Discontinuation of methylphenidate after long-term exposure in nonhuman primates.

Attention-deficit hyperactivity disorder (ADHD) is a common human neurobehavioral disorder that usually begins in early childhood. Methylphenidate (MPH) has been used extensively as a first-line medicine for the treatment of ADHD. Since ADHD is often diagnosed in early childhood and can persist for the entire lifespan, individuals may take MPH for many years. Given that in the course of one's lifetime a person may stop taking MPH for periods of time, or may implement lifestyle changes that may reduce the need for MPH entirely, it is important to understand how cessation of MPH affects the adult brain following long-term use of MPH. The blockage of the dopamine transporter (DAT) and the norepinephrine transporter (NET) by MPH may help with ADHD symptoms by boosting monoamine levels in the synapse. In the present study, microPET/CT was used to investigate possible neurochemical alterations in the cerebral dopamine system after cessation of long-term MPH administration in nonhuman primates. MicroPET/CT images were collected from adult male rhesus monkeys 6 months after they stopped receiving vehicle or MPH following 12 years of chronic treatment. The neurochemical status of brain dopaminergic systems was evaluated using the vesicular monoamine transporter 2 (VMAT2) ligand [18F]-AV-133 and a tracer for imaging dopamine subtype 2 (D2) and serotonin subfamily 2 (5HT2) receptors, [18F]-FESP. Each tracer was injected intravenously and ten minutes later microPET/CT images were obtained over 120 min. The binding potential (BP) of each tracer in the striatum was obtained using the Logan reference tissue model with the cerebellar cortex time activity curve (TAC) as an input function. Brain metabolism was also evaluated using microPET/CT images of [18F]-FDG. [18F]-FDG was injected intravenously, and ten minutes later, microPET/CT images were obtained over 120 min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum, and cerebellum were converted into standard uptake values (SUVs). Compared to the vehicle control group, the BPs of [18F] AV-133 and [18F]-FESP in the striatum were not significantly altered in MPH treated groups. Additionally, no significant differences were detected in the SUVs of [18F]-FDG in the MPH treated group compared with control. This study demonstrates that 6 months after cessation of long-term, chronic MPH treatment, there are no significant neurochemical or neural metabolic changes in the central nervous system (CNS) of non-human primates (NHPs) and suggests that microPET imaging is helpful in assessing the status of biomarkers of neurochemical processes linked to chronic CNS drug exposure. (Supported by NCTR).

MicroPET/CT assessment of neurochemical effects in the brain after long-term methylphenidate treatment in nonhuman primates.

Methylphenidate (MPH) is a psychostimulant approved by the FDA to treatment Attention-Deficit Hyperactivity Disorder (ADHD). MPH is believed to exert its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. We used a quantitative non-invasive PET imaging technique to study the effects of long-term methylphenidate use on the central nervous system (CNS). We conducted microPET/CT scans on young adult male rhesus monkeys to monitor changes in the dopaminergic system. We used [18F] AV-133, a ligand for the vesicular monoamine transporter 2 (VMAT2), and [18F]FESP a ligand for the D2 and 5HT2 receptors. In this study we evaluated the effects if chronic MPH treatment in the nonhuman primates (NHP). Two-year-old, male rhesus monkeys were orally administered MPH diluted in the electrolyte replenisher, Prang, twice a day, five days per week (M-F) over an 8-year period. The dose of MPH was gradually escalated from 0.15 mg/kg initially to 2.5 mg/kg/dose for the low dose group, and 1.5 mg/kg to 12.5 mg/kg/dose for the high dose group (Rodriguez et al., 2010). Scans were performed on Mondays, about 60 h after their last treatment, to avoid the acute effects of MPH. Tracers were injected intravenously ten minutes before microPET/CT scanning. Sessions lasted about 120 min. The Logan reference tissue model was used to determine the Binding Potential (BP) of each tracer in the striatum with the cerebellar cortex time activity curve as an input function. Both MP treatment groups had a lower [18F] AV-133 BP, although this failed to reach statistical significance. MPH treatment did not have a significant effect on The BP of [18F] FESP in the striatum. Long-term administration of MPH did not significant change any of the marker of monoamine function used here. These data suggest that, despite lingering concerns, long-term use of methylphenidate does not negatively impact monoamine function. This study also demonstrates that microPET imaging can distinguish differences in binding potentials of a variety of radiotracers in the CNS of NHPs. This approach may provide minimally-invasive biomarkers of neurochemical processes associated with chronic exposure to CNS medications. (Supported by NCTR).

MicroPET/CT assessment of FDG uptake in brain after long-term methylphenidate treatment in nonhuman primates.

Methylphenidate (MPH) is a psychostimulant commonly used for the treatment of Attention-Deficit Hyperactivity Disorder (ADHD). Since the long-term effects of this drug on the central nervous system (CNS) are not well understood, we conducted microPET/CT scans on young adult male rhesus monkeys (n=4/group) to gather information on brain metabolism using the uptake of [(18)F]Fluoro-2-deoxy-2-d-glucose (FDG) as a marker. Approximately two-year old, male rhesus monkeys were treated orally with MPH twice per day, five days per week (M-F) over a 6-year period. Subjects received MPH at either 2.5 or 12.5mg/kg/dose or vehicle (Prang). To minimize the acute effects of MPH on FDG uptake, microPET/CT scans were scheduled on Mondays before their first daily dosing of the week (approximately 68h since their last treatment). FDG (370±8.88MBq) was injected intravenously and 30min later microPET/CT images were obtained over 60min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum and cerebellum were converted into Standard Uptake Values (SUVs). Compared to the control group, the uptake of FDG in the cerebellum was significantly decreased in both the low and high dose groups. These preliminary data demonstrate that microPET imaging is capable of distinguishing differences in retention of FDG in the brains of NHPs treated chronically with MPH and suggests that this approach may provide a minimally invasive biomarker for exploring the effects of chronic MPH treatment on aspects of brain function.

MicroPET imaging of ketamine-induced neuronal apoptosis with radiolabeled DFNSH.

Recent reports indicate that 6-12 h of ketamine anesthesia can trigger neuronal apoptosis in postnatal day (PND) 7 rats. In vitro, ex vivo, and confocal fluorescent imaging studies suggest that dansyl compounds can accumulate within the cytoplasm of the apoptotic cell. High-resolution positron emission tomography (microPET) imaging has been proposed as a minimally invasive method for detecting apoptosis in the rat brain. Compared with [(18)F]-labeled annexin V, which binds to externalized phosphatidylserine (PS) on the outer membrane of apoptotic cells, intracellular uptake of the dansylhydrazone of p-fluorobenzaldehyde (DFNSH) may lead to improved target-to-background contrast ratios. In this study, the effect of ketamine on the uptake and retention of [(18)F]-DFNSH in the rat brain was investigated using microPET imaging. On PND 7, rat pups in the experimental group were exposed, at 2-h intervals, to six subcutaneous injections of ketamine (20 mg/kg) and control rat pups received six injections of saline. On PND 35, [(18)F]-DFNSH (37 MBq) was injected into the tail vein of rats and microPET images were obtained over 2 h following the injection. Radiolabeled tracer accumulation in the region of interest (ROI) in the frontal cortex was converted into standard uptake values (SUVs). The radiotracer was quickly distributed into the brains of both ketamine- and saline-treated rats. Compared with the control group, the uptake of [(18)F]-DFNSH was significantly increased in the ROI, frontal cortex area of ketamine-treated rats. In addition, the wash-out duration of the tracer was prolonged in the ketamine-treated animals. This study demonstrates that microPET imaging is capable of distinguishing differences in retention of [(18)F]-DFNSH in ROI and suggests that this compound may serve as a minimally invasive biomarker of neuronal apoptosis in rodents.

The effect of scatter and attenuation on aerosol deposition as determined by gamma scintigraphy.

Gamma scintigraphy is often used to quantify deposition patterns from aerosol inhalers. The errors caused by scatter and tissue attenuation in planar Tc-99m gamma scintigraphy were investigated based on the data collected from four subjects in this study. Several error correction methods were tested. The results from two scatter correction methods, Jaszczak's method and factor analysis of dynamic sequences (FADS), were similar. Scatter accounted for 20% of raw data in the whole lung, 20% in the oropharynx, and 43% in the central airways and esophagus. Three attenuation correction methods were investigated and compared. These were: uniform attenuation correction (UAC), a known method used for inhalation drug imaging work; the broad-beam attenuation correction used for organ imaging in nuclear medicine; and a narrow-beam inhomogeneous tissue attenuation correction proposed in this study. The three methods differed significantly (p < 0.05), but all indicated that attenuation is a severe quantification problem. The narrow beam attenuation correction with scatter correction, showed that raw data underestimated tracer deposition by 44% in the lung, 137% in the oropharynx, and 153% in the trachea/esophageal region. To quantify aerosol lung deposition using planar scintigraphy even in relative terms, corrections are necessary. Much of the literature concerning quantified aerosol dose distributions measured by gamma scintigraphy needs to be interpreted carefully.

Solid-phase analysis method for (S)-[18F]fluorocarazolol and its metabolites.

(S)-[18F]Fluorocarazolol is a radiopharmaceutical developed to quantitatively assess beta-adrenergic receptors in vivo via positron emission tomography imaging. Since radioactive metabolites of (S)-[18F]fluorocarazolol rapidly appear in the plasma, methods for conveniently and reliably evaluating plasma for (S)-[18F]fluorocarazolol content are required. Here we present methods and validation of an approach using commercial extraction cartridges that is faster and more convenient than an approach using internal-surface reverse-phase chromatography but yields comparable results.

The in vitro pharmacology of the beta-adrenergic receptor pet ligand (s)-fluorocarazolol reveals high affinity for cloned beta-adrenergic receptors and moderate affinity for the human 5-HT1A receptor.

RATIONALE: s-Fluorocarazolol [(S)-FCZ] is the major positron emission tomography (PET) ligand currently used to visualize central beta-adrenergic receptors in vivo, although its pharmacology is incompletely known. OBJECTIVE: Our objective was to comprehensively characterize the in vitro pharmacology of (S)- and (R)-FCZ to determine its suitability for study of central and peripheral beta-adrenergic receptors. METHODS: We characterized the in vitro pharmacology of (S)-FCZ at 42 biogenic amine receptors and transporters in vitro using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. RESULTS: As expected (R)- and (S)-FCZ had high affinities for beta-adrenergic receptors (Ki values=0.08-0.45 nM) and negligible affinities (Ki values>100 nM) for nearly all other tested receptors and transporters with the exception of the h5-HT1A receptor for which (S)-FCZ had high affinity (Ki=34 nM). Interestingly, (R)-FCZ had low affinity for the h5-HT1A receptor (Ki=342 nM). CONCLUSION: The high affinity of (S)-FCZ for the h5-HT1A receptor is not likely to interfere with studies of peripheral beta-adrenergic receptors, since 5-HT1A receptors are expressed at very low levels outside the central nervous system. Indeed, computer simulations predict that even at low ligand concentrations, 5-HT1A binding in brain regions like hippocampus are likely to be substantial. Thus, (S)-FCZ may not be a suitable PET ligand for studies of central nervous system beta-adrenergic receptors unless the contribution by 5-HT1A sites can be shown to be negligible.

Synthesis and properties of 18F-labeled potential myocardial blood flow tracers.

PET centers without particle accelerators make clinical PET widely available at reduced cost. For myocardial perfusion tracers, these satellite PET centers are limited to generator- produced 82Rb(+) and 62Cu[PTSM]. Their limitations motivate a search for transportable alternatives. In search of new tracers we have synthesized several 18F-labeled amines and quaternary ammonium salts. Among them, 4-[18F]fluorotri-N-methylanilinium ([18F]FTMA) has flow-tracing properties. The compound is functional, but has properties that justify a continued search.

Usefulness and pitfalls of planar gamma-scintigraphy for measuring aerosol deposition in the lungs: a Monte Carlo investigation.

UNLABELLED: Planar gamma-scintigraphy is often used to quantify pulmonary deposition patterns from aerosol inhalers. The results are quite different from those obtained using 3-dimensional PET and SPECT. The purpose of this study was to characterize the effects of scatter and tissue attenuation on the distribution of radiolabeled aerosol as measured by planar scintigraphy using Monte Carlo simulations. This study also investigated the applicability of a few correction methods used in inhalation studies. METHODS: Body density maps were derived from CT scans. Regions of interest-lungs, major airways, and esophagus-were defined from the same CT volume. Two radioactivity source distribution patterns in the lung, uniform and nonuniform, were used. A Monte Carlo program, SIMIND, was used to generate anterior and posterior gamma-images of the composed inhalation distributions for 2 energy windows, photopeak (127-153 keV) and scatter (92-125 keV). The effects of scatter and attenuation were estimated on the basis of the imaging components separated from the simulation. A scatter correction method and 2 attenuation correction methods, all applied to inhalation scintigraphy, were evaluated using the simulated images. RESULTS: The amount of scatter ranges from 24% to approximately 29% in the lungs and from 29% to approximately 35% in the central (airway or esophagus) region on the planar images. Significant differences were found among regions and between source distributions (P < 0.05). The fraction k used for dual-energy-based scatter correction also varied and was found to be less than the commonly used k = 0.5. The simplified narrow-beam attenuation correction and the effective (broad-beam) correction methods were found to either under- or overcorrect the regional activities. CONCLUSION: The amount of scatter and tissue attenuation in the thorax region depends on source distribution and body attenuation. In applying planar scintigraphy for aerosol inhalation studies, it is difficult to obtain precise quantitative measurements because of the uncertainties associated with scatter and attenuation corrections. Accurate corrections require knowledge of both source and density distributions.

Pulmonary distribution and kinetics of inhaled [11C]triamcinolone acetonide.

UNLABELLED: Triamcinolone acetonide (TAA) is an anti-inflammatory steroid used for topical treatment of allergic rhinitis and asthma. Drug deposition onto target tissues is an important parameter, so methods for accurate deposition measurement are needed. Lung deposition is especially problematic to measure because of the large field of view and low relative drug penetration. Our main objective was to use PET to measure the deposition and postdeposition kinetics of TAA in the lung after administration from the Azmacort inhaler. The second objective was to evaluate changes in distribution caused by the inhalation spacer that is built into the product. METHODS: 11C-labeled TAA was formulated as the Azmacort product, 5 healthy volunteers inhaled it, and PET scans were obtained of its distribution in the head and chest. Region-of-interest analysis with CT overlay was used to analyze the distribution and kinetics in the airway and lung. RESULTS: From 10% to 15% of the inhaled drug dose was deposited in target airway regions in a distally decreasing pattern. Deposition in the oral cavity was about 30% of the dose. Slow absorption or clearance of drug from target tissues was observed over time. Use of the inhalation spacer caused statistically significant increases in all target tissues (factor of 2-5) and a roughly 40% decrease in oral deposition. Measurable amounts of the drug remained in target regions throughout the scanning period. CONCLUSION: Local pulmonary distribution and kinetics of inhaled drugs can be measured accurately by PET for drug development. The integrated actuator-spacer significantly enhanced deposition of TAA in target tissues and reduced deposition in the oropharyngeal region.

Regional distribution and kinetics of inhaled pharmaceuticals.

Drug biodistribution is often secondary to drug action. However, drugs that have a topical action and are deposited into the airway by inhalation are dependent on effective deposition at the intended site of action. Measurement of the distribution of such drugs in the airway is a useful tool. Distribution data can help to interpret clinical results, to evaluate products relative to each other, to optimize a new drug formulation, and to choose effective drug delivery methods. Imaging of radiotracers is the only means available to measure drug deposition throughout the lungs, nasal passages, and sinuses. There are several approaches to imaging drug deposition. Planar imaging has been the most used method, but SPECT and PET imaging are beginning to be applied effectively. The properties of non-drug tracers, labeling of drugs, evaluation of distribution patterns, and quantification of deposited drugs are important issues that have been addressed. Imaging has been shown to be a powerful technique to evaluate and to speed development of inhaled drugs. This review explores the most recent advances and issues with an emphasis on drug development.

Mapping PET-measured triamcinolone acetonide (TAA) aerosol distribution into deposition by airway generation.

The three dimensional (3D) distribution of inhaled drugs was measured using Positron Emission Tomography (PET) (Berridge, M.S, Muswick, G.J., Lee, Z., Leisure, G.L., Nelson, A.D., Muzic, R.F. Jr., Miraldi, F., Heald, D.L., 1997. PET evaluation of Azmacort(R) ([C-11]triamcinolone acetonide) dose administration. J. Nucl. Med. 38 (5) Suppl., 4-5). Data analysis was based upon regional ratios or penetration indices. To improve the analytical usefulness and objectivity, labeled drug from dynamic PET images was mapped into 23 airway generations following a general framework from a SPECT-based methodology (Fleming, J.S., Nassim, M.A., Hashish, A.H., Bailey, A.G. , Conway, J., Holgate, S., Halson, P., Moore, E., Martonen, T.B., 1995. Description of pulmonary deposition of radiolabeled aerosol by airway generation using a conceptual three dimensional model of lung morphology. J. Aerosol Med. 8, 341-356). A recently developed airway network model was used in this study. Quantitative PET scans of [C-11]triamcinolone acetonide distribution in the lung were determined following administration of Azmacort(R), a commercial metered dose inhaler with an integrated spacer device. Distributions at varying time periods after drug administration were investigated to explore the dynamics and kinetics of the aerosolized drug. Initially, deposition of labeled drug on conducting airways (generations 1-14) was found to be higher than those on acinar airways (generation 15-23), 64% versus 36%. The distribution pattern changed slowly with time. By 47 min, 51% of the dose remaining in the lung was found on conducting airways while 49% was on acinar airways. This study illustrates the value of PET imaging for the evaluation and design of drug formulations.

Synthesis of [18F]fluoromethyl iodide, a synthetic precursor for fluoromethylation of radiopharmaceuticals.

[18F]fluoroiodomethane was labeled via nucleophilic substitution of diiodomethane with [18F]fluoride, and labeling conditions were optimized. The optimized labeling yield was 40 +/- 8% (decay-corrected). The synthesis and purification of [18F]fluoroiodomethane took 15 min. The reactions of [18F]fluoroiodomethane with amine, carboxylic acid, thiol and phenoxide groups produced fluoromethylated derivatives with various yields (12-95%). The results indicated that [18F]fluoroiodomethane is a valuable synthetic precursor for the introduction of an [18F]fluoromethyl group into radiopharmaceuticals.

Iterative optimal design of PET experiments for estimating beta-adrenergic receptor concentration.

To estimate in vivo myocardial beta-adrenergic receptor concentration with sufficient precision and to reduce the experimental complexities in positron emission tomography (PET), an iterative optimal design method is applied. An initial three-injection protocol, utilising [F-18]-labelled (R)- and (S)-fluorocarazolol and unlabelled (S)-fluorocarazolol, is optimised for ligand dosages and administration times to maximise the precision of all model parameters using the D-optimal criterion. Using this experimental protocol, PET data are collected in porcine studies, and model parameters are estimated. All model parameters are identified with satisfactory precision. The in vivo myocardial beta-receptor concentration is 7.5+/-0.6 pmol x ml(-1), which corresponds to the in vitro result of 10.1+/-1.3 pmol x ml(-1). With more accurate parameter values, a simplified two-injection protocol is optimally designed, utilising only radiolabelled and unlabelled (S)-fluorocarazolol, based on a new criterion to maximise the precision of the beta-receptor concentration. This revised optimum design predicts that the in vivo beta-receptor concentration can be estimated with good precision but reduced experiment complexity.

High yield synthesis of [11C]-acetone through selective quenching of methyl lithium.

Carbon-11 labeled acetone is a useful radiosynthetic precursor. Previously, strict control of no-carrier-added stoichiometry was required to prepare it from reaction of CO2 and methyl lithium. However, excess methyl lithium may be selectively quenched to avoid reaction with nascent acetone to give tert-butanol. We report a simple pKa-based strategy to sequentially and selectively quench MeLi and acetone to give yields of up to 100% acetone even in the presence of a large excess of MeLi. The method gives good yields of acetone under conditions that previously precluded its synthesis.

PET quantification of specific binding of carbon-11-nicotine in human brain.

UNLABELLED: Previous work on the PET measured uptake of (S)-[11C]nicotine presents conflicting findings as to whether it reflects specific binding. METHODS: We studied the uptake of (R)-[11C]nicotine and (S)-[11C]nicotine in normal volunteers at baseline conditions and after a challenge with unlabeled (S)-nicotine to decrease the concentration of free binding sites or with CO2 to increase perfusion. We analyzed the data using two- and three-compartment models. RESULTS: We found tissue pharmacokinetics of (R)- and (S)-[11C]nicotine are adequately described by the two-compartment model. (S)-nicotine challenge induced small but statistically significant reductions in distribution volume (DV) of both (R)- and (S)-[11C]nicotine. The changes in DV could not be attributed to perfusion changes because DV was not affected by CO2 challenge. Although the reduction in DV indicates sensitivity of [11C]nicotine to status of nicotinic binding sites, the small magnitude of the reduction suggests that most nicotine uptake is nonspecific. CONCLUSION: Although differences in DV attributable to specific binding were detected, (R)- and (S)-[11C]nicotine are relatively poor tracers for studying nicotinic binding sites using PET.

Biodistribution and kinetics of nasal carbon-11-triamcinolone acetonide.

UNLABELLED: PET is a technique with a strong potential for use in drug evaluation and development. In particular, the distribution and pharmacokinetics of locally administered drugs may be advantageously explored noninvasively using labeled compounds. This pilot study was performed to demonstrate the effectiveness of PET for drug development and to determine the human biodistribution and kinetics of triamcinolone acetonide, labeled with 11C, formulated and nasally administered as Nasacort AQ nasal inhalant. METHODS: Carbon-11-labeled triamcinolone acetonide was formulated as the commercial product, and PET scans of the heads of four volunteers were performed in a vertical orientation. Region-of-interest analysis with MRI coregistration was used to analyze the distribution and kinetics in nasal tissues. RESULTS: Deposition of the majority of the dose on target tissues was immediate. Penetration into sinuses was observed. There was moderate redistribution and slow migration of the drug through nasal passages to the throat. Significant amounts of the drug remained in target regions for several hours. CONCLUSION: PET is an effective means to determine local drug distribution and kinetics.

The use of 3-methoxymethyl-16 beta, 17 beta-epiestriol-O-cyclic sulfone as the precursor in the synthesis of F-18 16 alpha-fluoroestradiol.

We have prepared 3-methoxymethyl-16 beta, 17 beta-epiestriol-O-cyclic sulfone (1c) and used it as a substrate for the production of F-18 16 alpha-fluoroestradiol, via nucleophilic fluorination with fluoride ion. The compound is straightforward to make from the commercially available epiestriol and is a stable crystalline compound that can be stored for at least a year at room temperature. Reaction with fluorine-18 fluoride provides excellent yields; typically > 90% incorporation of the fluoride is achieved. Partial purification of the labeled product may be accomplished at this stage. Hydrolysis of the methoxymethyl protecting group and ring-opened sulfate occurs rapidly in ethanolic acid solution. In the presence of water the hydrolysis requires more vigorous conditions and additional time but still proceeds to completion. Labeled fluoroestradiol is isolated at the end of a 1-2 h synthesis, depending on the hydrolysis method of 30-45% chemical (decay corrected) yield with respect to fluoride, with a specific activity > 1 Ci per micromole.

PET measurement of neuroreceptor occupancy by typical and atypical neuroleptics.

UNLABELLED: The goal of this study was to use PET and 11C-N-methylspiperone (11C-NMSP) to measure the difference in relative occupancy of serotonin (5-hydroxytryptamine-2 or 5-HT2A) and dopamine-2 (D2) neuroreceptors in subjects being treated with typical or atypical antipsychotic drugs. METHODS: We used PET and single-dose 11C-NMSP to measure receptor indices and relative receptor occupancy of 5-HT2A receptors in frontal cortex and D2 receptors in basal ganglia in five subjects who were neuroleptic free, five subjects who were being treated with typical antipsychotic drugs and five subjects who were being treated with clozapine, an atypical antipsychotic drug. RESULTS: Among the three groups, there were significant differences in 5-HT2A indices, D2 indices and the ratio of 5-HT2A to D2 indices. With no overlap, the 5-HT2A index separated all subjects who received clozapine and the D2 index separated the remaining two groups. CONCLUSION: Typical antipsychotic and atypical antipsychotic subjects do have differing patterns of 5-HT2A and D2 relative receptor occupancy when measured with a single PET scan, single 11C-NMSP radiotracer dose and no separately injected "cold" pharmaceutical.