosphate buffer, pH 7.4, 21 °C.
Detailed Description:
Having tested a number of other modified phosphonate TS structures related to 3 without success, the present investigators decided to make a simple change in the linker of the hapten. A β-alanyl unit was appended at the linker terminus that afforded the GNL hapten 9, according to the synthetic method disclosed in Figure 3.
Mice were immunized with a GNL-KLH conjugate (immunogen) and the resultant mAbs were screened for the hydrolysis of ***e by following the release of benzoic acid using HPLC. Remarkably, catalysis was detected in -25% of the total mAbs tested (>3-fold over background in the initial rate; 20 μM mAb, 500 μM ***e), of which several were considered to have good activity (Figure 6). Significantly, as facilitated by the low Km value, the most efficient mAb, GNL3A6, was able to completely degrade all offered ***e (20 μM mAb, 900 μM ***e). Notably, the Km is the lowest reported to date for any ***e catalytic mAb at physiological pH. A low value for this parameter is an essential contributor to a high kcJKm, the apparent second-order rate constant for the reaction of antibody and ***e, that dictates mAb catalytic power.
The mAbs disclosed herein are similar in activity to all mAbs reported by Landry, except one, in which / cat was ~60-fold better than GNL23A6 and kcat/Km ~19-fold better than GNL3A6 (Yang, G. X.-Q.; et al. J. Am. Chem. Soc. 1996, 118, 5881 ). However, the conditions for the Landry mAbs were optimized, which required an increase to pH 8. Cashman et al. reported the most efficient mAb (k Km ~ 103 M"1 s"1) (Cashman, J. R.; et al. J. Pharmacol. Exp. Ther. 2000, 293, 952), however this was at pH 8.4 and 37 °C, so the value under the conditions herein would likely be reduced ~10-fold. What the results demonstrate is that, despite efforts by three laboratories involving numerous mAbs and methods, efficient clones are rare and new approaches will be required.
From the standpoint of the ***e hydrolysis problem, but also catalytic antibody technology in general, the effect incurred through a subtle change in the linker was of great interest. Based on the experience of the present investigators
with hapten designs for a variety of hydrolytic reactions, the linker lengths in 3 and 4 should be adequate to allow recognition of the ***e framework, and certainly the phosphonate moiety. However, since the β-alanyl fragment not only introduced a new amide functionality, but also increased the linker length, it was necessary to separate these characteristics and determine which contributed to the efficacy of 9. The hapten 13 (GNK) was synthesized in which the linker is an alkyl linker as in 3, but of the same length as in 9 (Figure 4).
Only one mAb from a panel of 19 clones derived from GNK-KLH was found with a significant rate above background (Figure 6). Even though the activity was low, the one clone and its catalysis was more than previously observed for GNP mAbs. Hence, the longer linker length possibly promotes some elicitation of catalytic activity. However, the internal amide bond seems principally responsible for the linker-directed effects that led to a "switching on" of an immune response that resulted in catalytic mAbs. In order to provide a positive internal control and further support for the hypothesis, a new β-alanyl linker was introduced at the nitrogen atom as in 4 to give the GNJ hapten 17 (Figure 5).
With GNJ-KLH three catalysts were found out of 24 tested (12.5%), fewer than with GNL-KLH. In addition, the best mAb, GNJ14G12, was less efficient than the GNL mAbs (Figure 6). But again, this single panel of mAbs, derived from one fusion to produce a set of hybridomas, contained several catalysts, where before the GNN hapten yielded nothing from many fusions and a large survey of candidates.
It is disclosed herein that an amide linkage allows for more favorable hapten-peptide fragment presentation by MHC II and/or recognition by the T-cell receptor. Perhaps more tangible, the hydrogen bonding of the linker amide bond at the antibody binding site of B-cell surface immunoglobulin seems to elicit amino acid residues for chemical catalysis akin to the principle of "bait and switch" (Lavey, B. J.; Janda, K. D. In Antibody Expression and Engineering; Wang, H. Y.; Imanaka, T., Eds.; ACS Symposium Series 604, 1995; Chapter 10). Notably, the
haptens of Landry et al. contain an amide-based linker, in which an amino terminus is capped with a succinyl unit, and their work has shown that mAb catalytic activity exceeds that expected from a correlation based only on TS stabilization (Yang, G. X.-Q.; et al. J. Am. Chem. Soc. 1996, 118, 5881 ).
Both spontaneous (Garrett, E. R.; Seyda, K. J. Pharm. Sci. 1983, 72, 258; Stewart, D. J.; et al. Clin. Pharmacol. Therapeut. 1979, 25, 464; Cunningham, K. A.; Lakoski, J. M. Neuropsychopharmacology 1990, 3, 41 ; Li, P.; et al. Helv. Chim. Ada 1999, 82, 85) and esterase-catalyzed (Stewart, D. J.; et al. Clin. Pharmacol. Therapeut. 1979, 25, 464; Brzezinski, M. R.; et al. Biochem. Pharmacol. 1994, 48, 1747; Boyer, C. S.; Petersen, D. R. J. Pharmacol. Exp. Then 1992, 260, 939; Matsubara, K.; et al. Forensic Sci. Intl. 1984, 26, 169) hydrolysis of ***e contribute to the short in vivo half-life of -30 minutes in human blood, comparable to that determined in the laboratory of the present investigators in rats (Carrera, M.; et al. Nature 1995, 378, 727). Yet, for an enzyme or catalytic antibody therapy to be effective, extensive clearance of ***e must take place within seconds. The administration of purified human plasma cholinesterase reduced ***e toxicity in mice (Hoffman, R. S.; et al. Clin. Toxicol. 1996, 34, 259). However, the catalytic power of this enzyme is not sufficient to achieve ***e clearance in the human condition at clinically manageable concentrations of enzyme (Xie, W.; et al. Mol. Pharmacol. 1999, 55, 83). Landry et al. also reported some positive results in animal models using their best catalytic mAb (Mets, B.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10176). However, high catalytic power is required to meet the demands of hydrolyzing ***e rapidly enough to alter its pharmacokinetic profile and psychoactive effects in the human condition.
An estimate can be made as to the requirements of an anti-***e catalytic mAb during a period of rehabilitation from ***e abuse. It is disclosed herein that an administered catalytic mAb, "humanized" or even "fully human" to minimize an immune response (James, K. In Handbook of Experimental Pharmacology : The Pharmacology of Monoclonal Antibodies; Rosenberg, M.; Moore, G. P., Eds.; Springer-Verlag: New York, 1994; Vol. 113, pp 3-19; Burton,
D. R.; Barbas, C. F. III. Adv. Immunol. 1994, 57, 191), that is circulating at a practical, long-term clinical level of -1 mg/mL (-15 μM in active sites for whole IgG) must have a minimum kcJKm - 104 M"1 s"1. A mAb operating with this rate constant affords sufficient clearance of a typical single dose of circulating ***e (-10 μM) from the bloodstream within a few seconds before transit into the brain. This activity is in the range of the esterase family of enzymes studied using various ester substrates, other than ***e, which again is indicative of the recalcitrant nature of ***e as a substrate and for hapten programming.
Detailed Description of Figures:
Figure 1 shows the antibody-catalyzed hydrolysis of ***e. The products of this reaction are benzoic acid and the non-psychoactive methyl ecgonine ester 2.
Figure 2 shows the principle of TS stabilization and haptens based on this concept.
Figure 3 shows the scheme used for the synthesis of hapten 9 GNL. Reagents and conditions: (a) 1.25 M HCI, reflux; (b) 2-trimethylsilylethyl- 6-bromohexanoate, NaOH, pyridine, 80 °C; (c) (i) LDA, (ii) 11 ; (d) (i) TFA, (ii) β-alanine benzyl ester, EDC, HOBt; (e) H2, Pd/C; (f) benzyl alcohol, NEt3; (g) PCI5, CHCl3, 40 °C.
Figure 4 is a scheme showing the synthetic steps used in the synthesis of hapten 13 GNK. Reagents and conditions: (a) benzyl 10-bromodecanoate, Bu4NOH, Bu4NI, DMF; (b) (i) LDA, (ii) 11; (c) H2, Pd/C.
Figure 5 is a scheme showing the synthetic steps used in the synthesis of hapten 17 GNJ. Reagents and conditions: (a) HCI, MeOH; (b) (i) LDA, (ii) 11; (c) (i) Troc-CI, NEt3, (ii) Zn, formic acid; (d) f-butyl 6-bromohexanoate, NEt3, CH3CN; (e) (i) TFA, (ii) β-alanine benzyl ester, EDC, HOBt; (iii) H2, Pd/C.
Figure 6 is a table showing the mAbs screened for the hydrolysis of ***e by following the release of benzoic acid using HPLC. Remarkably, catalysis was detected in -25% of the total mAbs tested (>3-fold over background in the initial rate; 20 μM mAb, 500 μM ***e), of which several were considered to have good activity (Table 1). Significantly, as facilitated by the low Km value,
the most efficient mAb, GNL3A6, was able to completely degrade all offered ***e (20 μM mAb, 900 μM ***e). Notably, the Km is the lowest reported to date for any ***e catalytic mAb at physiological pH. A low value for this parameter is an essential contributor to a high kc Km, the apparent second-order rate constant for the reaction of antibody and ***e, that dictates mAb catalytic power.