Enzyme immunoassay for nomegestrol acetate in human plasma.

Currently available chromatographic assays of the progestative drug nomegestrol acetate in human plasma are not suitable for monitoring drug kinetics more than 24 h after clinical dosage. A specific and sensitive enzyme immunoassay was therefore developed. A 3(O-carboxymethyl)oxime derivative of nomegestrol acetate was synthesized and coupled to bovine serum albumin in order to raise polyclonal antibodies in rabbits. The enzymatic tracer was obtained by coupling the 3(O-carboxymethyl)oxime derivative to acetylcholinesterase (E.C.3.1.1.7.). HPLC fractionation of human plasma samples followed by enzyme immunoassay revealed the presence of cross-reacting metabolites. An automated procedure of metabolite separation was developed using silica bonded with diol groups (Diol Bakerbond column). This procedure ensured assay specificity. The quantification limit in human plasma was 0.1 ng/ml. Mean repeatability (intra-assay variation) and reproducibility (inter-assay variation) were 9 and 15%, respectively. The enzyme immunoassay allowed monitoring of the kinetics of nomegestrol acetate 144 h after oral administration of a single 5 mg dose. Values for human samples were in excellent agreement with those assayable by HPLC followed by u.v. detection.

Properties of biospecific adsorbents, obtained by immobilization of oestradiol 7 alpha-derivatives, for purification of calf-uterine cytosol oestradiol receptor.

The properties of three types of adsorbents obtained by coupling oestradiol 7 alpha-derivatives to agarose were compared. The adsorbents examined were: oestradiol 7 alpha-decamethylene-agarose, oestradiol 7 alpha-decamethylene-poly(anayl-lysine)-agarose and oestradiol 7 alpha-trimethylene-poly(alanyl-lysine)-agarose. The following results were obtained. (1) All these adsorbents are stable at 0 degrees C for a least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximala receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin-Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is possible only with biospecific adsorbents containing low concentrations of coupled steroid (less than or equal to 0.2 mg/ml). The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr 60000). When starting from a low salt preparation containing the native 8-S receptor, partially purified by heparin-Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250--500-fold and led to a 50--90% pure protein (assuming one oestradiol binding site per molecule of Mr 70000).

New biospecific adsorbents for the purification of estradiol receptor.

The synthesis of biospecific adsorbents for the purification of the cytosol estrogen receptor from calf uterus is described. The characteristic of several estradiol derivatives, spacer chains, and insoluble matrix were systematically studied. Estradiol derivatives substituted at positions C2, 3, 4 7 alpha, 17 alpha, and 17 beta were tested for their affinities for the receptor; positions 7 alpha and 17 alpha were the most suibable. Acidic compounds had lower affinities than their methylester analogues. Long chain derivatives bound the receptor less firmly than corresponding shorter chains. However, when these ligands were attached to an insoluble matrix, the long spacer chain derivatives (greater than or equal to 14 atoms) were more efficient than the shorter ones. There was a satisfactory parallelism between affinities of free ligands and receptor binding to the respective biospecific adsorbents. On the basis of their stability in the presence of cytosol (no release of ligand), due to the absence of ester bonds, long chains were selected as spacers. Both acrylamide and agarose biospecific adsorbents displayed some ionic exchange capacity and consequently nonspecifically bound proteins; the influence of this nonspecific binding on the purification of the receptor was studied. On the basis of their stability, of their binding specificity, and of their selectivity for the receptor, the estradiol-7 alpha derivative adsorbents were selected for the purification of the receptor.

[Unexpected antibiotic properties of a cephalosporin]. / Propriétés antibiotiques inattendues dans le domaine des céphalosphorines.

The presence of an oximino group on the methylene of the (2-aminothiazol 4-yl)-acetyl side chain bound to the 7-amino cephalosporanic acid affords derivatives with syn or anti configuration. Whereas the anti isomers display low antibiotic activities, the syn isomers possess a surprising high efficiency against gram negative bacteria, enhanced a factor of 10 to 100 as compared with the cephalosporins known at the moment.

[Radioimmunoassay of synthetic steroids]. / Radioimmunoessai des stéroïdes de synthèse

The sensitivity of a radioimmunoassay depends on the intrinsic association constant of the interaction between ligand and antibody. Its specificity depends on the position of the chain which forms the link with the antigen. Thus, an antibody specific of estradiol has been obtained by coupling estradiol to albumin via a chain at position 7. For synthetic steroids the structure of which is sufficiency different from that of natural hormones, the requirements for a sensitive assay method not involving chromatography are simply maximum affinity and positioning of the couple at a site which does not undergo metabolic attack. These criteria were used to develop assays for R 2858 and R 2453 which obviate the need to administer radioactive product in clinical pharmacology. Cross-reaction with structural analogs may be used to assay competitors. Thus, R 2323 antibody, highly specific for endogenous steroids, may be used to assay other trienes such as R 1697 (trenbolone) and R 2010 (norgestrienone).