Formation of site and conformation specific antibodies to centchroman, a new non-steroidal contraceptive drug

Abstract

Centchroman [Ormeloxi fene, 3,4<i>-trans-</i>2,<i> </i>2-dimethyl-3-phenyl-4-(<i>p</i>-pyrrolidino ethoxy)phenyl-7 -methoxy-chroman (2a)] is a new non steroidal oral contraceptive for women. In an attempt to develop sensitive immunoassay to measure centchroman concentration in biological fluids, we describe production of antibodies against four hapten derivatives of centchroman, and evaluation of efficacy of these antibodies in developing sensitive and specific immunoassay of centchroman. Antigens were made by coupling four hapten derivatives, 3,4-<i>trans</i>-2,2-dimethyl-3-phenyl-4-(<i>p</i>-pyrrolidinoethoxy) phenyl-7-carboxymethoxyl-chroman (2c), 3,4-<i>trans</i>-2,2-dimethyl-3-phenyl-4-(<i>p</i>-succinoylethoxy)phenyl-7-methoxychroman (3a), 3, 4<i>-trans- </i>2,2-dimethyl-3-(<i>m</i>-hydroxyl)phenyl-4-(<i>p</i>-pyrro-lidinoethoxy)phenyl-7-methoxychroman

(5a), and 3,4-<i>trans</i>-2,2-dimethyl-3 -phenyl-4-(3'-amino,4' -pyrrolidinoethoxy) phenyl-7-methoxy-chroman (6a), to bovine serum albumin (BSA). Three enzyme tracers, made by coupling centchroman derivatives (2c, 3a, and 5a) to peroxidase (HRP), were utilized to monitor four antisera. Four homologous and heterologous enzyme immunoassay formats were developed and specificity of each antiserum was analyzed against anticipated metabolite of centchroman. Antiserum raised against centchroman derivative (6a) was found to be most specific, showing <4% crossreactivity with a putative metabolite, 7-desmethyl centchroman (2b). This antiserum, when used in solid phase EIA format, exhibited sensitivity of 250<i> </i>pg/ml , total assay variance of CV < 14%, and analytical recoveries between 82-106%. Comparison of the EIA with a RIA, which was developed by using same antibody, showed a close correlation (r=0.9, n=40). Specificity of all antisera, as determined by the respective enzyme immunoassays, followed an uncharacteristic pattern of crossreactivity towards <i>cis</i> centchroman. Especially, antiserum generatted against an immunogen (5b)<i> </i>showed high crossreaction with <i>cis </i>centchroman. Evidence provided in this study showed that immunogen (5b)<i> </i>produced a sub population of antibodies to a flipped conformation of the drug, which conformationally looks similar to <i>cis </i>centchroman. These conformation specific antibodies

accounted for high crossreactivity towards <i>cis </i>centchroman. In conclusion, we describe the formation of conformation specific antibodies and the significance of site of attachment of centchroman derivative to carrier protein on the specificity of antibodies towards anticipated metabolites. Furthermore, by using antiserum generated in this study, sensitive immunoassays were developed and validated for the measurement of centchroman in serum.