Translational neurophysiological markers for activity of the metabotropic glutamate receptor (mGluR2) modulator JNJ-40411813: Sleep EEG correlates in rodents and healthy men.

Alterations in rapid eye movement sleep (REM) have been suggested as valid translational efficacy markers: activation of the metabotropic glutamate receptor 2 (mGluR2) was shown to increase REM latency and to decrease REM duration. The present paper addresses the effects on vigilance states of the mGluR2 positive allosteric modulator (PAM) JNJ-40411813 at different circadian times in rats and after afternoon dosing in humans. Due to its dual mGluR2 PAM/serotonin 2A (5-HT2A) receptor antagonism in rodents, mGlu2R specificity of effects was studied in wild-type (WT) and mGluR2 (-/-) mice. 5-HT2A receptor occupancy was determined in humans using positron emission tomography (PET). Tolerance development was examined in rats after chronic dosing. EEG oscillations and network connectivity were assessed using multi-channel EEG. In rats, JNJ-40411813 increased deep sleep time and latency of REM onset but reduced REM time when administered 2 h after 'lights on' (CT2): this was sustained after chronic dosing. At CT5 similar effects were elicited, at CT10 only deep sleep was enhanced. Withdrawal resulted in baseline values, while re-administration reinstated drug effects. Parieto-occipital cortical slow theta and gamma oscillations were correlated with low locomotion. The specificity of functional response was confirmed in WT but not mGluR2 (-/-) mice. A double-blind, placebo-controlled polysomnographic study in healthy, elderly subjects showed that 500 mg of JNJ-40411813 consistently increased deep sleep time, but had no effect on REM parameters. This deep sleep effect was not explained by 5-HT2A receptor binding, as in the PET study even 700 mg only marginally displaced the tracer. JNJ-40411813 elicited comparable functional responses in rodents and men if circadian time of dosing was taken into account. These findings underscore the translational potential of sleep mechanisms in evaluating mGluR2 therapeutics when administered at the appropriate circadian time.

N-aryl piperazine metabotropic glutamate receptor 5 positive allosteric modulators possess efficacy in preclinical models of NMDA hypofunction and cognitive enhancement.

Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca(2+) mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.

Progress in the developement of positive allosteric modulators of the metabotropic glutamate receptor 2.

The metabotropic glutamate type 2 (mGlu2) receptor is a G-protein coupled receptor (GPCR) expressed on presynaptic nerve terminals where it negatively modulates glutamate and GABA release. Mixed mGlu2/mGlu3 orthosteric agonists such as LY354740 have shown activity in a range of preclinical animal models of anxiety and schizophrenia. Clinical work with LY354740 demonstrated activity in a CO(2) inhalation study suggesting application in the treatment of anxiety related disorders. Subsequently, a related prodrug LY2140023 demonstrated improvements in positive and negative symptoms in patients suffering from schizophrenia. These molecules exhibit combined mGlu2/mGlu3 activity although there is evidence from knock-out studies that preclinical anti-psychotic effects may be mediated via the mGlu2 receptor. An alternative avenue for modulating GPCRs is to act via allosteric mechanisms, binding at a different site from the orthosteric agonist. Since the first discovery of mGlu2 positive allosteric modulators (PAMs) such as 2,2,2-TEMPS and BINA, multiple families of mGlu2 modulators have been reported and several have entered into clinical development. This review focuses on recent advances in the development of novel mGlu2 PAMs by analysis of compounds disclosed in research articles and patent literature between 2007 and 2010.

Breed differences in clearance of porcine FSH in hypophysectomized rats.

Extracts of anterior pituitary (AP) glands were infused i.v. into hypophysectomized male rats followed by sequential sampling of blood for 120 min. Determination of follicle-stimulating hormone (FSH) concentrations established that FSH from Chinese Meishan males decreased in the circulation of rats more slowly than FSH in extracts of AP from crossbred occidental pigs (P<0.003). Additionally, FSH from AP extracts of castrated males disappeared somewhat more slowly (P<0.06) than FSH from extracts of boars. Evaluation of FSH by bioassay and radioimmunoassay yielded similar concentrations in AP from Meishan and crossbred boars. Serum testosterone concentrations increased with time through 90 min after infusion of AP, but the rate of increase of testosterone was not related to amount of luteinizing hormone (LH) that was administered indicating LH receptor saturation. Unexpectedly, the rate of increase in testosterone was more rapid with AP extracts from boars than with extracts from castrated males. Observations from the current study imply structural alterations of FSH in the AP of Meishan males relative to crossbred males allowing sustained concentrations in the circulation, and this FSH possesses similar activation of the FSH receptor. The amount of LH in the AP extracts saturated the LH receptors of the hypophysectomized male rats, but some factor in extracts of boars differed from those of castrated males.

Giving and receiving of gifts between pharmaceutical companies and medical specialists in Australia.

BACKGROUND: This study investigated the 'gift-relationship' between pharmaceutical companies and doctors. METHODS: The study was based on a survey questionnaire of 823 medical specialists from across Australia. The aim of this study was to investigate gifts offered to medical specialists in Australia by pharmaceutical companies, financial support actively sought by medical specialists for activities other than research and to consider what is ethically appropriate. RESULTS: A high percentage of specialists received offers of food (96%), items for the office (94%), personal gifts (51%) and journals or textbooks (50%). Most specialists were invited to product launches, symposia or educational events (75-84%) and 52% received offers of travel to conferences. A high proportion of offers were accepted (66-79%) except invitations to product launches (49%), sponsored symposia (53%) and offers of travel that included partners (27%). Fifteen per cent of specialists requested financial support from pharmaceutical companies for activities and items, including conferences, travel, educational activities, salaries and donations to specific funds. The study outlined guidelines on gifts from pharmaceutical companies and differing standards applying to gifts and grants for travel. We found that, although most gifts and requests for support complied with professional and pharmaceutical industry guidelines, some--including personal gifts, tickets to sporting events, entertainment and travel expenses for specialists' partners--did not. CONCLUSION: To ensure that physicians' judgements are free from real or perceived influence from industry and to maintain public trust, we support a shift towards more conservative standards on gifts and support for travel evident in recent guidelines.

Partial restoration of lutropin activity by an intersubunit disulfide bond: implications for structure/function studies.

Gonadal function is controlled by lutropins and follitropins, heterodimeric cystine knot proteins that have nearly identical alpha-subunits. These heterodimeric proteins are stabilized by a portion of the hormone-specific beta-subunit termed the "seatbelt" that is wrapped around alpha-subunit loop 2 (alpha 2). Here we show that replacing human chorionic gonadotropin (hCG) alpha 2 residue Lys51 with cysteine or alanine nearly abolished its lutropin activity, an observation that implies that alpha Lys51 has a key role in hormone activity. The activity of the heterodimer containing alpha K51C, but not that containing alpha K51A, was increased substantially when beta-subunit seatbelt residue beta Asp99 was converted to cysteine. As had been reported by others, heterodimers containing alpha K51C and beta D99C were crosslinked by a disulfide. The finding that an intersubunit disulfide restored some of the activity lost by replacing alpha Lys51 suggests that this residue is not crucial for receptor binding or signaling and also that hCG and related hormones may be particularly sensitive to mutations that alter interactions between their subunits. We propose the unique structures of hCG and related family members may permit some subunit movement in the heterodimer, making it difficult to deduce key residues involved in receptor contacts simply by correlating the activities of hormone analogs with their amino acid sequences.

Design and synthesis of novel 2,3-dihydro-1H-isoindoles with high affinity and selectivity for the dopamine D3 receptor.

Starting from the tetrahydroisoquinoline SB-277011 1, a novel series of 5-substituted-2,3-dihydro-1H-isoindoles has been designed. Subsequent optimisation resulted in identification of 19, which has high affinity for the dopamine D3 receptor (pKi 8.3) and > or = 100-fold selectivity over other aminergic receptors. In rat studies 19 was brain penetrant with an excellent pharmacokinetic profile (oral bioavailability 77%, t1/2 5.2h).

Effects of nitric oxide synthase inhibition on angiotensin receptors and metabolism in the pregnant hypertensive rat.

Endothelial dysfunction and a consequent decrease in nitric oxide production have been implicated in the pathogenesis of pre-eclampsia. A prominent feature of the pre-eclamptic syndrome is a loss of the pregnancy-induced refractoriness to infused pressor agents, such as angiotensin. In this study, we sought to determine whether a decrease in nitric oxide production might be linked via changes in angiotensin II receptors and angiotensin II metabolism to changes in pressor sensitivity to infused angiotensin II. Pregnant and non-pregnant spontaneously hypertensive rats (SHRs) were randomly allocated to receive 5 mg x kg(-1) x day(-1) N(G)-nitro-L-arginine methyl ester (L-NAME) in the drinking water or drinking water alone from days 7 to 14 of gestation. Steady-state metabolic clearance studies of angiotensin II were then performed, or tissues were harvested for angiotensin II receptor studies. Treatment with L-NAME caused an increase in systolic pressure (P<0.001) in both pregnant and non-pregnant rats, while urinary protein excretion increased only in the pregnant SHRs (P<0.001). Plasma angiotensin II levels were significantly increased in the L-NAME-treated SHRs compared with controls (non-pregnant, P<0.0005; pregnant, P<0.01). The metabolic clearance rate of angiotensin II was decreased by L-NAME treatment in non-pregnant SHRs (P<0.05), but was increased by L-NAME treatment in the pregnant rats (P<0.01). In the aorta, the angiotensin II receptor number increased after treatment with L-NAME in both non-pregnant (P<0.0005) and pregnant (P<0.05) SHRs, and the dissociation constant increased in the non-pregnant SHRs (P<0.005). Thus treatment of SHRs with L-NAME increased blood pressure, as well as the circulating angiotensin II concentration and vascular angiotensin II receptor expression. However, treatment with L-NAME did not affect pressor sensitivity to infused angiotensin II. We conclude, therefore, that although a decrease in nitric oxide production is associated with changes in angiotensin II concentrations and receptor numbers, it does not induce changes in pressor sensitivity to infused angiotensin II in the SHR.

Novel 2,3,4,5-tetrahydro-1H-3-benzazepines with high affinity and selectivity for the dopamine D3 receptor.

Starting from the dopamine D3 receptor antagonist SB-277011 1, a series of 2,3,4,5-tetrahydro-1H-3-benzazepines has been identified with high affinity for the dopamine D3 receptor and selectivity over the D2 receptor. The 3-acetamido-2-fluorocinnamide derivative 20 gave high D3 receptor affinity (pKi 8.4) with 130-fold selectivity over the 2, receptor.

Positive association between expression of follicle-stimulating hormone beta and activin betaB-subunit genes in boars.

This study tested our hypothesis that inhibin/activin (I/A) betaB subunit and not follistatin (FS) gene expression relates positively to plasma FSH concentrations in the anterior pituitary gland of boars. Mature crossbred boars (n = 12) were selected for divergence in plasma FSH concentrations, and their anterior pituitary glands were evaluated for expression of the FSHbeta, I/A ssB, FS, calmodulin, and GnRH receptor (GnRH-R) genes by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and/or RNase protection assays (RPAs). Expression of I/A ssB was greater (p < 0. 01) in the six boars with high FSH than in the six with low FSH; expression of the I/A betaB-subunit gene was positively correlated to that of the FSHbeta gene (RT-PCR: r = 0.96; p < 0.01; RPA: r = 0.68; p < 0.05). In contrast, expression of the FS (p > 0.10), GnRH-R (p > 0. 08), and calmodulin (p > 0.10) genes was similar in the two groups of boars. Additionally, expression of the FSHbeta gene was correlated positively with pituitary and plasma FSH concentrations (r = 0.69 and 0.88, respectively; p < 0.05). These results support the hypothesis that activin B is partially responsible for elevated FSH concentrations in boars. Furthermore, the expression difference of the calmodulin gene observed previously between Meishan and White Composite boars represents a breed difference unrelated to FSH.

Development of a new assay for measurement of total angiotensin-converting enzyme after inhibition.

1. We set out to develop a simple, rapid, highly sensitive and reproducible assay to quantify total angiotensin-converting enzyme (ACE) in serum and tissues after ACE inhibition. 2. Total ACE was measured by dissociating ACE inhibitor from the enzyme using zinc chelation (EDTA). The enzyme reconstituted with zinc ion (ZnSO4) was then measured by enzymatic fluorimetric assay. Angiotensin-converting enzyme inhibition was produced in vitro by incubation of enalaprilat or perindoprilat with human serum or cell membranes from rat heart. To achieve the highest recovery rate, we studied concentrations of EDTA from 1 to 300 mmol/L with a pH range from 4 to 13, where the incubation times of EDTA were between 1 and 12 h. After washing off ACE inhibitor and excess EDTA, the resulting concentrates and pellets were then resuspended with Tris buffer containing ZnSO4 to restore ACE activity. 3. The optimal assay conditions to dissociate the drug from the enzyme were 300 mmol/L EDTA in Tris buffer at pH 11 for 12 h with cell membranes and 23 mmol/L EDTA at the same pH incubated for 4 h with serum. The recovery of ACE activity was 81.7 +/- 15.8 and 97.3 +/- 2.9% in tissues and serum, respectively. Intra- and interassay variability coefficients were 5.6 and 12.8% in tissue, respectively, 3.2 and 13.0% in serum, respectively. The method allows quantification of ACE in 100 microliters or less serum and 20 mg or less tissue. 4. The total ACE concentration after ACE inhibition can be determined by this assay, which is suitable for micro-sampling studies of both tissue and serum ACE levels.

Epitope-specific focusing of the immune response to a minimized human chorionic gonadotropin analog.

Minimized proteins have long been used to elicit an immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. This approach works well for linear epitopes but poorly for conformational epitopes. Here we describe a homodimeric human chorionic gonadotropin (hCG) analog that retains the conformation of related parts of hCG and elicits high affinity specific antibodies. This novel immunogen displays the tertiary structure of selected loops of the protein but lacks structures that could elicit potentially undesirable antibodies.

Spectrum of 100-kyr glacial cycle: orbital inclination, not eccentricity.

Spectral analysis of climate data shows a strong narrow peak with period approximately 100 kyr, attributed by the Milankovitch theory to changes in the eccentricity of the earth's orbit. The narrowness of the peak does suggest an astronomical origin; however the shape of the peak is incompatible with both linear and nonlinear models that attribute the cycle to eccentricity or (equivalently) to the envelope of the precession. In contrast, the orbital inclination parameter gives a good match to both the spectrum and bispectrum of the climate data. Extraterrestrial accretion from meteoroids or interplanetary dust is proposed as a mechanism that could link inclination to climate, and experimental tests are described that could prove or disprove this hypothesis.

Nitric oxide synthase inhibition in a spontaneously hypertensive rat model of diabetic nephropathy.

1. To investigate the role of nitric oxide (NO) in diabetic nephropathy the effect of nitric oxide synthase (NOS) inhibition by NG-nitro-L-arginine methyl ester (L-NAME) was observed in a streptozotocin diabetic spontaneously hypertensive rat (SHR) model. 2. Two groups of SHR (n = 8) with streptozotocin-induced diabetes were studied. One group was given L-NAME 5 mg/kg bodyweight per day in the drinking water for 8 weeks while both groups received daily subcutaneous injections of Ultratard insulin. Creatinine clearance, urinary protein excretion, urinary nitrate concentration and systolic blood pressure were measured at fortnightly intervals. Rats were killed at 8 weeks and plasma angiotensin II (AngII) was measured by radioimmunoassay. 3. Renal function (endogenous creatinine clearance) remained stable in both groups. In the L-NAME group, however, there was a progressive increase in proteinuria that was highly significant at 6 weeks (22.1 +/- 2.9 compared with 6.5 +/- 0.7 mg/ 24 h per 100 g in control SHR diabetic rats P < 0.001). 4. Systolic blood pressure was significantly elevated in the L-NAME group throughout the study compared with the control group. 5. Plasma AngII was significantly elevated in the L-NAME group compared with controls (42.8 +/- 10.3 vs 15.1 +/- 1.9 pmol/L, respectively; P < 0.05). 6. Activation of the renin-angiotensin system may account, at least in part, for the resulting vasoconstrictor activity with chronic nitric oxide depletion.

Breed differences in expression of inhibin/activin subunits in porcine anterior pituitary glands.

Chinese Meishan (MS) boars have greater plasma FSH concentrations than European White Composite boars, but this difference does not occur in females of these breeds. To understand this disparity, we studied expression of the follistatin gene and of genes for the inhibin/activin alpha-, beta A-, and beta B-subunits in porcine anterior pituitary glands using quantitative reverse transcription-PCR and ribonuclease protection techniques. We found that 1) the inhibin/activin beta A- and beta B-subunits and follistatin were expressed in porcine pituitary, 2) the alpha-subunit was not detected in the porcine pituitary, but was highly expressed in porcine follicles; and 3) the beta B-subunit gene is more abundantly expressed (2-fold greater) in MS boar pituitaries than in pituitaries of White Composite boars. We conclude that this is not due to a breed difference, because the expression levels of this gene were similar in pituitaries of females of these breeds. No breed differences were detected for other genes screened in this study. From these observations, we propose that activin B, a dimer of beta B-subunits and a stimulator of FSH secretion, may be partially responsible for the elevated plasma FSH concentrations in MS boars, and intrapituitary inhibin plays no or a very minimal role.

Overexpression of beta-subunit of thyroid-stimulating hormone in Meishan swine identified by differential display.

Anterior pituitary gland RNA isolated from Meishan (MS) and White Composite (WC) sexually mature boars was compared by a newly developed method of differential expression cloning called differential display. A number of cDNAs were identified that differed distinctly in expression between these breeds. The gene for beta-subunit of thyroid-stimulating hormone (TSH-beta) was initially identified as more highly expressed in MS than in WC boars. Subsequently, RNase protection assays and TSH RIA were used to quantify the magnitude of differences in transcription and translation in both males and females. Mature MS pigs had threefold greater expression of TSH-beta (P < .05) and greater plasma TSH concentrations than mature WC pigs (P < .01). The cDNA cloning and sequence analyses indicate identity in TSH-beta mRNA between MS and WC males. Elevated plasma TSH concentration may contribute MS pigs reaching sexual maturity earlier than WC or other European breeds. The method used in this study provides a useful molecular tool 1) to detect differentially expressed genes, 2) to study genetic variation that occurs at the transcription level between individuals, populations, or breeds; and additionally, 3) to identify candidate genes that control economically important quantitative traits in livestock.

Changes in angiotensin II metabolism contribute to the increased pressor response to angiotensin after chronic treatment with L-NAME in the spontaneously hypertensive rat.

1. Administration of nitric oxide (NO) synthase inhibitors, such as L-NAME, is associated with an increase in blood pressure and an increase in pressor responsiveness to infused angiotensin II (AngII). The present study was designed to investigate the contribution of changes in the metabolism of AngII to the enhanced pressor response to AngII in the spontaneously hypertensive rat (SHR; 14 weeks old) chronically treated with L-NAME. 2. Group I rats received L-NAME for 7 days (5 mg/kg per day) in their drinking water. Group II rats received water only. On day 7, rats were anaesthetized and metabolic clearance studies were performed. AngII concentrations in plasma and infusate were measured by radioimmunoassay. 3. Urinary NO2 was unchanged after L-NAME treatment, while NO3 decreased compared with control. Mean arterial pressure (MAP) was higher in the L-NAME treated rats than in control. After 30 min infusion of AngII, MAP increased significantly in both groups, although the increase was larger in L-NAME-treated than control rats. The metabolic clearance rate of AngII was significantly lower in L-NAME-treated rats than in the control group. 4. We conclude that chronic NO synthase inhibitors, such as L-NAME, cause a decrease in the rate at which AngII is metabolized. This decrease, in combination with the increase in the number of vascular AngII receptors, may account for the reported increase in pressor responsiveness to infused AngII.

A study of angiotensin II receptors after chronic inhibition of nitric oxide synthase in the spontaneously hypertensive rat.

1. Nitric oxide (NO) synthase inhibition, induces a sustained increase in blood pressure and amplifies the pressor response to infused angiotensin II (AngII). This study was designed to investigate the contribution of AngII receptors in the elevated blood pressure and enhanced pressor response to AngII in the spontaneously hypertensive rat (SHR) chronically treated with N(G)-nitro-L-arginine-methyl ester (L-NAME). 2. Two groups of 13 week old female SHR were housed four to a box. Group I rats received L-NAME for 7 days (2.5 mg/kg per day) in their drinking water. Group II rats received water only. Blood pressure was monitored daily by tail-cuff plethysmography. Plasma AngII was measured by radioimmunoassay. Aortic and uterine receptor binding was determined by saturation analysis using [125I]-Sar8, Ile1)AngII. Data was analysed using the computer program LIGAND. 3. Mean systolic blood pressure was significantly elevated in rats treated with L-NAME compared with the control group. Plasma AngII concentration was slightly decreased in rats treated L-NAME compared with control. Densities of both aortic and uterine AngII receptors increased significantly following NO synthase inhibition. Receptor affinity in the aorta decreased in the L-NAME group compared with control. However, uterine AngII receptor affinity was unchanged. 4. We conclude that the increased blood pressure and enhanced pressor responsiveness that occurs with chronic inhibition of NO synthesis may result partly from increased vascular AngII receptor expression.