Neurogenic dural protein extravasation induced by meta-chlorophenylpiperazine (mCPP) involves nitric oxide and 5-HT2B receptor activation.

The compound m-chlorophenylpiperazine (mCPP), which is known to trigger migraine-like head pain in some subjects, was evaluated for its ability to induce dural plasma protein extravasation (PPE) in guinea pigs. Intravenous mCPP dose-dependently increased PPE. This effect was inhibited by non-selective 5-HT2 receptor antagonists (methysergide, LY53857, LY215840), by a peripherally restricted 5-HT2 receptor antagonist (xylamidine) and by a 5-HT2B selective receptor antagonist (LY202146). These data suggests that peripheral 5-HT2B receptors mediate mCPP-induced PPE. The nitric oxide synthase inhibitor L-NAME and 5-HT1 agonist sumatriptan also blocked mCPP-induced PPE, suggesting a role for nitric oxide (NO) and the trigeminal system, respectively. NO release has been linked to activation of the 5-HT2B receptor on the vascular endothelium. However, LY202146 did not block PPE induced by electrical stimulation of the trigeminal ganglion. These data are consistent with activation of peripheral 5-HT2B receptors initiating PPE and the theory that selective 5-HT2B antagonists might be effective prophylactic therapies for migraine.

N-[3-(2-Dimethylaminoethyl)-2-methyl-1H- indol-5-yl]-4-fluorobenzamide: a potent, selective, and orally active 5-HT(1F) receptor agonist potentially useful for migraine therapy.

Recent studies have demonstrated that selective 5-HT(1F) receptor agonists inhibit neurogenic dural inflammation, a model of migraine headache, indicating that these compounds may be effective therapies for the treatment of migraine pain. This communication describes the synthesis and discovery of a novel compound, N-[3-(2-(dimethylamino)ethyl)-2-methyl-1H-indol-5-yl]-4-fluorobenzamide (4), which possesses high binding affinity and selectivity at the 5-HT(1F) receptor relative to more than 40 other serotonergic and nonserotonergic receptors examined.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Kinetic analysis of LY320236: competitive inhibitor of type I and non-competitive inhibitor of type II human steroid 5alpha-reductase.

Type I and type II steroid 5alpha-reductases (5alpha-R) catalyze the conversion of testosterone (T) to dihydrotestosterone (DHT). LY320236 is a benzoquinolinone (BQ) that inhibits 5alpha-R activity in human scalp skin (Ki(typeI)=28.7+/-1.87 nM) and prostatic homogenates (Ki(typeII)=10.6+/-4.5 nM). Lineweaver-Burk, Dixon, and non-linear analysis methods were used to evaluate the kinetics of 5alpha-R inhibition by LY320236. Non-linear modeling of experimental data evaluated V(max) in the presence or absence of LY320236. Experimental data modeled to the following equation 1v=+ fixing the In0c value equal to 1.0 or 0 are consistent with non-competitive or competitive inhibition, respectively. LY320236 is a competitive inhibitor of type I 5alpha-R (In0c=0, Ki=3.39+/-0.38, RMSE = 1.300) and a non-competitive inhibitor of type II 5alpha-R (In0c=1, Ki=29. 7+/-3.4, RMSE = 0.0592). These data are in agreement with linear transformation of the data using Lineweaver-Burk and Dixon analyses. These enzyme kinetic data support the contention that the BQ LY320236 is a potent dual inhibitor with differing modes of activity against the two known human 5alpha-reductase isozymes. LY320236 represents a class of non-steroidal 5alpha-R inhibitors with potential therapeutic utility in treating a variety of androgen dependent disorders.

N-Methyl-5-tert-butyltryptamine: A novel, highly potent 5-HT1D receptor agonist.

It has been observed that reported 5-HT1D receptor agonists have at least one heteroatom (N, O, or S) on the 5-substituent of the indole. This has led to the hypothesis that a 5-substituent capable of participating in hydrogen bonding is critical for conveying high affinity. This article describes the synthesis and biological evaluation of a new series of 5-alkyltryptamine analogues, which does not have a heteroatom in the 5-substituent group. In contrast to the hypothesis, 5-alkyltryptamines all exhibit high binding affinities for the human 5-HT1D receptor. The size of the lipophilic alkyl group at the 5-position of the indole has significant impact on the 5-HT1D binding affinity. Compounds with a tert-butyl group at the 5-position such as 9d, 10, and 11 were identified. These analogues display high binding affinity (Ki < 1 nM) and moderate receptor selectivity in comparison with known antimigraine agents such as sumatriptan, naratriptan, rizatriptan, and VML-251.

Synthesis and 5 alpha-reductase inhibitory activity of 8-substituted benzo[f]quinolinones derived from palladium mediated coupling reactions.

Benzoquinolinones have been shown to be potent, selective inhibitors of the Type I 5 alpha-reductase enzyme, which is responsible for the production of dihydrotestosterone from testosterone localized in the scalp. In an effort to identify compounds that demonstrate inhibition of both 5 alpha-reductase isozymes, we have employed 8-bromobenzoquinolinone as an advanced intermediate for participation in a variety of palladium mediated carbon-carbon bond forming reactions. By varying the 8-substituent it is possible to alter the selectivity profile of the series.

5-HT1F receptor agonists inhibit neurogenic dural inflammation in guinea pigs.

The serotonin (5-HT) receptor subtype mediating inhibition of neurogenic dural inflammation in guinea pigs was investigated using a series of serotonin agonists with differing affinities for the 5-HT1B, 5-HT1D and 5-HT1F receptors. When agonist potencies for inhibiting neurogenic inflammation were compared with affinities for these receptor subtypes, a significant positive correlation was seen only with the 5-HT1F receptor. The potency of agonists in inhibiting adenylate cyclase in cells transfected with human 5-HT1F receptor was also highly correlated with their potency in the animal model of migraine. In situ hybridization demonstrated 5-HT1F receptor mRNA in guinea pig trigeminal ganglion neurons. These data suggest that the 5-HT1F receptor is a rational target for migraine therapeutics.

Amyloid beta-mediated oxidative and metabolic stress in rat cortical neurons: no direct evidence for a role for H2O2 generation.

H2O2 and free radical-mediated oxidative stresses have been implicated in mediating amyloid beta (1-40) [A beta (1-40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2-scavenging enzyme catalase protects neurons in culture against A beta-mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. A beta-mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection. Three intracellular biochemical markers of H2O2-mediated oxidative stress were unchanged by A beta treatment: (a) glyceraldehyde-3-phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. lonspray mass spectra of A beta in the incubation medium indicated that A beta itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24-h exposure of neurons to A beta. Our results challenge a pivotal role for H2O2 generation in mediating A beta toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.

Potent, selective tetrahydro-beta-carboline antagonists of the serotonin 2B (5HT2B) contractile receptor in the rat stomach fundus.

A series of potent, selective 5HT2B receptor antagonists has been identified based upon yohimbine, with SAR studies resulting in a 1000-fold increase in 5HT2B receptor affinity relative to the starting structure (-log KBS > 10.0 have been obtained). These high-affinity tetrahydro-beta-carboline antagonists are able to discriminate among the 5HT2 family of serotonin receptors, with members of the series showing selectivities of more than 100-fold versus both the 5HT2A and 5HT2C receptors based upon radioligand binding and functional assays. As the first compounds reported with such selectivity and enhanced receptor affinity, these tetrahydro-beta-carboline antagonists are useful tools for elucidating the role of serotonin acting at the 5HT2B receptor in normal and disease physiology.

LY191704 inhibits type I steroid 5 alpha-reductase in human scalp.

Conversion of testosterone to dihydrotestosterone (DHT) has been demonstrated to be catalyzed by two isoforms of steroid 5 alpha-reductase, designated types I and II. Although several classes of steroid-based inhibitors of the type II isoform have been identified, these agents have not demonstrated highly selective pharmacological activity against human type I 5 alpha-reductase. LY191704 is representative of a series of nonsteroidal agents that have potent [apparent inhibitory constant (Ki) = 11.3 nM] inhibitory activity in human scalp skin homogenates (pH 7.5), a source of type I 5 alpha-reductase. [3H]-DHT production in the presence and absence of LY191704 is consistent with a noncompetitive mode of inhibition. In human prostatic homogenates (pH 5.5), a source of type II 5 alpha-reductase, LY191704 is virtually inactive as an inhibitor [concentration of inhibitor producing 50% inhibition of enzymatic activity (IC50) > 1,000 nM] of [3H]-DHT formation. LY191704 does not inhibit the type I or type II isoforms of rat 5 alpha-reductase, nor does the compound compete for binding to the murine androgen receptor expressed in SF9 cells using a baculo virus expression system. The benzoquinolinones, as exemplified by LY191704, possess exquisite pharmacological selectivity and provide a tool to understand the role of human type I 5 alpha-reductase in normal and pathophysiological states. These agents may also find clinical utility in treating androgen-dependent dermatological conditions.

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

The conversion of testosterone (T) to dihydrotestosterone (DHT) has been demonstrated to be catalysed by at least two isoforms of human steroid 5 alpha-reductase, designated types I and II. Type II 5 alpha-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 [14C]-T and demonstrated the time-dependent formation of [14C]-DHT. Oxidative metabolism (conversion of [14C]-T to [14C]-androstenedione) and the formation of conjugated androgen metabolites occurred at a relatively low rate in the DU145 cells. Using human type I 5 alpha-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 5 alpha-reductase II cDNA failed to reveal expression of the type II isoform. The expression of functional type I activity has been confirmed pharmacologically using isoform-selective 5 alpha-reductase inhibitors. Reductive metabolism of [3H]-T in the DU145 cells was inhibited in a concentration-dependent manner by LY306089, a potent non-steroidal type I-selective inhibitor (IC50 = 10.0 nM). SKF105657, a steroidal type II-specific inhibitor was distinctly less active at inhibiting [3H]-DHT formation. LY306089 was a non-competitive inhibitor of type I 5 alpha-reductase in DU145 cellular homogenates with an apparent Ki value of 4.0 nM. These studies have identified and pharmacologically defined type I 5 alpha-reductase activity in an androgen-insensitive prostatic cancer cell line and provide the basis for additional investigations into the significance of type I 5 alpha-reductase to human prostatic pathophysiology.

Responses of LNCaP prostatic adenocarcinoma cell cultures to LY300502, a benzoquinolinone human type I 5alpha-reductase inhibitor.

We evaluate the metabolic inhibitory, antiproliferative, and antisecretory effects of LY300502, a benzoquinolinone human-specific type I-selective steroid 5alpha-reductase inhibitor in LNCaP human prostatic adenocarcinoma cell cultures. Reductive metabolism of [3H-T] in the LNCaP cells was inhibited in a concentration-dependent manner by LY300502 (IC50 approximately 5.77 nM). The proliferative responses of LNCaP cells to LY300502 were examined in the presence of 0.1 NM testosterone (T), a concentration that stimulates maximal LNCaP cell numbers 40% above control levels. LY300502 significantly anatagonized T-induced stimulation of LNCaP cellular proliferation at concentrations greater that 10 nM (P<0.05), and at 1,000 nMcompletely blocked the mitogenic effects of T on LNCaP cells. In the absence of androgen, LY300502 had no effect on LNCaP cellular proliferation. In the presence of 100 nM T, an androgen concentration that maximally stimulates in vitro PSA production, LY300502 significantly antagonized T-induced PSA secretion at a concentration equal to or greater than 30 nM (P<0.05). These studies provide the basis for additional investigations into the pathophysiologic significance of type I 5alpha-reductase to prostatic cancer and the potential utility of selective inhibitors as therapeutic agents.

Comparison of desmethylsertraline with sertraline as a monoamine uptake inhibitor in vivo.

1. Desmethylsertraline, a metabolite of the antidepressant drug sertraline, was compared with sertraline for its ability to produce effects characteristic of inhibitors of the serotonin transporter in vivo. Desmethylsertraline antagonized brain serotonin depletion by p-chloroamphetamine, a depletion dependent upon the serotonin transporter, being less potent than sertraline in rats but almost as potent as sertraline in mice. Desmethylsertraline was a weak antagonist of 6-hydroxydopamine-induced depletion of heart norepinephrine in mice; sertraline had no effect at the doses studied. 2. Desmethylsertraline decreased brain concentrations of 5-hydroxyindoleacetic acid (5HIAA) in rats as did sertraline, the duration of the effect after both drugs being at least 24 hrs but less than 48 hrs. 3. After sertraline injection, desmethylsertraline was present in rat brain at higher concentrations than the parent drug at 8 hrs and thereafter. 4. In rats, repeated injections of sertraline, at doses previously shown to diminish beta-adrenergic receptor-mediated responses, led to marked accumulation of desmethylsertraline in brain and to inhibition of the catecholamine transporters. 5. In mice, brain concentrations of desmethylsertraline were higher than those of parent drug within 7 hrs after sertraline injection and probably contributed importantly to the antagonism of p-chloroamphetamine effects. 6. These data show that desmethylsertraline is less potent than sertraline as a serotonin uptake inhibitor in vivo, as the in vitro data would have predicted, but that desmethylsertraline may nonetheless contribute to the prolonged inhibition of the serotonin transporter after sertraline administration, perhaps more in mice than in rats.

N(1)-substituted ergolines and tryptamines show species differences for the agonist-labeled 5-HT2 receptor.

Previous studies indicated that selected ergolines and tryptamines showed species differences for affinity to the antagonist-labeled 5-HT2 receptor. The present study examined these same compounds for affinity at the agonist-labeled 5-HT2 receptor in rat and squirrel monkey cortical homogenates using [125I]DOI ([125I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane). As seen with the antagonist-labeled 5-HT2 receptor, N(1) alkyl substitution of either the ergolines or tryptamines resulted in a slight increase or no effect on their affinity for the agonist-labeled rat 5-HT2 receptor. In contrast, these same N(1) substitutions resulted in significant decreases in affinity for the agonist-labeled monkey 5-HT2 receptor. It was also noted that N(1)-unsubstituted ergolines and tryptamines (such as ergonovine, LY86057, LY193525 and 5-methoxytryptamine) tended to have higher affinity for the monkey versus the rat agonist-labeled receptor. However, the N(1) alkyl-substituted ergolines and tryptamines (such as mesulergine, LY53857, amesergide, N(1)-isopropyltryptamine and N(1)-isopropyl-5-methoxytryptamine) showed significantly lower affinity for the monkey versus the rat 5-HT2 receptor. These data suggest that, at least in relation to the N(1) position, ergolines and tryptamines bind in a similar orientation. These results are also discussed in terms of what amino acid differences between species may account for this structure-activity relationship.

Species differences in the pharmacology of the 5-hydroxytryptamine2 receptor: structurally specific differentiation by ergolines and tryptamines.

Species differences in the recognition of a series of ergolines by the 5-hydroxytryptamine2 (5-HT2, serotonin2) receptor were investigated in four species, the rat, pig, squirrel monkey and human. In pig frontal cortical membranes the initial studies showed that the ergolines gave shallow displacement curves against [3H]ketanserin binding. The component of [3H]ketanserin binding having low affinity for the ergolines was determined to be the result of [3H]ketanserin binding to alpha-1 adrenergic receptors. Thus, in all subsequent assays prazosin was used to mask [3H]ketanserin binding to alpha-1 adrenergic receptors. Examination of a series of ergolines revealed a distinct pattern in the species selectivity. Compounds that were unsubstituted at the N1 position of the ergoline nucleus showed higher affinity for the pig, squirrel monkey and human 5-HT2 receptors than for the rat. Conversely, compounds that had an N1-isopropyl substituent showed higher affinity for the rat receptor compared to the pig, squirrel monkey and human 5-HT2 receptors. For example, LY53857, a widely used 5-HT2 antagonist, has an isopropyl substituent at position N1 of the ergoline nucleus and exhibited a 4- to 5-fold higher affinity for the rat 5-HT2 receptor, whereas its N1-unsubstituted homologue, LY86057, had more than 10-fold higher affinity for the pig, squirrel monkey and human 5-HT2 receptors. Similar results were seen with three additional ergoline pairs, each having different substituents at the C8 position compared to LY53857. Even an N1-substitution on LY53857 as small as a methyl group, LY108742, resulted in the compound having higher affinity for the rat 5-HT2 receptor compared to the other species.(ABSTRACT TRUNCATED AT 400 WORDS)

LY191704: a selective, nonsteroidal inhibitor of human steroid 5 alpha-reductase type 1.

Androgens, in particular dihydrotestosterone (DHT), play a key role in differentiation, growth, and maintenance of the mammalian prostate. Production of DHT from testosterone is catalyzed by two distinct membrane-bound steroid 5 alpha-reductase [5 alpha-reductase; 3-oxo-5 alpha-steroid delta 4-dehydrogenase; 3-oxo-5 alpha-steroid:(acceptor) delta 4-oxidoreductase, EC 1.3.99.5] isozymes designated types 1 and 2. Benign prostatic hyperplasia (BPH), a disease that occurs almost universally in males, is characterized by obstructive and irritative urinary voiding symptoms and has been associated with an overproduction of DHT. Recently, steroidal inhibitors of 5 alpha-reductase type 2 have been used successfully for treatment of BPH. Described here is a nonsteroidal inhibitor of 5 alpha-reductase type 1, LY191704 (8-chloro-4-methyl-1,2,3,4,4a,5,6,10b-octaahydro-benzo[f]quinol in-3(2H)-one). This compound was identified based on its capacity to inhibit 5 alpha-reductase activity in a human genital skin fibroblast cell line (Hs68). Surprisingly, LY191704 is inactive when tested in freshly isolated prostate cells obtained from subjects with BPH, whereas previously described 4-azasteroids are active. LY191704 is, however, a potent inhibitor of the 5 alpha-reductase activity of BPH cells that have been maintained in culture. Analysis of human and rat 5 alpha-reductases expressed from transfected cDNAs in simian COS cells indicates that LY191704 is a specific noncompetitive inhibitor of the human 5 alpha-reductase type 1. Taken together, the results suggest that prostate cells have the capacity to express both 5 alpha-reductase isozymes and that LY191704 may be useful in treatment of human endocrine disorders associated with overproduction of DHT by 5 alpha-reductase type 1.

Pharmacological characteristics of the newly cloned rat 5-hydroxytryptamine2F receptor.

The rat 5-hydroxytryptamine (5-HT)2F (serotonin2F) receptor is a newly cloned member of the 5-HT2/1C receptor family. The pharmacology of the 5-HT2F receptor was explored using a variety of structurally different compounds in a radioligand binding assay. In addition, the 5-HT2F receptor was shown to stimulate production of inositol 1,4,5-trisphosphate in the transformed cells. Based on the affinities of the compounds tested, their known affinities for certain of the other 5-HT receptors, and the fact that activation of the cloned 5-HT2F receptor stimulates inositol 1,4,5-trisphosphate production, the 5-HT2F receptor was determined to be a novel receptor and a member of the 5-HT2/1C receptor family. In addition, several agonists and partial agonists were evaluated for contractile activity in the rat stomach fundus, and these activities were correlated with their binding affinities at the 5-HT2F receptor. A highly significant correlation was found, providing additional evidence that is consistent with the 5-HT2F receptor being the stomach fundal contractile receptor. [3H]5-HT had high affinity for this receptor both at 37 degrees and at 0 degree (Kd = 7.87 +/- 0.55 and 0.12 +/- 0.02 nM, respectively). The difference in affinity for [3H]5-HT at the two temperatures prompted an investigation of potential temperature-dependent differences in the binding affinities of agonists versus antagonists. Agonists such as 5-HT, 5-methoxytryptamine, etc., showed higher affinity for the 5-HT2F receptor at 0 degree than at 37 degrees, whereas antagonists such as methysergide, 1-naphthylpiperazine, etc., showed no difference in affinity for this receptor at the two different temperatures. Therefore, the affinity of a compound for the 5-HT2F receptor at 37 degrees versus 0 degree was shown to be useful for predicting agonist or antagonist activity. Additionally, information is provided about some of the structural requirements for the affinity of certain tryptamines at the 5-HT2F receptor.