Synthesis and biological activity of some known and putative duloxetine metabolites.

Several putative phase I duloxetine metabolites, 4-hydroxy-, 5-hydroxy-, 6-hydroxy-, 5-hydroxy-6-methoxy-, 6-hydroxy-5-methoxy-, 5,6-dihydroxy-, and 4,6-dihydroxyduloxetine were synthesized, and their phase II metabolite as glucuronide or sulfate conjugates were also synthesized. Their in vitro binding activities were compared to that of parent compound duloxetine.

Comparative affinity of duloxetine and venlafaxine for serotonin and norepinephrine transporters in vitro and in vivo, human serotonin receptor subtypes, and other neuronal receptors.

The blockade of serotonin (5-HT) and norepinephrine (NE) transporters in vitro and in vivo by the dual 5-HT/NE reuptake inhibitors duloxetine and venlafaxine was compared. Duloxetine inhibited binding to the human NE and 5-HT transporters with K(i) values of 7.5 and 0.8 nM, respectively, and with a K(i) ratio of 9. Venlafaxine inhibited binding to the human NE and 5-HT transporters with K(i) values of 2480 and 82 nM, respectively, and with a K(i) ratio of 30. Duloxetine inhibited ex vivo binding to rat 5-HT transporters and NE transporters with ED(50) values of 0.03 and 0.7 mg/kg, respectively, whereas venlafaxine had ED(50) values of 2 and 54 mg/kg, respectively. The depletion of rat brain 5-HT by p-chloramphetamine and depletion of rat hypothalamic NE by 6-hydroxydopamine was blocked by duloxetine with ED(50) values of 2.3 and 12 mg/kg, respectively. Venlafaxine had ED(50) values of 5.9 and 94 mg/kg for blocking p-chloramphetamine- and 6-hydroxydopamine-induced monoamine depletion, respectively. Thus, duloxetine more potently blocks 5-HT and NE transporters in vitro and in vivo than venlafaxine.

Expedited discovery of second generation NK-1 antagonists: identification of a nonbasic aryloxy substituent.

Solution-phase, parallel-synthesis techniques were used to optimize a series of nonbasic NK-1 antagonists, resulting in the identification of (R)-26, an orally bioavailable compound with subnanomolar potency.

Novel radiotracers for imaging the serotonin transporter by positron emission tomography: synthesis, radiosynthesis, and in vitro and ex vivo evaluation of (11)C-labeled 2-(phenylthio)araalkylamines.

A series of four 2-(phenylthio)araalkylamines have been radiolabeled with (11)C and evaluated as potential radiotracers for imaging the serotonin transporter (SERT) by positron emission tomography (PET). All four candidates display high affinity for SERT and low affinity for the dopamine or norepinephrine transporters using in vitro binding assays. Biodistribution studies in rats demonstrated that tail-vein injection of the (11)C-labeled radiotracers resulted in high brain uptake of radioactivity with a preferential distribution in brain regions known to be rich in SERT such as hypothalamus and thalamus. The most promising candidate, 16, had hypothalamus-to-cerebellum ratios of 9:1, 1 h postinjection, an indication of high specific to nonspecific binding. Ex vivo pharmacological studies demonstrated that uptake in SERT-rich brain regions was both saturable and selective for SERT. Two of the tested radiotracers, 15 and 16, have highly favorable properties for imaging SERT and will be used in pilot human PET imaging studies.

3-Aryl-1,2-diacetamidopropane derivatives as novel and potent NK-1 receptor antagonists.

Early structure-activity studies on racemic tryptophan ester and amide NK-1 antagonists 5-7 led to the discovery that the potency of the series could be markedly increased by moving the carbonyl function in these molecules to an off-chain position as in the 3-aryl-1,2-diacetamidopropane 9. Further medicinal chemistry incorporating this change resulted in the discovery of a novel series of highly potent aryl amino acid derived NK-1 antagonists of the R stereoisomeric series (IC50's = 100 pM to > 5 microM). Compounds in this series were shown to be competitive antagonists using an in vitro NK-1 smooth muscle assay, and this data correlated well with observed human NK-1 binding affinities. Two of these agents, (R)-25 and (R)-32, blocked intrathecal NK-1 agonist-driven [Ac-[Arg6,Sar9,Met(O2)11]- substance P 6-11 (Ac-Sar9)] nociceptive behavior in mice. Both compounds potently blocked the neurogenic dural inflammation following trigeminal ganglion stimulation in the guinea pig after intravenous administration. Further, upon oral administration in this model, (R)-32 was observed to be very potent (ID50 = 91 ng/kg) and have a long duration of action (> 8 h at 1 micrograms/kg). Compound (R)-32, designated LY303870, is currently under clinical development as an NK-1 antagonist with a long duration of action.

Pharmacological characterization of LY303870: a novel, potent and selective nonpeptide substance P (neurokinin-1) receptor antagonist.

LY303870 [(R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4- (piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane] is a new, potent and selective nonpeptide neurokinin-1 (NK-1) receptor antagonist. LY303870 bound selectively and with high affinity to human peripheral (Ki = 0.15 nM) and central (Ki = 0.10 nM) NK-1 receptors. LY303870 inhibited [125I]substance P (SP) binding to guinea pig brain homogenates with similar affinity; however, it had approximately 50-fold lesser affinity for rat NK-1 sites. The less active enantiomer, LY306155 [(S)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4- (piperidin-1-yl)piperidin-1-yl)acetyl)amino]-propane], was 1,000- to 15,000-fold less potent in all the species examined. LY303870 antagonized in vitro NK-1 receptor effects as demonstrated by blockade of SP-stimulated phosphoinositide turnover in UC-11 MG human astrocytoma cells (Ki = 1.5 nM) and interleukin-6 secretion from U-373 MG human astrocytoma cells (Ki = 5 nM). In addition, LY303870 inhibited SP-induced rabbit vena cava contractions (pA2 = 9.4) with high (50,000-fold) selectivity vs. NK-2 or NK-3 receptor-mediated responses. In vivo, LY303870 inhibited [Sar9,Met(O2)11]-SP induced guinea pig bronchoconstriction (ED50 = 75 micrograms/kg i.v.) and pulmonary microvascular leakage in the bronchi (ED50 = 12.8 micrograms/kg i.v.) and trachea (ED50 = 18.5 micrograms/kg i.v.). Therefore, LY303870 is a potent and selective NK-1 receptor antagonist in vitro and in vivo. The use of LY303870 will facilitate a better understanding of NK-1 receptors in physiological processes.

Cyclosporin A is a substance P (tachykinin NK1) receptor antagonist.

The immunosuppressive cyclic undecapeptide, cyclosporin A, inhibited the binding of [125I]substance P to tachykinin NK1 receptors expressed by human IM-9 lymphoblastoid cells, U-373 MG human astrocytoma cells and guinea pig lung parenchyma with IC50 values of 425 +/- 58, 783 +/- 180, and 784 +/- 163 nM respectively. The dihydro derivative of cyclosporin A (dihydro-cyclosporin A) was an equally effective inhibitor, but the O-acetylated derivative (cyclosporin A-OAc) was 3-4 fold less potent. The cyclosporin compounds also inhibited [125I]neurokinin A binding to human NK2 receptors with potencies slightly less than at NK1 sites. In contrast, they were 8-20-fold less effective inhibitors of [125I]MePhe7-neurokinin B binding to guinea pig NK3 receptors (p < 0.001). Thus, the cyclosporin A compounds showed selectivity for NK1 and NK2 receptors. The structure-activity pattern for the effects of cyclosporin A compounds at tachykinin receptors differs from the pattern previously described for their immunosuppressive activity. All three compounds inhibited substance P induced interleukin-6 (IL-6) secretion from U-373 MG astrocytoma cells with potencies similar to their NK1 receptor binding affinities. In addition, cyclosporin A blocked substance P induced phosphatidylinositol (PI) turnover in U-373 MG cells without blocking the corresponding response to histamine. This novel pharmacological profile of the cyclosporin A compounds as NK1 receptor antagonists does not appear to correlate with other known in vitro cyclosporin A functions.

Dopamine receptor affinities in vitro and neurochemical effects in vivo of pergolide and its metabolites.

Pergolide (LY127809, CAS 66104-23-2), pergolide sulfone and pergolide sulfoxide inhibited 3H-dopamine binding to dopamine receptors in bovine striatal membranes with Ki values of 2.5, 4.6 and 15.5 nmol/l, respectively. Despropyl pergolide and despropyl pergolide sulfoxide are not as potent, with Ki values of 58.6 and 158.8 nmol/l, respectively. The derivatives of pergolide inhibit the binding of antagonist ligands 3H-Sch23390 and 3H-Spiperone to D1 and D2 dopamine receptors, respectively, in bovine striatal membranes with higher Ki concentrations. Upon administration to rats, pergolide sulfoxide and sulfone were as effective as pergolide in increasing acetylcholine levels and decreasing the metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in striatum, while the despropyl derivatives of pergolide and pergolide sulfoxide were only marginally effective in increasing acetylcholine levels in striatum.

Affinities of fluoxetine, its enantiomers, and other inhibitors of serotonin uptake for subtypes of serotonin receptors.

The new antidepressant drugs, fluoxetine (and its enantiomers), citalopram, indalpine, paroxetine, and femoxetine show relatively weak affinities for 5-HT receptors as measured by radioligand binding to 5-HT-1(A,B,C and D), 5-HT-2, and 5-HT-3 subtypes. Fluoxetine and R(-)-fluoxetine, at near micromolar concentrations, inhibit 3H-mesulergine binding to 5-HT-1C receptors in bovine choroid plexus, and the R(-) enantiomer is 23 times more potent than the S(+) enantiomer. However, the near nanomolar potencies of these drugs as inhibitors of 5-HT uptake most likely account for their pharmacologic effects in animals.

Higher density of serotonin-1A receptors in the hippocampus and cerebral cortex of alcohol-preferring P rats.

Saturable [3H]-8OHDPAT binding to 5HT-1A receptors in membranes prepared from hippocampus and frontal cerebral cortex of alcohol-preferring P rats and of alcohol-nonpreferring NP rats has been compared. The Bmax values or densities of recognition sites for 5HT-1A receptors in both brain areas of the P rats are 39 and 131 percent, respectively, higher than those in the NP rats. The corresponding KD values are 38 and 44 percent lower in the P rats than in the NP rats, indicating higher affinities of the recognition sites for the 5HT-1A receptors in hippocampus and cerebral cortex of the P rats. These findings indicate either an enrichment of 5HT-1A receptor density during selective breeding for alcohol preference or an upregulation of 5HT-1A receptors developed as an adaptation to lower presynaptic concentrations of 5HT found in these brain areas of P rats as compared with the NP rats.

Suppression of food intake in rats by fluoxetine: comparison of enantiomers and effects of serotonin antagonists.

R- and S-enantiomers of fluoxetine lowered food intake in meal-fed rats and in 2-deoxyglucose-induced hyperphagic rats. In both feeding paradigms, the S-enantiomer was slightly more potent. The potency of the two enantiomers of fluoxetine in producing anorectic effects paralleled their potency as inhibitors of 5-hydroxytryptamine (5HT) uptake in vivo. Both enantiomers were selective inhibitors of 5HT uptake in vitro and showed only weak affinity for 5HT-1, 5HT-1A and 5HT-2 receptors or for other receptors in rat brain. The anorectic effect of fluoxetine in meal-fed rats was not reversed by either centrally or peripherally acting 5HT-2 receptor antagonists (ritanserin, LY53857, xylamidine, BW 501C67) or a nonspecific 5HT receptor antagonist, metergoline. However, the serotonergic mechanism involved in the anorexic effect of fluoxetine is discussed.

Serotonergic and adrenergic receptors in alcohol-preferring and non-preferring rats.

Serotonergic and adrenergic receptors in brain areas of the alcohol-preferring P and alcohol-nonpreferring NP rats were compared by radioligand-binding assays. Binding of 3H-serotonin (3H-5HT) to 5HT-1 receptors in membranes of cerebral cortex and hippocampus was significantly higher in density (B max values) and affinity (Kd values) in the P than in the NP rats, whereas B max values in membranes from the brain stem of the P rats were lower than those of the NP rats. No significant difference between the P and NP lines was observed when the binding of 3H-ketanserin to 5HT-2 receptors and of 3H-WB4101, 3H-clonidine and 3H-dihydoalprenolol to alpha-1, alpha-2 and beta-adrenergic receptors was compared. The increase of 3H-5HT binding probably indicates up-regulation or supersensitivity of 5HT-1 receptors as a compensatory mechanism to the lower levels of 5HT in brain areas of the P rats (Murphy, et al., 1982).

Chronic effects of fluoxetine, a selective inhibitor of serotonin uptake, on neurotransmitter receptors.

Fluoxetine administration to rats at a dose of 10 mg/kg i.p. daily up to 12 or 24 days failed to change the concentration-dependent binding of [3H]WB4101, [3H]clonidine and [3H]dihydroalprenolol to alpha 1-, alpha 2- and beta-adrenergic receptors, respectively; [3H]quinuclidinyl benzilate to muscarinic receptors; [3H]pyrilamine to histamine H1 receptors and [3H]naloxone to opiate receptors. Persistent and significant decreases in receptor number (Bmax value) without changes in the dissociation constant (KD value) of [3H]5-HT binding in cortical membranes were observed upon chronic treatment with fluoxetine administered either by intraperitoneal injection or incorporation in the diet. A detectable reduction of 5-HT1 receptor number occurred after once-daily injections of fluoxetine at 10 mg/kg i.p. within 49 hours. After pretreatment for 3 days with p-chlorophenylalanine, an inhibitor of 5-HT synthesis, followed by repeated administration of fluoxetine, 5-HT1 receptor numbers were higher than those of normal rats, suggesting a dependence on synaptic concentration of 5-HT for fluoxetine to affect a receptor down-regulation. These studies provide further evidence for the selectivity of fluoxetine as an inhibitor of 5-HT reuptake, resulting in a selective down-regulation of 5-HT1 receptors in the cerebral cortex of rat brain.

Saturable binding of 35S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with GABAergic agents.

35S-t-Butylbicyclophosphorothionate (35S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25 degrees C, in 250 mM NaCl, specific binding of 35S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. 35S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase 3H-GABA binding. Pharmacological studies show that 35S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and tracazolate, and a diaryltriazine, LY81067, are also potent inhibitors of 35S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin and LY81067 on the saturable binding of 35S-TBPS in cortical membranes are compared. The present findings are consistent with the interpretation that 35S-TBPS binds at or near the picrotoxin-sensitive anion recognition sites of the GABA/benzodiazepine/picrotoxin receptor complex.

Altered behavioral response to a D2 agonist, LY141865, in spontaneously hypertensive rats exhibiting biochemical and endocrine responses similar to those in normotensive rats.

LY141865, a dopamine agonist selective for D2 dopamine receptors, caused hypomotility at low doses (0.01 and 0.1 mg/kg) and hypermotility at high doses (1 and 10 mg/kg) after i.p. injection into normotensive rats (WKY). In age-matched hypertensive rats (SHR), LY141865 caused hypomotility (not hypermotility) at all of these doses. The basal locomotor activity was higher in SHR than in WKY, but striatal concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and of serotonin and its metabolite, 5-hydroxyindoleacetic acid, were not different between the two groups of rats. The specific binding of a dopamine receptor radioligand, tritiated pergolide, in striatum and mesolimbic regions, did not differ in SHR compared with WKY. In contrast to the lack of locomotor stimulation in SHR, other dopaminergic responses to LY141865 occurred in SHR as well as WKY. For instance, LY141865 decreased striatal and mesolimbic concentrations of 3,4-dihydroxyphenylacetic acid and homovanillic acid, increased striatal and mesolimbic concentrations of acetylcholine, decreased hypothalamic concentrations of epinephrine, increased serum corticosterone concentration and decreased serum prolactin concentration in SHR as in WKY. Because radioligand-labeled dopamine receptors and several LY141865 responses mediated by dopamine receptors did not differ appreciably in SHR compared with WKY, the lack of behavioral hypermotility in response to LY141865 in SHR may be due to abnormalities in some synaptic mechanisms beyond dopamine receptor activation that are involved in the expression of increased locomotion in response to the dopamine agonist.

Fluoxetine and two other serotonin uptake inhibitors without affinity for neuronal receptors.

Fluoxetine and nine other antidepressant drugs which interact with brain receptors for neurotransmitters were studied in vitro using radioligand-binding techniques and transmitter-coupled adenylate cyclase assays. Tricyclic antidepressant drugs (desipramine, imipramine, clomipramine, amitriptyline and doxepin) had marked affinity for alpha-adrenergic, muscarinic cholinergic and histaminergic H1 receptors, and lesser affinity for serotonin and dopamine receptors. Mianserin was relatively similar to some of the tricyclic compounds, whereas trazodone had less affinity for most receptors except serotonin and alpha-adrenergic receptors. Fluoxetine had little affinity for any of these receptors, and the same was true for zimelidine and fluvoxamine, two other selective inhibitors of serotonin uptake. None of the compounds showed much affinity for beta-adrenergic receptors, opiate receptors, gamma-aminobutyric acid receptors, or benzodiazepine receptors. The present findings with fluoxetine are consistent with the virtual absence of anticholinergic or other side effects often observed with tricyclic antidepressant drugs in animal models or during the treatment of depressed patients.

A new inhibitor of norepinephrine uptake devoid of affinity for receptors in rat brain.

LY135252, (+/-)-N-methyl-gamma-(2-methylphenoxy) phenylpropylamine hydrochloride, is a competitive inhibitor of norepinephrine uptake in synaptosomes of rat hypothalamus. The resolved optical (-)-isomer, LY139603, is 2 and 9 times more effective than the racemate and the (+)-isomer, LY139602, with inhibitor constants (Ki) of 1.9, 3.4 and 16.8 nM, respectively. All three compounds are relatively weak in the inhibition of dopamine and serotonin uptake, with Ki values at least two orders of magnitude greater. The racemate and the two optical isomers in vivo are potent inhibitors of norepinephrine uptake with relative effectiveness being parallel with their K1 values. The most potent and long-acting compound was the (-)-isomer, LY139603, which inhibited norepinephrine uptake ex vivo with an ED50 value of 2.2 mg/kg i.p, and a half-life of 6.4 hr. In comparison with the tricyclic antidepressants desipramine and imipramine, LY139603 is a relatively weak ligand for alpha-1, alpha-2, and beta adrenergic receptors, acetylcholine-muscarinic receptors, histaminergic H1 receptors and the receptors of gamma-aminobutyric acid and benzodiazepines. Thus, LY139603 is a remarkably specific inhibitor of norepinephrine uptake. Its potential as an antidepressant is discussed.