ABSTRACT:Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (ϳ96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC 50 values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.
Introduction
A radioligand for measuring the density of corticotrophin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF1 receptor ligands based on a core 3-(phenylamino)pyrazin-2(1H)-one scaffold.
Methods
CRF1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF receptors (displacement of [125I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF1 receptor density (Bmax) was assessed in rhesus brain cortical and cerebellum membranes with the CRF receptor ligand, [3H]BMS-728300.
Results
The three ligands selected for development showed high binding affinity (IC50 values, 0.3–8 nM) at CRF1 receptors and moderate lipophilicity (LogD, 2.8–4.4). [3H]BMS-728300 and the two 18F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF1 receptor Bmax in rhesus brain was found to be 50–120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [18F]BMS-721313, [18F]BMS-732098 and [11C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro.
Conclusions
Candidate CRF1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF1 receptors.
SummaryThe syntheses of ðSÞ-3-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-[18 F]-fluoro-6-(trifluoromethyl)-1H-indol-2-one was accomplished by the microwave assisted carrier-added 18 F fluorination of ðR; SÞ-3-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-chloro-6-(trifluoromethyl)-1H-indol-2-one, followed by chiral HPLC separation to afford the desired 18 F-labeled enantiomer in radiochemical yields of 5-15% (EOS) and synthesis and purification times of 60-67 min. Biodistribution studies in rodents were consistent with previously reported studies using racemic 18 F-radiolabeled material.
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