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Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Phospholipidosis (PLD) is an accumulation of phospholipids in lysosome-derived multilamellar vesicles. More than 50 commercial drugs are known to cause PLD. In vitro screening assays were developed in HepG2 cells, rat primary hepatocytes, and rhesus monkey hepatocytes using the fluorescent-labeled phospholipid probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) or Nile Red lipid stain. The assays were qualified using amiodarone and fluoxetine as positive controls and precocene and valproic acid as negative controls. NBD-PE accumulation and Nile Red staining were first measured using fluorescence microscopy with morphometric analysis, and the throughput of the NBD-PE assay in HepG2 cells was increased by measuring fluorescence with a multiwell spectrofluorometer. The PLD potential values obtained for the tested compounds from the morphometric analysis were similar to the values obtained from the spectrofluorometer, suggesting the plate reader assay was effective at measuring the induction of NBD-PE accumulation. Fifteen commercial compounds were evaluated using the NBD-PE assay in HepG2 cells, rat primary hepatocytes, and rhesus monkey hepatocytes. The relative NBD-PE accumulation and PLD potentials of the evaluated compounds were similar and comparable to the values observed from other in vitro PLD assays referenced in the literature using different probes and cell lines. NBD-PE accumulations observed in rat hepatocytes after drug treatments were similar to those in HepG2 cells. NBD-PE accumulation potential observed in rhesus monkey hepatocytes after drug treatment was different for tamoxifen, perhexiline, clomipramine, and haloperidol. These agents caused potent NBD-PE accumulation in HepG2 cells, but minimal or no activity was observed in rhesus monkey hepatocytes. These data suggest that the NBD-PE spectrofluorometer assay in HepG2 cells has the speed and throughput to sensitively and quantitatively determine the PLD potential of various drug candidates. In addition, these data demonstrate the species differences in PLD potential between rat and monkey hepatocytes.

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