Inhibition of serotonin receptor type 1 in acute myeloid leukemia impairs leukemia stem cell functionality: a promising novel therapeutic target.

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous neoplasia with poor outcome, organized as a hierarchy initiated and maintained by a sub-population with differentiation and self-renewal capacities called leukemia stem cells (LSCs). Although currently used chemotherapy is capable of initially reducing the tumor burden producing a complete remission, most patients will ultimately relapse and will succumb to their disease. As such, new therapeutic strategies are needed. AML cells differentially expressed serotonin receptor type 1 (HTR1) compared with healthy blood cells and the most primitive hematopoietic fraction; in fact, HTR1B expression on AML patient samples correlated with clinical outcome. Inhibition of HTR1s activated the apoptosis program, induced differentiation and reduced the clonogenic capacity, while minimal effect was observed on healthy blood cells. In vivo regeneration capacity of primary AML samples was disrupted upon inhibition of HTR1. The self-renewal capacity remaining in AML cells upon in vivo treatment was severely reduced as demonstrated by serial transplantation. Thus, treatment with HTR1 antagonists showed antileukemia effect, especially anti-LSC activity while sparing healthy blood cells. Our results highlight the importance of HTR1 in leukemogenesis and LSC survival and identify this receptor family as a new target for therapy in AML with prognostic value.

Prophylactic effects of asiaticoside-based standardized extract of Centella asiatica (L.) Urban leaves on experimental migraine: Involvement of 5HT1A/1B receptors.

The present study aimed at evaluation of prophylactic efficacy and possible mechanisms of asiaticoside (AS) based standardized extract of Centella asiatica (L.) Urban leaves (INDCA) in animal models of migraine. The effects of oral and intranasal (i.n.) pretreatment of INDCA (acute and 7-days subacute) were evaluated against nitroglycerine (NTG, 10 mg·kg(-1), i.p.) and bradykinin (BK, 10 µg, intra-arterial) induced hyperalgesia in rats. Tail flick latencies (from 0 to 240 min) post-NTG treatment and the number of vocalizations post-BK treatment were recorded as a measure of hyperalgesia. Separate groups of rats for negative (Normal) and positive (sumatriptan, 42 mg·kg(-1), s.c.) controls were included. The interaction of INDCA with selective 5-HT1A, 5-HT1B, and 5-HT1D receptor antagonists (NAN-190, Isamoltane hemifumarate, and BRL-15572 respectively) against NTG-induced hyperalgesia was also evaluated. Acute and sub-acute pre-treatment of INDCA [10 and 30 mg·kg(-1) (oral) and 100 µg/rat (i.n.) showed significant anti-nociception activity, and reversal of the NTG-induced hyperalgesia and brain 5-HT concentration decline. Oral pre-treatment with INDCA (30 mg·kg(-1), 7 d) showed significant reduction in the number of vocalization. The anti-nociceptive effects of INDCA were blocked by 5-HT1A and 5-HT1B but not 5-HT1D receptor antagonists. In conclusion, INDCA demonstrated promising anti-nociceptive effects in animal models of migraine, probably through 5-HT1A/1B medicated action.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 11. (1) reversed enantioselectivity of 1,4-dioxane derivatives in α1-adrenergic and 5-HT1A receptor binding sites recognition.

5-HT(1A) receptor and α(1)-adrenoreceptor (α(1)-AR) binding sites recognized by the 1,4-dioxanes 2-4 display reversed stereochemical requirements. (S)-2 proved to be a potent 5-HT(1A) receptor agonist highly selective over α(1)-AR subtypes. Chirality influenced the anticancer activity of 3 and 4 in human prostate cancer cells (PC-3): (R)-4, eutomer at the α(1d)-AR subtype, was the most potent. The decreased effect of 4 and (R)-4 in α(1d)-AR silenced PC-3 cells confirmed that their anticancer activity was α(1d)-AR-dependent.

N-(4-Methoxy-2-nitrophenyl)hexadecanamide, a palmitoylethanolamide analogue, reduces formalin-induced nociception.

AIMS: To investigate the local antinociceptive effect as well as the possible mechanisms of action of a novel analogue of palmitoylethanolamide (PEA) N-(4-methoxy-2-nitrophenyl)hexadecanamide (HD) in the rat formalin test. MAIN METHODS: The formalin test was used to assess the antinociceptive activity of HD in vivo. The hydrolysis of anandamide catalyzed by fatty acid amide hydrolase (FAAH) was used to determine the action of HD on FAAH activity in vitro. KEY FINDINGS: Local peripheral ipisilateral, but not contralateral, administration of HD (10-100µg/paw) produced a dose-dependent antinociceptive effect in rats. The CB(1) and CB(2) receptor antagonists AM281 (0.3-30µg/paw) and SR144528 (0.3-30µg/paw), respectively, reduced the antinociceptive effect of HD (100µg/paw). In addition, methiothepin (0.03-0.3µg/paw) and naloxone (5-50µg/paw) significantly reduced HD-induced antinociception (100µg/paw). In vitro, HD reduced only to a minor extent the hydrolysis of anandamide catalyzed by FAAH. SIGNIFICANCE: HD local administration produces antinociception that probably results from an indirect activation of peripheral CB(1) and CB(2) cannabinoid receptors. Data suggest that 5-HT(1) and opioid receptors also participate in the antinociceptive effect of this compound. HD may have potential as analgesic drug.

Effect of chronic l-DOPA treatment on 5-HT(1A) receptors in parkinsonian monkey brain.

After chronic use of l-3,4-dihydroxyphenylalanine (l-DOPA), most Parkinson's disease (PD) patients suffer from its side effects, especially motor complications called l-DOPA-induced dyskinesia (LID). 5-HT(1A) agonists were tested to treat LID but many were reported to worsen parkinsonism. In this study, we evaluated changes in concentration of serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) and of 5-HT(1A) receptors in control monkeys, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys, dyskinetic MPTP monkeys treated chronically with l-DOPA, low dyskinetic MPTP monkeys treated with l-DOPA and drugs of various pharmacological activities: Ro 61-8048 (an inhibitor of kynurenine hydroxylase) or docosahexaenoic acid (DHA) and dyskinetic MPTP monkeys treated with l-DOPA+naltrexone (an opioid receptor antagonist). Striatal serotonin concentrations were reduced in MPTP monkeys compared to controls. Higher striatal 5-HIAA/serotonin concentration ratios in l-DOPA-treated monkeys compared to untreated monkeys suggest an intense activity of serotonin axon terminals but this value was similar in dyskinetic and nondyskinetic animals treated with or without adjunct treatment with l-DOPA. As measured by autoradiography with [(3)H]8-hydroxy-2-(di-n-propyl) aminotetralin (8-OH-DPAT), a decrease of 5-HT(1A) receptor specific binding was observed in the posterior/dorsal region of the anterior cingulate gyrus and posterior/ventral area of the superior frontal gyrus of MPTP monkeys compared to controls. An increase of 5-HT(1A) receptor specific binding was observed in the hippocampus of MPTP monkeys treated with l-DOPA regardless to their adjunct treatment. Cortical 5-HT(1A) receptor specific binding was increased in the l-DOPA-treated MPTP monkeys alone or with DHA or naltrexone and this increase was prevented in low dyskinetic MPTP monkeys treated with l-DOPA and Ro 61-8048. These results highlight the importance of 5-HT(1A) receptor alterations in treatment of PD with l-DOPA.

µ1- and 5-HT1-dependent mechanisms in the anterior pretectal nucleus mediate the antinociceptive effects of retrosplenial cortex stimulation in rats.

AIM: This study examines if injection of cobalt chloride (CoCl(2)) or antagonists of muscarinic cholinergic (atropine), µ(1)-opioid (naloxonazine) or 5-HT(1) serotonergic (methiothepin) receptors into the dorsal or ventral portions of the anterior pretectal nucleus (APtN) alters the antinociceptive effects of stimulating the retrosplenial cortex (RSC) in rats. MAIN METHOD: Changes in the nociceptive threshold were evaluated using the tail flick or incision pain tests in rats that were electrically stimulated at the RSC after the injection of saline, CoCl(2) (1 mM, 0.10 µL) or antagonists into the dorsal or ventral APtN. KEY FINDINGS: The injection of CoCl(2), naloxonazine (5 µg/0.10 µL) or methiothepin (3 µg/0.10 µL) into the dorsal APtN reduced the stimulation-produced antinociception from the RSC in the rat tail flick test. Reduction of incision pain was observed following stimulation of the RSC after the injection of the same substances into the ventral APtN. The injection of atropine (10 ng/0.10 µL) or ketanserine (5 µg/0.10 µL) into the dorsal or ventral APtN was ineffective against the antinociception resulting from RSC stimulation. SIGNIFICANCE: µ(1)-opioid- and 5-HT(1)-expressing neurons and cell processes in dorsal and ventral APtN are both implicated in the mediation of stimulation-produced antinociception from the RSC in the rat tail flick and incision pain tests, respectively.

Physiological, pathophysiological and therapeutic impact of the enteric serotonergic system.

Serotonin (5-hydroxytryptamine, 5-HT) induces various effects in the central nervous system, cardiovascular system and gastrointestinal tract. The response depends primarily on the nature of the 5-HT receptors involved. In the light of the current knowledge about the anatomy and physiology of the serotonergic system and the distribution of the various 5-HT receptors in the gut, the established and potential therapeutic impact of 5-HT receptor ligands are discussed. In particular, selective 5-HT receptor ligands influencing intestinal motility and pain perception such as the 5-HT4 receptor agonist prucalopride appear promising for the treatment of irritable bowel syndrome.

Synthesis, biological activities and bioavailability of moschamine, a safflomide-type phenylpropenoic acid amide found in Centaurea cyanus.

Moschamine is a phenylpropenoic acid amide found in plants. In this article, the synthesis and two biological activities (serotoninergic and cyclooxygenase (COX) inhibitory activities) and bioavailability of moschamine were described. Moschamine was synthesised and confirmed using NMR spectroscopic methods. Using the moschamine synthesised, serotoninergic and COX inhibitory activities were investigated. At the concentration of 10 µmol L⁻¹, moschamine was able to inhibit forskolin-stimulated cAMP formation by 25% (p < 0.015), via inhibiting serotonin receptors in the OK cells. The inhibition was repressed by two 5-HT1 antagonists (Nan-190 and spiperone), suggesting that moschamine may suppress cAMP formation via binding to 5-HT1 receptors in the cells. Also, moschamine was a very potent compound that is able to inhibit COX-I by 58% (p < 0.012) and COX-II by 54% (p < 0.014), at the concentration of 0.1 µmol L⁻¹. The oral bioavailability of moschamine was also determined in mice.

5-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-2(1H)-quinolinones and 3,4-dihydro-2(1H)-quinolinones: dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors. Part 3.

5-{2-[4-(2-Methyl-5-quinolinyl)-1-piperazinyl]ethyl}-2(1H)-quinolinones and 3,4-dihydro-2(1H)-quinolinones have been identified with different combinations of 5-HT(1) autoreceptor antagonist and hSerT potencies and excellent rat PK profiles. The availability of tool compounds with a range of profiles at targets known to play a key role in the control of synaptic 5-HT levels will allow exploration of different pharmacological profiles in a range of animal behavioral and disease models.

Involvement of 5-hydroxytryptaminergic transmission for the Mesobuthus tamulus venom-induced depression of spinal reflexes in neonatal rat in vitro.

Mesobuthus tamulus (MBT) venom is shown to depress the spinal reflexes through a mechanism unrelated to the NMDA receptors. 5-Hydroxytryptamine (5-HT) is another excitatory transmitter in the spinal cord therefore, the present study was undertaken to examine the involvement of 5-HT in the venom-induced depression of reflexes. The experiments were performed on isolated hemisected spinal cords from 4 to 6-day-old rats. Stimulation of a dorsal root with supramaximal strength evoked monosynaptic reflex (MSR) and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. MBT venom (0.3 microg/ml) depressed the spinal reflexes in a time-dependent manner and the maximal depression was seen at 10 min. The time to produce 50% depression (T-50) of MSR and PSR was 8.1+/-1.41 and 6.8+/-0.5 min, respectively. Pretreatment with pindolol (1 microM; 5-HT(1A/1B) receptor antagonist) blocked the reflexes up to 15 min. On the other hand, ketanserin (10 microM; 5-HT(2A/2C) receptor antagonist) or ondansetron (0.1 microM; 5-HT(3) receptor antagonist) blocked the venom-induced depression of MSR and PSR during entire exposure time (30 min). The 5-HT concentration of the cords exposed to venom (1.6+/-0.04 microg/g tissue) was significantly greater than the control group (0.98+/-0.08 microg/g tissue). The results indicate that venom-induced depression of spinal reflexes is mediated via 5-HTergic transmission involving 5-HT(1A/1B), 5-HT(2A/2C) and 5-HT(3) receptors.

Inverse agonist and neutral antagonist actions of synthetic compounds at an insect 5-HT1 receptor.

BACKGROUND AND PURPOSE: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT(1) receptor of an insect model for neurobiology, physiology and pharmacology. EXPERIMENTAL APPROACH: A cDNA encoding for the Periplaneta americana 5-HT(1) receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. KEY RESULTS: The P. americana 5-HT(1) receptor (Pea5-HT(1)) shares pronounced sequence and functional similarity with mammalian 5-HT(1) receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT(1) was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. CONCLUSIONS AND IMPLICATIONS: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT(1) receptor. The results presented here should facilitate further analyses of 5-HT(1) receptors in mediating central and peripheral effects of 5-HT in insects.

Attenuation of somatodendritic responses to 8-hydroxy-2-di-npropylamino tetralin following long-term dietary sugar consumption in rats.

OBJECTIVE: To determine changes in response to a selective serotonin-1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) following long-term consumption of sugar as part of meal in rats. STUDY DESIGN: Experimental study. PLACE AND DURATION OF STUDY: The Department of Biochemistry, University of Karachi, from June to August 2005. METHODOLOGY: The study was conducted on 24 male albino Wistar rats. Sugar containing diet was prepared by mixing standard rodent diet and table sugar in the ratio of 3:1 (w/w) and rats were fed freely on this diet. Control rats were fed freely on standard rodent diet. After five weeks of treatment, control and sugar diet treated animals were injected with 8-OH-DPAT, at a dose of 0.5 mg/ml/kg, to monitor the effects of drug on food intake and brain serotonin (5-hydroxytryptamine, 5-HT) metabolism. Dissected neural tissue was analyzed electrochemically and findings were compared by Newman-Keuls test. RESULTS: Administration of 8-OH-DPAT elicited hyperphagia and decreased 5-HT metabolism in normal diet treated rats. The neurochemical and hyperphagic responses to 8-OH-DPAT were smaller in sugar than normal diet treated animals suggesting a downregulation of somatodendritic responses in sugar diet treated animals. CONCLUSION: A decrease in serotonin metabolism but not an increase in the responsiveness of somatodendritic 5-HT-1A receptors is involved in sugar-rich diet induced hyperphagia.

Characterization of serotonin receptors in pregnant human myometrium.

The monoamine, 5-hydroxytryptamine (5-HT), stimulates contraction of human uterine smooth muscle (myometrium), but the receptor subtypes involved have not been characterized. We studied the effects of a range of 5-HT receptor subtype-selective agonists and antagonists in isolated strips of myometrium obtained at the time of caesarean section. The 5-HT(1A) receptor agonist, 8-hydroxy-2-dipropylaminotetralin, produced an increase in contractions that was highly variable, of low potency, and was not significantly inhibited by the 5-HT(1A) antagonist WAY100635 [[O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide]. The 5-HT(2) receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT), produced a strong, consistent, and concentration-dependent stimulation of contractions (pEC(50) = 7.60 +/- 0.10, n = 5). The 5-HT(2A) receptor antagonist, ketanserin [3-[2-[4-(4-fluoro benzoyl)-piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione], caused a parallel shift in the response to alpha-Me-5-HT, with a pK(B) value consistent with its known affinity for the 5-HT(2A) receptor (pK(B) = 8.47 +/- 0.16, n = 5), but it had no effect on the response to oxytocin. The 5-HT(2B) and 5-HT(2C) receptor agonists, BW723C86 [(alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine)] and Ro-60-01-75 [(S)-2-(6-chloro-5-fluoro-indol-1-yl)-1-methyl-ethylamine fumarate], produced inconsistent responses at potencies that were lower than expected for activation of their cognate receptors. The response to alpha-Me-5-HT was unaffected by the 5-HT(2B) and 5-HT(2C) receptor antagonists, SB204741 [(N-(1-methyl-1H-indolyl-5-yl)-N-(3-methyl-5-isothiazolyl)urea)] and RS102221 [8-[5-(2,4-dimethoxy-5-(4-trifluoromethyl phenylsulphonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione]. The 5-HT(1B/1D) receptor agonist, sumatriptan [1-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-methyl-methanesulfonamide], the 5-HT(4) agonist, cisapride [4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)propyl]-3-methoxy-4-piperidyl]-2-methoxy-benzamide], and the 5-HT(7) agonist, AS19 [(2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin], all had no effect on myometrial contractility. 5-HT(2A) receptor mRNA and immunoreactivity were identified using reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. Specific binding of [(3)H]ketanserin was demonstrated. This study provides strong evidence for the expression of contractile 5-HT(2A) receptors in pregnant human myometrium, and this receptor is a potential target for novel uterotonic therapies.

Studies on a series of potent, orally bioavailable, 5-HT(1) receptor ligands--part II.

A series of 5-(piperidinylethyloxy)quinoline 5-HT(1) receptor ligands have been studied by elaboration of the series of dual 5-HT(1)-SSRIs reported previously. These new compounds display a different in vitro pharmacological profile with potent affinity across the 5-HT(1A), 5-HT(1B) and 5-HT(1D) receptors and selectivity against the serotonin transporter. Furthermore, they have improved pharmacokinetic profiles and CNS penetration.

Intracerebroventricular serotonin reduces the degree of acute hypoxic ventilatory depression in peripherally chemodenervated rabbits.

Hypoxia causes changes in the rate of synthesis or release of neurotransmitters in the brain. The accumulation of serotonin (5-HT) in the central nervous system might cause hypoxic respiratory depression. In the present study, we aimed to examine the role of central 5-HT on normoxic and acute hypoxic ventilatory depression (AHVD) in peripheral chemoreceptors denervated rabbits. All experiments were performed in peripherally chemodenervated rabbits anesthetized with intravenous injection of urethane (400 mg/kg) and alpha-chloralose (40 mg/kg). For intracerebroventricular (ICV) administration of 5-HT (20 microg/kg) and ketanserin (10 microg/kg), a cannula was placed in left lateral ventricle by stereotaxic method. Respiratory frequency (fR), tidal volume (VT), ventilation minute volume (VE) and systemic arterial bood pressure (BP) were recorded in each experimental phases and mean arterial pressure was calculated (MAP). Heart rate (HR) was also determined from the pulsation of BP. The effects of ICV serotonin and ICV ketanserin on the indicated parameters during air breathing (normoxia) and breathing of hypoxia (8% O2--92% N2) were investigated. During hypoxia, fR, VT, VE, MAP and HR decreased, and AHVD was thus obtained. ICV injection of 5-HT during normoxia caused significant increases in VT (P < 0.001) and in VE (P < 0.01). On the other hand, ICV 5-HT injection reduced the degree of AHVD in peripherally chemodenervated rabbits during hypoxia (fR; P < 0.05, VT; P < 0.05 and VE; P < 0.01). After ICV injection of ketanserin, the enhancement of 5-HT on VE was prevented during normoxia. On the breathing of hypoxic gas after ICV ketanserin, the degree of AHVD was augmented. In conclusion, our findings suggested that central 5-HT increases normoxic ventilation and reduces the degree of AHVD during hypoxia and that ICV ketanserin prevents the stimulatory effect of 5-HT on respiration and augments AHVD.

Synthesis and biological affinity of new imidazo- and indol-arylpiperazine derivatives: further validation of a pharmacophore model for alpha(1)-adrenoceptor antagonists.

In the continuing search for selective alpha(1)-adrenoceptor (AR) antagonists, new alkoxyarylpiperazinylalkylpyridazinone derivatives were designed and synthesized. The new compounds were tested for their affinity toward alpha(1)-AR, alpha(2)-AR and 5-HT(1A) receptors. The ability of these compounds to inhibit the serotonin transporters (SERT) was also determined. The pharmacological data confirm that increasing the size of the ortho alkoxy substituent on the phenyl ring of the arylpiperazine moiety afforded compounds with enhanced affinity toward the alpha(1)-AR. The isopropoxy group, the largest group evaluated, led the best alpha(1)-AR affinity profile. In contrast, the compounds which have an amide group within of the o-alkoxy-phenylpiperazine fragment showed low affinity toward the receptors studied. Similar results were obtained when the amide group was present in the linker of the junction between the two major constituents of the molecule.

Norepinephrine-mediated regulation of 5HT1 receptor functioning in human platelets.

Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.