Synthesis, binding affinity and SAR of new benzolactam derivatives as dopamine D3 receptor ligands

A series of new benzolactam derivatives was synthesized and the derivatives were evaluated for theiraffinities at the dopamine D1, D2, and D3 receptors. Some of these compounds showed high D2 and/orD3 affinity and selectivity over the D1 receptor. The SAR study of these compounds revealed structuralcharacteristics that decisively influenced their D2 and D3 affinities. Structural models of the complexesbetween some of the most representative compounds of this series and the D2 and D3 receptors wereobtained with the aim of rationalizing the observed experimental results. Moreover, selected compoundsshowed moderate binding affinity on 5-HT2A which could contribute to reducing the occurrence of extrapyramidalside effects as potential antipsychotics.

Impact of genetic SLC28 transporter and ITPA variants on ribavirin serum level, hemoglobin drop and therapeutic response in patients with HCV infection.

BACKGROUND & AIMS: In the last decade, pegylated interferon-α (PegIFN-α) plus ribavirin (RBV) was the standard treatment of chronic hepatitis C for genotype 1, and it remains the standard for genotypes 2 and 3. Recent studies reported associations between RBV-induced anemia and genetic polymorphisms of concentrative nucleoside transporters such as CNT3 (encoded by SLC28A3) and inosine triphosphatase (encoded by ITPA). We aimed at studying genetic determinants of RBV kinetics, efficacy and treatment-associated anemia. METHODS: We included 216 patients from two Swiss study cohorts (61% HCV genotype 1, 39% genotypes 2 or 3). Patients were analyzed for SLC28A2 single nucleotide polymorphism (SNP) rs11854484, SLC28A3 rs56350726, and SLC28A3 rs10868138 as well as ITPA SNPs rs1127354 and rs7270101, and followed for treatment-associated hemoglobin changes and sustained virological response (SVR). In 67 patients, RBV serum levels were additionally measured during treatment. RESULTS: Patients with SLC28A2 rs11854484 genotype TT had higher dosage- and body weight-adjusted RBV levels than those with genotypes TC or CC (p=0.02 and p=0.06 at weeks 4 and 8, respectively). ITPA SNP rs1127354 was associated with hemoglobin drop ≥3 g/dl during treatment, in genotype (relative risk (RR)=2.1, 95% CI 1.3-3.5) as well as allelic analyses (RR=2.0, 95%CI 1.2-3.4). SLC28A3 rs56350726 was associated with SVR in genotype (RR=2.2; 95% CI 1.1-4.3) as well as allelic analyses (RR=2.0, 95% CI 1.1-3.4). CONCLUSIONS: The newly identified association between RBV serum levels and SLC28A2 rs11854484 genotype, as well as the replicated association of ITPA and SLC28A3 genetic polymorphisms with RBV-induced anemia and treatment response, may support individualized treatment of chronic hepatitis C and warrant further investigation in larger studies.

A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice.

The high Km glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic beta-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic beta-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.

Differential effects of glutamate transporter inhibitors on the global electrophysiological response of astrocytes to neuronal stimulation

Astrocytes are responsible for regulating extracellular levels of glutamate and potassium during neuronal activity. Glutamate clearance is handled by glutamate transporter subtypes glutamate transporter 1 and glutamate-aspartate transporter in astrocytes. DL-threo-beta-benzyloxyaspartate (TBOA) and dihydrokainate (DHK) are extensively used as inhibitors of glial glutamate transport activity. Using whole-cell recordings, we characterized the effects of both transporter inhibitors on afferent-evoked astrocyte currents in acute cortical slices of 3-week-old rats. When neuronal afferents were stimulated, passive astrocytes responded by a rapid inward current followed by a persistent tail current. The first current corresponded to a glutamate transporter current. This current was inhibited by both inhibitors and by tetrodotoxin. The tail current is an inward potassium current as it was blocked by barium. Besides inhibiting transporter currents, TBOA strongly enhanced the tail current. This effect was barium-sensitive and might be due to a rise in extracellular potassium level and increased glial potassium uptake. Unlike TBOA, DHK did not enhance the tail current but rather inhibited it. This result suggests that, in addition to inhibiting glutamate transport, DHK prevents astrocyte potassium uptake, possibly by blockade of inward-rectifier channels. This study revealed that, in brain slices, glutamate transporter inhibitors exert complex effects that cannot be attributed solely to glutamate transport inhibition.

Resistance to diet-induced obesity and associated metabolic perturbations in haploinsufficient monocarboxylate transporter 1 mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 (+/-) mice developed normally. However, when fed high fat diet (HFD), MCT1 (+/-) mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 (+/+) mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 (+/-) mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O2 consumption and CO2 production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 (+/+) mice when fed HFD, were reduced in MCT1 (+/-) mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 (+/+) mice under high fat diet was prevented in the liver of MCT1 (+/-) mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.

Regional variations in ABC transporter expression along the mouse intestinal tract.

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

An Overview of the association between schizotypy and dopamine

Schizotypy refers to a constellation of personality traits that are believed to mirror the subclinical expression of schizophrenia in the general population. Evidence from pharmacological studies indicates that dopamine is involved in the aetiology of schizophrenia. Based on the assumption of a continuum between schizophrenia and schizotypy, researchers have begun investigating the association between dopamine and schizotypy using a wide range of methods. In this article, we review published studies on this association from the following areas of work: (1) Experimental investigations of the interactive effects of dopaminergic challenges and schizotypy on cognition, motor control and behaviour, (2) dopaminergically supported cognitive functions, (3) studies of associations between schizotypy and polymorphisms in genes involved in dopaminergic neurotransmission, and (4) molecular imaging studies of the association between schizotypy and markers of the dopamine system. Together, data from these lines of evidence suggest that dopamine is important to the expression and experience of schizotypy and associated behavioural biases. An important observation is that the experimental designs, methods, and manipulations used in this research are highly heterogeneous. Future studies are required to replicate individual observations, to enlighten the link between dopamine and different schizotypy dimensions (positive, negative, cognitive disorganisation), and to guide the search for solid dopamine-sensitive behavioural markers. Such studies are important in order to clarify inconsistencies between studies. More work is also needed to identify differences between dopaminergic alterations in schizotypy compared to the dysfunctions observed in schizophrenia.

Gene deletion of dopamine beta-hydroxylase and alpha1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling.

In vitro studies have shown that stimulation of alpha1-adrenoceptors (ARs) directly induces proliferation, hypertrophy, and migration of arterial smooth muscle cells and adventitial fibroblasts. In vivo studies confirmed these findings and showed that catecholamine trophic activity becomes excessive after experimental balloon injury and contributes to neointimal growth, adventitial thickening, and lumen loss. However, past studies have been limited by selectivity of pharmacological agents. The aim of this study, in which mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase (DBH-/-)] or deficient in alpha1-AR subtypes expressed in murine carotid (alpha1B-AR-/- and alpha1D-AR-/-) were used, was to test the hypothesis that catecholamines contribute to wall hypertrophy after injury. At 3 wk after injury of wild-type mice, lumen area and carotid circumference increased significantly, and hypertrophy of media and adventitia was in excess of that needed to restore circumferential wall stress to normal. In DBH-/- and alpha1B-AR-/- mice, increases in lumen area, circumference, and hypertrophy of the media and adventitia were reduced by 50-91%, resulting in restoration of wall tension to nearly normal (DBH-/-) or normal (alpha1B-AR-/-). In contrast, in alpha1D-AR-/- mice, increases in lumen area, circumference, and wall hypertrophy were unaffected and wall thickening remained in excess of that required to return tension to normal. When examined 5 days after injury, proliferation and leukocyte infiltration were inhibited in DBH-/- mice. These studies suggest that the trophic effects of catecholamines are mediated primarily by alpha1B-ARs in mouse carotid and contribute to hypertrophic growth after vascular injury.

Interaction of alpha-melanotropin with central dopamine systems: role of hormonal state and molecular structure

The tubero-infundibular and nigrostriatal DA neurone systems of rats respond to systemic (i.p.) injection of alpha-MSH (2-100 microgram/kg). The response of the tubero-infundibular (arcuate) DA neurones, an increase in cellular fluorescence intensity which can be interpreted as a sign of increased neuronal activity, is essentially the same in males, estrogen-progesterone-pretreated ovariectomized females and hypophysectomized males, whereas the type of response elicited by alpha-MSH in the nigral DA neurones depends upon the hormonal state of the animal. Differences between the two DA neurone groups exist also with regard to the effects of peptide fragments containing the two active sites of the alpha-MSH molecule. Results of lesion experiments in the lower brainstem (area postrema) and of blockade of muscarinic mechanisms by atropine further point to differences in the mechanisms underlying the peptide effects on the two neurone systems. The reaction of the tubero-infundibular DA system (which controls the pars intermedia of the pituitary) can be considered to reflect the activation of a feedback mechanism on MSH secretion, while the functional counterpart of the changes observed in the nigral system remains unknown at the present time.

Requirement of glucose metabolism for regulation of glucose transporter type 2 (GLUT2) gene expression in liver.

Previous studies have shown that glucose increases the glucose transporter (GLUT2) mRNA expression in the liver in vivo and in vitro. Here we report an analysis of the effects of glucose metabolism on GLUT2 gene expression. GLUT2 mRNA accumulation by glucose was not due to stabilization of its transcript but rather was a direct effect on gene transcription. A proximal fragment of the 5' regulatory region of the mouse GLUT2 gene linked to a reporter gene was transiently transfected into liver GLUT2-expressing cells. Glucose stimulated reporter gene expression in these cells, suggesting that glucose-responsive elements were included within the proximal region of the promoter. A dose-dependent effect of glucose on GLUT2 expression was observed over 10 mM glucose irrespective of the hexokinase isozyme (glucokinase K(m) 16 mM; hexokinase I K(m) 0.01 mM) present in the cell type used. This suggests that the correlation between extracellular glucose and GLUT2 mRNA concentrations is simply a reflection of an activation of glucose metabolism. The mediators and the mechanism responsible for this response remain to be determined. In conclusion, glucose metabolism is required for the proper induction of the GLUT2 gene in the liver and this effect is transcriptionally regulated.

Immunodensity and mRNA expression of A2A adenosine, D2 dopamine, and CB1 cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment.

RATIONALE: Dopamine D2 receptors are the main target of antipsychotic drugs. In the brain, D2 receptors coexpress with adenosine A2A and CB1 cannabinoid receptors, leading to functional interactions. OBJECTIVES: The protein and messenger RNA (mRNA) contents of A2A, D2, and CB1 receptors were quantified in postmortem prefrontal cortex of subjects with schizophrenia. MATERIALS AND METHODS: The study was performed in subjects suffering schizophrenia (n=31) who mainly died by suicide, matched with non-schizophrenia suicide victims (n=13) and non-suicide controls (n=33). The density of receptor proteins was evaluated by immunodetection techniques, and their relative mRNA expression was quantified by quantitative real-time polymerase chain reaction. RESULTS: In schizophrenia, the densities of A2A (90+/-6%, n=24) and D2-like receptors (95+/-5%, n=22) did not differ from those in controls (100%). Antipsychotic treatment did not induce changes in the protein expression. In contrast, the immunodensity of CB1 receptors was significantly decreased (71+/-7%, n=11; p<0.05) in antipsychotic-treated subjects with schizophrenia but not in drug-free subjects (104+/-13%, n=11). The relative mRNA amounts encoding for A2A, D2, and CB1 receptors were similar in brains of drug-free, antipsychotic-treated subjects with schizophrenia and controls. CONCLUSIONS: The findings suggest that antipsychotics induce down-regulation of CB1 receptors in brain. Since A2A, D2, and CB1 receptors coexpress on brain GABAergic neurons and reductions in markers of GABA neurotransmission have been identified in schizophrenia, a lower density of CB1 receptor induced by antipsychotics could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release, contributing to the normalization of cognitive functions in the disorder.

Thermogenic and metabolic effects of dopamine in healthy men.

OBJECTIVE: To assess the thermogenic response of dopamine at three different infusion rates and to analyze its effects on various biochemical variables. DESIGN: Randomized sequential experimental treatment bracketed by control periods. PATIENTS: Eight young healthy male volunteers with normal body weight (51 to 89 kg). INTERVENTIONS: Three experimental periods during which dopamine was administered iv in a randomized order at rates of 2.5, 5, or 10 micrograms/kg.min with one preinfusion baseline and two recovery periods in between. MEASUREMENTS AND MAIN RESULTS: A significant (p less than .01) increase in resting energy expenditure was observed in response to the two highest dopamine infusion rates (5 and 10 micrograms/kg.min), corresponding to 6% and 15% median increases, respectively, as compared with preinfusion values. At the lowest dopamine infusion rate, no variation in resting energy expenditure was observed. Dopamine induced a significant (p less than .01) increase in hyperglycemia at all three infusion rates, and, at the highest infusion rate, dopamine induced a significant (p less than .05) increase of plasma free fatty acid concentrations. Insulin plasma concentrations were significantly (p less than .05 to p less than 0.1) increased at the three dopamine infusion rates. CONCLUSIONS: Dopamine infusion produces a dose-dependent thermogenic effect and induces various metabolic actions in man.

Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

C/EBPbeta couples dopamine signalling to substance P precursor gene expression in striatal neurones.

Dopamine-induced changes in striatal gene expression are thought to play an important role in drug addiction and compulsive behaviour. In this study we report that dopamine induces the expression of the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBP)-beta in primary cultures of striatal neurones. We identified the preprotachykinin-A (PPT-A) gene coding for substance P and neurokinin-A as a potential target gene of C/EBPbeta. We demonstrated that C/EBPbeta physically interacts with an element of the PPT-A promoter, thereby facilitating substance P precursor gene transcription. The regulation of PPT-A gene by C/EBPbeta could subserve many important physiological processes involving substance P, such as nociception, neurogenic inflammation and addiction. Given that substance P is known to increase dopamine signalling in the striatum and, in turn, dopamine increases substance P expression in medium spiny neurones, our results implicate C/EBPbeta in a positive feedback loop, changes of which might contribute to the development of drug addiction.