(3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752) -a Novel Cortical-Preferring Catecholamine Transmission- and Cognition-Promoting Agent.

Here we describe for the first time the distinctive pharmacological profile for (3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752), a new phenyl-pyrrolidine derivative with regioselective central nervous system transmission-enhancing properties. IRL752 (3.7-150 µmol/kg, s.c.) was characterized through extensive in vivo studies using behavioral, tissue neurochemical, and gene expression as well as microdialysis methods. Behaviorally, the compound normalized tetrabenazine-induced hypoactivity, whereas it was unable to stimulate basal locomotion in normal animals or either accentuate or reverse hyperactivity induced by amphetamine or MK-801. IRL752 induced but minor changes in monoaminergic tissue neurochemistry across noradrenaline (NA)- and dopamine (DA)-dominated brain regions. The expression of neuronal activity-, plasticity-, and cognition-related immediate early genes (IEGs), however, increased by 1.5-fold to 2-fold. Furthermore, IRL752 dose-dependently enhanced cortical catecholamine dialysate output to 600%-750% above baseline, whereas striatal DA remained unaltered, and NA rose to ∼250%; cortical and hippocampal dialysate acetylcholine (ACh) increased to ∼250% and 190% above corresponding baseline, respectively. In line with this cortically preferential transmission-promoting action, the drug was also procognitive in the novel object recognition and reversal learning tests. In vitro neurotarget affinity and functional data coupled to drug exposure support the hypothesis that 5-hydroxytryptamine 7 receptor and α2(C)-adrenoceptor antagonism are key contributors to the in vivo efficacy and original profile of IRL752. The cortical-preferring facilitatory impact on catecholamine (and ACh) neurotransmission, along with effects on IEG expression and cognition-enhancing features, are in line with the potential clinical usefulness of IRL752 in conditions wherein these aspects may be dysregulated, such as in axial motor and cognitive deficits in Parkinson disease. SIGNIFICANCE STATEMENT: This report describes the distinctive preclinical profile of (3S)-3-(2,3-difluorophenyl)-3-methoxypyrrolidine (IRL752). Its in vivo neurochemical, behavioral, microdialysis, and gene expression properties are consistent with a cortically regioselective facilitatory impact on catecholaminergic and cholinergic neurotransmission accompanied by cognitive impairment-reversing features. The pharmacological characteristics of IRL752 are in line with the clinical usefulness of IRL752 in conditions wherein these aspects may be dysregulated, such as in axial motor and cognitive deficits in Parkinson disease.

Monitoring pulmonary perfusion by electrical impedance tomography: an evaluation in a pig model.

BACKGROUND: Electrical impedance tomography (EIT) is a non-invasive technique that generates images of impedance distribution. Changes in the pulmonary content of air and blood are major determinants of thoracic impedance. This study was designed to evaluate EIT in monitoring pulmonary perfusion in a wide range of cardiac output. METHODS: Eight anaesthetised, mechanically ventilated pigs were fitted with a 16-electrode belt at the mid-thoracic level to generate EIT images that were analysed to determine pulse-synchronous systolic changes in impedance (DeltaZ(sys)). Stroke volume (SV) was derived using a pulmonary artery catheter. Reductions in cardiac pre-load, and thus pulmonary perfusion, were induced either by inflating the balloon of a Fogarty catheter positioned in the inferior caval vein or by increasing the positive end-expiratory pressure (PEEP). All measurements were performed in a steady state during a short apnoea. RESULTS: Pulse-synchronous changes in DeltaZ(sys) were easily discernable during apnoea. Balloon inflation reduced SV to 36% of the baseline, with a corresponding decrease in DeltaZ(sys) to 45% of baseline. PEEP reduced SV and DeltaZ(sys) to 52% and 44% of the baseline, respectively. Significant correlations between SV and DeltaZ(sys) were demonstrated during all measurements (rho=0.62) as well as during balloon inflation (rho=0.73) and increased PEEP (rho=0.40). A Bland-Altman comparison of relative changes in SV and DeltaZ(sys) demonstrated a bias of -7%, with 95% limits of agreement at -51% and 36%. CONCLUSIONS: EIT provided beat-to-beat approximations of pulmonary perfusion that significantly correlated to a wide range of SV values achieved during both extra and intrapulmonary interventions to change cardiac output.

Electrical impedance tomography and heterogeneity of pulmonary perfusion and ventilation in porcine acute lung injury.

BACKGROUND: The heterogeneity of pulmonary ventilation (V), perfusion (Q) and V/Q matching impairs gas exchange in an acute lung injury (ALI). This study investigated the feasibility of electrical impedance tomography (EIT) to assess the V/Q distribution and matching during an endotoxinaemic ALI in pigs. METHODS: Mechanically ventilated, anaesthetised pigs (n=11, weight 30-36 kg) were studied during an infusion of endotoxin for 150 min. Impedance changes related to ventilation (Z(V)) and perfusion (Z(Q)) were monitored globally and bilaterally in four regions of interest (ROIs) of the EIT image. The distribution and ratio of Z(V) and Z(Q) were assessed. The alveolar-arterial oxygen difference, venous admixture, fractional alveolar dead space and functional residual capacity (FRC) were recorded, together with global and regional lung compliances and haemodynamic parameters. Values are mean+/-standard deviation (SD) and regression coefficients. RESULTS: Endotoxinaemia increased the heterogeneity of Z(Q) but not Z(V). Lung compliance progressively decreased with a ventral redistribution of Z(V). A concomitant dorsal redistribution of Z(Q) resulted in mismatch of global (from Z(V)/Z(Q) 1.1+/-0.1 to 0.83+/-0.3) and notably dorsal (from Z(V)/Z(Q) 0.86+/-0.4 to 0.51+/-0.3) V and Q. Changes in global Z(V)/Z(Q) correlated with changes in the alveolar-arterial oxygen difference (r(2)=0.65, P<0.05), venous admixture (r(2)=0.66, P<0.05) and fractional alveolar dead space (r(2)=0.61, P<0.05). Decreased end-expiratory Z(V) correlated with decreased FRC (r(2)=0.74, P<0.05). CONCLUSIONS: EIT can be used to assess the heterogeneity of regional pulmonary ventilation and perfusion and V/Q matching during endotoxinaemic ALI, identifying pivotal pathophysiological changes.

Regionally differentiated fibrinolytic responses during volume-resuscitated acute endotoxemia in pigs.

BACKGROUND: Microcirculatory dysfunction and formation of microthrombi are common in sepsis as a result of a procoagulant state. A profibrinolytic change has however, been reported in early sepsis. This study investigates systemic and regional (pulmonary, preportal, hepatic, renal) fibrinolytic capacity as mirrored by fluxes of tissue-type plasminogen activator (t-PA) in response to acute endotoxemia and volume resuscitation. METHODS: Eight anaesthetized, ventilated pigs (24-29 kg) were instrumented for systemic and regional haemodynamic monitoring. Aortic, pulmonary arterial, portal, hepatic and renal venous blood samples were collected. Following baseline stabilisation, Escherichia coli endotoxin was infused for 120 min. During the last 30 min of infusion, volume resuscitation was initiated targeting baseline cardiac output, and animals were observed for 3 h. Total tPA was analyzed by ELISA calibrated for porcine tPA. Net organ tPA fluxes were calculated based on in/outflowing plasma concentrations and regional blood flows. RESULTS: Preportal release and hepatic extraction of tPA was observed at baseline. Pulmonary and renal net fluxes of tPA were not significantly different from zero. Endotoxemia increased plasma tPA levels in all investigated vascular beds. Preportal tPA release increased approximately 10-fold and hepatic extraction increased approximately 12-fold in non-resuscitated acute endotoxemia. No significant changes in pulmonary or renal tPA fluxes were observed. Volume resuscitation restored net fluxes to baseline values while plasma levels remained elevated approximately twofold compared with baseline. CONCLUSION: Acute endotoxemia induces a prompt, marked and regionally differentiated pro-fibrinolytic response that cannot be discerned based on systemic levels of circulating tPA and that was normalized during volume resuscitation.

Pulmonary net release of tissue-type plasminogen activator during porcine primary and secondary acute lung injury.

BACKGROUND: Tissue-type plasminogen activator (tPA) is a key mediator of fibrinolysis. Matching of pulmonary perfusion and ventilation is a critical denominator of oxygenation in acute lung injury (ALI). This study investigates pulmonary venoarterial plasma tPA gradients in association with acute ALI induced by bronchoalveolar lavage (BAL) and endotoxinemia (ETX). METHODS: Twenty-one anaesthetized, ventilated pigs were allocated to control (CTRL, n=5), bronchoalveolar saline lavage (BAL, n=8) or infusion of Escherichia coli endotoxin (ETX, n=8). Total tPA was analyzed in plasma (ELISA calibrated for porcine tPA). The inflammatory response was assessed by TNFa levels (ELISA). All variables were assessed at baseline and 2 h following ALI. RESULTS: Bronchoalveolar lavage and ETX induced similar increases in pulmonary shunt whereas pulmonary vascular resistance was significantly more increased in ETX animals. Cardiac output remained stable in BAL animals but decreased in ETX animals. The pulmonary venoarterial tPA plasma gradient increased in ETX animals, yielding a positive pulmonary net flux of tPA, which was absent in BAL animals. TNFalpha levels increased in ETX, but not in BAL, animals. A significant correlation was observed between TNFalpha and tPA plasma levels in ETX animals. All variables remained unchanged in CTRL animals. CONCLUSION: Plasma changes of tPA levels support a pulmonary release of tPA in early experimental ALI induced by acute ETX but not lavage, and are related to the inflammatory response. Despite increased vascular fibrinolytic capacity in ETX animals, pulmonary dysfunction was not different from BAL animals. The results demonstrate the close relation between inflammation and coagulation in early ALI.

Microsatellite loci are not abundant in all arthropod genomes: analyses in the hard tick, Ixodes scapularis and the yellow fever mosquito, Aedes aegypti.

Plasmid libraries enriched for microsatellites were generated in the tick, Ixodes scapularis and in the mosquito Aedes aegypti. Libraries were enriched for genomic DNA containing (AC)n, (AG)n, (ATG)n, (CAG)n, (TAG)n, (AAT)n, (CTGY)n or (GATA)n motifs. Clones containing each motif were sequenced in both species for PCR primer design. In I. scapularis, most primers amplified a single locus and alleles varied in the number of microsatellite repeats and segregated as codominant markers. In contrast (AC)n, (TAG)n and (GATA)n microsatellite loci extracted from Ae. aegypti appeared to be members of multigene families. A primer pair designed to amplify a particular TAG locus instead amplified many independently segregating loci, some of which did not contain TAG microsatellites. Alleles at the TAG loci segregated as dominant markers and there was limited evidence for length variation among alleles. These results suggest that microsatellite loci are not universally abundant in arthropod genomes nor do alleles always segregate as codominant markers.