Amyloid and tau imaging, neuronal losses and function in mild cognitive impairment.

OBJECTIVES: Establish new approaches for early diagnosis of dementia, based on imaging amyloid and tau pathology, cell losses and neuronal function, in subjects with mild cognitive impairment (MCI),. The overall aim is to develop effective tools for monitoring disease progression in the living patient to facilitate discovery of early therapeutic interventions to modify the course of the disease. DESIGN: Use 2-(1-{6-[(2-[F- 18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile ([F-18]FDDNP) in combination with positron emission tomography (PET) to produce dynamic images for quantification of regional cortical brain deposition in MCI patients and compare them with controls subjects and patients with Alzheimer's disease (AD). Comparison with other molecular imaging probes for neuronal losses and function were also made. SETTING: Patients are positioned supine in the tomograph bed with his/her head in the detector ring field. Upon injection of the molecular imaging probe (e.g., [F-18]FDDNP) images are obtained at very short time intervals for up to two hours. This results in dynamic sequences of brain distribution of the probe. PARTICIPANTS: Patients with clinical diagnosis of AD, MCI and control subjects. MEASUREMENTS: Subjects in the categories established above were scanned with [F-18]FDDNP-PET and quantification performed using Logan parametric graphical analysis to measure relative quantitative amyloid loads throughout the brain within patient groups. These results were compared in the same patients with cell losses in hippocampus using 4-[F-18]fluoro-N-{2-[4-(2-methoxyphenyl)- 1-piperazinyl]ethyl}-N-(2-pyridinyl)benzamide,([F-18]MPPF) and regional cerebral glucose metabolic rates using 2-deoxy-2-[F-18]fluoro-2-deoxy-D-glucose (2-[F-18]FDG). RESULTS: [F-18]FDDNP reliably follows neuropathological progression (amyloid plaques [SP]; neurofibrillary tangles [NFT]) in the living brain of AD patients and those with MCI. The distribution of [F-18]FDDNP brain cortical accumulation correlates well with behavioral measures (e.g., MMSE scores) and follows known patterns of pathological distribution observed at autopsy. We have also established conversion of controls to MCI and MCI to AD with precision and sensitivity in patients and control subjects in follow-up studies. Moreover, we have established that hemispheric cortical surface mapping of [F-18]FDDNP binding is a powerful tool for assessment and visualization of the rate of brain pathology deposition. A strong correlation of [F-18]FDDNP binding, cell losses in hippocampus and decreased glucose utilization ([F-18]FDG PET) in several neocortical regions was found in the same AD and MCI subjects. CONCLUSIONS: The combined evaluation of [F-18]FDDNP PET (targeting NFT and_SP) with neuronal losses in the hippocampus and with [F-18]FDG PET (targeting neuronal function) offers the opportunity for reliable, noninvasive detection of MCI patients at risk for AD. The approach offers a glimpse to the molecular and cellular mechanisms associated with dementia and provides a means for their assessment in the living patient. Monitoring disease progression in MCI patients demonstrates the usefulness of this imaging approach for early diagnosis and provides a means for evaluation of neuroprotective agents and drugs aimed at prevention and modification of disease progression.

PET of brain amyloid and tau in mild cognitive impairment.

BACKGROUND: Amyloid senile plaques and tau neurofibrillary tangles are neuropathological hallmarks of Alzheimer's disease that accumulate in the cortical regions of the brain in persons with mild cognitive impairment who are at risk for Alzheimer's disease. Noninvasive methods to detect these abnormal proteins are potentially useful in developing surrogate markers for drug discovery and diagnostics. METHODS: We enrolled 83 volunteers with self-reported memory problems who had undergone neurologic and psychiatric evaluation and positron-emission tomography (PET). On the basis of cognitive testing, 25 volunteers were classified as having Alzheimer's disease, 28 as having mild cognitive impairment, and 30 as having no cognitive impairment (healthy controls). PET was performed after injection of 2-(1-{6-[(2-[F-18]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile (FDDNP), a molecule that binds to plaques and tangles in vitro. All subjects also underwent 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) PET, and 72 underwent magnetic resonance imaging (MRI). RESULTS: Global values for FDDNP-PET binding (average of the values for the temporal, parietal, posterior cingulate, and frontal regions) were lower in the control group than in the group with mild cognitive impairment (P<0.001), and the values for binding in the group with mild cognitive impairment were lower than in the group with Alzheimer's disease (P<0.001). FDDNP-PET binding differentiated among the diagnostic groups better than did metabolism on FDG-PET or volume on MRI. CONCLUSIONS: FDDNP-PET scanning can differentiate persons with mild cognitive impairment from those with Alzheimer's disease and those with no cognitive impairment. This technique is potentially useful as a noninvasive method to determine regional cerebral patterns of amyloid plaques and tau neurofibrillary tangles.

In vitro detection of (S)-naproxen and ibuprofen binding to plaques in the Alzheimer's brain using the positron emission tomography molecular imaging probe 2-(1-[6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene)malononitrile.

Epidemiological studies have suggested that the chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the relative risk of Alzheimer's disease (AD). The possible neuroprotection by NSAIDs in AD is generally attributed to anti-inflammatory activity. An additional mode of drug action may involve anti-aggregation of beta-amyloid (Abeta) peptides by commonly used NSAIDs. We utilized in vitro competition assays, autoradiography, and fluorescence microscopy with AD brain specimens to demonstrate concentration-dependent decreases in the binding of the in vivo molecular imaging probe, 2-(1-[6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene)malononitrile ([(18)F]FDDNP), against (S)-naproxen and (R)- and (S)-ibuprofen (but not diclofenac) to Abeta fibrils and ex vivo Abeta senile plaques. Conversely, in vitro amyloid dyes Congo Red and Thioflavine T were demonstrated in the same experiments not to bind to the FDDNP binding site. FDDNP and the NSAIDs that share the same binding site also exhibit anti-aggregation effects on Abeta peptides, suggesting that the shared binding site on Abeta fibrils and plaques may be a site of anti-aggregation drug action. Our results indicate for the first time the binding of select NSAIDs to plaques, specifically to the binding site of the molecular imaging probe [(18)F]FDDNP. Our understanding of the molecular requirements of FDDNP binding may help in the optimization of the Abeta anti-aggregation potency of experimental drugs. [(18)F]FDDNP has been used to image plaques in vivo with positron emission tomography (PET), and investigations into the influence of Abeta anti-aggregation on the risk-reduction effects of NSAIDs on AD could utilize [(18)F]FDDNP and PET in determining the occupancy rate of NSAIDs and experimental drugs in plaques in the living brain of AD patients.

Monitoring adenoviral DNA delivery, using a mutant herpes simplex virus type 1 thymidine kinase gene as a PET reporter gene.

Current gene therapy protocols often suffer from an inability to monitor the site, level and persistence of gene expression following somatic DNA delivery. Herpes simplex virus 1 thymidine kinase (HSV1-tk) is currently under intensive investigation as a reporter gene for in vivo imaging of reporter gene expression. The presence of the HSV1-tk reporter gene is repetitively and non-invasively monitored by systemic injection of positron-emitting, radionuclide-labeled thymidine analogues or acycloguanosine HSV1-TK substrates and subsequent detection, by positron emission tomography, of trapped, phosphorylated product. To improve the efficacy of the HSV1-tk PET reporter gene system, both alternative substrates and mutations in the HSV1-tk gene have been described. We used a replication defective adenovirus to deliver the HSV1-sr39tk mutant enzyme and the wild-type HSV1-tk enzyme to mice. HSV1-sr39TK demonstrates greater sensitivity than wild-type HSV1-TK enzyme in vivo, using 9-[(4-[(18)F]fluoro-3-hydroxymethylbutyl)guanine as probe, following adenovirus-mediated hepatic expression in mice. Using this adenoviral delivery system, the location, magnitude and duration of HSV1-sr39tk PET reporter gene expression could be non-invasively, quantitatively and repetitively monitored for over 3 months by microPET.

Imaging and therapeutics: the role of neuronal transport in the regional specificity of L-DOPA accumulation in brain.

PURPOSE: To investigate the in vitro regional accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) in brain tissue. PROCEDURE: Neuronal membrane transport of L-DOPA was investigated in rat and squirrel monkey brain tissue. The kinetics of L-DOPA regional transport was characterized, and the effect of amino acids on transport was evaluated using isolated nerve terminals from striatum and cerebral cortex. RESULTS: When L-DOPA uptake was measured in modified Krebs-Ringer medium, transport occurred in both synaptosome preparations. In the presence of dilute protein-free plasma, uptake of L-DOPA was significantly present in striatal nerve terminals (P < 0.005), but was completely inhibited in terminals isolated from the cortex. L-DOPA transport in striatal synaptosomes was primarily inhibited by large neutral aromatic L-amino acids, in contrast to that in cortical synaptosomes that was mainly affected by large neutral aliphatic L-amino acids. A saturable component of influx was detected in both synaptosome preparations, where kinetic analysis revealed that the relative affinity of L-DOPA was greater for the carrier in the striatum than in the cortex. Based on the distribution of neuronal cell types within the two regions and the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesioning in squirrel monkeys, the striatal specific accumulation of L-DOPA likely occurs within dopaminergic terminals. CONCLUSIONS: These results demonstrate that the in vivo regional specificity of L-DOPA localization in brain tissue is primarily controlled by neuronal transport.

Tantalum [18O]water target for the production of [18F]fluoride with high reactivity for the preparation of 2-deoxy-2-[18F]fluoro-D-glucose.

PURPOSE: To develop a new tantalum [18O]water target for the routine production of reactive, no-carrier-added [18F]fluoride ion in Curie amounts for the synthesis of radiopharmaceuticals. PROCEDURES: The tantalum target body was filled with 0.86 mL of 95% enriched [18O]water and irradiated with 10.2 MeV protons on target with beam currents of 26-40 microA for 60-90 min. [18F]Fluoride ion produced is trapped in an anion exchange resin cartridge and the expensive [18O]water is recovered for recycling. The [18F]fluoride ion is released from the resin by elution with a dilute solution of K(2)CO(3) and utilized in the synthesis of 2-deoxy-2-[18F]fluoro-D-glucose (FDG). RESULTS: Using the tantalum target, Curie levels of no-carrier-added [18F]fluoride ion (specific activity: >30,000 Ci/mmol) were reliably and reproducibly produced (n > 100), from which FDG was synthesized in 70% radiochemical yield. CONCLUSION: Tantalum [18O]water target body for the production of [18F]fluoride ion has the best characteristics from the standpoint of [18F]fluoride ion recovery and its radiochemical reactivity and low induced activation. This new target has the potential to replace the currently used silver and titanium [18O]targets for the routine production of [18F]fluoride ion.

Binding characteristics of radiofluorinated 6-dialkylamino-2-naphthylethylidene derivatives as positron emission tomography imaging probes for beta-amyloid plaques in Alzheimer's disease.

Senile plaques (SPs) and neurofibrillary tangles (NFTs) are hallmark pathologies accompanying the neurodegeneration involved in Alzheimer's disease (AD), for which beta-amyloid (Abeta) peptide is a major constituent of SPs. Our laboratories previously developed the hydrophobic, fluorescent molecular-imaging probe 2-(1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl)ethylidene)malononitrile ([(18)F]FDDNP), which crosses the blood-brain barrier and determines the localization and load of SPs and NFTs in vivo in AD patients. In this report, we used fluorimetric and radioactive binding assays to determine the binding affinities of FDDNP and its analog, 1-(6-[(2-[(18)F]fluoroethyl)(methyl)amino]naphthalen-2-yl)ethanone ([(18)F]FENE), to synthetic fibrils of Abeta(1-40). FDDNP and FENE both appeared to bind to two kinetically distinguishable binding sites on Abeta(1-40) fibrils. Fluorescence titrations yielded apparent K(d) values of 0.12 and 0.16 nm for high-affinity binding sites for FDDNP and FENE, respectively, and apparent K(d) values of 1.86 and 71.2 nm for the low-affinity binding sites. The traditional radioactive binding assays also produced apparent K(d) values in the low nanomolar range. The presence of two kinetically distinguishable binding sites for FDDNP and FENE suggests multiple binding sites for SPs and identifies the parameters that allow for the structural optimization of this family of probes for in vivo use. The high-affinity binding of the probes to multiple binding sites on fibrils are consistent with results obtained with digital autoradiography, immunohistochemistry, and confocal fluorescence microscopy using human brain specimens of AD patients.

Novel observations with FDOPA-PET imaging after early nigrostriatal damage.

Striatal 6-[18F]fluoro-L-DOPA (FDOPA) kinetic rate constants were measured by positron emission tomography (PET) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys. After scanning, stereological counts of dopaminergic neurons were done in substantia nigra, and dopamine (DA) and metabolite concentrations were determined in the caudate, putamen, and substantia nigra. Graded doses of MPTP produced animals with mild to moderate reductions (10-35%) in dopaminergic neurons, where the percent of cell loss was proportional to the amount of MPTP given. Striatal DA and metabolite concentrations were relatively unchanged in animals given 1.0 and 1.5 mg/kg of MPTP, but were significantly reduced after 2.0 mg/kg of MPTP. All animals injected with a single dose of MPTP showed no overt signs of parkinsonism. In contrast, DA and metabolite concentrations in the substantia nigra were significantly reduced for all MPTP-treated animals. Reduction of dopaminergic indices in the substantia nigra did not parallel reductions in the striatum, indicating differential sensitivity of the nigrostriatal pathway to the neurotoxic effects of MPTP. The percent change in FDOPA uptake (Ki) and decarboyxlation (k3) after MPTP showed significant positive correlations to striatal DA levels, but not to the number of dopaminergic neurons. This suggests that FDOPA is a good index of striatal DA levels.

Quantitative imaging of gene induction in living animals.

Methods to repeatedly, non-invasively, and quantitatively image gene expression in living animals are rapidly emerging and should fundamentally change studies of gene expression in vivo. We previously developed assays utilizing positron emission tomography (PET) to image reporter gene expression. In this paper we: (1) describe a new bi-directional, tetracycline-inducible system that can be used to pharmacologically induce target gene expression and to quantitatively image induced expression by using a PET reporter gene; (2) demonstrate the potential of this system in transient and stable cell transfection assays; and (3) demonstrate the ability to repetitively and quantitatively image tetracycline and tetracycline analog induction of gene expression in living animals. We utilize the dopamine type-2 receptor (D(2)R) and the mutant herpes-simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter genes to validate this system. We utilize microPET technology to show that quantitative tomographic imaging of gene induction is possible. We find a high correlation (r(2) = 0.98) between 'target' and reporter gene expression. This work establishes a new technique for imaging time-dependent variation of gene expression both from vectors with inducible promoters and in transgenic animals in which pharmacologic induction of gene expression must be monitored. These techniques may be applied both in gene therapy and for the study of gene expression in transgenic animals.

Blood-brain barrier and neuronal membrane transport of 6-[18F]fluoro-L-DOPA.

The transport of 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]FDOPA) across the blood-brain barrier (BBB) and neuronal membranes was compared with that of L-3,4-dihydroxyphenylalanine (L-DOPA) in rats. The carotid injection method was used as a direct measurement of [18F]FDOPA, 1-[14C]-L-DOPA, and 3-[14C]-L-DOPA transport across the BBB, while isolated nerve terminals were used to examine neuronal membrane transport of [3H]-L-DOPA. [18F]FDOPA appeared to use the same large neutral amino acid carrier for BBB transport as L-DOPA and L-phenylalanine. In addition, carbidopa [L-alpha-hydrazino-alpha-methyl-beta-(3,4-dihydroxyphenyl)propionic acid] was found not to have direct interference with the transport carrier on the BBB, but indirectly inhibited aromatic L-amino acid decarboxylase (AAAD) activity in brain endothelium by depletion of pyridoxal phosphate, a necessary cofactor of the enzyme. In striatal and cortical synaptosomes, [3H]-L-DOPA uptake was inhibited by non-radioactive L-DOPA, FDOPA, and 6-fluoro-L-meta-tyrosine (6-FMT). The inhibition was significantly greater in terminals isolated from the striatum than in those from the cerebral cortex. FDOPA, 6-FMT, and L-DOPA equally inhibited the neuronal transport of [3H]-L-DOPA. This suggests that FDOPA and 6-FMT compete with L-DOPA at similar transport sites at the neuronal membrane.

Monitoring gene therapy with reporter gene imaging.

Rapid advances in imaging technologies and gene transfer strategies offer a great opportunity to optimize clinical trials of human gene therapy. Reporter genes are emerging as very powerful tools to monitor the delivery, magnitude, and time variation of therapeutic gene transfer in vivo. Several reporter genes, such as the herpes simplex virus type 1 thymidine kinase, the dopamine type 2 receptor, and the somatostatin receptor type 2, are currently being successfully used with gamma camera, single photon emission computed tomography, and positron emission tomography imaging. These reporter genes can be coupled with a therapeutic gene of interest to indirectly monitor the expression of the therapeutic gene. Finally, applications of the reporter gene technology to other areas, such as cell trafficking studies and transgenic animal models, are now possible.

Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction.

The dopamine D2 receptor (D2R) has been used in adenoviral delivery systems and in tumor cell xenografts as an in vivo reporter gene. D2R reporter gene expression has been non-invasively, repetitively and quantitatively imaged by positron emission tomography (PET), following systemic injection of a positron-labeled ligand (3-(2'-[18F]-fluoroethyl)-spiperone; FESP) and subsequent D2R-dependent sequestration. However, dopamine binding to the D2R can modulate cyclic AMP levels. For optimal utilization of D2R as a reporter gene, it is important to uncouple ligand-binding from Gi-protein-mediated inhibition of cAMP production. Mutation of Asp80 or Ser194 produces D2Rs that still bind [3H]spiperone in transfected cells. The D2R80A mutation completely eliminates the ability of the D2R to suppress forskolin-stimulated cAMP accumulation in response to dopamine, in cells transfected with a D2R80A expression plasmid and in cells infected with replication-defective adenovirus expressing D2R80A. The D2R194A mutation substantially reduces, but does not completely eliminate, dopamine modulation of cAMP levels. Cultured cells infected with adenoviruses expressing D2R and D2R80A demonstrated equivalent [3H]spiperone binding activity. Moreover, hepatic FESP sequestration is equivalent, following intravenous injection of adenoviruses expressing D2R and D2R80A. The D2R80A mutant, which can no longer modulate cAMP levels following ligand binding, has full capability as a PET reporter gene.

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors.

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

Human pharmacokinetic and dosimetry studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression.

UNLABELLED: 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]FHBG) has been used as a reporter probe to image expression of herpes simplex virus type-1 thymidine kinase (HSV1-tk) reporter gene in living animals. Our aim was to study the kinetics, biodistribution, stability, dosimetry, and safety of [(18)F]FHBG in healthy human volunteers, preparatory to imaging patients undergoing HSV1-tk gene therapy. METHODS: [(18)F]FHBG was synthesized with a specific activity of 37,000--444,000 GBq/mmol and a radiochemical purity > 99%. Ten healthy volunteers consented to participate in the study. A transmission scan was obtained before bolus injection of 70.3--229.4 MBq [(18)F]FHBG into a hand vein, followed by dynamic PET imaging with 4 consecutive emission scans. Warmed hand-vein blood was withdrawn at various times after injection for blood time--activity measurements. Electrocardiography, blood pressure, and blood and urine pharmacologic parameters were measured before and after injection of the [(18)F]FHBG tracer (n = 5). The stability of [(18)F]FHBG in the urine was analyzed. Attenuation-corrected images were reconstructed using the ordered-subsets expectation maximization algorithm. Image region-of-interest time-activity data were used with the MIRD program to estimate absorbed radiation dosages. RESULTS: [(18)F]FHBG had rapid blood clearance; only 8.42% +/- 4.76% (mean +/- SD) of the peak blood activity remained at approximately 30 min. The average ratio of plasma activity to whole-blood activity during the study was 0.91 +/- 0.04. Penetration of [(18)F]FHBG across the blood-brain barrier was not observed. The primary routes of clearance were renal and hepatobiliary. High activities were observed in the bladder, gut, liver, and kidneys, but <0.0002% of the injected dose per gram was observed in other tissues. In the urine, 83% of activity 180 min after injection was stable [(18)F]FHBG. Blood and urine pharmacologic parameters did not change significantly after injection of the [(18)F]FHBG tracer. The bladder absorbed the highest radiation dose. CONCLUSION: [(18)F]FHBG has the desirable in vivo characteristics of stability, rapid blood clearance, low background signal, biosafety, and acceptable radiation dosimetry in humans. This study forms the foundation for using [(18)F]FHBG in applications to monitor HSV1-tk reporter gene expression.

8-[18F]Fluoropenciclovir: an improved reporter probe for imaging HSV1-tk reporter gene expression in vivo using PET.

UNLABELLED: We have synthesized and evaluated 8-[18F]fluoropenciclovir (FPCV) and compared it with 8-[18F]fluoroganciclovir (FGCV) for monitoring the expression of herpes simplex virus type 1 thymidine kinase (HSV1 -tk) reporter gene in cell culture and in vivo. METHODS: C6 rat glioma cells stably transfected with HSV1-tk (C6-stb-tk+) and control C6 cells were evaluated for their ability to accumulate FGCV versus FPCV. For in vivo studies, 15 mice were injected by tail vein with increasing levels of an adenoviral vector carrying HSV1-tk. Forty-eight hours later the mice were injected with FPCV and killed 3 h later. The percentage injected dose per gram (%ID/g) liver was then determined. Two additional mice were studied by microPET and autoradiography using FPCV to image adenoviral-mediated hepatic HSV1-tk reporter gene expression. A tumor-bearing mouse (C6 control and C6-stb-tk+) was imaged with FDG, FGCV, and FPCV. Two mice carrying tumors expressing two different reporter genes, HSV1-tk and dopamine type 2 receptor (D2R), were also imaged by microPET using FPCV (day 1) and 3-(2'-[18F]fluoroethyl)spiperone (FESP) (day 2). RESULTS: FPCV shows a significantly greater accumulation in C6-stb-tk+ cells than does FGCV (P < 0.05). Over identical ranges of adenoviral administration, mouse liver shows a higher %ID/g liver for FPCV (0%-9%) compared with our previously reported results with FGCV (0%-3%). In C6 control and C6-stb-tk+ tumor-bearing mice, FPCV has a greater accumulation than does FGCV for equal levels of HSV1-tk gene expression. In mice carrying tumors expressing either HSV1-tk or D2R reporter genes, there is a corresponding retention of FPCV and FESP, respectively. CONCLUSION: These results indicate that FPCV is a better reporter probe than is FGCV for imaging lower levels of HSV1 -tk gene expression in vivo. The results also reveal the ability to monitor the expression of two distinct reporter genes in the same animal using reporter probes specific for each gene.

Distribution volume of 3-O-methyl-6.

The distribution volume (DV) of 6-[F-18]fluoro-L-DOPA (FDOPA) in the cerebellum recently has been linked using positron emission tomography (PET) to plasma large neutral amino acid (LNAA) concentrations in monkeys. In this article the authors provide additional experimental support for this relation by directly measuring the DV as the steady-state tissue to plasma radioactivity ratio in rats using a labeled LNAA analog 3-O-methyl-6-[F-18]FDOPA (OMFD), a compound that has no known specific enzyme or receptor interactions in brain tissue. The measured DV for OMFD (tissue OMFD concentration/plasma OMFD concentration) was found to be inversely related to plasma LNAA concentrations. The relation (DV = 1.5-0.00094*[LNAA], R--2 = 0.79) resulted in an 8% DV decrease per 100 nmol/mL plasma LNAA increase within the observed range of 330 to 510 nmol/mL. This was similar to recent noninvasive observations with FDOPA PET in vervet monkeys and with 6-[F-18]Fluoro-m-tyrosine PET in squirrel monkeys. The OMFD striatum to cerebellum (Str/Cb) ratio was greater than 1.0 for all measurements, averaging 1.09 +/- 0.04, and was approximately equal to the Str/Cb LNAA ratio of 1.12 +/- 0.05. This current study verifies the variation of DV of OMFD or FDOPA as a function of plasma LNAA concentrations and suggests the possibility of using OMFD for measuring cerebral LNAA noninvasively with PET.

Imaging transgene expression with radionuclide imaging technologies.

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.

Striatal kinetic modeling of FDOPA with a cerebellar-derived constraint on the distribution of volume of 30MFD: a PET investigation using non-human primates.

The peripherally born metabolite of FDOPA, 3-O-Methyl-FDOPA (3OMFD), crosses the blood-brain barrier, thus complicating positron emission tomography-FDOPA (PET-FDOPA) data analysis. In previous reports the distribution volume (DV) of 3OMFD was constrained to unity. We have recently shown that the forward transport rate-constant of FDOPA (K(S1)) and the cerebellum-to-plasma ratio (C(b)/C(p)), a measure for the DV of 3OMFD, are functions of plasma large neutral amino acid (LNAA) concentration. Given large interstudy and intersubject differences in plasma LNAA levels, variations in the DV of 3OMFD are significant. In this report, the authors propose a constraint on the DV of 3OMFD that accounts for these variations. Dynamic PET-FDOPA scans were performed on 12 squirrel monkeys and 12 vervet monkeys. Two sets of constraints were employed on the compartmental model--M1 or M2. In M1, the striatal DV of 3OMFD was constrained to unity; in M2, the striatal DV of 3OMFD was constrained to an estimate derived from the cerebellum. Striatal and cerebellar time-activity curves were fitted using FDOPA and 3OMFD plasma input functions. The estimate of K(S1) and that of the compartmental FDOPA uptake-constant (K(i)), both obtained using M2, were adjusted to values corresponding to average LNAA levels. Finally, K(i) was compared with the graphical uptake-constant (PK(j)). With the use of constraint M2, intersubject variability of squirrel monkey k(S3) and K(i) was reduced by 45% and 53%, respectively; and for vervet monkeys, by 54% and 44%, respectively. Intersubject variability of K(1) and K(i) was further reduced after correction for variations in intersubject plasma LNAA levels (for squirrel monkeys, by 67% and 41%; for vervet monkeys, by 40% and 36%, respectively). K(i) correlation to PK(i) was enhanced to identity. Finally, average cerebellar k(C2) estimates were more than 2.5-fold higher than striatal k(S2) estimates (P < 0.0001). In modeling of PET-FDOPA data, it cannot be assumed that the DV of 3OMFD is unity. The cerebellar-derived constraint furnishes a reliable estimate for the DV of 3OMFD. Invoking the constraint and correcting for variations in plasma LNAA significantly reduced interstudy and intersubject variations in parameter estimates.

Nigrostriatal reduction of aromatic L-amino acid decarboxylase activity in MPTP-treated squirrel monkeys: in vivo and in vitro investigations.

Aromatic L-amino acid decarboxylase (AAAD) activity was examined in vivo with positron emission tomography (PET) using 6-[18F]fluoro-L-DOPA (FDOPA) in squirrel monkeys lesioned with graded doses of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In vitro biochemical determinations of AAAD activity in caudate, putamen, substantia nigra, and nucleus accumbens were performed in the same animals to establish a direct comparison of in vivo and in vitro measurements. In vivo and in vitro AAAD activities in caudate/ putamen were substantially reduced in animals treated with the highest dose of MPTP (2.0 mg/kg). The percent change in the striatal FDOPA uptake (K(i)) and decarboxylation rate constant (k3) values resulting from MPTP treatment showed highly significant correlations with in vitro-determined AAAD activities. However, decarboxylase rates within individual animals presented as approximately 10-fold difference between in vivo and in vitro values. Lower in vivo k3 measurements may be attributed to several possibilities, including transport restrictions limiting substrate availability to AAAD within the neuron. In addition, reductions in AAAD activity in the substantia nigra did not parallel reductions in AAAD activity within the striatum, supporting the notion of a nonlinear relationship between nigrostriatal cell degeneration and terminal losses. This work further explores the role of AAAD in Parkinson's disease, a more important factor than previously thought.