High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus.

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Remote, semiautomated production of 6-[18F]fluoro-L-dopa for human studies with PET.

Regioselective radiofluorodemercuration of the 6-mercurio derivative 5 with [18F]acetylhypofluorite afforded, after acidic hydrolysis, 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-FD, 1) with a radiochemical yield of 11% (decay corrected and based on the total amount of [18F]F2 recovered from the target). 6-FD was obtained with a chemical and radiochemical purity of greater than 99% and with a level of mercury in the final preparation of less than 20 ppb. Utilization of a remote, semiautomated production system, resulted in the preparation of a sterile, pyrogen-free product suitable for human injection after a synthesis time of 50 min.

Evaluation of positron-emitting SCH 23390 analogs as tracers for CNS dopamine D1 receptors.

Two positron-emitting analogs of SCH 23390, one labelled with 75Br (or 76Br) and another with 11C, were evaluated as potential PET tracers for central dopamine D1 receptors. In vivo studies were performed to assess the time course of the biodistribution of these tracers in mice and to determine whether dopamine receptors mediated their uptake in the brains of these animals. Results show that indeed cerebral uptake was consistent with dopamine receptor innervation, i.e. uptake and clearance was regionally consistent with the target receptors and that specific uptake was saturable. Because of the relatively rapid pharmacokinetics of this drug, 11C-labelled SCH 23390 would be best suited for PET imaging although the metabolism of this compound needs to be further examined.

Synthesis of [11C]SCH 23390 for dopamine D1 receptor studies.

The optimal conditions for the synthesis of 11C-labelled SCH 23390 by radio-methylation of its desmethyl precursor, SCH 24518, with [11C]iodomethane are described. Isocratic reversed phase HPLC was used for the purification of [11C]SCH 23390. The specific activity range in 30 runs was 10-235 Ci/mmol and average radiochemical yield was 72% based on [11C]iodomethane. Mean synthesis time was 40-60 min from the end of bombardment. Preliminary animal studies indicate that [11C]SCH 23390 would be useful in visualizing D1 receptors in a living brain by positron tomography.

Exogenous l-dopa alters spiroperidol binding, in vivo, in the mouse striatum.

The effect of exogenous l-dopa on the binding of tritiated spiroperidol, in vivo, in the murine striatum was investigated. The results obtained demonstrated that the binding of 3H-spiroperidol can be altered by the administration of l-dopa. This effect appears to be related to both the dose of spiroperidol and l-dopa given. Since l-dopa raised striatal dopamine levels in a dose dependent manner, these results suggest that the estimates of receptor parameters obtained using positron emission tomography and analogs of spiroperidol may be affected by the differences in endogenous dopamine concentrations among subjects.