Nicotine haptens, immunoconjugates and their uses
A technology of immunoconjugates and haptens, which is applied in the field of immunoconjugates and addictive substances, and can solve the problem that the long-term success rate is less than 20%.
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Embodiment 1
[0177] Example 1 Starting materials, compound synthesis and schemes
[0178] raw material. Unless otherwise noted, all reactions were performed under an inert atmosphere using dry reagents, solvents, and flame-dried glassware. Both (-)-nicotine and (-)-cotinine were purchased from Sigma-Aldrich (St. Louis, MO). Trans-3'-hydroxymethylnicotine was purchased from Toronto Research Chemicals Inc. (NorthNork, ON). All other chemicals were purchased from large suppliers and used without further purification. Compounds were purified by reverse preparative high performance liquid chromatography (HPLC) (Grace, Vydac 218TP C 18 10-15μm). All compounds were characterized using a Bruker 500 MHz NMR instrument and an Agilent LC-MS (ESI) mass spectrometer.
[0179] Nicotine hapten. Racemic NIC nicotine haptens were prepared by reaction between nornicotine and appropriate linker fragments as previously described (Scheme 1). The AM1 nicotine hapten was synthesized according to Scheme ...
Embodiment 2
[0189] Example 2 Synthesis of Nicotine Hapten AM1 and Immunoconjugates
[0190] AM1 is along figure 1 3' of the general formula shown - substituted unconstrained hapten. Compared to those haptens proposed by Langone and Pentel (Langone et., Biochemistry 12:5025-5030, 1973; and Hieda et al., Int J Immunopharmacol 22:809-819, 2000), AM1 has a hapten design Advantage. The presence of unstable ester bonds in the Langone-designed hapten would cause the antigen to spontaneously fall off from the carrier protein, thereby losing the anti-nicotine immunogenicity; while such a design would not hinder the production of monoclonal antibodies, it would seriously Affect the potency of live vaccines. Spontaneous ester hydrolysis under physiological conditions is a known phenomenon, and the increase in hapten stabilization by ester-amide exchange has been successfully studied previously for the immunopharmaceutical effects of cocaine (Carrera el al., Proc Natl Acad. Sci 98; 1988-1992, 2...
Embodiment 3
[0194] Example 3 Immunogenicity of Immunoconjugates Based on Nicotine Hapten AM1
[0195] This example describes the immunogenicity of nicotine hapten immunoconjugates and the generation of antibodies in mice. In an effort to elucidate the optimal vehicle for delivery of immunizations, the AM1 hapten was coupled to three different carrier proteins, namely, KLH, TT and CRM. The efficacy of all AM1 hapten-protein immunoconjugates was achieved by vaccination up to 129GI using standard immunization protocols x In vivo evaluation in mice. Specifically, 100 μg of the hapten conjugate was mixed with the desired adjuvant and immediately injected into mice (ip administration, n = 4 per hapten conjugate group). Three test groups were included, AM1-KLH, AM1-TT and AM1-CRM, plus three vehicle-only control groups. In all cases, the vaccines were formulated with AS-03, an emulsion-based adjuvant patented by GlaxoSmithKline. The immunization schedule was set as follows: injections were...
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