Despite irreversible binding, PET tracer [11C]-SA5845 is suitable for imaging of drug competition at sigma receptors-the cases of ketamine and haloperidol

Many psychotropic compounds bind to sigma receptors and several new sigma ligands are in development for psychiatric indications such as anxiety, attention deficit hyperactivity disorder, depression and psychosis. Of special interest for drug development are tomographic methods that can quantify the binding of promising sigma ligands in a regional manner. Here we present the development of such a method and the first evaluation of sigma ligand [11C]-SA5845 in a primate. Extensive pharmacokinetic modeling was done on tissue curves and a heart lumen curve. The effects of pretreatment and challenge with haloperidol were studied as well as those of pretreatment with +/- -ketamine. The tracer had a plasma half-life of 77+/-1.7min and was rapidly taken up by all brain areas. The binding pattern was consistent with binding to sigma receptors and compartment modeling showed there was considerable specific binding that was irreversible. We therefore calculated the net influx rate, Ki, with the Gjedde-Patlak linearization, as a measure of free receptors. As expected, Ki was very sensitive to the presence of competing ligands - -ketamine and/or haloperidol. Summarizing, the tracer is well suited for visualizing sigma receptors in the brain and moreover, the presented method is able to quantify, on a regional basis, specific binding of unlabeled ligands to sigma receptors.     

Anaerobic energy metabolism of the European eel,Anguilla anguilla L.

Summary Eels, acclimated the 15°C and aerated water (P _{O 2} 130 mm Hg) were exposed to hypoxia (P _{O 2} lowered from 130 to 8 mm Hg in 4 h) and to complete anoxia until loss of equilibrium. Experiments were carried out at night. The mean survival time (LT₅₀) during anoxic conditions proved to be 5.7 h. ATP, ADP, AMP, IMP, CrP, glycogen, lactate, pyruvate, -ketoglutarate, malate, succinate, alanine, aspartate, glutamate and ammonia levels were determined in skeletal muscle and liver of control, hypoxic and anoxic fish. Some of the mentioned parameters were also measured in heart muscle and blood. Hypoxia causes declines of aspartate (muscle), CrP (muscle) and glycogen (liver, heart), and increases of alanine (blood, liver) and lactate (blood, liver, heart). During anoxia, muscle CrP stores are almost completely exhausted and adenylates are partially broken down to IMP. A decrease of glycogen and an accumulation of lactate were observed in all tissues examined. The energy charge of muscle and heart did not drop below 0.79, but in liver tissue it decreased from 0.65 to 0.17. Liver cytoplasm became significantly reduced during anoxia, but such a change of redox state did not occur in muscle. Eels seem to lack the capacity for anaerobic fermentation of glycogen to ethanol, as observed in goldfish. Lactate glycolysis and creatine phosphate breakdown appear to be the main energy producing pathways during anaerobiosis.Abbreviations ALA alanine - ASP aspartate - CrP creatine phosphate - EC (adenylate) energy charge - GLU glutamate - GLC glucose - GLY glycogen - IMP inosine-5-monophosphate - KG ketoglutarate - LAC lactate - MAL malate - PYR pyruvate - SUC succinate - TAN total pool of adenine nucleotides     

Quantification of an C-labelled β-adrenoceptor ligand, S-(−)CGP 12177, in plasma of humans and rats

β-Adrenoceptors in human lungs and heart can be imaged with the radioligand 4-[3-[(1,1-dimethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-¹¹C-one (CGP 12177, [¹¹C]I). For quantification of receptor density with compartment models by adjustment of rate constants, an ‘input function’ is required which consists of the integral of the concentration of unmodified ligand in arterial plasma over time. A discrepancy in the literature regarding metabolic stability of [¹¹C]I prompted us to study metabolism in rats by reversed-phase HPLC (RP-HPLC) of trichloroacetic acid extracts of arterial plasma after i.v. injection of [¹¹C]I (> 11.1 TBq/mmol, 11 MBq/kg). Some plasma samples were also directly applied to an internal-surface reversed-phase (ISRP) column. In parallel experiments, tritiated [¹¹C]I was employed and methanol extracts of arterial plasma were analyzed by straight-phase TLC. The three methods were in excellent agreement. Unmodified [¹¹C]I decreased from > 98.5% (³H) or > 99.9% (¹¹C) initially to 57 ± 7% at 80 min post injection to formation of two polar metabolites. Using the RP-HPLC method, no metabolism was detectable in humans up to 30 min after injection of [¹¹C]I (1851 MBq). Deproteinization of plasma with acetonitrile resulted in the formation of a radioactive species (artifact) which eluted immediately after the void volume in RP-HPLC and which could be mistakenly interpreted as a metabolite. Plasma protein binding was low (ca. 30%) in both humans and rats. Association of the radioligand to blood cells suggested that equilibrium between receptor-bound and free radioligand was reached within 15 min after high-specific-activity injections, but only after more than 30 min after low-specific-activity injections.     

16S and 23S plastid rDNA phylogenies of Prototheca species and their auxanographic phenotypes

Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships.     

Diversity and demography in Beringia: multilocus tests of paleodistribution models reveal the complex history of arctic ground squirrels

To assess effects of historical climate change on northern species, we quantified the population history of the arctic ground squirrel (Spermophilus parryii), an arctic-adapted rodent that evolved in Beringia and was strongly influenced by climatic oscillations of the Quaternary. Competing hypotheses for the species' population history were derived from patterns of mitochondrial (mtDNA) structure and a bioclimatic envelope model (BEM). Hypotheses invoked (1) sequential isolation of regional populations beginning with the Arctic, (2) deep isolation only across central Alaska, and (3) widespread panmixia, and were tested using coalescent methods applied to eight nuclear (nDNA) loci. The data rejected strict interpretations of all three hypotheses, but perspectives underlying each encompassed aspects of the species' history. Concordance between mtDNA and nDNA geographic structure revealed three semi-independently evolving phylogroups, whereas signatures of gene flow at nDNA loci were consistent with a historical contact between certain populations as inferred by the BEM. Demographic growth was inferred for all regions despite expectations of postglacial habitat contraction for parts of Beringia. Our results highlight the complementary perspectives on species' histories that multiple lines of evidence provide, and underscore the utility of multilocus data for resolving complex population histories relevant to understanding effects of climate change.     

In vivo evaluation of 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-D-glucopyronuronate: a positron emission tomographic tracer for imaging β-glucuronidase activity in a tumor/inflammation rodent model

β-Glucuronidase (β-GUS) plays an important role in inflammation and degenerative processes. The enzyme has also been investigated as a target in prodrug therapy for cancer. To investigate the role of β-GUS in pathologies and to optimize β-GUS-based prodrug therapies, we recently developed a positron emission tomographic (PET) tracer, 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-D-glucopyronuronate ([18F]FEAnGA), which proved to be selectively cleaved by β-GUS. Here we present the in vivo evaluation of [18F]FEAnGA for imaging of β-GUS in a tumor/inflammation model. Ex vivo biodistribution of [18F]FEAnGA was conducted in healthy rats. PET imaging and pharmacokinetic modeling were performed in Wistar rats bearing C6 tumors of different sizes and sterile inflammation. The biodistribution studies of [18F]FEAnGA indicated low uptake in major organs and rapid excretion through the renal pathway. MicroPET studies revealed three times higher uptake in the viable part of larger C6 gliomas than in smaller C6 gliomas. Uptake in inflamed muscle was significantly higher than in control muscle. The distribution volume of [18F]FEAnGA in the viable part of the tumor correlated well with the cleavage of the tracer to [18F]fluoroethylamine and the spacer 4-hydroxy-3-nitrobenzyl alcohol. [18F]FEAnGA is a PET tracer able to detect increased activity of β-GUS in large solid tumors and in inflamed tissues.     

Synthesis and preliminary evaluation of (S)-[11C]-exaprolol, a novel beta-adrenoceptor ligand for PET

Positron-emitting beta-adrenoceptor ligands for the CNS could allow determination of changes in beta-adrenoceptor availability after treatment of patients with norepinephrine reuptake inhibitors or tricyclic antidepressants, and differential diagnosis between multiple sclerosis and other brain disorders in an early stage of the disease. No ligands suitable for this purpose are available for human use. In order to prepare a tracer for human studies, we labeled the biologically active enantiomer of the beta-blocker exaprolol with (11)C. Exaprolol has the appropriate lipophilicity (log P + 1.6) for entry of the CNS and is claimed to be a very potent beta-adrenoceptor antagonist. (S)-Desisopropyl-exaprolol was synthesized by reaction of 2-hexylphenol with (S)-glycidyl-nosylate followed by ring opening using ammonia gas. The desisopropyl precursor was reacted with (11)C-acetone in methanol to produce (S)-[(11)C]-exaprolol. Radiochemical purification was performed with RP-HPLC and was followed by Sep-Pak formulation. The labeled product was i.v. injected into male Wistar rats. Brain images were acquired using a microPET Focus 220 and the biodistribution of (11)C was assessed. The radiochemical yield of (S)-[(11)C]-exaprolol was 7% with a total synthesis time of 30 min. Specific activities were >10 GBq/micromol. Brain uptake of the tracer reached a maximum after 15 min. Standardized uptake values were moderate (0.5-0.9) but sufficient for imaging. However, beta-blockade (propranolol, 2.5mg/kg body weight) did not lower tracer uptake in any CNS region and washout from the brain was not accelerated when propranolol was administered 40 min after injection of (S)-[(11)C]-exaprolol. Tracer binding in lung, spleen and erythrocytes was lowered after beta-blockade, but the myocardial uptake of radioactivity was not affected. These data indicate that (S)-[(11)C]-exaprolol is not a suitable beta-adrenoceptor ligand for PET, probably because the in vivo affinity of exaprolol to beta-adrenoceptors is in the nM rather than the sub-nM range. The observed inhibition of tracer uptake in lung, spleen and erythrocytes seems due to an interaction of propranolol with amine transporters rather than beta-adrenoceptors.