Joseph H. Krushinski

LY248686, A New Inhibitor of Serotonin and Norepinephrine Uptake

LY248686 is an inhibitor of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) uptake in synaptosomal preparations of hypothalamus and cerebral cortex, and 5-HT uptake in human blood platelets, with inhibitor constants near nanomolar concentrations. Upon administration to rats 1 hour before sacrifice, LY248686 caused dose-dependent and parallel decreases of 5-HT and NE uptake in hypothalamus homogenates ex vivo. LY248686 is a positive enantiomer and was slightly more potent than its negative isomer, LY248685, as an inhibitor of 5-HT uptake. Both isomers were only weak inhibitors of dopamine (DA) uptake in striatal synaptosomes. The inhibitory effects on 5-HT and NE uptake after a single administration of LY248686 followed similar time courses and simultaneously persisted for as long as 6 hours. LY248686 in vivo could effectively antagonize the p-chloroamphetamine-induced decreases of 5-HT uptake and levels of 5-HT and 5-hydroxyindoleacetic acid in cerebral cortex, and block the accumulation of 14C-NE in rat hearts. In food deprived rats, LY248686 suppressed food intake synergistically with 5-hydroxytryptophan, a precursor amino acid of 5-HT. Because of its lack of affinity for receptors of 5-HT, NE, DA, acetylcholine, histamine and naloxone, and its ability to inhibit 5-HT and NE uptake simultaneously, LY248686 has a favorable pharmacological profile as a potential antidepressant drug.

Norfluoxetine enantiomers as inhibitors of serotonin uptake in rat brain

Like fluoxetine, the N-demethylated metabolite norfluoxetine exists in R- and S-enantiomeric forms. S-Norfluoxetine inhibited serotonin (5-HT) uptake and Pillperoxetine binding to 5-HT uptake sites with a pKi of 7.86 and 8.88 or 14 and 1.3 nM, respectively, whereas R-norfluoxetine was 22 and 20 times, respectively, less potent. R-and S-Norfluoxetine were less potent than the corresponding enantiomers of fluoxetine as inhibitors of norepinephrine uptake and [3H]tomoxetine binding to norepinephrine uptake sites. Ex vivo studies showed that S-norfluoxetine inhibited 5-HT uptake with an ED50 of 3 mg/kg intraperitoneally, 4.7 mg/kg subcutaneously, and 9 mg/kg orally (7.3, 11.4 and 21.9 μmol/kg, respectively), while the ED50 for R-norfluoxetine exceeded 20 mg/kg intraperitoneally (48.6 μmol/kg). Inhibition of 5-HT uptake in cerebral cortex ex vivo and decrease in 5-HIAA levels in hypothalamus persisted for 24 hours after administration of S-norfluoxetine as demonstrated with the administration of fluoxetine. Thus, S-norfluoxetine is the active N-demethylated metabolite responsible for the persistently potent and selective inhibition of 5-HT uptake in vivo.

The PET Radioligand [11C]MePPEP Binds Reversibly and with High Specific Signal to Cannabinoid CB1 Receptors in Nonhuman Primate Brain

The cannabinoid CB1 receptor is one of the most abundant G protein-coupled receptors in the brain and is a promising target of therapeutic drug development. Success of drug development for neuropsychiatric indications is significantly enhanced with the ability to directly measure spatial and temporal binding of compounds to receptors in central compartments. We assessed the utility of a new positron emission tomography (PET) radioligand to image CB1 receptors in monkey brain. [11C]MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) has high CB1 affinity (Kb=0.574±0.207 nM) but also moderately high lipophilicity (measured LogD7.4=4.8). After intravenous injection of [11C]MePPEP, brain activity reached high levels of almost 600% standardized uptake value (SUV) within 10–20 min. The regional uptake was consistent with the distribution of CB1 receptors, with high radioactivity in striatum and cerebellum and low in thalamus and pons. Injection of pharmacological doses of CB1-selective agents confirmed that the tracer doses of [11C]MePPEP reversibly labeled CB1 receptors. Preblockade or displacement with two CB1 selective agents (ISPB; (4-(3-cyclopentyl-indole-1-sulfonyl)-N-(tetrahydro-pyran-4-ylmethyl)-benzamide) and rimonabant) showed that the majority (>89%) of brain uptake in regions with high receptor densities was specific and reversibly bound to CB1 receptors in the high binding regions. [11C]MePPEP was rapidly removed from arterial plasma. Regional brain uptake could be quantified as distribution volume relative to the concentration of parent radiotracer in plasma. The P-glycoprotein (P-gp) inhibitor DCPQ ((R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol) did not significantly increase brain uptake of [11C]MePPEP, suggesting it is not a substrate for this efflux transporter at the blood–brain barrier. [11C]MePPEP is a radioligand with high brain uptake, high specific signal to CB1 receptors, and adequately fast washout from brain that allows quantification with 11C (half-life=20 min). These promising results in monkey justify studying this radioligand in human subjects.

Comparison of norfluoxetine enantiomers as serotonin uptake inhibitors in vivo

Norfluoxetine, the N-desmethyl metabolite of fluoxetine, has been reported to resemble fluoxetine in being a potent and selective inhibitor of the serotonin uptake carrier. The enantiomers of norfluoxetine have now been compared as serotonin uptake inhibitors in vivo, based on their antagonism of p-chloroamphetamine-induced depletion of serotonin in brain and their lowering of concentrations of the metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) in brain. In rats, S-norfluoxetine (ED50 3.8 mg/kg) was more potent than R-norfluoxetine (ED50 > 20 mg/kg) in blocking the depletion of serotonin by p-chloroamphetamine after intraperitoneal administration. The S enantiomer decreased concentrations of 5-HIAA in whole brain after doses of 2.5-20 mg/kg, whereas the R enantiomer did not. The concentrations of both enantiomers in brain increased in proportion to dose and the R enantiomer disappeared from the brain at a slightly slower rate than the S enantiomer. The relative inability of the R enantiomer to block the uptake of serotonin was therefore not a result of smaller concentrations of drug in the brain. In mice, S-norfluoxetine was also more potent than R-norfluoxetine in blocking depletion of serotonin by p-chloroamphetamine (ED50 values 0.82 and 8.3 mg/kg, respectively). Thus, in contrast to the relatively similar potencies of the enantiomers of fluoxetine in blocking the uptake of serotonin, the enantiomers of norfluoxetine have markedly different potencies as inhibitors of the uptake of serotonin.

Synthesis, Ex Vivo Evaluation, and Radiolabeling of Potent 1,5-Diphenylpyrrolidin-2-one Cannabinoid Subtype-1 Receptor Ligands as Candidates for In Vivo Imaging

We have reported that [methyl- (11)C] (3 R,5 R)-5-(3-methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one ([(11)C] 8, [(11)C]MePPEP) binds with high selectivity to cannabinoid type-1 (CB 1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely, the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy- d 2 (21, FMPEP- d 2), and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high, selective potency at CB 1 receptors in vitro (K b < 1 nM). Each ligand (30 microg/kg, iv) was injected into rats under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand uptakes were high (22.6-48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced (6.5-17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB 1 receptors, we then established and here describe methods for producing [(11)C] 8, [(11)C] 16, and [(18)F] 20- 22 in adequate activities for evaluation as candidate PET radioligands in vivo.

Novel halogenated analogs of tomoxetine that are potent and selective inhibitors of norepinephrine uptake in brain

Halogenated analogs of the potent norepinephrine (NE) uptake inhibitor, tomoxetine, were synthesized and their affinities for the serotonin (5HT) and NE uptake sites evaluated. One of the most potent was the 2-iodo substituted analog (289306) that inhibited [3H]tomoxetine binding to rat cerebral cortex with a Ki of 0.37 nM. The compound also inhibited the uptake of [3H]NE into rat hypothalamic synaptosomes with a Ki of 3.5 nM. This analog was significantly less potent at the 5HT uptake site, as exhibited by a Ki of 25 nM in the inhibition of [3H]paroxetine binding and a Ki of 121 nM in [3H]5HT uptake. The resolved (R) enantiomer (303926) was 10 times more potent as a [3H]NE uptake inhibitor and 29 times more potent as an inhibitor of [3H]tomoxetine binding than the (S) enantiomer (303884). Administration of 289306 to rats prior to an i.c.v. injection of 6-hydroxydopamine prevented the depletion of hypothalamic NE and Epi with ED50 values of 0.28 and 0.47 mg/kg, respectively. Thus, 289306 was a potent inhibitor of NE uptake in vitro and in vivo. In addition, these compounds provide structures for potential ligands for the study of NE uptake sites by autoradiography, PET or SPECT imaging.

Localization of ATP sensitive potassium channels in the rat brain using a novel radioligand, [125I]iodoglibenclamide

In order to provide a radioligand for the rapid autoradiographic localization of the ATP-sensitive potassium channels we have developed and characterized an 125 I-labelled, iodinated analogue of glibenclamide as a novel radioligand; in this analogue, the chlorine atom of glibenclamide has been replaced with iodine, and we will refer to this molecule in this report as «iodoglibenclamide»

Characterization of type I 5α-reductase activity in DU145 human prostatic adenocarcinoma cells

Abstract The conversion of testosterone (T) to dihydrotestosterone (DHT) has been demonstrated to be catalysed by at least two isoforms of human steroid 5α-reductase, designated types I and II. Type II 5α-reductase expression predominates in human accessory sex tissues, localized to the fibromuscular stromal compartment. The type I isoform predominates in skin, prostatic epithelia and, to a lesser extent, in prostatic fibromuscular stroma. The significance of the type I isoform to prostatic cellular growth and function remains undefined. In cultured DU145 cells, we evaluated the metabolism of [ 14 C]-T and demonstrated the time-dependent formation of [ 14 C]-DHT. Oxidative metabolism (conversion of [ 14 C]-T to [ 14 C]-androstenedione) and the formation of conjugated androgen metabolites occurred at a relatively low rate in the DU145 cells. Using human type I 5α-reductase cDNA, Northern blot analysis of DU145 cell mRNA revealed high levels of type I isoform expression. Analogous probing of the DU145 cells with a human 5α-reductase II cDNA failed to reveal expression of the type II isoform. The expression of functional type I activity has been confirmed pharmacologically using isoformselective 5α-reductase inhibitors. Reductive metabolism of [ 3 H]-T in the DU145 cells was inhibited in a concentration-dependent manner by LY306089, a potent non-steroidal type I-selective inhibitor (IC 50 = 10.0 nM). SKF105657, a steroidal type II-specific inhibitor was distinctly less active at inhibiting [ 3 H]-DHT formation. LY306089 was a non-competitive inhibitor of type I 5α-reductase in DU145 cellular homogenates with an apparent K i value of 4.0 nM. These studies have identified and pharmacologically defined type I 5α-reductase activity in an androgen-insensitive prostatic cancer cell line and provide the basis for additional investigations into the significance of type I 5α-reductase to human prostatic pathophysiology.

(R)-thionisoxetine, a potent and selective inhibitor of central and peripheral norepinephrine uptake

Inhibitors of neuronal norepinephrine (NE) uptake are useful for the treatment of a variety of diseases including depression and urinary incontinence. In the present study, we synthesized and evaluated a novel analog of the potent and selective NE uptake inhibitor, nisoxetine. Thionisoxetine more potently inhibited the uptake of [3H]-NE into hypothalamic synaptosomes and [3H]-nisoxetine binding to the NE transporter than (R)-nisoxetine. The (R) enantiomer of this compound was significantly more potent than the (S) enantiomer, having a Ki of 0.20 nM in [3H]-nisoxetine binding. The (R) enantiomer was approximately 70-fold more potent in inhibiting [3H]-NE uptake when compared to [3H]-5HT uptake. In rats, (R)-thionisoxetine prevented hypothalamic NE depletion by 6-hydroxydopamine with an ED50 of 0.21 mg/kg. Depletion of NE in peripheral nerves was accomplished by the administration of metaraminol to rats. In this paradigm, (R)-thionisoxetine prevented the depletion of heart NE with an ED50 of 3.4 mg/kg and urethral NE with an ED50 of 1.2 mg/kg. Thus, (R)-thionisoxetine is a potent and selective inhibitor of NE uptake in both central and peripheral tissues.

An Allosteric Potentiator of the Dopamine D1 Receptor Increases Locomotor Activity in Human D1 Knock-In Mice without Causing Stereotypy or Tachyphylaxis

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3–20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30–240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.