Quantitative Autoradiographic Study Research Articles

Geoffery Burnstock's influence on the evolution of P2X3 receptor pharmacology.

I first met Geoffery Burnstock in 1985 while working as a young post-doctoral fellow for Michael Williams at CIBA-Geigy Corporation. During that time, we were researching the role of adenosine in stroke and cardiovascular disease. These research efforts crystallized into a drug discovery program aimed at developing selective adenosine receptor agonists as cardioprotective agents. The first potent and selective A2A receptor agonist, CGS-21680, was discovered as part of this effort [1]. This high-affinity agonist was successfully radiolabeled [2] and quantitative receptor autoradiography studies demonstrated that A2A receptors were discretely localized in the rat basal ganglia [3]. These data were considered controversial at the time since other radioligand binding studies with less selective ligands had indicated a much broader expression of the A2A receptor in the central nervous system [4]. During my first discussions with Geoff, I had the opportunity to share some of these early data. Needless to say, that as a newcomer to the field, I was a bit nervous about discussing our work with such a pioneering scientist. I remember Geoff’s supportive enthusiasm for our new data and his encouragement not to let existing dogma hinder our exploration into this area of purine pharmacology. His open-minded approach and unfettered excitement for new ideas has served as an inspirational guidepost throughout my research career. Shortly after I joined the Neuroscience Discovery team at Abbott Laboratories in 1995, a new drug discovery effort targeting novel analgesic mechanisms as potential non-opioid interventions for chronic neuropathic and inflammatory pain was initiated. During an internal scientific research governance meeting, a photomicrograph from Geoff’s UCL team showing the discrete expression of P2X3 mRNA selectively localized on peripherin-positive small diameter sensory nerves was shared ahead its publication [5]. Geoff’s willingness to share these exciting data indicating a potential specific role for a single member of the emerging P2X receptor family in nociceptive neurotransmission provided sufficient evidence to initiate a specific drug discovery research effort on the nociceptive role of P2X3 receptors. In retrospect, the notion that highly selective expression of individual members of the emerging purine receptor superfamilies (e.g., A2A receptors in brain and P2X3 receptors on peripheral nerves) could drive physiologically relevant neuromodulation is striking. Furthermore, it served as the scientific foundation of much of my drug discovery research activities for the better part of two decades. Below is a brief overview of the historical development P2X3 receptor pharmacology, much of which was directly driven by Geoff’s interactions with a wide variety of academic and industrial purinergic investigators over the years. Specific questions and insights provided by Geoff on our work are highlighted to illustrate the significant impact Geoff had on our work.

Abstract 3976: Molecular imaging of epigenetic regulation mediated by HDACs 4, 5 using PET/CT/MRI with 18F-TFAHA in a rat model of human glioma

Abstract Histone deacetylases (HDACs) are involved in the pathogenesis of cancer through modulation of the expression of various genes involved in cellular proliferation, migration, angiogenesis and apoptosis. HDAC class IIa enzymes interact with tumor suppressor proteins including HIF-1a, GATA-1, and PLZF-RARa. Given the importance of HDACs class IIa in epigenetic regulation of cancer development, progression, and maintenance, there is a pressing need for non-invasive imaging approaches for monitoring the expression-activity of HDACs in vivo. To address this need, our laboratory has developed 6-(tri-fluoroacetamido)-1-hexanoicanilide (18F-TFAHA) for imaging HDAC class IIa enzymes. Previous studies show that 18F-TFAHA is enzymatically cleaved specifically by HDAC class IIa, predominantly by HDACs 4 and 5. To quantitatively visualize the expression-activity of HDACs 4 and 5 in brain gliomas, immunocompromised rats (NTac:NIH-Foxn1rnu, Taconic Biosciences, NY) were implanted intracerebrally (i.c.) with U87-tdRluc cells (4×105 cells in 20 uL), that have been lentivirally-transfected to express GFP-luciferase and tdTomato fluorescent protein fusion reporter genes. Bioluminescence images (BLI) of GBMs were obtained after administration of luciferin (10 μl/g i.p.) using an InVivo Xtreme system (Carestream, Toronto, Canada) at 7 and 14 days post U87-tdRluc cell implantation. Gross tumor morphology and progression was evaluated with T2 MRI on day 14 after tumor implantation using a CliniScan 7T MRI system (Bruker, UK). Following 15-20 days, the rats were administered with 18F-TFAHA (500 μCi/animal, i.v.) and imaged using microPET R4 and Inveon CT (Siemens, TN). After PET/CT imaging, the animals were sacrificed and their brains extracted for immunohistochemical (IHC) and quantitative autoradiographic (QAR) studies (Typhoon 7000, General Electric, CT). The 18F-TFAHA accumulation in regions of interest (ROI) at 20-30 minutes post i.v. administration and was quantified using Logan graphical analysis, which demonstrated a significant increase in 18F-TFAHA accumulation in tumors versus surrounding normal cortex and white matter (p <0.05), outside the structures with normally-increased HDAC IIa activity (e.g., hippocampus, amygdala, periaqueductal gray, n. accumbens). PET/CT/MR imaging results were validated by IHC of brain tissue sections that demonstrated a significant hypoacetylation of histones H2A, H2B, and H4 in tumor tissue with increased 18F-TFAHA accumulation. The ongoing studies in brain tumor-bearing rats undergoing treatment with HDAC inhibitor vorinostat are aimed to assess the feasibility of PET/CT/MRI with 18F-TFAHA for pharmacodynamic monitoring therapies with HDAC class IIa inhibitors. Ultimately, we aim to translate 18F-TFAHA PET/CT/MR imaging into the clinic. Support: NIH RO1 DA030333-06, NCI CCGS Core Grant CA 016672, NIH RC2 DA028912-01, NIH P30 CA022453 Citation Format: Maxwell T. Laws, Robin E. Bonomi, Swatabdi Kamal, David Gelovani, Jeremy Llanguez, Vadim Popov, Xin Lu, Srinivasu Kallakuri, Thomas Mangner, Juri G. Gelovani. Molecular imaging of epigenetic regulation mediated by HDACs 4, 5 using PET/CT/MRI with 18F-TFAHA in a rat model of human glioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3976.

Brain penetrant small molecule 18F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats

IntroductionThe gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood–brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [18F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these 18F-labeled compounds in brain of living rats. MethodsWe synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for 18F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo. ResultsA palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The 18F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5–15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the 18F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo. ConclusionsThe novel small molecule 18F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro, and may prove useful for quantitative autoradiographic studies in vitro. The compounds were permeable to the blood–brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain.

Changes in D1 but not D2 dopamine or mu-opioid receptor expression in limbic and motor structures after lateral hypothalamus electrical self-stimulation: A quantitative autoradiographic study

Intracranial self-stimulation (ICSS) of the lateral hypothalamus (LH) is involved in the activation of neuroanatomical systems that are also associated with the processing of natural and other artificial rewarding stimuli. Specific components of this behavior (hedonic impact, learning, and motor behavior) may involve changes in different neurotransmitters, such as dopamine and opioids. In this study, quantitative autoradiography was used to examine changes in mu-opioid and D1/D2-dopamine receptor expression in various anatomical regions related to the motor and mesolimbic reward systems after intracranial self-stimulation of the LH. Results of the behavioral procedure and subsequent radiochemical assays show selective changes in D1 but not D2 or mu receptors in Accumbens-Shell, Ventral Pallidum, Caudate–Putamen, and Medial Globus Pallidus. These findings are discussed in relation to the different psychobiological components of the appetitive motivational system, identifying some dissociation among them, particularly with respect to the involvement of the D1-dopamine subsystem (but not D2 or mu receptors) in goal-directed behaviors.

Tu1827 Yh4808, a Potent, Highly Selective K+-Competitive Acid Blocker, Suppressed Rat Acute Reflux Esophagitis More Effectively Than Esomeprazole

Introduction: Proton pump inhibitors (PPIs) are now widely used for the treatment of acidrelated diseases, such as peptic ulcer and gastroesophageal reflux disease (GERD). PPIs which are weak-base prodrugs are activated in an acidic condition and only inhibit H+/K+ATPase that has already been activated. Therefore, the effect of PPIs on acid-related diseases is slow onset and their maximum efficacy can be achieved after several days of dosing, leading to unmet medical needs such as nocturnal acid breakthrough and unsatisfied symptom relief. Methods: We investigated the pharmacological profiles of YH4808, a novel potent, highly selective K+-competitive acid blocker (P-CAB), in terms of hog gastric H+/K+-ATPase activity, gastric acid secretion, and acute gastroesophageal lesions in animal models, in comparison with esomeprazole. In addition, a quantitative whole body autoradiography (QWBA) study was performed with [14C]YH4808 (10 mg/kg) in rats to characterize tissue distribution. Results: YH4808 inhibited H+/K+-ATPase activity in a concentration-dependent manner, with an IC50 value of 3.4 nM which is more potent than that of esomeprazole. The inhibitory mechanism of YH4808 on H+/K+-ATPase was a competitive antagonism to the K+-binding site of H+/K+-ATPase, and it was also a reversible inhibition. In a selectivity assay, YH4808 showed 3,000-fold greater selectivity against Na+/K+-ATPase. In pylorus-ligated rats, oral administration of YH4808 inhibited basal acid secretion in a dose-dependent manner with the ID50 value of 1.1 mg/kg. Anti-secretory activity of YH4808 against histamine-stimulated acid secretion in rats after IV injection (ID50=0.1 mg/kg) was stronger than that of esomeprazole (ID50=0.9 mg/kg). In Heidenhain pouch dogs, IV administration of YH4808 inhibited histamine-stimulated acid secretion in a dose-dependent manner with almost complete inhibition at the dose of 0.6 mg/kg. In a reflux esophagitis model, intraduodenal administration of YH4808 prevented esophageal lesions by 30, 82 and 91% at a dose of 1, 3, and 10 mg/kg, respectively while esomeprazole reduced the lesion by 47% at 10 mg/kg. A QWBA study in rats showed YH4808 accumulated and was retained in the gastric tissue for more than 24h after oral administration. Conclusion: YH4808, a novel selective P-CAB exerted a potent, longer-lasting anti-secretory efficacy through high accumulation in the gastric tissue, which translated into the more effective inhibition of reflux esophagitis than esomeprazole. These findings indicate that YH4808 can provide significant clinical benefits to patients with acid-related diseases.

Autoradiographic evaluation of [3H]CUMI-101, a novel, selective 5-HT1AR ligand in human and baboon brain

[11C]CUMI-101 is the first selective serotonin receptor (5-HT1AR) partial agonist radiotracer for positron emission tomography (PET) tested in vivo in nonhuman primates and humans. We evaluated specific binding of [3H]CUMI-101 by quantitative autoradiography studies in postmortem baboon and human brain sections using the 5-HT1AR antagonist WAY-100635 as a displacer. The regional and laminar distributions of [3H]CUMI-101 binding in baboon and human brain sections matched the known distribution of [3H]8-OH-DPAT and [3H]WAY-100635. Prazosin did not measurably displace [3H]CUMI-101 binding in baboon or human brain sections, thereby ruling out [3H]CUMI-101 binding to α1-adrenergic receptors. This study demonstrates that [11C]CUMI-101 is a selective 5-HT1AR ligand for in vivo and in vitro studies in baboon and human brain.

Comparison of 18F-fluoro-deoxy-glucose, 18F-fluoro-methyl-choline, and 18F-DPA714 for positron-emission tomography imaging of leukocyte accumulation in the aortic wall of experimental abdominal aneurysms

Abdominal aortic aneurysm (AAA) is a frequent form of atherothrombotic disease, whose natural history is to enlarge and rupture. Indicators other than AAA diameter would be useful for preventive surgery decision-making, including positron-emission tomography (PET) methods permitting visualization of aortic wall leukocyte activation relevant to prognostic AAA evaluation. In this study, we compare three PET tracers of activated leukocytes, 18F-fluoro-deoxy-glucose (FDG), 18F-fluoro-methyl-choline (FCH), and 18F-DPA714 (a peripheral benzodiazepine receptor antagonist) for in vivo PET quantification of aortic wall inflammation in rat experimental AAAs, in correlation with histopathological studies of lesions. AAAs were induced by orthotopic implantation of decellularized guinea pig abdominal aorta in 46 Lewis rats. FDG-PET (n = 20), FCH-PET (n = 8), or both (n = 12) were performed 2 weeks to 4 months after the graft, 1 hour after tracer injection (30 MBq). Six rats (one of which had FDG-PET) underwent 18F-DPA714-PET. Rats were sacrificed after imaging; AAAs and normal thoracic aortas were cut into axial sections for quantitative autoradiography and histologic studies, including ED1 (macrophages) and CD8 T lymphocyte immunostaining. Ex vivo staining of AAAs and thoracic aortas with 18F-DPA714 and unlabeled competitors was performed. AAAs developed in 35 out of 46 cases. FCH uptake in AAAs was lower than that of FDG in all cases on imaging, with lower AAA-to-background maximal standardized uptake value (SUV(max)) ratios (1.78 ± 0.40 vs 2.71 ± 0.54; P < .01 for SUV(max) ratios), and lower AAA-to-normal aorta activity ratios on autoradiography (3.52 ± 1.26 vs 8.55 ± 4.23; P < .005). FDG AAA-to-background SUV(max) ratios correlated with the intensity of CD8 + ED1 staining (r = .76; P < .03). FCH AAA-to-background SUV(max) ratios correlated with the intensity of ED1 staining (r = .80; P < .03). 18F-DPA714 uptake was similar in AAAs and in normal aortas, both in vivo and ex vivo. In rat experimental AAA, characterized by an important aortic wall leukocytes activity, FDG-PET showed higher sensitivity than FCH-PET and 18F-DPA714-PET to detect activated leukocytes. This enhances potential interest of this tracer for prognostic evaluation of AAA in patients.

Auditory Hallucinations as the Only Presenting Symptom of Right-Parietal Spontaneous Hemorrhage: FDG-PET Evidence of Corpus Callosum Hyperactivity

Back to table of contents Previous article Next article LettersFull AccessAuditory Hallucinations as the Only Presenting Symptom of Right-Parietal Spontaneous Hemorrhage: FDG-PET Evidence of Corpus Callosum HyperactivityMassimiliano Godani, M.D., Andrea Ciarmiello, M.D., Cesare Capellini, M.D., and Massimo Del Sette, M.D.Massimiliano GodaniSearch for more papers by this author, M.D., Andrea CiarmielloSearch for more papers by this author, M.D., Cesare CapelliniSearch for more papers by this author, M.D., and Massimo Del SetteSearch for more papers by this author, M.D.Published Online:1 Jan 2012https://doi.org/10.1176/appi.neuropsych.11020054AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: Auditory hallucinations are very rare in stroke patients. We report FDG-PET findings in a case of hemorrhagic stroke presenting with auditory hallucinations.Case ReportA 67-year-old woman was admitted to the emergency room of our hospital for acute onset of complex auditory hallucinations; she reported hearing loud music and voices of known persons speaking “filthy.” She had never suffered any psychiatric disorders in the past, and she was taking atenolol for many years as the only treatment for chronic arterial hypertension.Neurological examination did not show any focal neurological signs, and blood tests were normal. Brain CT scan revealed a right-parietal hemorrhage involving both superficial and deep territory of the posterior branches of middle cerebral artery (Figure 1 [D]). EEG showed absence of focal paroxystic epyleptiform discharges. Brain MRI and MR-angio confirmed the intraparenchimal hemorrhage, excluding the presence of aneurysm or artero-venous malformations. FDG-PET scan showed a significant increase in glucose metabolism (versus normal control database) in the middle and posterior areas of corpus callosum as well as in the precuneus and cuneus cortex (Figure 1 [A, B, C]).The auditory hallucinations gradually resolved in a few days without any treatment. She was discharged after 18 days from onset, and neurological examination showed visuospatial deficit as the consequence of right-parietal damage.DiscussionAuditory hallucinations are uncommon phenomena in patients with acute stroke; in particular, they have been reported in acute ischemic lesions of the brainstem but very rarely reported after cortical ischemic strokes.1 To our knowledge, this is the first case of auditory hallucinations as the only presenting symptom of a spontaneous hypertensive parenchymal hemmorrhage.In our case FDG-PET scan showed high relative glucose metabolic rates in the middle and posterior areas of corpus callosum. Hyperactivity of corpus callosum has already been reported in schizophrenic patients with auditory hallucinations.2,3 In particular, abnormalities in the posterior part of corpus callosum has been associated to schizophrenic positive symptoms.4 We speculate that in our patient the acute cortico-subcortical hemorrhage caused, through rapid development of peri-lesional edema and release of excitatory mediators such as calcium ions and amino-acids, an electrode-like effect,5 which abnormally activated the posterior part of corpus callosum, with consequent development of auditory hallucinations. We believe that a better understanding of corpus callosum pathophysiology could help in clarifying the mechanism of perceptive disturbances of various origin.Neurology UnitS. Andrea HospitalLA Spezia, ItalyCorrespondence: Massimiliano Godani, M.D.; massimiliano.[email protected]liguria.it1. Yair L , Mordechai L , Ronit G, et al.: Auditory hallucinations in acute stroke. Behav Neurol 2005; 16:211–216Crossref, Medline, Google Scholar2. Buchsbaum MS , Buchsbaum BR , Hazlett EA, et al.: Relative glucose metabolic rate higher in white matter in patients with schizophrenia. Am J Psychiatry 2007; 164:1072–1081Crossref, Medline, Google Scholar3. Hubl D , Koenig T , Strik W, et al.: Pathways that make voices. Arch Gen Psychiatry 2004; 61:658–668Crossref, Medline, Google Scholar4. Kubicki M , Styner M , Bouix S, et al.: Reduced interhemispheric connectivity in schizophrenia: tractography base segmentation of the corpus callosum. Schizophr Res 2008; 106(2–3):125–131Crossref, Medline, Google Scholar5. Weber B , Fouad K , Burger C, et al.: White-matter glucose metabolism during intracortical electrostimulation: a quantitative fluorodeoxyglucose autoradiography study in the rat. Neuroimage 2002; 16:993–998Crossref, Medline, Google Scholar FiguresReferencesCited byDetailsCited byJournal of the Neurological Sciences, Vol. 398Frontiers in Psychiatry, Vol. 8PET and SPECT Findings in Patients with Hallucinations29 January 2014 Volume 24Issue 1 Winter 2012Pages E28-E29 Metrics PDF download History Published online 1 January 2012 Published in print 1 January 2012

Toward in Vivo Imaging of Heart Disease Using a Radiolabeled Single-Chain Fv Fragment Targeting Tenascin-C

Antibodies specific to a particular target molecule can be used as analytical reagents, not only for in vitro immunoassays but also for noninvasive in vivo imaging, e.g., immunoscintigraphies. In the latter case, it is important to reduce the size of antibody molecules in order to achieve suitable in vivo "diagnostic kinetics" and generate higher-resolution images. For these purposes, single-chain Fv fragments (scFvs; M(r) < 30 kDa) have greater potential than intact immunoglobulins (~150 kDa) or Fab (or Fab') fragments (~50 kDa). Our recent observation of enhanced tenascin-C (Tnc) expression at sites of cardiac repair after myocardial infarction prompted us to develop a radiolabeled scFv against Tnc for in vivo imaging of heart disease. We cloned the genes encoding the heavy and light chain variable domains of the mouse anti-Tnc monoclonal antibody 4F10, and combined them to create a single gene. The resulting scFv-4F10 gene was expressed in E. coli cells to produce soluble scFv proteins. scFv-4F10 has an affinity for Tnc (K(a) = 3.5 × 10(7) M(-1)), similar to the Fab fragment of antibody 4F10 (K(a) = 1.3 × 10(7) M(-1)) and high enough to be of practical use. A cysteine residue was then added to the C-terminus to achieve site-specific (111)In labeling via a chelating group. The resulting (111)In-labeled scFv was administered to a rat model of acute myocardial infarction. Biodistribution and quantitative autoradiographic studies indicated higher uptake of the radioactivity at the infarcted myocardium than the noninfarcted one. Single photon emission computed tomography (SPECT) provided in vivo cardiac images that coincided with the ex vivo observations. Our results will promote advances in diagnostic strategies for heart disease.

Laminar distributions of fibrillar β-amyloid and activated astrocytes in Alzheimer's disease autopsy brain: A quantitative autoradiography study

Laminar distributions of fibrillar β-amyloid and activated astrocytes in Alzheimer's disease autopsy brain: A quantitative autoradiography study

MRI and quantitative autoradiographic studies following bolus injections of unlabeled and 14C‐labeled gadolinium‐diethylenetriaminepentaacetic acid in a rat model of stroke yield similar distribution volumes and blood‐to‐brain influx rate constants

In previous studies on a rat model of transient cerebral ischemia, the blood and brain concentrations of gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) following intravenous bolus injection were repeatedly assessed by dynamic contrast-enhanced (DCE)-MRI, and blood-to-brain influx rate constants (K(i)) were calculated from Patlak plots of the data in areas with blood-brain barrier (BBB) opening. For concurrent validation of these findings, after completing the DCE-MRI study, radiolabeled sucrose or α-aminoisobutyric acid was injected intravenously, and the brain disposition and K(i) values were calculated by quantitative autoradiography (QAR) assay employing the single-time equation. To overcome two of the shortcomings of this comparison, the present experiments were carried out with a radiotracer virtually identical to Gd-DTPA, Gd-[(14)C]DTPA, and K(i) was calculated from both sets of data by the single-time equation. The protocol included 3 h of middle cerebral artery occlusion and 2.5 h of reperfusion in male Wistar rats (n = 15) preceding the DCE-MRI Gd-DTPA and QAR Gd-[(14)C]DTPA measurements. In addition to K(i) , the tissue-to-blood concentration ratios, or volumes of distribution (V(R) ), were calculated. The regions of BBB opening were similar on the MRI maps and autoradiograms. Within them, V(R) was nearly identical for Gd-DTPA and Gd-[(14)C]DTPA, and K(i) was slightly, but not significantly, higher for Gd-DTPA than for Gd-[(14)C]DTPA. The K(i) values were well correlated (r = 0.67; p = 0.001). When the arterial concentration-time curve of Gd-DTPA was adjusted to match that of Gd-[(14)C]DTPA, the two sets of K(i) values were equal and statistically comparable with those obtained previously by Patlak plots (the preferred, less model-dependent, approach) of the same data (p = 0.2-0.5). These findings demonstrate that this DCE-MRI technique accurately measures the Gd-DTPA concentration in blood and brain, and that K(i) estimates based on such data are good quantitative indicators of BBB injury.

3H]4‐(dimethylamino)‐N‐(4‐(4‐(2‐methoxyphenyl)piperazin‐1‐yl) butyl)benzamide: A selective radioligand for dopamine D3 receptors. II. Quantitative analysis of dopamine D3 and D2 receptor density ratio in the caudate‐putamen

4-(Dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzamide (WC-10), a N-phenyl piperazine analog, displays high affinity and moderate selectivity for dopamine D(3) receptors versus dopamine D(2) receptors (Chu et al. [2005] Bioorg Med Chem 13:77-87). In this study, WC-10 was radiolabeled with tritium (specific activity = 80 Ci/mmol), and quantitative autoradiography studies were conducted using rhesus monkey and Sprague-Dawley rat brain sections. K(d) values for the binding of [3H]WC-10 to D(3) receptors obtained from quantitative autoradiography with rhesus monkey and rat brain sections are in agreement with K(d) values obtained from cloned human and rat receptors (Xu et al. [2009] Synapse 63:717-728). The D(2) selective antagonist [3H]raclopride binds with 11-fold higher affinity to human HEK D(2L) (K(d) = 1.6 nM) than HEK D(3) (K(d) = 18 nM) receptors; [3H]raclopride binds to rat Sf9 rD(2L) receptors with a K(d) of 6.79 nM, a value that is 4-fold lower than binding to human HEK D(2L) receptors and 2.5-fold higher than binding to rat Sf9 rD(3) receptors. In vitro quantitative autoradiography studies with [3H]WC-10 and [3H]raclopride were conducted on adult rat and rhesus monkey brain sections. A mathematical model for calculating the absolute densities of dopamine D(2) and D(3) receptors based on the in vitro receptor binding data of [3H]WC-10 and [3H]raclopride was developed.

Agonist binding fraction of dopamine D 2/3 receptors in rat brain: A quantitative autoradiographic study

There has arisen considerable interest in the study of dopamine D 2/3 agonist binding sites by positron emission tomography (PET), based on the claim that agonist sites represent a functional subset of the total number of sites labeled by more conventional antagonist ligands. To test the basis of this claim, we used quantitative autoradiography to measure the abundance of binding sites of a dopamine D 2/3 agonist ([ 3H]NPA) and an antagonist ([ 3H]raclopride) in cryosections of rat brain. Saturation binding studies revealed that the B max for [ 3H]NPA was nearly identical to that of [ 3H]raclopride in dorsal brain regions, but was 25% less in the ventral striatum and 56% less in the olfactory tubercle. We also tested the displacement of the two ligands by the hallucinogen LSD, which is known to have dopamine agonist properties. Whereas displacement of [ 3H]raclopride by increasing LSD concentrations was monophasic, displacement of [ 3H]NPA was biphasic, suggesting an action of LSD via a subset of dopamine D 2/3 agonist binding sites. Addition of the stable GTP analogue Gpp(NH)p to the medium abolished 90% of the [ 3H]NPA binding, and increased [ 3H]raclopride binding by 10%, with a shift to the right in the LSD competition curve, suggesting retention of endogenous dopamine in washed cryostat sections. Thus [ 3H]NPA and [ 3H]raclopride binding sites have nearly identical abundances in rat dorsal striatum, but are distinct in the ventral striatum, and with respect to their displacement by LSD.

Vascular Endothelial-Cadherin Targeted Alpha-Particle Mediated Vascular Killing and Remodeling Augments Subsequent Chemotherapeutic Efficacy.

Abstract 3054Poster Board II-1030Cancer therapies targeting tumor endothelium have shown promising results when combined with chemotherapeutics. Vascular endothelial (VE) cadherin is an endothelial cell specific molecule that is expressed constitutively throughout the vasculature and participates in the formation of adherens junctions between adjacent endothelial cells. The monoclonal antibody E4G10 specifically binds to a cryptic epitope exposed only on the monomeric unengaged form of VE-Cadherin found in neovasculature. We chemically conjugated E4G10 with the cytotoxic, short-range αa-particle emitter, 225Ac, and show that the agent dampens tumor growth as a single agent and this effect is enhanced when given in combination with chemotherapy in a xenograft mouse model. The therapeutic efficacy of the combination therapy is sequence dependent and most pronounced when 225Ac-E4G10 is administered prior to the chemotherapy. Immunohistochemical and immunofluorescence studies for endothelial cells (Meca32), vascular basement membrane (Collagen IV), and pericytes (alpha-smooth muscle actin (alpha-SMA), nerve/glial antigen 2 (NG2) ) showed that the vasculature of 225Ac-E4G10 treated tumors was depleted and the remaining vessels appeared normalized as evidenced by morphological changes, increased pericyte density and coverage. Fluorescence studies with Hoechst 33342 dye i.v. injected showed more homogenous distribution of the dye within 225Ac-E4G10 treated tumors, suggesting improved perfusion, and biodistribution studies with i.v. injected 111In-Diethylene triamine pentaacetic acid (DTPA), a freely diffusible small molecule, showed significantly (p=0.03) increased passive tumor accumulation of 111In-DTPA in 225Ac-E4G10-treated tumors when compared to controls. Quantitative autoradiographic studies on tumor sections also showed both stronger and more homogenous signal distribution in 225Ac-E4G10-treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and remodeling of the tumor vasculature allowing improved delivery of subsequent small molecules, such as chemotherapeutics, and thereby improves therapeutic outcome. DisclosuresMcDevitt:Actinium Pharma Inc.: Patents & Royalties. Scheinberg:Actinium Pharma Inc.: Patents & Royalties.

Quantitative autoradiographic study on receptor regulation in the basal ganglia in rat model of levodopa-induced motor complications

In order to study neurotransmitter receptor regulation in the basal ganglia involved in the functional changes underlying levodopa-induced motor complications, quantitative autoradiography was used to observe receptor bindings of dopamine D1 and D2, N-methyl-D-aspartate (NMDA), amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) and amino butyric acid (GABA) in the basal ganglia of rats that had unilateral nigrostriatal lesions and had been chronically treated with levodopa until motor complications developed. The rats were randomly assigned to three groups: normal, denervated and treatment-complicated groups. The results showed that response duration to levodopa became progressively shorter and abnormal involuntary movement (AIM) score was progressively increased during the course of levodopa treatment. Chronic treatment augmented D1 receptors more than denervation, and reduced D2 receptors that were also increased by dopamine denervation. Striatal NMDA receptors were substantially up-regulated in the treatment-complicated group. Levodopa treatment did not change receptors of nigral AMPA, pallidal GABA, and subthalamic GABA, which remained the same as that in denervation group. However, chronic treatment reversed the increase of nigral GABA receptors caused by the lesion. It was concluded that a shortening of response duration and AIM mimicked levodopa-induced motor complications of Parkinson's patients. These data suggested that up-regulation of dopamine D1 and NMDA receptors in the striatum leads to an imbalance of stimulation through the striatal output pathways, which is associated with levodopa-induced motor complications.

Species Differences in the Relative Densities of D1- and D2-Like Dopamine Receptor Subtypes in the Japanese Quail and Rats: An in vitro Quantitative Receptor Autoradiography Study

Evidence has accumulated that the regulation of male sexual behavior by dopamine might not be the same in Japanese quail (and perhaps all birds) as it is in mammals. For example, the non-selective dopamine receptor agonist, apomorphine (APO), facilitates male sexual behavior in rats but inhibits it in quail. Although the general organization of the dopamine system is similar in birds and mammals, it is possible that the relative distribution and/or density of binding sites are different. We therefore compared the relative densities of D1-like and D2-like receptor subtypes in Japanese quail and rats, with the use of in vitro quantitative receptor autoradiography. Brain sections from 8 male rats and 8 male quail were labeled with [³H]SCH-23390 and [³H]Spiperone. In general we found a systematic species difference in the relative density of D1- vs. D2-like receptors such that the D2/D1 ratio is higher in quail than in rats in areas, known to be important target sites for dopamine action such as striatal regions or the preoptic area, which is also associated with activation of sexual behavior. This difference might explain the variation in the behavioral effectiveness of APO in rats as compared to quail; with a higher relative density of D2-like receptors in quail, a similar dose of APO would be more likely to activate inhibitory processes in quail than in rats.

Chronic nicotine alters cannabinoid-mediated locomotor activity and receptor density in periadolescent but not adult male rats

A significant number of youths use cigarettes, and more than half of the youths who smoke daily also use illicit drugs. The focus of these studies is on how exposure to nicotine affects subsequent responses to both nicotine and cannabinoids in adolescents compared with adults. We have shown previously that chronic treatment with nicotine produces sensitization to its locomotor-activating effects in female and adult rats but not male adolescent rats. To better understand the effects of nicotine on adolescent and adult rats, rats were injected with nicotine or saline for 7 days and, on day 8, either challenged with delta-9-tetrahydrocannabinol (Δ9-THC) or the cannabinoid agonist CP 55,940 and tested for locomotor activity, or the brains were removed for quantitative autoradiography studies of the cannabinoid1 receptor. A separate group of rats was treated with nicotine plus the cannabinoid antagonist AM 251 and then challenged with CP 55,940. In adolescent male rats, nicotine administration led to sensitization to the locomotor-decreasing effects of both Δ9-THC and CP 55,940, but in adult male rats, the response to either drug was unchanged compared to controls. The effect of nicotine on CP 55,940-mediated locomotor activity was blocked by co-administration of AM 251 with the nicotine. Further, cannabinoid receptor density was increased in the prelimbic prefrontal cortex, ventral tegmental area, and select regions of the hippocampus in adolescent male rats pretreated with nicotine compared to vehicle-treated controls. There were no significant changes in cannabinoid receptor binding, however, in any of the brain regions examined in adult males pretreated with nicotine. The prelimbic prefrontal cortex and the hippocampus have been shown previously to be involved in stimulant reinforcement; thus it is possible that these changes contribute to the unique behavioral effects of chronic nicotine and subsequent drug administration in adolescents compared with adults.

Use of 11C-(+)-α-dihydrotetrabenazine for the assessment of dopaminergic innervation in animal models of Parkinson's disease

This study evaluates the utility of (11)C-(+)-alpha -dihydrotetrabenazine ((11)C-(+)DTBZ) in the quantification of dopaminergic innervation by positron emission tomography (PET) in rat and monkey, two animal species used as animal models of Parkinson's disease. Healthy control animals (n = 10) and the effect of 6-hydroxidopamine (6-OHDA) neurotoxic were studied in rats. (18)F-DOPA PET studies and digital quantitative autoradiography were also carried out. Studies with Macaca fascicularis were performed in control and 1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine (MPTP) treated animals. In both species high quality images were generated in which clear uptake of (11)C-(+)DTBZ was found in the striatum. (11)C-(+)DTBZ uptake quantification was estimated by creating parametric images and binding potential (BP) calculation. BP in control rats was 1.10 +/- 0.16 (mean +/- standard deviation [SD], whereas 6-OHDA produced a decrease in the uptake depending on the lesion degree. Images obtained with (18)F-DOPA were not adequate for the analysis as they did not discriminate the stratum whereas digital quantitative autoradiography studies confirmed the high affinity of striatum by (11)C-(+)DTBZ. In monkeys, final BP values were 1.31 and 1.06 and MPTP treatment reduced uptake by 40 %. The quality of PET images and the decrease of uptake in 6-OHDA and MPTP lesions show that (11)C-(+)DTBZ is an adequate radiotracer for the study of dopaminergic innervation in these animal models.

Expression of gonadotrophin-releasing hormone binding sites in somatic tissues of the gilthead seabream (Sparus aurata): a quantitative autoradiographic study

In this study, we have analysed the expression of gonadotrophin-releasing hormone (GnRH) binding sites in somatic tissues (intestine, liver, gill, skeletal muscle, ovary, heart, stomach, kidney and spleen) of the gilthead seabream, Sparus aurata using 3-[125I]iodototyrosyl5-mammalian GnRH and auto-radiographic techniques. The qualitative and quantitative analysis showed the existence of a basal expression of specific GnRH binding sites in intestine, skeletal muscle, ovary, stomach and spleen. Furthermore, our data suggest that the level of expression of GnRH binding sites can be significantly enhanced by GnRH treatment in intestine, gill, heart, stomach, kidney and spleen. This study shows that GnRH can exert direct effects in both reproductive and non-reproductive somatic tissues of the gilthead seabream.

99m Tc-Annexin-V Functional Imaging of Luminal Thrombus Activity in Abdominal Aortic Aneurysms

The mural thrombus of abdominal aortic aneurysms (AAA) is involved in aneurysm progression via several interdependent biological processes including platelet activation. 99mTc-annexin V (ANX) is a scintigraphic tracer that binds to phosphatidylserine exposed on activated platelets and apoptotic cells. Here, we evaluated the potential of ANX imaging to assess mural thrombus biological activity in an experimental AAA model. The clinical applicability was further tested ex vivo on human samples of excised AAA thrombi. Experimental AAA was created by infusing elastase into infrarenal abdominal aorta in 17 rats, and 6 sham-operated rats were used as controls. Abdominal ANX scintigraphy was performed 2 weeks later followed by quantitative autoradiography and histological studies. Among the 13 rats which developed AAA, 11 displayed intense ANX uptake within AAA by scintigraphy. ANX uptake in the aneurysms on planar and single-photon emission computed tomography (SPECT) imaging was higher than that observed in infrarenal aorta of sham-operated controls (target/background ratio: 5.7+/-0.9 versus 1.33+/-0.21; P<0.005 for SPECT). Aneurysm-to-background activity ratios obtained by scintigraphy correlated with ANX activity in corresponding autoradiograms (R=0.69; P<0.02). This activity was located in the thrombus area where activated platelets and polymorphonuclear leukocytes accumulated. Similar patterns were also found in all of the 7 human AAA thrombi harvested during surgery. ANX imaging may assess mural thrombus renewal activity linked to permanent flowing blood interface.