PET Imaging a MPTP-Induced Mouse Model of Parkinson’s Disease Using the Fluoropropyl-Dihydrotetrabenazine Analog [18F]-DTBZ (AV-133)

Parkinson’s disease (PD) is characterized by the loss of dopamine-producing neurons in the nigrostriatal system. Numerous researchers in the past have attempted to track the progression of dopaminergic depletion in PD. We applied a quantitative non-invasive PET imaging technique to follow this degeneration process in an MPTP-induced mouse model of PD. The VMAT2 ligand 18F-DTBZ (AV-133) was used as a radioactive tracer in our imaging experiments to monitor the changes of the dopaminergic system. Intraperitoneal administrations of MPTP (a neurotoxin) were delivered to mice at regular intervals to induce lesions consistent with PD. Our results indicate a significant decline in the levels of striatal dopamine and its metabolites (DOPAC and HVA) following MPTP treatment as determined by HPLC method. Images obtained by positron emission tomography revealed uptake of 18F-DTBZ analog in the mouse striatum. However, reduction in radioligand binding was evident in the striatum of MPTP lesioned animals as compared with the control group. Immunohistochemical analysis further confirmed PET imaging results and indicated the progressive loss of dopaminergic neurons in treated animals compared with the control counterparts. In conclusion, our findings suggest that MPTP induced PD in mouse model is appropriate to follow the degeneration of dopaminergic system and that 18F-DTBZ analog is a potentially sensitive radiotracer that can used to diagnose changes associated with PD by PET imaging modality

Genetic Incorporation of Human Metallothionein into the Adenovirus Protein IX for Non-Invasive SPECT Imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of 99mTc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a 99mTc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo

Construction and Radiolabeling of Adenovirus Variants that Incorporate Human Metallothionein into Protein IX for Analysis of Biodistribution

Using adenovirus (Ad)-based vectors is a promising strategy for novel cancer treatments; however, current tracking approaches in vivo are limited. The C-terminus of the Ad minor capsid protein IX (pIX) can incorporate heterologous reporters to monitor biodistribution. We incorporated metallothionein (MT), a low-molecular-weight metal-binding protein, as a fusion to pIX. We previously demonstrated 99mTc binding in vitro to a pIX-MT fusion on the Ad capsid. We investigated different fusions of MT within pIX to optimize functional display. We identified a dimeric MT construct fused to pIX that showed significantly increased radiolabeling capacity. After Ad radiolabeling, we characterized metal binding in vitro. We explored biodistribution in vivo in control mice, mice pretreated with warfarin, mice preimmunized with wild-type Ad, and mice that received both warfarin pretreatment and Ad preimmunization. Localization of activity to liver and bladder was seen, with activity detected in spleen, intestine, and kidneys. Afterwards, the mice were euthanized and selected organs were dissected for further analysis. Similar to the imaging results, most of the radioactivity was found in the liver, spleen, kidneys, and bladder, with significant differences between the groups observed in the liver. These results demonstrate this platform application for following Ad dissemination in vivo

Visual interpretation, not SUV ratios, is the ideal method to interpret 18F-DOPA PET scans to aid in the cure of patients with focal congenital hyperinsulinism.

IntroductionCongenital hyperinsulinism is characterized by abnormal regulation of insulin secretion from the pancreas causing profound hypoketotic hypoglycemia and is the leading cause of persistent hypoglycemia in infants and children. The main objective of this study is to highlight the different mechanisms to interpret the 18F-DOPA PET scans and how this can influence outcomes.Materials and methodsAfter 18F-Fluoro-L-DOPA was injected intravenously into 50 subjects' arm at a dose of 2.96-5.92 MBq/kg, three to four single-bed position PET scans were acquired at 20, 30, 40 and 50-minute post injection. The radiologist interpreted the scans for focal and diffuse hyperinsulinism using a visual interpretation method, as well as determining the Standard Uptake Value ratios with varying cut-offs.ResultsVisual interpretation had the combination of the best sensitivity and positive prediction values.ConclusionsIn patients with focal disease, SUV ratios are not as accurate in identifying the focal lesion as visual inspection, and cases of focal disease may be missed by those relying on SUV ratios, thereby denying the patients a chance of cure. We recommend treating patients with diazoxide-resistant hyperinsulinism in centers with dedicated multidisciplinary team comprising of at least a pediatric endocrinologist with a special interest in hyperinsulinism, a radiologist experienced in interpretation of 18F-Fluoro-L-DOPA PET/CT scans, a histopathologist with experience in frozen section analysis of the pancreas and a pancreatic surgeon experienced in partial pancreatectomies in patients with hyperinsulinism

Gel filtration column profile of 99mTc binding to adenovirus containing a pIX-MT fusion protein.

<p>After incubation with^99mTc glucoheptonate, the Ad-tGFP-pIX-MT virus containing a pIX-MT fusion protein and the Ad-CMV-EGFP virus containing a wild-type pIX protein were purified by gravity gel filtration using 20 mL Sephacryl S-200 columns. Each point represents the specific activity in mBq/v.p of individual 10 drop fractions collected.</p

Construction and analysis of a pIX-MT containing adenovirus.

<p>(A) Schematic representation of pIX-MT and a CMV-tGFP expression cassette inserted into the Ad5 genome. (B) Expression of GFP in cells infected with Ad-tGFP-pIX-MT shows formation of an individual plaque. (C) Detection of adenovirus fiber and metallothionein proteins incorporated into the purified Ad-tGFP-pIX-MT and Ad-CMV-EGFP viral particles by western blot analysis.</p

SPECT imaging analysis of a mouse injected with an adenovirus containing a capsid-incorporated pIX-MT fusion protein.

<p>C57BL6 mice were injected intravenously with approximately 1 mCi of ^99mTc in 0.3 mL PBS, and scanned at 30 min after the injection. Afterwards, 3D renderings of SPECT images in blue-purple-red-yellow scale against CT projections in grey scale were obtained. Shown are: (A) coronal image of a normal mouse injected with ^99mTc-pertechnetate; (B) coronal image of a mouse injected with ^99mTc-glucoheptonate; (C) coronal and sagittal images of a normal mouse injected with ^99mTc-Ad-tGFP-pIX-MT; and (D) coronal and sagittal images of a mouse pretreated with warfarin injected with ^99mTc-Ad-tGFP-pIX-MT.</p

Pancreatic uptake and radiation dosimetry of 6-[18F]fluoro-L-DOPA from PET imaging studies in infants with congenital hyperinsulinism.

After injecting 25.6 ± 8.8 MBq (0.7 ± 0.2 mCi) of 18F-Fluoro-L-DOPA intravenously, three static PET scans were acquired at 20, 30, and 40 min post injection in 3-D mode on 10 patients (6 male, 4 female) with congenital hyperinsulinism. Regions of interest (ROIs) were drawn over several organs visible in the reconstructed PET/CT images and time activity curves (TACs) were generated. Residence times were calculated using the TAC data. The radiation absorbed dose for the whole body was calculated by entering the residence times in the OLINDA/EXM 1.0 software.The mean residence times for the 18F-Fluoro-L-DOPA in the liver, lungs, kidneys, muscles, and pancreas were 11.54 ± 2.84, 1.25 ± 0.38, 4.65 ± 0.97, 17.13 ± 2.62, and 0.89 ± 0.34 min, respectively. The mean effective dose equivalent for 18F-Fluoro-L-DOPA was 0.40 ± 0.04 mSv/MBq. The CT scan used for attenuation correction delivered an additional radiation dose of 5.7 mSv. The organs receiving the highest radiation absorbed dose from 18F-Fluoro-L-DOPA were the urinary bladder wall (2.76 ± 0.95 mGy/MBq), pancreas (0.87 ± 0.30 mGy/MBq), liver (0.34 ± 0.07 mGy/MBq), and kidneys (0.61 ± 0.11 mGy/MBq). The renal system was the primary route for the radioactivity clearance and excretion.The estimated radiation dose burden from 18F-Fluoro-L-DOPA is relatively modest to newborns

Thermostability of Ad-tGFP-pIX-MT compared to Ad-CMV-EGFP and Ad-IX-EGFP.

<p>The thermostability of each virus was determined by incubation at 45°C for various times and quantified in terms of infectious titer. Each point represents the mean ± SE of three replicate samples.</p