Julie F. Falcone

Radioreceptor binding profile of the atypical antipsychotic olanzapine

The affinities of olanzapine, clozapine, haloperidol, and four potential antipsychotics were compared on binding to the neuronal receptors of a number of neurotransmitters. In both rat tissues and cell lines transfected with human receptors olanzapine had high affinity for dopamine D1, D2, D4, serotonin (5HT)2A, 5HT2C, 5HT3, α1-adrenergic, histamine H1, and five muscarinic receptor subtypes. Olanzapine had lower affinity for α2-adrenergic receptors and relatively low affinity for 5HT1 subtypes, GABAA, β-adrenergic receptors, and benzodiazepine binding sites. The receptor binding affinities for olanzapine was quite similar in tissues from rat and human brain. The binding profile of olanzapine was comparable to the atypical antipsychotic clozapine, while the binding profiles for haloperidol, resperidone, remoxipride, Org 5222, and seroquel were substantially different from that of clozapine. The receptor binding profile of olanzapine is consistent with the antidopaminergic, antiserotonergic, and antimuscarinic activity observed in animal models and predicts atypical antipsychotic activity in man.

Antagonism of estrogen action with a new benzothiophene derived antiestrogen

A new benzothiophene derived antiestrogen, LY139481, inhibited the uterotropic action of estradiol in a dose related fashion, and at 1 mg per day suppressed more than 90 percent of estradiols activity in immature rats. LY139481 induced minimal uterotropic activity, and that activity declined in relation to dose. The relative binding affinity of LY139481 for rat uterine cytosol estrogen receptors was greater than that of estradiol in competitive assays and increased in relation to temperature (2.9 +/- 0.5 x estradiol at 30 degrees C). LY139481 caused estradiol-induced uterine hypertrophy to regress in a manner similar to that which resulted from withdrawal of estradiol treatment. Three successive daily injections of LY139481 slightly increased uterine weight, and blocked additional uterotropic action in response to estradiol and LY139481 administration on subsequent days. Furthermore, ten daily injections of estradiol alone did not increase uterine weight in animals pretreated with LY139481 for three days. In contrast, LY139481 did not prevent the partial uterotropic action of tamoxifen administration.

Antagonism by olanzapine of dopamine D1, serotonin2, muscarinic, histamine H1 and α1-adrenergic receptors in vitro

The atypical antipsychotic olanzapine has relatively high affinity for a number of neuronal receptors in radioreceptor binding assays. The ability of olanzapine to activate or antagonize a number of neuronal receptors was investigated in vitro, in cell lines transfected selectively with receptor subtypes and in receptor-selective isolated tissue studies. Olanzapine had no agonist activity at any of the receptors examined. However, olanzapine was a potent antagonist of 5-HT-stimulated increases in IP3 in cell lines transfected with 5-HT2A or 5-HT2B receptors with IC50 values of 30-40 nM. Olanzapine weakly blocked 5-HT-induced formation of IP3 in cell lines transfected with 5-HT2c receptors, but in this cell line potently inhibited 5-HT-stimulated [35S]GTP gamma S binding with a Ki value of 15 nM. Olanzapine blocked dopamine-stimulated adenylyl cyclase in rat retina with modest potency (Ki = 69 nM), consistent with its relatively low affinity for dopamine D1 receptors. Olanzapine blocked agonist-induced activities at the muscarinic receptor subtypes M1, M2, M3, and M5 with Ki values of 70, 622, 126, and 82 nM, respectively. In studies using cell lines transfected with muscarinic M4 receptors, olanzapine and the atypical antipsychotic clozapine did not have agonist activities as determined with cAMP inhibition and stimulation assays, arachidonic acid release and [35S]GTP gamma S binding assays. However, olanzapine antagonized agonist-induced effects in muscarinic M4 cells with a Ki value of 350 nM. In isolated tissue studies, olanzapine potently blocked agonist-induced effects at alpha 1-adrenergic and histamine H1 receptors (KB = 9 and 19 nM, respectively). Thus, olanzapine was an antagonist at all receptors investigated and was a particularly potent antagonist at 5-HT2A, 5-HT2B, 5-HT2C, alpha 1-adrenergic and histamine H1 receptors. Olanzapine was a weaker antagonist at muscarinic and dopamine D1 receptors.

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a Novel Metabotropic Glutamate 2 Potentiator with Potential Anxiolytic/Antidepressant Properties: In Vivo Profiling Suggests a Link between Behavioral and Central Nervous System Neurochemical Changes

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC50 = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

Inhibition of central nervous system aromatase activity: A mechanism for fenarimol-induced infertility in the male rat

Fenarimol (alpha-(2-chlorophenyl)-alpha(4-chlorophenyl)-5-pyrimidine-methanol), a pyrimidine carbinol agricultural fungicide, was previously reported to cause a dose-related decrease in fertility in rats (K. S. Hirsch, E. R. Adams, D. G. Hoffman, J. K. Markham, and N. V. Owen (1986), Toxicol. Appl. Pharmacol. 86, 391-399). Based on the results of a number of reproduction studies (K. S. Hirsch, E. R. Adams, D. G. Hoffman, J. K. Markham, and N. V. Owen (1986), Toxicol. Appl. Pharmacol. 86, 391-399), the infertility appeared to be associated with an impairment of male sexual behavior. When [14C]fenarimol was administered to the dam, high concentrations of radioactivity were observed in the neonatal hypothalamus, which functions in the development and subsequent expression of male sexual behavior. In the present studies fenarimol exhibited neither antiandrogenic nor antiestrogenic activities. The compound did, however, prevent the increase in nuclear estrogen receptors in the brain which normally occurs in the male during the early postnatal period. These results suggested that fenarimol might be acting to inhibit estrogen biosynthesis (via the aromatase enzyme complex) within the central nervous system. [3H]Testosterone was administered to neonatal rats, and the tritiated metabolites were isolated. Testosterone and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) concentrations were similar in all treatment groups. Tritiated estrogens were detected in the brain cell nuclei from control neonates but not in neonates exposed to fenarimol. Fenarimol was also observed to inhibit rat ovarian aromatase activity in vitro. These data indicate that the decrease in male sexual behavior and the infertility associated with exposure to fenarimol were, most likely, due to inhibition of aromatase activity within the central nervous system.

Platelet Derived Growth Factor (Pdgf), Androgens and Inflammation: Possible Etiologic Factors in the Development of Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is characterized by varying degrees of epithelial and stromal hyperplasia in association with inflammation. Although androgens are known to be important for the growth and function of the prostate, their role in the development of BPH is unclear. The release of platelet-derived growth factor (PDGF) in response to inflammation suggests that PDGF may participate in the development of BPH. Cultured prostate cells derived from patients with BPH were examined for the presence of functional PDGF and androgen receptors. The cells expressed PDGF receptors and responded to PDGF stimulation by the activation of the PDGF signal transduction pathway and a dose-dependent stimulation of cell proliferation. Even though the cells expressed androgen receptors, dihydrotestosterone failed to elicit a mitogenic response. While the role of androgens in BPH remains unclear, these results suggest that inflammation and, specifically, PDGF may be important etiologic factors in the development of BPH.

Investigations into the physiological role of muscarinic M2 and M4 muscarinic and M4 receptor subtypes using receptor knockout mice.

Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.

Decreased binding affinity of olanzapine and clozapine for human muscarinic receptors in intact clonal cells in physiological medium

The binding affinity of olanzapine, clozapine and atropine for muscarinic receptor subtypes in clonal Chinese hamster ovary (CHO) cell lines was compared in intact cells in physiological media to disrupted cells in hypotonic buffer. The affinity of olanzapine and clozapine, but not atropine, for muscarinic receptor subtypes (M(1)-M(5)) was significantly reduced in intact cells in physiological medium compared to disrupted cells in hypotonic buffer. The affinity of olanzapine for muscarinic M(1) receptors was most affected with a reduction of K(i) value from 2.5 to 73 nM in intact cells. These data suggest that the affinity of olanzapine and clozapine for muscarinic receptors have been significantly overestimated.

In Vitro and in Vivo Evidence for a Lack of Interaction with Dopamine D2 Receptors by the Metabotropic Glutamate 2/3 Receptor Agonists 1S,2S,5R,6S-2-Aminobicyclo[3.1.0]hexane-2,6-bicaroxylate Monohydrate (LY354740) and (−)-2-Oxa-4-aminobicyclo[3.1.0] Hexane-4,6-dicarboxylic Acid (LY379268)

Some recently published in vitro studies with two metabotropic glutamate 2/3 receptor (mGluR2/3) agonists [(−)-2-oxa-4-aminobicyclo[3.1.0] hexane-4,6-dicarboxylic acid (LY379268) and 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate (LY354740)] suggest that these compounds may also directly interact with dopamine (DA) D2 receptors. The current in vitro and in vivo studies were undertaken to further explore this potential interaction with D2 receptors. LY379268 and LY354740 failed to inhibit D2 binding in both native striatal tissue homogenates and cloned receptors at concentrations up to 10 μM. LY379268 and LY354740 (up to 10 μM) also failed to stimulate [35S]GTPγS binding in D2L- and D2S-expressing clones in the presence of NaCl or N-methyl-d-glucamine. In an in vivo striatal D2 receptor occupancy assay, LY379268 (3–30 mg/kg) or LY354740 (1–10 mg/kg) failed to displace raclopride (3 μg/kg i.v.), whereas aripiprazole (10–60 mg/kg) showed up to 90% striatal D2 receptor occupancy. LY379268 (10 mg/kg) and raclopride (3 mg/kg) blocked d-amphetamine and phencyclidine (PCP)-induced hyperactivity in wild-type mice. However, the effects of LY379268 were lost in mGlu2/3 receptor knockout mice. In DA D2 receptor-deficient mice, LY379268 but not raclopride blocked both PCP and d-amphetamine-evoked hyperactivity. In the striatum and nucleus accumbens, LY379268 (3 and 10 mg/kg) was without effect on the DA synthesis rate in reserpinized rats and also failed to prevent S-(−)-3-(3-hydroxyphenyl)-N-propylpiperidine-induced reductions in DA synthesis rate. Taken together, the current data fail to show evidence of direct DA D2 receptor interactions of LY379268 and LY354740 in vitro or in vivo. Instead, these results provide further evidence for a novel antipsychotic mechanism of action for mGluR2/3 agonists.