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Lorlatinib (PF-06463922) is a next-generation small-molecule inhibitor of the orphan receptor tyrosine kinase c-ros oncogene 1 (ROS1), which has a kinase domain that is physiologically related to anaplastic lymphoma kinase (ALK), and is undergoing Phase I/II clinical trial investigations for non-small cell lung cancers. An early goal is to measure the concentrations of this drug in brain tumour lesions of lung cancer patients, as penetration of the blood-brain barrier is important for optimal therapeutic outcomes. Here we prepare both 11 C- and 18 F-isotopologues of lorlatinib to determine the biodistribution and whole-body dosimetry assessments by positron emission tomography (PET). Non-traditional radiolabelling strategies are employed to enable an automated multistep 11 C-labelling process and an iodonium ylide-based radiofluorination. Carbon-11-labelled lorlatinib is routinely prepared with good radiochemical yields and shows reasonable tumour uptake in rodents. PET imaging in non-human primates confirms that this radiotracer has high brain permeability.

The Massachusetts General Hospital Radiochemistry Program, in collaboration with Pfizer, has developed unique 11 C and 18 F-labeling strategies to synthesize isotopologs of lorlatinib (PF-06463922) which is undergoing phase III clinical trial investigations for treatment of non-small-cell lung cancers with specific molecular alterations. A major goal in cancer therapeutics is to measure the concentrations of this drug in the brain metastases of patients with lung cancer, and penetration of the blood-brain barrier is important for optimal therapeutic outcomes. Our recent publication in Nature Communications employed radiolabeled lorlatinib and positron emission tomography (PET) studies in preclinical models including nonhuman primates (NHPs) that demonstrated high brain permeability of this compound. Our future work with radiolabeled lorlatinib will include advanced PET evaluations in rodent tumor models and normal NHPs with the goal of clinical translation.

Unlabelled: This was a first-in-human study of the novel phosphodiesterase-2A (PDE2A) PET ligand (18)F-PF-05270430. The primary goals were to determine the appropriate tracer kinetic model to quantify brain uptake and to examine the within-subject test-retest variability., Methods: In advance of human studies, radiation dosimetry was determined in nonhuman primates. Six healthy male subjects participated in a test-retest protocol with dynamic scans and metabolite-corrected input functions. Nine brain regions of interest were studied, including the striatum, white matter, neocortical regions, and cerebellum. Multiple modeling methods were applied to calculate volume of distribution (VT) and binding potentials relative to the nondisplaceable tracer in tissue (BPND), concentration of tracer in plasma (BPP), and free tracer in tissue (BPF). The cerebellum was selected as a reference region to calculate binding potentials., Results: The dosimetry study provided an effective dose of less than 0.30 mSv/MBq, with the gallbladder as the critical organ; the human target dose was 185 MBq. There were no adverse events or clinically detectable pharmacologic effects reported. Tracer uptake was highest in the striatum, followed by neocortical regions and white matter, and lowest in the cerebellum. Regional time-activity curves were well fit by multilinear analysis-1, and a 70-min scan duration was sufficient to quantify VT and the binding potentials. BPND, with mean values ranging from 0.3 to 0.8, showed the best intrasubject and intersubject variability and reliability. Test-retest variability in the whole brain (excluding the cerebellum) of VT, BPND, and BPP were 8%, 16%, and 17%, respectively., Conclusion: (18)F-PF-05270430 shows promise as a PDE2A PET ligand, albeit with low binding potential values., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Importance: In Huntington disease (HD) striatal neuron loss precedes and predicts motor signs or symptoms. Current imaging biomarkers lack adequate sensitivity for assessing the early stages of HD. Developing an imaging biomarker for HD spanning the time of onset of motor signs remains a major unmet research need. Intracellular proteins whose expression is altered by the mutant huntingtin protein may be superior markers for early HD stages., Objective: To evaluate whether [18F]MNI-659 (2-(2-(3-(4-(2-[18F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione), a novel phosphodiesterase 10 positron emission tomography (PET) ligand, is a sensitive marker for striatal changes in early HD., Design, Setting, and Participants: A cohort of individuals with HD, including premanifest (pre-HD) or manifest with motor signs (mHD), underwent clinical assessments, genetic determination, [18F]MNI-659 PET imaging, and brain magnetic resonance imaging. Age-matched healthy volunteers (HVs) also received clinical assessments and PET and magnetic resonance imaging., Main Outcomes and Measures: Binding potentials (BPnds) were estimated for brain regions of interest, specifically within the basal ganglia, and compared between participants with HD and the HVs and correlated with markers of HD severity and atrophy of basal ganglia nuclei., Results: Eleven participants with HD (8 mHD and 3 pre-HD) and 9 HVs participated. Ten of 11 HD participants had known huntingtin CAG repeat length, allowing determination of a burden of pathology (BOP) score. One individual with HD declined CAG determination. All participants with mHD had relatively early-stage disease (4 with stage 1 and 4 with stage 2) and a Unified Huntington's Disease Rating Scale (UHDRS) total Motor subscale score of less than 50. The HD cohort had significantly lower striatal [18F]MNI-659 uptake than did the HV cohort (mean, -48.4%; P < .001). The HD cohort as a whole had a reduction in the basal ganglia BPnd to approximately 50% of the level in the HVs (mean, -47.6%; P < .001). The 3 pre-HD participants had intermediate basal ganglia BPnds. Striatal [18F]MNI-659 uptake correlated strongly with the severity of disease measured by the clinical scale (UHDRS Motor subscale; R = 0.903; P < .001), the molecular marker (BOP; R = 0.908; P < .001), and regional atrophy (R = 0.667; P < .05)., Conclusions and Relevance: As a promising striatal imaging biomarker, [18F]MNI-659 is potentially capable of assessing the extent of disease in early mHD. Furthermore, [18F]MNI-659 may identify early changes in medium spiny neurons and serve as a marker to predict conversion to mHD. Additional studies with larger, stratified cohorts of patients with HD and prospective studies of individuals with pre-HD are warranted.

Unlabelled: Clinical molecular imaging of apoptosis is a highly desirable yet unmet challenge. Here we provide the first report on (18)F-labeled 5-fluoropentyl-2-methyl-malonic acid ((18)F-ML-10), a small-molecule, (18)F-labeled PET tracer for the imaging of apoptosis in vivo; this report includes descriptions of the synthesis, radiolabeling, and biodistribution of this novel apoptosis marker. We also describe the use of (18)F-ML-10 for small-animal PET of neurovascular cell death in experimental cerebral stroke in mice., Methods: (18)F-ML-10 was synthesized by nucleophilic substitution from the respective mesylate precursor, and its biodistribution was assessed in healthy rats. Permanent occlusion of the middle cerebral artery (MCA) was induced in mice, and small-animal PET was performed 24 h later., Results: Efficient radiolabeling of ML-10 with (18)F was achieved. Biodistribution studies with (18)F-ML-10 revealed rapid clearance from blood (half-life of 23 min), a lack of binding to healthy tissues, and rapid elimination through the kidneys. No significant tracer metabolism in vivo was observed. Clear images of distinct regions of increased uptake, selectively in the ischemic MCA territory, were obtained in the in vivo small-animal PET studies. Uptake measurements ex vivo revealed 2-fold-higher uptake in the affected hemisphere and 6- to 10-fold-higher uptake in the region of interest of the infarct. The cerebral uptake of (18)F-ML-10 was well correlated with histologic evidence of cell death. The tracer was retained in the stroke area but was cleared from blood and from intact brain areas., Conclusion: (18)F-ML-10 is useful for noninvasive PET of neurovascular histopathology in ischemic cerebral stroke in vivo. Such an assessment may assist in characterization of the extent of stroke-related cerebral damage and in the monitoring of disease course and effect of treatment.

Sigma-1 receptors are widely expressed in the mammalian brain and also in organs of the immune, endocrine and reproductive systems. Based on behavioral and pharmacological assessments, sigma-1 receptors are important in memory and cognitive processes, and are thought to be involved in specific psychiatric illnesses, including schizophrenia, depression, and drug addiction. It is thought that specific neuroactive steroids are endogenous ligands for these sites. In addition, several sigma-1 receptor binding steroids including progesterone, dihydroepiandrosterone (DHEA), and testosterone are being examined clinically for specific therapeutic purposes; however, their mechanisms of action have not been clearly defined. We previously described the high affinity sigma-1 receptor selective PET tracer [(18)F]FPS. This study examines the effect of neuroactive steroids on [(18)F]FPS binding in vitro and in vivo. Inhibition constants were determined in vitro for progesterone, testosterone, DHEA, estradiol, and estriol binding to the [(18)F]FPS labeled receptor. The affinity order (K(i) values) for these steroids ranged from 36 nM for progesterone to >10,000 nM for estrodiol and estriol. Biodistribution studies revealed that i.v. coadministration of progesterone (10 mg/kg), testosterone (20 mg/kg), or DHEA (20 mg/kg) significantly decreased [(18)F]FPS uptake (%ID/g) by up to 50% in nearly all of eight brain regions examined. [(18)F]FPS uptake in several peripheral organs that express sigma-1 receptors (heart, spleen, muscle, lung) was also reduced (54-85%). These studies clearly demonstrate that exogenously administered steroids can occupy sigma-1 receptors in vivo, and that [(18)F]FPS may provide an effective tool for monitoring sigma-1 receptor occupancy of specific therapeutic steroids during clinical trials.

Noninvasive tomographic imaging methods including positron emission tomography (PET) and single photon emission computed tomography (SPECT) are extremely sensitive and are capable of measuring biochemical processes that occur at concentrations in the nanomolar range. Inherent to neurodegenerative processes is neuronal loss. Thus, PET or SPECT monitoring of biochemical processes altered by neuronal loss (changes in neurotransmitter turnover, alterations in receptor, transporter or enzyme concentrations) can provide unique information not attainable by other methods. Such imaging techniques can also be used to longtitudinally monitor the effects of neuroprotective treatments. This review highlights current imaging probes used to evaluate patients with specific neurodegenerative disorders (e.g., Alzheimer's Disease, Parkinson's Disease, Huntington's Chorea), including those that image receptors of the dopaminergic, cholinergic and glutamatergic systems. Areas of future research focus are also defined. It is clear that monitoring the progression of neurodegenerative disorders and the impact of neuroprotective treatments are two different but related goals for which noninvasive imaging via PET and SPECT methods plays a powerful and unique role.

Sigma receptors have been implicated in a myriad of cellular functions, biological processes and diseases. While the precise biological functions of sigma receptors have not been elucidated, recent work has shed some light on to these enigmatic systems. Sigma receptors have recently been a target of drug development related to psychiatric and neurological disorders. Sigma ligands have also been shown to modulate endothelial cell proliferation and can control angiogenesis which makes them a promising target for oncology applications. Other areas currently being investigated include treatment of gastrointestinal, cardiovascular, endocrine and immune system disorders. Of interest is that the human sigma-1 receptor gene contains a steroid binding component, and several gonadal steroids, including progesterone, testosterone and dehydroepiandrosterone (DHEA), interact with sigma-1 receptors. Of the steroids examined thus far, progesterone binds with the highest affinity to human sigma-1 receptors, with a reported affinity (Ki) as high as 30 nM while the other steroids exhibit lower affinity. For this and other reasons, sigma-1 receptors have been proposed as a link between the central nervous system and the endocrine and reproductive systems. Taken together, the above information highlights an important yet largely unexplored but promising area of research to examine the biological function and therapeutic potential of sigma receptors. This review provides an overview of the current knowledge of these sites with a focus on specific areas where in vivo sigma receptor imaging is currently being investigated.

[(18)F]1-(2-Fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]SFE) is a novel, selective, high-affinity sigma-1 receptor radioligand that has been preclinically well characterized in rodents. To support an investigational new drug (IND) application for the first evaluation of [(18)F]SFE in humans, single-organ and whole-body radiation adsorbed doses associated with [(18)F]SFE injection were estimated from rat distribution data. In addition, single- and multiple-dose toxicity studies were conducted in rabbits and in dogs. Multiple-dose toxicity studies in rabbits and single-dose toxicity studies in beagles suggest at least a 100-fold safety margin for humans studies at a mass dose limit of 4.0 mug per intravenous injection, based on the combined no observable adverse effect levels (NOAEL, mg/m(2)) measured in these species. Radiation dosimetry estimates obtained from rat biodistribution analyses of [(18)F]SFE suggest that most tissues would receive about 0.010-0.020 mGy/MBq, while the adrenal glands, brain, bone, liver, lungs, and spleen would receive slightly higher doses (0.024-0.044 mGy/MBq). The adrenal glands were identified as the critical organ, because they received the highest adsorbed radiation dose. The total exposure resulting from a 5 mCi administration of [(18)F]SFE is well below the FDA-defined limits for yearly cumulative and per-study exposures to research participants. These combined results support the expectation that [(18)F]SFE will be safe for use in human positron emission tomography (PET) imaging studies with the administration of 5 mCi and a mass dose equal to or less than 4.0 mug SFE per injection.

[123I]TPCNE (1(trans-[123I]iodopropen-2-yl)-4-[(4-cyanophenoxy)methyl] piperidine; Ki = 0.67 nM; log P = 3.36) is a novel sigma-1 receptor SPET ligand. In this study, we developed an optimized labeling method for [123I]TPCNE and investigated the kinetics, binding characteristics, and whole-body distribution of this tracer for the first time in humans. We also performed a challenge with the sigma-1 receptor antagonist haloperidol against [123I]TPCNE. Seven healthy volunteers were recruited. Dynamic brain SPET scans were performed following i.v. administration of 185 MBq [123I]TPCNE in all seven subjects. Three of the subjects were given oral haloperidol (2.5 mg) approximately 1 h before the scan. The dynamic data were analyzed with both reversible and irreversible compartmental models.[123I]TPCNE showed high uptake in brain and liver. All non-haloperidol-treated subjects showed a high whole-brain uptake (average: 8.7% of injected activity). No significant clearance of the tracer was seen up to 30 h post injection. In the haloperidol-treated subjects, the time-activity curves clearly demonstrated clearance of the tracer from the brain. Regional radioactivity concentrations were reduced by haloperidol from 42% in the cerebellum to 73% in the thalamus.[(123)I]TPCNE demonstrated high brain uptake, with highest binding found in the posterior cingulate. A region in which binding was unaffected by haloperidol pretreatment could not be identified, and the time-activity data were best described by an irreversible model.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.

Introduction: Sigma-1 receptors are expressed throughout the mammalian central nervous system (CNS) and are implicated in several psychiatric disorders, including schizophrenia and depression. We have recently evaluated the high-affinity (K(D)=0.5+/-0.2 nM, log P=2.9) sigma-1 receptor radiotracer [(18)F]1-(3-fluoropropyl)-4-(4-cyanophenoxymethyl)piperidine, [(18)F]FPS, in humans. In contrast to appropriate kinetics exhibited in baboon brain, in the human CNS, [(18)F]FPS does not reach pseudoequilibrium by 4 h, supporting the development of a lower-affinity tracer [Waterhouse RN, Nobler MS, Chang RC, Zhou Y, Morales O, Kuwabara H, et al. First evaluation of the sigma-1 receptor radioligand [(18)F]1-3-fluoropropyl-4-((4-cyanophenoxy)-methyl)piperidine ([(18)F]FPS) in healthy humans. Neuroreceptor Mapping 2004, July 15-18th, Vancouver, BC Canada 2004]. We describe herein the in vivo evaluation in rats of [(18)F]1-(2-fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]SFE) (K(D)=5 nM, log P=2.4), a structurally similar, lower-affinity sigma-1 receptor radioligand., Methods: [(18)F]SFE was synthesized (n=4) as previously described in good yield (54+/-6% EOB), high specific activity (2.1+/-0.6 Ci/micromol EOS) and radiochemical purity (98+/-1%) and evaluated in awake adult male rats., Results: Similar to [(18)F]FPS, regional brain radioactivity concentrations [percentage of injected dose per gram of tissue (%ID/g), 15 min] for [(18)F]SFE were highest in occipital cortex (1.86+/-0.06 %ID/g) and frontal cortex (1.76+/-0.38 %ID/g), and lowest in the hippocampus (1.01+/-0.02%ID/g). Unlike [(18)F]FPS, [(18)F]SFE cleared from the brain with approximately 40% reduction in peak activity over a 90-min period. Metabolite analysis (1 h) revealed that [(18)F]SFE was largely intact in the brain. Blocking studies showed a large degree (>80%) of saturable binding for [(18)F]SFE in discrete brain regions., Conclusions: We conclude that [(18)F]SFE exhibits excellent characteristics in vivo and may provide a superior PET radiotracer for human studies due to its faster CNS clearance compared to [(18)F]FPS.

In this study, in vivo evaluation in mice and rabbits of [123I]-4-iodo-N-(4-(4-(2-methoxyphenyl)-piperazin-1-yl)butyl)-benzamide ([123I]-BPB), a potential radioligand for visualisation of the sigma receptor by single photon emission computed tomography (SPECT), is reported. The compound possesses appropriate lipophilicity (log P=2.2) and binds sigma-1 and sigma-2 receptors (pKi=6.51 and 6.79, respectively). In mice, this new radioiodinated tracer exhibited high brain uptake (4.99% ID/g tissue at 10 min postinjection) and saturable binding (3.06% ID/g tissue at 10 min postinjection) as determined by pretreatment with unlabeled [123I]-BPB. A metabolite study demonstrated no (less than 5%) labeled metabolites in the brain. In rabbits, regional brain distribution was investigated and the tracer displayed high, homogeneous central nervous system uptake. Selectivity was assessed by competition experiments with known sigma ligands. Metabolite analysis showed no (less than 8%) labeled metabolites in the rabbit brain. In conclusion, our findings indicate that [123I]-BPB is not a suitable tracer for visualisation of D3 receptors while its potential for sigma receptor imaging is severely hampered by its affinity for dopamine receptors.

The development of imaging methods to measure changes in NMDA ion channel activation would provide a powerful means to probe the mechanisms of drugs and device based treatments (e.g., ECT) thought to alter glutamate neurotransmission. To provide a potential NMDA/PCP receptor PET tracer, we synthesized the radioligand [11C]GMOM (ki = 5.2 +/-0.3 nM; log P = 2.34) and evaluated this ligand in vivo in awake male rats and isoflurane anesthetized baboons. In rats, the regional brain uptake of [11C]GMOM ranged from 0.75+/-0.13% ID/g in the medulla and pons to 1.15+/-0.17% ID/g in the occipital cortex. MK801 (1 mg/kg i.v.) significantly reduced (24-28%) [11C]GMOM uptake in all regions. D-serine (10 mg/kg i.v.) increased [11C]GMOM %ID/g values in all regions (10-24%) reaching significance in the frontal cortex and cerebellum only. The NR2B ligand RO 25-6981 (10 mg/kg i.v.) reduced [11C]GMOM uptake significantly (24-38%) in all regions except for the cerebellum and striatum. Blood activity was 0.11+/-0.03 %ID/g in the controls group and did not vary significantly across groups. PET imaging in isoflurane-anesthetized baboons with high specific activity [11C]GMOM provided fairly uniform regional brain distribution volume (VT) values (12.8-17.1 ml g(-1)). MK801 (0.5 mg/kg, i.v., n = 1, and 1.0 mg/kg, i.v., n = 1) did not significantly alter regional VT values, indicating a lack of saturable binding. However, the potential confounding effects associated with ketamine induction of anesthesia along with isoflurane maintenance must be considered because both agents are known to reduce NMDA ion channel activation. Future and carefully designed studies, presumably utilizing an optimized NMDA/PCP site tracer, will be carried out to further explore these hypotheses. We conclude that, even though [11C]GMOM is not an optimized PCP site radiotracer, its binding is altered in vivo in awake rats as expected by modulation of NMDA ion channel activity by MK801, D-serine or RO 25-6981. The development of higher affinity NMDA/PCP site radioligands is in progress.

Compound lipophilicity is a fundamental physicochemical property that plays a pivotal role in the absorption, distribution, metabolism, and elimination (ADME) of therapeutic drugs. Lipophilicity is expressed in several different ways, including terms such as Log P, clogP, delta Log P, and Log D. Often a parabolic relationship exists between measured lipophilicity and in vivo brain penetration of drugs, where those moderate in lipophilicity often exhibit highest uptake. Reduced brain extraction of more lipophilic compounds is associated with increased non-specific binding to plasma proteins. More lipophilic compounds can also be more vulnerable to P450 metabolism, leading to faster clearance. Very polar compounds normally exhibit high water solubility, fast clearance through the kidneys, and often contain ionizable functional groups that limit blood-brain barrier (BBB) penetration. The brain penetration and specific to non-specific binding ratios exhibited in vivo by positron emission tomography (PET) and single photon emission computed tomography (SPECT) radiotracers involves a complex interplay between many critical factors, including lipophilicity, receptor affinity, metabolism, molecular size and shape, ionization potential, and specific binding to BBB efflux pumps or binding sites on albumin or other plasma proteins. This paper explores situations in which lipophilicity is a good predictor of BBB penetration, as well as those where this correlation is poor. The more commonly used methods for measuring lipophilicity are presented, and the various terms often found in the literature outlined. An attempt is made to describe how this information can be used in optimizing the development of PET and SPECT tracers that target the central nervous system (CNS).

The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenous substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and "open" state, but not when it is in the inactive or "closed" state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed.

[(18)F]1-(Fluoropropyl)-4-[(4-cyanophenoxy)methyl]piperidine ([(18)F]FPS) is a novel high affinity (KD = 0.5 nM) sigma receptor radioligand that exhibits saturable and selective in vivo binding to sigma receptors in rats, mice and non-human primates. In order to support an IND application for the characterization of [(18)F]FPS through PET imaging studies in humans, single organ and whole body radiation adsorbed doses associated with [(18)F]FPS injection were estimated from distribution data obtained in rats. In addition, acute toxicity studies were conducted in rats and rabbits and limited toxicity analyses were performed in dogs. Radiation dosimetry estimates obtained using rat biodistribution analysis of [(18)F]FPS suggest that most organs would receive around 0.012-0.015 mGy/MBq. The adrenal glands, brain, kidneys, lungs, and spleen would receive slightly higher doses (0.02-0.03 mGy/MBq). The adrenal glands were identified as the organs receiving the greatest adsorbed radiation dose. The total exposure resulting from a 5 mCi administration of [(18)F]FPS is well below the FDA defined limits for yearly cumulative and per study exposures to research participants. Extended acute toxicity studies in rats and rabbits, and limited acute toxicity studies in beagle dogs suggest at least a 175-fold safety margin in humans at a mass dose limit of 2.8 microg per intravenous injection. This estimate is based on the measured no observable effect doses (in mg/m(2)) in these species. These data support the expectation that [(18)F]FPS will be safe for use in human PET imaging studies at a maximum administration of 5 mCi and a mass dose equal to or less than 2.8 microg FPS per injection.

The synthesis and evaluation of [(11)C]zolpidem, an imidazopyridine with agonist properties at central benzodiazepine receptors, is reported herein. The reaction of desmethylzolpidem with [(11)C] methyl iodide afforded the title compound [(11)C]zolpidem in a yield of 19.19 +/- 3.23% in 41 +/- 2 min in specific activities of 0.995-1.19 Ci/micromol (1.115 +/- 0.105 Ci/micromol) (n = 3; decay corrected, EOB). The amount of radioactivity in the brain after tail vein injection in male Wistar rats was low, and the regional distribution was homogeneous and not consistent with the known distribution of the central benzodiazepine receptors. The frontal cortex/cerebellum ratio was not significantly greater than one (1.007 +/- 0.266 at 5 min) and did not increase from 5 to 40 min post-injection. A PET brain imaging study in one baboon confirmed the results obtained in rats. Therefore, it can be concluded that [(11)C]zolpidem is not a suitable tracer for in vivo visualization of central benzodiazepine receptors.

New glutamatergic drugs are being developed as potential therapies for neurodegenerative disorders, anxiety disorders, and psychoses. The development of effective mGluR radiotracers would provide essential tools with which to probe these sites in living humans, providing critical information about certain disease processes involving the glutamaterigic system and its regulation in humans. As a first step towards this goal, the tritiated form of the high affinity group II metabotropic glutamate receptor (mGluR) antagonist LY341495 [K(D) (mGluR(2)) = 1.67 +/- 0.20 nM, K(D) (mGluR(3)) = 0.75 +/- 0.43 nM] was evaluated to determine its potential to label mGluRs in vivo. Dissection analysis of the regional brain distribution over time of [(3)H]LY341495 in male rats revealed low brain uptake and no significant demonstrable saturable binding of this tracer. A group II mGluR tracer possessing higher affinity than [(3)H]LY341495 and an absence of carboxylic acid groups is likely required for in vivo PET imaging purposes.

Alterations in normal NMDA receptor composition, densities and function have been implicated in the pathophysiology of certain neurological and neuropsychiatric disorders such as Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. In our first effort to provide PET ligands for the NMDA/glycine site, we reported the synthesis of a novel high affinity glycine site ligand, 3-[2-[(3-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxylic acid ((3MPICA), Ki = 4.8 +/- 0.9 nM) and the corresponding carbon-11 labeled PET ligand, [11C]3MPICA. We report here the in vivo evaluation of [11C]3MPICA in rats. Biodistribution analysis revealed that [11C]3MPICA exhibited low degree of brain penetration and high blood concentration. The average uptake at two minutes was highest in the cerebellum (0.19 +/- 0.04 %ID/g) and thalamus (0.18 +/- 0.05 %ID/g) and lower in the hippocampus (0.13 +/- 0.03) and frontal cortex (0.11 +/- 0.04 %ID/g). The radioactivity cleared quickly from all brain regions examined. Administration of unlabeled 3MPICA (1 mg/kg, i.v.) revealed at 60 minutes a small general reduction in regional brain radioactivity concentrations in treated animals versus controls, however, the blood radioactivity concentration was also lowered, confounding the assessment of the degree of saturable binding. Warfarin co-administration (100 mg/kg, i.v.) significantly lowered blood activity at 5 minutes post-injection (-27%, P < 0.01) but failed to significantly increase the brain uptake of the radiotracer. In view of these results, and especially considering the low brain penetration of this tracer, [11C]3MPICA does not appear to be a promising PET radiotracer for in vivo use.

A series of N,N'-diphenyl and N-naphthyl-N'-phenyl guanidine derivatives was synthesized as potential N-methyl-D-aspartate (NMDA) receptor positron emission tomography (PET) ligands. The affinity of the different compounds was determined using in vitro receptor binding assays, and their log P values were estimated using HPLC analysis. The effect of N'-3 and N'-3,5 substitution on affinity and lipophilicity was examined. The K(i) values ranged from 1.87 to 839nM, while log P values between 1.22 and 2.88 were observed.

Positron emission tomography (PET) provides a powerful means to non-invasively image and quantify protein expression and biochemical changes in living subjects at nano- and picomolar levels. As the field of molecular imaging develops, and as advances in the biochemistry, pharmacology, therapeutics, and molecular biology of disease are made, there is a corresponding increase in the number of clinically relevant, novel disease-associated biomarkers that are brought to the attention of those developing imaging probes for PET. In addition, due to the high specificity of the PET radiotracers being developed, there is a demand for PET cameras with higher sensitivity and resolution. This manuscript reviews advances over the past five years in clinical and pre-clinical PET instrumentation and in new PET probes and imaging methods associated with the latest trends in the molecular imaging of cancer. Included in the PET tracer review is a description of new radioligands for steroid receptors, growth factor receptors, receptor tyrosine kinases, sigma receptors, tumor-associated enzymes, gene reporter probes, markers for tumor hypoxia and metabolism, and sites associated with angiogenesis and cellular proliferation. The use of PET imaging in drug development, including the monitoring of cancer chemotherapy, also is discussed.

The radioiodinated tetrapeptide delta-opioid antagonist [(125)I]ITIPP(psi) [H-Tyr(3'I)-Ticpsi[CH2NH]Phe-Phe-OH] (Ki(delta) = 2.08 nM; Ki(micro)/Ki(delta) = 1280) has been synthesized and evaluated as a potential lung tumour imaging agent. [(125)I]ITIPP(psi) was obtained, via electrophilic iodination, in 46% yield (>44,000 MBq/micromol) from the parent TIPP(psi). The biodistribution of [(125)I]ITIPP(psi) in nu/nu mice bearing SCLC-SW210.5 xenographs revealed good uptake and prolonged retention of radioactivity in organs known to possess delta-opioid receptors. Metabolite analysis showed that [(125)I]ITIPP(psi) was largely unmetabolized at 25 min PI and blocking studies showed significant reduction of uptake of the tracer in the brain, liver, intestine and tumor indicating that the iodinated tetrapeptide binds to delta opioid receptors in vivo.

It has been shown that N-, P- and S-deficiencies result in major reductions of root hydraulic conductivity (Lpr) which may lead to lowered stomatal conductance, but the relationship between the two conductance changes is not understood. In a variety of species, Lpr decreases in the early stages of NO3-, H2PO4(2-) and SO4(2-) deprivation. These effects can be reversed in 4-24 h after the deficient nutrient is re-supplied. Diurnal fluctuations of root Lpr have also been found in some species, and an example of this is given for Lotus japonicus. In nutrient-sufficient wheat plants, root Lpr is extremely sensitive to brief treatments with HgCl2; these effects are completely reversible when Hg is removed. The low values of Lpr in N- or P-deprived roots of wheat are not affected by Hg treatments. The properties of plasma membrane (PM) vesicles from wheat roots are also affected by NO3(-)-deprivation of the intact plants. The osmotic permeability of vesicles from N-deprived roots is much lower than that of roots adequately supplied with NO3-, and is insensitive to Hg treatment. In roots of L. japonicus, gene transcripts are found which have a strong homology to those encoding the PIP1 and PIP2 aquaporins of Arabidopsis. There is a very marked diurnal cycle in the abundance of mRNAs of aquaporin gene homologues in roots of L. japonicus. The maxima and minima appear to anticipate the diurnal fluctuations in Lpr by 2-4 h. The temporal similarity between the cycles of the abundance of the mRNAs and root Lpr is most striking. The aquaporin encoded by AtPIP1 is known to have its water permeation blocked by Hg binding. The lack of Hg-sensitivity in roots and PMs from N-deprived roots provides circumstantial evidence that lowered root Lpr may be due to a decrease in either the activity of water channels or their density in the PM. It is concluded that roots are capable, by means completely unknown, of monitoring the nutrient content of the solution in the root apoplasm and of initiating responses that anticipate by hours or days any metabolic disturbances caused by nutrient deficiencies. It is the incoming nutrient supply that is registered as deficient, not the plant's nutrient status. At some point, close to the initiation of these responses, changes in water channel activity may be involved, but the manner in which monitoring of nutrient stress is transduced into an hydraulic response is also unknown.

The hydraulic conductivity of excised roots (Lp(r)) of the legume Lotus japonicus (Regel) K. Larsen grown in mist (aeroponic) and sand cultures, was found to vary over a 5-fold range during a day/night cycle. This behaviour was seen when Lp(r) was measured in roots exuding, either under root pressure (osmotic driving force), or under an applied hydrostatic pressure of 0.4 MPa which produced a rate of water flow similar to that in a transpiring plant. A similar daily pattern of variation was seen in plants grown in natural daylight or in controlled-environment rooms, in plants transpiring at ambient rates or at greatly reduced rates, and in plants grown in either aeroponic or sand culture. When detached root systems were connected to a root pressure probe, a marked diurnal variation was seen in the root pressure generated. After excision, this circadian rhythm continued for some days. The hydraulic conductivity of the plasma membrane of individual root cells was measured during the diurnal cycle using a cell pressure probe. Measurements were made on the first four cell layers of the cortex, but no evidence of any diurnal fluctuation could be found. It was concluded that the conductance of membranes of endodermal and stelar cells may be responsible for the observed diurnal rhythm in root Lp(r). When mRNAs from roots were probed with cDNA from the Arabidopsis aquaporin AthPIP1a gene, an abundant transcript was found to vary in abundance diurnally under high-stringency conditions. The pattern of fluctuations resembled closely the diurnal pattern of variation in root Lp(r). The plasma membranes of root cells were found to contain an abundant hydrophobic protein with a molecular weight of about 31 kDa which cross-reacted strongly to an antibody raised against the evolutionarily conserved N-terminal amino acid sequence of AthPIP1a.

The development of sigma (sigma) receptor radioligands has become the focus of research over the past few years due to their potential uses in nuclear medicine. It has been shown that a wide variety of human tumor cell lines express sigma receptors, including malignant melanoma and tumors of the colon, lung, brain, breast and testes. To provide potential probes for the in vivo SPECT examination of sigma receptor densities, we have synthesized a series of halogenated 4-(phenoxymethyl)piperidines and related compounds as high affinity sigma receptor ligands. Four of these have been labeled with I-123 and evaluated in vivo in mouse tumor models. All four radioligands were synthesized no-carrier-added using oxidative radioiododestannylation methods and specific activities > 74,000 MBq/mumol were obtained. Radiochemical yields were 55-83% EOS and radiochemical purities were > 98%. All four tracers were initially screened in vivo using distribution studies in nude mice with B16 melanoma tumors (8-12 mm diameter in the flank). In all four studies, high uptake (up to 0.90 +/- 0.42 %ID, 12.99 +/- 4.28 %ID/g at 48 h) and excellent retention of radioactivity in tumor tissues was exhibited for as long as 48 h post-injection (PI). In the B16 melanoma model, the most promising results were obtained with [123I]-1-(2-hydroxyethyl)-4-(iodophenoxymethyl)piperidine (123I-3), for which tumor/tissue ratios were significantly > 1.0 by 4 h PI for most organs and increased thereafter. Tumor/tissue ratios at 48 h were as follows: blood, 68.4; muscle 31.7; brain, 7.4; lung, 6.3; liver, 1.3. In subsequent studies, 123I-3 was evaluated in nude mice with A375 human malignant melanoma. As in the B16 model, high uptake and prolonged retention of radioactivity in tumor tissues was noted. These results indicate that 123I-3 shows promise as a SPECT ligand for the detection of malignant melanoma.

Several halogenated 4-(4-phenoxymethyl)piperidines were synthesized as potential sigma receptor ligands. The affinity and selectivity of these compounds were determined using in vitro receptor binding assays, and their log P values were estimated using HPLC analysis. The effect of various N-substituents on the sigma-1 and sigma-2 dissociation constants was examined. These substituents included fluoroalkyl, hydroxyalkyl, iodopropenyl, and selected ortho-, meta-, and para-substituted benzyl groups. Also determined were the effects of various moieties on the phenoxy ring; specifically 4-iodo, 4-bromo, 4-nitro, 4-cyano, 3-bromo, and pentafluoro substituents were examined. The ranges in the dissociation constants of these compounds for sigma-1 and sigma-2 receptors were 0.38-24.3 and 3.9-361 nM, respectively. The ratio of Ki (sigma-2/sigma-1) ranged from 1.19 to 121. One of the most promising of the iodinated ligands, 1-(trans-iodopropen-2-yl)-4-[(4-cyanophenoxy)methyl]piperidi ne (4), was labeled with 123I and studied in vivo in adult male rats. High uptake and good retention of radioactivity was observed in the brain and many other organs known to possess sigma receptors. Blocking studies revealed high specific binding of [123I]-4 to sigma receptors in the brain, lung, kidney, heart, muscle, and other organs known to possess these sites. These results indicate that [123I]-4 and other halogenated 4-(phenoxymethyl)piperidines of this series may provide useful probes for in vivo tomographic studies of sigma receptors.

1-(3-Fluoropropyl)-4-(4-cyanophenoxymethyl)piperidine (1) has been synthesized as a selective high affinity (Ki = 4.3 nM) ligand for sigma-1 receptors (Ki sigma-1/sigma2 = 0.03). The corresponding radioligand, 18F-1, was synthesized via nucleophilic [18F]fluoride displacement from the appropriate N-alkylmesylate precursor. After HPLC purification, 18F-1 was obtained in 56-70% EOB (n = 5) with a specific activity > 74,000 MBq/mumol. In vivo distribution and pharmacological blocking studies using 18F-1 were performed in male Australian Albino Wistar rats. 18F-1 exhibited high brain uptake (2.47 +/- 0.37% ID at 20 min PI) with no significant loss of radioactivity from the brain over the course of the study (4 h). The uptake of radioactivity in the brain, lung, heart, and kidney was reduced significantly by the pre-administration sigma receptor-binding ligands, indicating the in vivo specificity of the ligand. The radiotracer also exhibited high uptake (11.14 +/- 1.99% ID/g) in B16 melanoma tumours in nude mice. The mean tumour/ tissue ratios at 4 h for the blood, muscle, lung and brain were 123.8, 7.2, 2.5 and 2.6, respectively. In view of these results, 18F-1 shows promise for the in vivo tomographic evaluation of sigma receptors.

1-(4-Cyanobenzyl)-4-[[(trans-iodopropen-2-yl)oxy]methyl]pipe ridine (2) has been synthesized as a novel iodinated ligand for sigma receptors. This new compound exhibited high affinity (Ki = 0.38 nM) for the sigma-1 receptor and selectivity for sigma-1 vs. sigma-2 receptors (Ki sigma-1/sigma-2 = 0.02) using in vitro receptor binding assays. Moderate affinity for muscarinic M1 (Ki = 322 nM) and M2 (Ki = 178 nM) receptors and low affinity (Ki = 1,460 nM) for dopamine D2 receptors was also noted. The lipophilicity of 2 (log P7.5 = 3.24) is moderate, indicating that the ligand should readily cross the blood/brain barrier. The corresponding radioiodinated ligand, 123I-2, was synthesized from the appropriate trans vinyl tributylstannane precursor under acidic conditions using oxidative iododestannylation methods. HPLC purification provided the radioligand in yields ranging between 63 and 75% EOS (n = 5) and with > 99% radiochemical purity and a specific activity > 77,000 MBq/mumol. Preliminary in vivo distribution and pharmacological blocking studies using 123I-2 were performed in male Australian Albino Wistar rats. 123I-2 exhibited good brain uptake (1.11 +/- 0.14% ID at 20 min PI) with no significant loss of radioactivity from the brain over the course of the study (4 h). The gross regional brain distribution of the radioligand showed highest uptake in the posterior cortex and frontal cortex, with slightly lower uptake in other brain regions examined. Most of the uptake of radioactivity in the brain, lung, heart, muscle, and kidney was prevented by pre-administration of compounds with affinity for sigma receptors, indicating the in vivo specificity of the ligand. In view of these results, 123I-2 shows promise for the in vivo tomographic evaluation of sigma receptor densities.

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.

Cell numbers of two morphogenic forms of Bacillus subtilis (the cell-walled parental and the derived stable cell wall-deficient L-form) have been compared by two methods: DNA hybridization (i.e. deduced genome numbers) and viable cell counts (i.e. number of colony-forming units (cfu)). The DNA hybridization method was shown to be a reliable and reproducible method for estimating genome numbers. Comparison of different L-form populations showed that the two methods of enumeration gave different values, with the deduced genome numbers much higher (by several orders of magnitude) than cell numbers deduced from viable cell counts. In contrast, when a culture of the cell-walled form was enumerated, the discrepancy between the two methods was low (by a factor of about 6) The combination of a high number of L-form genomes detected by DNA hybridization and a relatively low number of cfu was thought to be a consequence of a diminished co-ordination between the DNA replication and cell division processes in L-form bacteria. This suggestion was further substantiated by assessing the stability of plasmid pPL608 in a transformed B. subtilis L-form cell line, where even in the presence of continued kanamycin selection, 25% of the population lost kanamycin resistance. The results are discussed with particular reference to cell division in cell wall-deficient, stable L-form bacteria.

Bioluminescence from developing L-form colonies of the plant pathogen, Pseudomonas syringae pv. phaseolicola, was monitored using the enhanced light-detecting capabilities of a charge-coupled device. During L-form induction, the bacteria entered a prolonged period during which the level of light output and hence metabolic activity, was very low. A relatively small number of highly bioluminescent L-form colonies were then observed to develop against a background of non-bioluminescent bacteria. When these colonies were sub-cultured and examined microscopically, typical L-form morphology was observed and continued high bioluminescence was detectable from derived colonies.

Bioluminescence (lux) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola-specific phage (phi 11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola phi 11P, 19H and P. aeruginosa phi PLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux+ bacteria were inoculated onto French bean leaf (Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux+ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.

We have developed a method which enables the cloning and identification of procaryotic repetitive DNA suitable for use as DNA fingerprinting probes. The method involves shotgun cloning of restricted genomic DNA with subsequent selection of clones containing repetitive DNA by reverse-probed genomic hybridizations, in which the plasmid DNA clones are probed with labelled genomic DNA. Confirmation that the clones contained repeated sequences was by Southern hybridization, gene copy equivalence, and DNA sequencing. The sequences were used for highly specific and sensitive detection of bacteria and as target sequences for the mediation of chromosomal integration of reporter gene constructs.

Three different methods of DNA isolation (organic deproteinization, potassium acetate deproteinization, and the use of cetyltrimethylammonium bromide) have been used to prepare DNA from Bacillus subtilis. Subsequent hybridization with an rDNA probe (DNA coding for rRNA) produces different patterns, which mirror those previously reported to indicate an rDNA deletion.

Charge-coupled device (CCD) image-enhancement was used for rapid screening of genomic libraries and allowed selection of promoters with differing characteristics. In addition, both the CCD and luminometry were used to monitor and characterize the expression from two Pseudomonas syringae pv. phaseolicola promoters. The same pattern of gene expression was indicated by the two methods but the CCD enabled the rapid non-destructive in situ monitoring of a microbial population, over a prolonged period.

Genes encoding bioluminescence from Vibrio harveyi were cloned into Pseudomonas syringae pv. phaseoli-cola, resulting in high levels of bioluminescence. After inoculation of sterile and nonsterile soil slurries with bioluminescent P. syringae, cells could not be identified by conventional light microscopy. However, when we used charge coupled device-enhanced microscopy, bioluminescent single cells were detected easily in dark fields despite masking by soil particulate matter, and in addition, the extent of competition from indigenous soil bacteria could be monitored. The technique which we describe offers great potential for tracking and determining the spatial distribution of genetically marked microorganisms in the environment.