Hank F. Kung

Increased striatal dopamine transporter in adult patients with attention deficit hyperactivity disorder: effects of methylphenidate as measured by single photon emission computed tomography.

Ten previously untreated adults with attention deficit hyperactivity disorder (ADHD) were investigated before and after 4 weeks of treatment with a dose of 3x5 mg methylphenidate/d by single photon emission computed tomography (SPECT) with [Tc-99m]TRODAT-1, the first Tc-99m labelled SPECT ligand specifically binding to the dopamine transporter (DAT). For semiquantitative evaluation of the DAT, specific binding ([STR-BKG]/BKG) was calculated in the striatum (STR) with the cerebellum used as background (BKG). The patients with ADHD presented with increased specific binding of Tc-99m-TRODAT-1 to the DAT as compared with age and sex matched controls ([STR-BKG]/BKG 1.43+/-0.18 vs. 1.22+/-0.05, P<0.001). After treatment with methylphenidate specific binding decreased in all patients ([STR-BKG]/BKG 1.02+/-0.23, P<0.001). Thus, for the first time it could be demonstrated using SPECT that methylphenidate lowers increased striatal DAT availability in adults suffering from ADHD.

In Vivo Imaging of Amyloid Deposition in Alzheimer Disease Using the Radioligand 18F-AV-45 (Flobetapir F 18)

An 18F-labeled PET amyloid-β (Aβ) imaging agent could facilitate the clinical evaluation of late-life cognitive impairment by providing an objective measure for Alzheimer disease (AD) pathology. Here we present the results of a clinical trial with (E)-4-(2-(6-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl benzenamine (18F-AV-45 or flobetapir F 18). Methods: An open-label, multicenter brain imaging, metabolism, and safety study of 18F-AV-45 was performed on 16 patients with AD (Mini-Mental State Examination score, 19.3 ± 3.1; mean age ± SD, 75.8 ± 9.2 y) and 16 cognitively healthy controls (HCs) (Mini-Mental State Examination score, 29.8 ± 0.45; mean age ± SD, 72.5 ± 11.6 y). Dynamic PET was performed over a period of approximately 90 min after injection of the tracer (370 MBq [10 mCi]). Standardized uptake values and cortical-to-cerebellum standardized uptake value ratios (SUVRs) were calculated. A simplified reference tissue method was used to generate distribution volume ratio (DVR) parametric maps for a subset of subjects. Results: Valid PET data were available for 11 AD patients and 15 HCs. 18F-AV-45 accumulated in cortical regions expected to be high in Aβ deposition (e.g., precuneus and frontal and temporal cortices) in AD patients; minimal accumulation of the tracer was seen in cortical regions of HCs. The cortical-to-cerebellar SUVRs in AD patients showed continual substantial increases through 30 min after administration, reaching a plateau within 50 min. The 10-min period from 50 to 60 min after administration was taken as a representative sample for further analysis. The cortical average SUVR for this period was 1.67 ± 0.175 for patients with AD versus 1.25 ± 0.177 for HCs. Spatially normalized DVRs generated from PET dynamic scans were highly correlated with SUVR (r = 0.58–0.88, P < 0.005) and were significantly greater for AD patients than for HCs in cortical regions but not in subcortical white matter or cerebellar regions. No clinically significant changes in vital signs, electrocardiogram, or laboratory values were observed. Conclusion: 18F-AV-45 was well tolerated, and PET showed significant discrimination between AD patients and HCs, using either a parametric reference region method (DVR) or a simplified SUVR calculated from 10 min of scanning 50–60 min after 18F-AV-45 administration.

Preclinical Properties of 18F-AV-45: A PET Agent for Aβ Plaques in the Brain

β-amyloid plaques (Aβ plaques) in the brain, containing predominantly fibrillary Aβ peptide aggregates, represent a defining pathologic feature of Alzheimer disease (AD). Imaging agents targeting the Aβ plaques in the living human brain are potentially valuable as biomarkers of pathogenesis processes in AD. (E)-4-(2-(6-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl benzenamine (18F-AV-45) is such as an agent currently in phase III clinical studies for PET of Aβ plaques in the brain. Methods: In vitro binding of 18F-AV-45 to Aβ plaques in the postmortem AD brain tissue was evaluated by in vitro binding assay and autoradiography. In vivo biodistribution of 18F-AV-45 in mice and ex vivo autoradiography of AD transgenic mice (APPswe/PSEN1) with Aβ aggregates in the brain were performed. Small-animal PET of a monkey brain after an intravenous injection of 18F-AV-45 was evaluated. Results: 18F-AV-45 displayed a high binding affinity and specificity to Aβ plaques (Kd, 3.72 ± 0.30 nM). In vitro autoradiography of postmortem human brain sections showed substantial plaque labeling in AD brains and not in the control brains. Initial high brain uptake and rapid washout from the brain of healthy mice and monkey were observed. Metabolites produced in the blood of healthy mice after an intravenous injection were identified. 18F-AV-45 displayed excellent binding affinity to Aβ plaques in the AD brain by ex vivo autoradiography in transgenic AD model mice. The results lend support that 18F-AV-45 may be a useful PET agent for detecting Aβ plaques in the living human brain.

Altered depression-related behaviors and functional changes in the dorsal raphe nucleus of serotonin transporter-deficient mice

BACKGROUND As a key regulator of serotonergic activity and target of many antidepressant treatments, the serotonin transporter (SERT) represents a potential mediator of anxiety- and depression-related behaviors. Using mice lacking the SERT (SERT KO), we examined the role of SERT function in anxiety- and depression-related behaviors and serotonergic neuron function. METHODS Serotonin transporter knockout mice were evaluated in paradigms designed to assess anxiety-, depression-, and stress-related behaviors. Dorsal raphe nucleus (DRN) function was assessed by quantitative serotonergic cell counting and extracellular electrical recording of neuronal firing properties. RESULTS Serotonin transporter knockout mice showed an increase in latency to feed in a novel situation, more immobility in a forced swim, increased escape latency in a shock escape paradigm, and decreased immobility in tail suspension. No differences in anxiety-related behaviors were seen in the open field and the elevated plus maze. Serotonin transporter knockout mice exhibit a 50% reduction in serotonergic cell number and a fourfold decrease in firing rate in the DRN. CONCLUSIONS Developmental loss of SERT produces altered behaviors in models of depression that are generally opposite to those produced by antidepressant treatment. The reduced serotonergic cell number and firing rate in the DRN of adult SERT KO mice suggest a mechanism for these altered behaviors.

MICRODIALYSIS AND SPECT MEASUREMENTS OF AMPHETAMINE-INDUCED DOPAMINE RELEASE IN NONHUMAN PRIMATES

The competition between endogenous transmitters and radiolabeled ligands for in vivo binding to neuroreceptors might provide a method to measure endogenous transmitter release in the living human brain with noninvasive techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT). In this study, we validated the measure of amphetamine‐induced dopamine release with SPECT in nonhuman primates. Microdialysis experiments were conducted to establish the dose‐response curve of amphetamine‐induced dopamine release and to document how pretreatment with the dopamine depleter alpha‐methyl‐para‐tyrosine (αMPT) affects this response. SPECT experiments were performed with two iodinated benzamides, [123I]IBZM and [123I]IBF, under sustained equilibrium condition. Both radio‐tracers are specific D2 antagonists, but the affinity of [123I]IBZM (KD = 0.4 nM) is lower than that of [123I]IBF (KD = 0.1 nM). With both tracers, we observed a prolonged reduction in binding to D2 receptors following amphetamine injection. [123I]IBZM binding to D2 receptors was more affected than [123I]IBF by high doses of amphetamine, indicating that a lower affinity increases the vulnerability of a tracer to endogenous competition. With [123I]IBZM, we observed an excellent correlation between reduction of D2 receptor binding measured with SPECT and peak dopamine release measured with microdialysis after various doses of amphetamine. Pretreatment with αMPT significantly reduced the effect of amphetamine on [123I]IBZM binding to D2 receptors, confirming that this effect was mediated by intrasynaptic dopamine release. Together, these results validate the use of this SPECT paradigm as a noninvasive measurement of intrasynaptic dopamine release in the living brain. Synapse 25:1–14, 1997.

Imaging of dopamine transporters in humans with technetium-99m TRODAT-1.

Technetium-99m TRODAT-1, a tropane derivative, has shown promise as a tracer for the imaging of dopamine transporters in preliminary studies in rats and baboons. The present report concerns the first study of the use of [99mTc]TRODAT 1 for the same purpose in humans. The specific uptake of [99mTc]TRODAT1 in dopamine transporter sites located in the basal ganglia area was confirmed: the best contrast between the basal ganglia and the occipital area, which is devoid of dopamine transporters, was achieved at 120–140 min following injection. The development of a99mTc-based agent bypasses the need for cyclotron-produced radionuclides, which will be of benefit for routine clinical studies.

2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine (ADAM): an improved serotonin transporter ligand

Serotonin transporters (SERT) are target-sites for commonly used antidepressants, such as fluoxetine, paroxetine, sertraline, and so on. Imaging of these sites in the living human brain may provide an important tool to evaluate the mechanisms of action as well as to monitor the treatment of depressed patients. Synthesis and characterization of an improved SERT imaging agent, ADAM (2-((2-((dimethylamino)methyl)phenyl)thio)-5-iodophenylamine)(7) was achieved. The new compound, ADAM(7), displayed an extremely potent binding affinity toward SERT ( K(i)=0.013 nM, in membrane preparations of LLC-PK(1)-cloned cell lines expressing the specific monoamine transporter). ADAM(7) also showed more than 1,000-fold selectivity for SERT over norepinephrine transporter (NET) and dopamine transporter (DAT) ( K(i)=699 and 840 nM, for NET and DAT, respectively). The radiolabeled compound [(125)I]ADAM(7) showed an excellent brain uptake in rats (1.41% dose at 2 min post intravenous [IV] injection), and consistently displayed the highest uptake (between 60-240 min post IV injection) in hypothalamus, a region with the highest density of SERT. The specific uptake of [(125)I]ADAM(7) in the hypothalamus exhibited the highest target-to-nontarget ratio ([hypothalamus - cerebellum]/cerebellum was 3.97 at 120 min post IV injection). The preliminary imaging study of [(123)I]ADAM in the brain of a baboon by single photon emission computed tomography (SPECT) at 180-240 min post IV injection indicated a specific uptake in midbrain region rich in SERT. These data suggest that the new ligand [(123)I]ADAM(7) may be useful for SPECT imaging of SERT binding sites in the human brain.

Congo red and thioflavin-T analogs detect Aβ oligomers

Several small molecule ligands for amyloid‐β (Aβ) fibrils deposited in brain have been developed to facilitate radiological diagnosis of Alzheimer’s disease (AD). Recently, the build‐up of Aβ oligomers (AβO) in brain has been recognized as an additional hallmark of AD and may play a more significant role in early stages. Evidence suggests that quantitative assessment of AβO would provide a more accurate index of therapeutic effect of drug trials. Therefore, there is an urgent need to develop methods for efficient identification as well as structural analysis of AβO. We found that some well established amyloid ligands, analogs of Congo red and thioflavin‐T (ThT), bind AβO with high affinity and detect AβO in vitro and in vivo. Binding studies revealed the presence of binding sites for Congo red‐ and thioflavin‐T‐analogs on AβO. Furthermore, these ligands can be used for imaging intracellular AβO in living cells and animals and as positive contrast agent for ultrastructural imaging of AβO, two applications useful for structural analysis of AβO in cells. We propose that by improving the binding affinity of current ligands, in vivo imaging of AβO is feasible by a ‘signal subtraction’ procedure. This approach may facilitate the identification of individuals with early AD.

18F Stilbenes and Styrylpyridines for PET Imaging of Aβ Plaques in Alzheimer’s Disease: A Miniperspective

Alzheimer’s disease (AD) is a neurodegenerative disease of the brain, characterized by a slowly progressive dementia. This insidious disease is growing in importance because it affects millions of older patients. Clinical symptoms of AD include cognitive decline, irreversible memory loss, disorientation and language impairment. Major neuropathology observations of postmortem AD brain include the presence of senile plaques containing β-amyloid (Aβ) aggregates and neurofibrillary tangles containing highly phosphorylated tau proteins (Figure 1A).1,2 Several genomic factors have been linked to AD. Familial AD (or early onset AD) has been reported to have mutations in genes encoding β-amyloid precursor protein (APP), presenilin 1, presenilin 2 and Apolipoprotein E (APOE).3 The exact mechanisms of these mutations, which lead to the development of AD, are not fully understood; however, formation of plaques comprised of Aβ peptide in the brain is a pivotal event in the pathology of Alzheimer’s disease. Significant evidence suggests that accumulation and aggregation of Aβ peptides may play a major causative role in AD pathogenesis.2,4 The excessive burden of Aβ, produced by various mechanisms, may represent the starting point of neurodegenerative events, and may initiate a cascade of events (β-amyloid cascade, Figure 1B) that includes gliosis, inflammatory changes, neuritic/synaptic change, tangles and transmitter loss.2 Currently, there is no definitive method to diagnose AD, except by postmortem evaluation and staining of the brain tissue, which demonstrates the existence of Aβ plaques. Figure 1 A. Processes (β-amyloid cascade) participating in AD pathogenesis. Aβ peptides produced by neurons aggregate into a variety of assemblies, some of which may impair synapses and neuronal dendrites. Build-up of pathogenic Aβ aggregates ... Recent reports have suggested that β-amyloid aggregates in the brain play a key role in a cascade of events leading to AD.2,5 Thus, the development of diagnostic imaging agents targeting Aβ aggregates is very important in the diagnosis and treatment of AD. Novel PET imaging agents specifically targeting the Aβ plaques may lead to early detection of AD pathology, differential diagnosis of patients with dementia, and for monitoring patients who are undergoing drug treatment designed to reverse the Aβ buildup in the brain. Indeed, diagnosis and treatment of AD have been hampered by the absence of reliable non-invasive markers for the underlying pathology. Diagnosis based on consensus criteria is approximately 81% sensitive and 70% specific by comparison to the gold standard of pathology at autopsy.6 In addition to errors of misdiagnosis in patients with AD, there is significant under diagnosis; approximately 10% of community dwelling elderly still have undiagnosed dementia, and community physicians may fail to diagnose up to 33% of mild dementia cases.7 Thus, there is a need for a biomarker that can be applied in the community setting and can help physicians separate those patients who do not have AD from those who have pathological signs and should be evaluated further. Additionally, there are a large number of patients who, upon comprehensive diagnostic testing, are found to have cognitive impairment but are not demented and thus, do not meet diagnostic criteria for AD (e.g., patients with mild cognitive impairment, MCI). Some, but not all of these patients will go on to develop AD within 3-5 years. A reliable biomarker might aid prognostic evaluation by documenting the presence or absence of AD related pathology. Based on the definitions of AD endorsed by the American Academy of Neurology, American Psychiatric Association (DSM-IV) and others, patients without abnormal amyloid plaque levels do not meet currently accepted neuropathological criteria for AD. This definition of AD, which includes amyloid plaques as a required feature, is supported by more than 100 years of autopsy data. Therefore, based on this widely-endorsed definition of AD, the use of a test for ruling-out the presence of amyloid plaque pathology in subjects with clinical signs and symptoms of cognitive impairment will, effectively, rule-out the diagnosis of AD, and lead to more careful evaluation and appropriate treatment for alternative causes of cognitive deficits. Moreover, the use of a test for ruling-in the presence of abnormal levels of Aβ plaques in the brain of subjects with signs and symptoms of cognitive impairment will lead to the selection of patients who warrant more detailed work-up for the possible diagnosis of AD or MCI. The differential diagnosis for AD includes a large number of other diseases. At early stages of disease (e.g. MCI), frequent confounds include cognitive impairment as a result of underlying depression, effects of CNS active medications, inadequately treated or end stage medical conditions affecting other organ systems, and even normal age-related changes. At later stages of disease, more common confounds include vascular dementia, frontal temporal lobar dementia (FTLD) complex, dementia with Lewy bodies (DLB) as well as rarer neurodegenerative diseases such as Creutzfeld Jacob Disease (CJD). Importantly, AD subjects will always have Aβ plaques, whereas amyloid is seen not at all, or only sporadically in most of these other diseases. In each case, appropriate prognosis and treatment requires accurate diagnostic assessment.

[99mTc]TRODAT-1 : a novel technetium-99m complex as a dopamine transporter imaging agent

Technetium-99m is the most commonly used radionuclide in routine nuclear medicine imaging procedures. Development of99mTc-labeled receptor-specific imaging agents for studying the central nervous system is potentially useful for evaluation of brain function in normal and disease states. A novel99mTc-labeled tropane derivative, [99mTc]TRODAT 1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. After i.v. injection into rats, [99mTc]TRODAT-1 displayed specific brain uptake in the rat striatal region (striatum-cerebellum/cerebellum ratio 1.8 at 60 min), where dopamine neurons are concentrated. The specific striatal uptake could be blocked by pretreating rats with a dose of competing dopamine transporter ligand, ß-CIT (or RTI-55, i.v., 1 mg/kg). However, the specific striatal uptake of [99mTc]TRODAT-] was not affected by co-injection of excess free ligand (TRODAT-1, up to 200 μg per rat) or by pretreating the rats with haloperidol (i.v., 1 mg/kg). The specific uptake in striatal regions of rats that had prior 6-hydroxydopamine lesion in the substantia nigra area showed a dramatic reduction. The radioactive material recovered from the rat striatal homogenates at 60 min after i.v. injection of [99mTc]TRODAT-1 showed primarily the original compound (>95%), a good indication of in vivo stability in brain tissue. Similar and comparable organ distribution patterns and brain regional uptakes of [99mTc]TRODAT-1 were obtained for male and female rats. Ex vivo autoradiography results of rat brain sections further confirmed the high uptake and retention of [99mTc]TRODAT-1 in the striatal region. In vitro binding studies measuring the affinity to dopamine transporters for the free ligand, TRODAT-1, and a nonradioactive rhenium derivative, Re-TRODAT-1, showed Ki values of 9.7 nM and 14.1 nM, respectively. Behavioral studies in rats using the free ligand, TRODAT-1 and Re-TRODAT-1 indicated that, unlike other tropane derivatives, they displayed no effect on locomotor activity, suggesting low toxicity. These results strongly support the conclusions that this novel99mTc radioligand binds selectively to dopamine transporters in the brain and that is is potentially useful for in vivo assessment of the loss of dopamine neurons in Parkinsons and other neurodegeneralive diseases.