In-vivo Intradermal Delivery of Co-57 labeled Vitamin B-12, and Subsequent Comparison with Standard Subcutaneous Administration.

Vitamin B-12 (cobalamin) deficiency in humans is a worldwide problem emanating from varied causes such as insufficient dietary intake or malabsorption of the micronutrient due to an underlying condition (absence or failure of intrinsic factor, atrophic gastritis, post-operative bariatric surgery, inflammatory bowel disease, cobalt deficiency etc.). As oral supplementation is limited by its bioavailability due to the absorptive property of intrinsic factor, clinicians often prescribe parenteral forms of administration to replenish diminished levels rapidly. The gold standard in parenteral delivery of cobalamin is subcutaneous and/or intramuscular injections. The relatively large molecular size of cobalamin (1355.39 Da) makes passive transdermal patch-based delivery via the stratum corneum quite challenging. Hence, the primary goal of this study is to investigate the feasibility of intradermal (ID) delivery of Vitamin B-12 via an almost painless microneedle injection and subsequent comparison with standard subcutaneous (SC) delivery. This work reports on a custom-made microneedle device built from a commercial insulin needle and it's use to perform ID delivery of Co-57 radiolabeled Vitamin B-12 in-vivo in rabbits. The pharmacokinetic profile and bioavailability were studied and compared with SC delivery. It is the first comprehensive study, to our best knowledge, that compares a micronutrient (eg. Vitamin B-12) delivery via ID and SC routes in-vivo. While the bioavailability for the SC route is found to be slightly higher compared to the ID route (99% vs. 96%), the Tmax for both are almost identical. Thus, ID delivery of Vitamin B-12 using a microneedle injection could be a viable and minimally invasive alternative to existing parenteral options.

Position statement on radiopharmaceutical production for clinical trials.

The EU regulation 536/2014 aims to facilitate the experimental use of diagnostic radiopharmaceuticals in particular for GMP requirements and needs to be applied in EU countries. As definitely clarified by this survey, the application is still far from being completed due to national restrictions that are conflicting with the content of the above EU regulation. Although the nuclear medicine centers are obliged to be compliant with national regulatory, national authorities have to be required to work towards full application of the regulation. On the other hand, an update of 536/2014 that includes therapeutic radiopharmaceuticals would also be beneficial to a rational and safe advance of nuclear medicine.

Preclinical evaluation of [(18)F]JNJ42259152 as a PET tracer for PDE10A.

Phosphodiesterase-10A (PDE10A) is implicated in several neuropsychiatric disorders involving basal ganglia neurotransmission, such as schizophrenia, obsessive-compulsive disorder and Huntington's disease. To confirm target engagement and exposure-occupancy relationships of clinical candidates for treatment, and to further explore the in vivo biology of PDE10A, non-invasive imaging using a specific PET ligand is warranted. Recently we have reported the in vivo evaluation of [(18)F]JNJ41510417 which showed specific binding to PDE10A in rat striatum, but with relatively slow kinetics. A chemically related derivative JNJ42259152 was found to have a similar in vivo occupancy, but lower lipophilicity and lower PDE10A in vitro inhibitory activity compared to JNJ41510417. (18)F-labeled JNJ42259152 was therefore evaluated as a potential PDE10A PET radiotracer. Baseline PET in rats and monkey showed specific retention in the PDE10A-rich striatum, and fast wash-out, with a good contrast to non-specific binding, in other brain regions. Pretreatment and chase experiments in rats with the selective PDE10A inhibitor MP-10 showed that tracer binding was specific and reversible. Absence of specific binding in PDE10A knock-out (KO) mice further confirmed PDE10A specificity. In vivo radiometabolite analysis using high performance liquid chromatography (HPLC) showed presence of polar radiometabolites in rat plasma and brain. In vivo imaging in rat and monkey further showed faster brain kinetics, and higher striatum-to-cerebellum ratios for [(18)F]JNJ42259152 compared to [(18)F]JNJ41510417. The arterial input function corrected for radiometabolites was determined in rats and basic kinetic modeling was established. For a 60-min acquisition time interval, striatal binding potential of the intact tracer referenced to the cerebellum showed good correlation with corresponding binding potential values of a Simplified Reference Tissue Model and referenced Logan Plot, the latter using a population averaged reference tissue-to-plasma clearance rate and offering the possibility to generate representative parametric binding potential images. In conclusion we can state that in vivo imaging in PDE10A KO mice, rats and monkey demonstrates that [(18)F]JNJ42259152 provides a PDE10A-specific signal in the striatum with good pharmacokinetic properties. Although presence of a polar radiometabolite in rat brain yielded a systematic but reproducible underestimation of the striatal BPND, a Logan reference tissue model approach using 60 min acquisition data is appropriate for quantification.

Equivalent dynamic human brain NK1-receptor occupancy following single-dose i.v. fosaprepitant vs. oral aprepitant as assessed by PET imaging.

The type 1 neurokinin receptor (NK1R) antagonist aprepitant and its i.v. prodrug fosaprepitant have been approved for prevention of acute and delayed nausea and vomiting associated with chemotherapy. This study evaluated the magnitude and duration of brain NK1R occupancy over a period of 5 days after single-dose i.v. infusion of 150-mg fosaprepitant and single-dose oral administration of 165-mg aprepitant, using serial [(18)F]MK-0999 positron emission tomography (PET) in 16 healthy subjects. Each subject underwent three scans. Brain NK1R occupancy rates after i.v. fosaprepitant at time to peak concentration (T(max); ~30 min), 24, 48, and 120 h after the dose were 100, 100, ≥97, and 41-75%, respectively. After aprepitant, NK1R occupancy rates at these time points (T(max) ~4 h) were ≥99, ≥99, ≥97, and 37-76%, respectively. Aprepitant plasma concentration profiles were comparable for the two dosage forms. The study illustrates the utility of PET imaging in determining central bioequivalence in a limited number of subjects.

Metabolic and type 1 cannabinoid receptor imaging of a transgenic rat model in the early phase of Huntington disease.

Several lines of evidence imply early alterations in metabolic and endocannabinoid neurotransmission in Huntington disease (HD). Using [(18)F]MK-9470 and small animal PET, we investigated for the first time cerebral changes in type 1 cannabinoid (CB1) receptor binding in vivo in pre-symptomatic and early symptomatic rats of HD (tgHD), in relation to glucose metabolism, morphology and behavioral testing for motor and cognitive function. Twenty-three Sprague-Dawley rats (14 tgHD and 9 wild-types) were investigated between the age of 2 and 11 months. Relative glucose metabolism and parametric CB1 receptor images were anatomically standardized to Paxinos space and analyzed voxel-wise. Volumetric microMRI imaging was performed to assess HD neuropathology. Within the first 10 months, bilateral volumes of caudate-putamen and lateral ventricles did not significantly differ between genotypes. Longitudinal- and genotype evolution showed that relative [(18)F]MK-9470 binding progressively decreased in the caudate-putamen and lateral globus pallidus of tgHD rats (-8.3%, p≤1.1×10(-5) at 5 months vs. -10.9%, p<1.5×10(-5) at 10 months). In addition, relative glucose metabolism increased in the bilateral sensorimotor cortex of 2-month-old tgHD rats (+8.1%, p≤1.5×10(-5)), where it was positively correlated to motor function at that time point. TgHD rats developed cognitive deficits at 6 and 11 months of age. Our findings point to early regional dysfunctions in endocannabinoid signalling, involving the lateral globus pallidus and caudate-putamen. In vivo CB1 receptor measurements using [(18)F]MK-9470 may thus be a useful early biomarker for HD. Our results also provide evidence of subtle motor and cognitive deficits at earlier stages than previously described.

MRK-409 (MK-0343), a GABAA receptor subtype-selective partial agonist, is a non-sedating anxiolytic in preclinical species but causes sedation in humans.

MRK-409 binds to α1-, α2-, α3- and α5-containing human recombinant GABA(A) receptors with comparable high affinity (0.21-0.40 nM). However, MRK-409 has greater agonist efficacy at the α3 compared with α1 subtypes (respective efficacies relative to the full agonist chlordiazepoxide of 0.45 and 0.18). This compound readily penetrates the brain in rats and occupies the benzodiazepine site of GABA(A) receptors, measured using an in vivo [(3)H]flumazenil binding assay, with an Occ(50) of 2.2 mg/kg p.o. and a corresponding plasma EC(50) of 115 ng/mL. Behaviourally, the α3-preferring agonist efficacy profile of MRK-409 produced anxiolytic-like activity in rodent and primate unconditioned and conditioned models of anxiety with minimum effective doses corresponding to occupancies, depending on the particular model, ranging from ∼35% to 65% yet there were minimal overt signs of sedation at occupancies greater than 90%. In humans, however, safety and tolerability studies showed that there was pronounced sedation at a dose of 2 mg, resulting in a maximal tolerated dose of 1 mg. This 2 mg dose corresponded to a C(max) plasma concentration of 28 ng/mL, which, based on the rodent plasma EC(50) for occupancy of 115 ng/mL, suggested that sedation in humans occurs at low levels of occupancy. This was confirmed in human positron emission tomography studies, in which [(11)C]flumazenil uptake following a single dose of 1 mg MRK-409 was comparable to that of placebo, indicating that occupancy of GABA(A) receptor benzodiazepine binding sites by MRK-409 was below the limits of detection (i.e. <10%). Taken together, these data show that MRK-409 causes sedation in humans at a dose (2 mg) corresponding to levels of occupancy considerably less than those predicted from rodent models to be required for anxiolytic efficacy (∼35-65%). Thus, the preclinical non-sedating anxiolytic profile of MRK-409 did not translate into humans and further development of this compound was halted.

Preclinical and clinical pharmacology of TPA023B, a GABAA receptor α2/α3 subtype-selective partial agonist.

In the accompanying paper we describe how MRK-409 unexpectedly produced sedation in man at relatively low levels of GABA(A) receptor occupancy (∼10%). Since it was not clear whether this sedation was mediated via the α2/α3 or α1 GABA(A) subtype(s), we characterized the properties of TPA023B, a high-affinity imidazotriazine which, like MRK-409, has partial agonist efficacy at the α2 and α3 subtype but is an antagonist at the α1 subtype, at which MRK-409 has weak partial agonism. TPA023B gave dose- and time-dependent occupancy of rat brain GABA(A) receptors as measured using an in vivo [(3)H]flumazenil binding assay, with 50% occupancy corresponding to a respective dose and plasma drug concentration of 0.09 mg/kg and 19 ng/mL, the latter of which was similar to that observed in mice (25 ng/mL) and comparable to values obtained in baboon and man using [(11)C]flumazenil PET (10 and 5.8 ng/mL, respectively). TPA023B was anxiolytic in rodent and primate (squirrel monkey) models of anxiety (elevated plus maze, fear-potentiated startle, conditioned suppression of drinking, conditioned emotional response) yet had no significant effects in rodent or primate assays of ataxia and/or myorelaxation (rotarod, chain-pulling, lever pressing), up to doses (10 mg/kg) corresponding to occupancy of greater than 99%. In man, TPA023B was well tolerated at a dose (1.5 mg) that produced occupancy of >50%, suggesting that the sedation previously seen with MRK-409 is due to the partial agonist efficacy of that compound at the α1 subtype, and highlighting the importance of antagonist efficacy at this particular GABA(A) receptor population for avoiding sedation in man.

Different regional brain activity during physiological gastric distension compared to balloon distension: a H2 15O-PET study.

BACKGROUND: Stepwise gastric balloon distension progressively activates a 'visceral pain neuromatrix', ultimately inducing discomfort and pain. On the other hand, normal meal ingestion requires gastric volume expansion without induction of pain. The aim was to test the hypothesis that physiological gastric distension (liquid meal infusion) until maximal satiation elicits brain responses similar to balloon distension at discomfort threshold. METHODS: Brain H(2) (15) O-positron emission tomography (PET) was performed in two different groups of healthy volunteers (both n=14) during continuous and stepwise infusion of a liquid meal through a nasogastric tube, until maximal satiation. Brain (de)activation patterns were compared with historical controls in which discomfort was elicited using gastric balloon distension. This latter reference group was acquired on the same scanner using the same acquisition protocol; all data were analyzed using statistical parametric mapping (SPM2). Within each group, brain activity at maximal distension was compared to baseline activity and between-group comparisons were made. KEY RESULTS: Intragastric volumes and satiation/gastric sensation scores at endpoint were similar in all groups. Continuous and stepwise nutrient infusion was associated with progressive deactivations in key areas of the 'visceral pain neuromatrix' that were activated during balloon distension. Additionally, stepwise infusion progressively activated prefrontal areas and showed deactivations in 'default network' brain regions also found to be deactivated during balloon distension. CONCLUSIONS & INFERENCES: Compared to gastric balloon distension, physiological gastric distension using nutrient infusion elicits opposite brain responses in the 'visceral pain neuromatrix', but similar responses in other areas. We interpret this finding as a prerequisite for tolerance of normal meal volumes in health.

Synthesis of 18F-labelled 2-(4'-fluorophenyl)-1,3-benzothiazole and evaluation as amyloid imaging agent in comparison with [11C]PIB.

2-(4'-[(18)F]fluorophenyl)-1,3-benzothiazole was synthesized as a fluorine-18 labelled derivative of the Pittsburg Compound-B (PIB), which has known affinity for amyloid beta and promising characteristics as tracer for in vivo visualisation of amyloid deposits in patients suffering from Alzheimer's disease (AD). Both the nitro-precursor 2-(4'-nitrophenyl)-1,3-benzothiazole and the non-radioactive reference compound were synthesized using a 1-step synthesis pathway. Labelling was achieved by direct aromatic nucleophilic substitution of the nitro-precursor using [(18)F]fluoride by heating for 20 min at 150 degrees C and with a radiochemical yield of 38%. The reference compound showed high affinity for amyloid in an in vitro competition binding study using human AD brain homogenates (K(i)=9.0 nM) and fluorescence imaging of incubated transgenic APP mouse brain slices confirmed binding to amyloid plaques. A biodistribution study in normal mice showed a high brain uptake at 2 min pi (3.20%ID/g) followed by a fast washout (60 min pi: 0.21%ID/g). A dynamic microPET study was performed in a transgenic APP and normal WT mouse, but, similar to [(11)C]PIB, no difference was seen in tracer retention between both kind of mice. The new (18)F-labelled 2-phenylbenzothiazole showed excellent preclinical characteristics comparable with those of the (11)C-labelled PIB.

11C-labelled PIB analogues as potential tracer agents for in vivo imaging of amyloid beta in Alzheimer's disease.

Pittsburgh Compound-B (PIB) is currently being evaluated clinically for in vivo visualization of amyloid plaques in patients with Alzheimer's disease (AD). We have synthesized three structural isomers of 6-hydroxy-2-(4'-aminophenyl)-1,3-benzothiazole, performed radiolabelling with carbon-11 and investigated their in vivo and in vitro properties. Specific binding to amyloid plaques was demonstrated in vitro using post-mortem brain homogenates of AD patients, transgenic AD mice brain sections and post-mortem human AD brain sections. In normal mice, initial brain uptake (at 2 min p.i.) was high and was followed by a fast wash-out. The three structural analogues have a high potential as tracer agents for in vivo visualization of amyloid plaques in AD patients.

Cortical deactivations during gastric fundus distension in health: visceral pain-specific response or attenuation of 'default mode' brain function? A H2 15O-PET study.

Gastric distension activates a cerebral network including brainstem, thalamus, insula, perigenual anterior cingulate, cerebellum, ventrolateral prefrontal cortex and potentially somatosensory regions. Cortical deactivations during gastric distension have hardly been reported. To describe brain areas of decreased activity during gastric fundus distension compared to baseline, using data from our previously published study (Gastroenterology, 128, 2005 and 564). H(2) (15)O-brain positron emission tomography was performed in 11 healthy volunteers during five conditions (random order): (C(1)) no distension (baseline); isobaric distension to individual thresholds for (C(2)) first, (C(3)) marked, (C(4)) unpleasant sensation and (C(5)) sham distension. Subtraction analyses were performed (in SPM2) to determine deactivated areas during distension compared to baseline, with a threshold of P(uncorrected_voxel_level) < 0.001 and P(corrected_cluster_level) < 0.05. Baseline-maximal distension (C(1)-C(4)) yielded significant deactivations in: (i) bilateral occipital, lateral parietal and temporal cortex as well as medial parietal lobe (posterior cingulate and precuneus) and medial temporal lobe (hippocampus and amygdala), (ii) right dorsolateral and dorso- and ventromedial PFC, (iii) left subgenual ACC and bilateral caudate head. Intragastric pressure and epigastric sensation score correlated negatively with brain activity in similar regions. The right hippocampus/amygdala deactivation was specific to sham. Gastric fundus distension in health is associated with extensive cortical deactivations, besides the activations described before. Whether this represents task-independent suspension of 'default mode' activity (as described in various cognitive tasks) or an visceral pain/interoception-specific process remains to be elucidated.

The role of somatosensory cortical regions in the processing of painful gastric fundic distension: an update of brain imaging findings.

Painful gastric distension is processed in a network consisting of brainstem, thalamus, insula, anterior cingulate cortex, (lateral) orbitofrontal and prefrontal cortex, superior temporal cortex and cerebellum. However, the role of primary and secondary somatosensory cortical regions (SI/SII) in the processing of visceral sensation or pain in general and gastric sensation in particular remains unclear. The aim of this study was to localize activations in the SI/SII area from our previously published functional brain imaging studies on gastric distension more precisely, using newly available cytoarchitectonic probability maps of SI/SII, implemented in the SPM Anatomy toolbox. In healthy volunteers, we found two clusters to be overlapping with SII (mainly the OP4 subregion) and, to a lesser extent, SI, although this overlap was small in size. In functional dyspepsia patients, we found two clusters to be overlapping with SII (mainly OP4), of which the cluster in the right hemisphere also overlapped with SI. These findings were confirmed in a conjunction analysis of both groups. Activation in right SI/SII was significantly higher in healthy volunteers when formally compared to patients. These results provide more detailed information on the brain processing of gastric sensation, supporting the hypothesis that SI/SII are involved. This is in line with some previously published studies on visceral sensation, but at variance with some other studies. Methodological differences between the brain imaging studies on gastric distension may account for these somewhat discrepant findings.

Synthesis and evaluation of a (99m)Tc-BAT-phenylbenzothiazole conjugate as a potential in vivo tracer for visualization of amyloid beta.

We have conjugated S,S'-bis-trityl-N-BOC-N'-acetic acid-1,2-ethylenedicysteamine, a protected bis-amino-bis-thiol (BAT) tetraligand, with 2-(4'-aminophenyl)-1,3-benzothiazole, a derivative of thioflavin-T with known affinity for amyloid. The conjugate was efficiently labelled with (99m)Tc by heating of the protected precursor in diluted hydrochloric acid followed by neutralization and heating in the presence of (99m)Tc-tartrate. It was demonstrated that the (99m)Tc-BAT-phenylbenzothiazole conjugate binds in vitro to amyloid beta present in postmortem brain slices of Alzheimer's patients. Despite its high lipophilicity and neutral character, the radiolabelled conjugate did not cross the blood-brain barrier to a sufficient degree and therefore is not useful for detection of Alzheimer's disease. Further evaluation of this (99m)Tc-labelled tracer agent could elucidate its potential usefulness to visualize amyloid plaques in peripheral amyloidosis.

[11C]methionine PET, histopathology, and survival in primary brain tumors and recurrence.

BACKGROUND AND PURPOSE: [(11)C]Methionine (MET) PET imaging is a sensitive technique for visualizing primary brain tumors and recurrence/progression after therapy. The aim of this study was to evaluate the relationship between the uptake of MET and histopathologic grading and to investigate the prognostic value of the tracer, in both settings. METHODS: Cerebral uptake of MET was determined in 52 patients: in 26 patients for primary staging (group A) and 26 patients with suspected brain tumor recurrence/progression after therapy (group B). Semiquantitative methionine uptake indices (UI) defined by the tumor (maximum)-to-background ratio was correlated with tumor grade and final outcome. RESULTS: Overall median survival was 34.9 months. MET showed pathologically increased uptake in 41 of 52 scans. Although a weak linear correlation between MET uptake and grading was observed (R = 0.38, P = .028), analysis of variance showed no significant differences in MET UI between tumor grades for either group A or B. Benign and grade I lesions showed significant difference in MET uptake in comparison with higher grade lesions (P = .006). Using Kaplan-Meier survival analysis, no thresholds could be found at which MET was predictive for survival. Proportional hazard regression showed that only WHO grading class (low versus high) was predictive of survival (P = .015). CONCLUSION: Interindividual MET uptake variability does not allow noninvasive grading on an individual patient basis. Moreover, there is no significant prognostic value in studying maximal methionine UI in brain tumors. The clinical use of MET should therefore be primarily focused on questions such as detection of recurrence, biopsy guidance, and radiation therapy target volume delineation.

Alzheimer dementia caused by a novel mutation located in the APP C-terminal intracytosolic fragment.

Since the first report showing that Alzheimer disease (AD) might be caused by mutations in the amyloid precursor protein gene (APP), 20 different missense mutations have been reported. The majority of early-onset AD mutations alter processing of APP increasing relative levels of Abeta42 peptide, either by increasing Abeta42 or decreasing Abeta40 peptide levels or both. In a diagnostic setting using direct sequence analysis, we identified in one patient with familial early-onset AD a novel mutation in APP (c.2172G>C), predicting a K724N substitution in the intracytosolic fragment. The mutation is located downstream of the epsilon-cleavage site of APP and is the furthermost C-terminal mutation reported to date. In vitro expression of APP K724N cDNA showed an increase in Abeta42 and a decrease in Abeta40 levels resulting in a near three-fold increase of the Abeta42/Abeta40 ratio. Further, in vivo amyloid positron emission tomography (PET) imaging revealed significantly increased cortical amyloid deposits, supporting that in human this novel APP mutation is likely causing disease.

Evaluation of hypoxia in an experimental rat tumour model by [(18)F]fluoromisonidazole PET and immunohistochemistry.

This study aimed to evaluate tumour hypoxia by comparing [(18)F]Fluoromisonidazole uptake measured using positron emission tomography ([(18)F]FMISO-PET) with immunohistochemical (IHC) staining techniques. Syngeneic rhabdomyosarcoma (R1) tumour pieces were transplanted subcutaneously in the flanks of WAG/Rij rats. Tumours were analysed at volumes between 0.9 and 7.3 cm(3). Hypoxic volumes were defined using a 3D region of interest on 2 h postinjection [(18)F]FMISO-PET images, applying different thresholds (1.2-3.0). Monoclonal antibodies to pimonidazole (PIMO) and carbonic anhydrase IX (CA IX), exogenous and endogenous markers of hypoxia, respectively, were used for IHC staining. Marker-positive fractions were microscopically measured for each tumour, and hypoxic volumes were calculated. A heterogeneous distribution of hypoxia was observed both with histology and [(18)F]FMISO autoradiography. A statistically significant correlation (P<0.05) was obtained between the hypoxic volumes defined with [(18)F]FMISO-PET and the volumes derived from the PIMO-stained tumour sections (r=0.9066; P=0.0001), regardless of the selected threshold between 1.4 and 2.2. A similar observation was made with the CA IX staining (r=0.8636; P=0.0006). The relationship found between [(18)F]FMISO-PET and PIMO- and additionally CA IX-derived hypoxic volumes in rat rhabdomyosarcomas indicates the value of the noninvasive imaging method to measure hypoxia in whole tumours.

Dopamine transporter SPECT using fast kinetic ligands: 123I-FP-beta-CIT versus 99mTc-TRODAT-1.

A comparative study was carried out on two promising presynaptic dopamine transporter single-photon emission tomography (SPECT) radioligands with a fast pharmacokinetic profile, 123I-FP-beta-CIT (FP) and 99mTc-TRODAT-1 (TR), in order to assess their differential diagnostic power in early parkinsonism and their sensitivity for detection of disease progression. This cross-sectional study was conducted on 96 patients with early-stage parkinsonism referred in a tertiary clinical setting. Mean disease duration was 2.0+/-1.3 years, and patients had a modified Hoehn and Yahr (H&Y) stage of 1-2 (average 1.2). Forty-seven patients received TR, and 49 received FP. In both groups, ten patients with normal presynaptic function were included as a control population; all other patients were clinically diagnosed as having idiopathic Parkinson's disease. Groups were matched for gender, age, disease duration and modified H&Y stage. Triple-head gamma camera SPECT was analysed using a semiquantitative index of transporter binding (BI). Discriminant analysis with cross-validation resulted in a maximal classification accuracy for FP of 93% (sensitivity 95% and specificity 86%) for the contralateral putamen BI. For TR, the corresponding values were 87% accuracy, 92% sensitivity and 70% specificity. For FP, disease duration was correlated with both the putamen BI (-8.8%/year, rho=-0.41, P=0.025) and the putamen/caudate ratio (-7.4%/year, rho=-0.51, P=0.004), but for TR no significant correlation was found (all P values >0.5). In conclusion, both FP and TR show high sensitivity in a clinically relevant setting, but FP has superior accuracy for early differential diagnosis of idiopathic parkinsonism and non-degenerative extrapyramidal disorders, as well as better sensitivity for disease follow-up.

RP-HPLC separation of the diastereomers of technetium-99m labelled tropanes and identity confirmation using radio-LC-MS.

99mTc-TRODAT-1 (technetium(V)-oxo-2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]]amino]-ethanethiolato(3-)) and 99mTc-TRODAT-M, the 4-methylphenyl derivative of 99mTc-TRODAT-1, are at this moment being evaluated in clinical trials as imaging agents for the central nervous dopamine transporter system. Both compounds are formed as a mixture of two major diastereomers. As the tracer concentration in preparations for clinical investigations is very low (30-150 pmol/ml), identification of these 99mTc-complexes was, up to now, carried out indirectly using X-ray diffraction analysis of the corresponding rhenium complexes which can be synthesized in gram amounts. In this study, we developed a convenient and practical reversed phase HPLC-method for purification and isolation of the respective diastereomers of 99mTc-TRODAT-1 and three of its derivatives using mixtures of solvents which are compatible with biological studies, i.e. aqueous buffers and ethanol. Furthermore, direct identity confirmation of the 99mTc-complexes using radio-LC-MS was successfully elaborated.

An efficient HPLC method for the analysis of isomeric purity of technetium-99m-exametazime and identity confirmation using LC-MS.

99mTc-exametazime (99mTc-d,l-HMPAO, 99mTc-d,l-hexamethylpropyleneamine oxime) is a neutral rather unstable complex of short-lived 99mTc (t(1/2)=6 h) with the d,l-isomer (mixture of D,D- and L,L-isomers) of a bis-amine bis-oxime tetraligand. It is widely used for measurement of regional cerebral perfusion in nuclear medicine. The meso-isomer (D,L-form) should not be present in a preparation as it is not retained in brain and thus does not provide clinically useful information. Meso-HMPAO is removed from the ligand during the synthesis procedure by repeated recrystallization, but can still be present as impurity in d,l-isomer. Due to the lack of a suitable chromatographic method for analysis of the isomeric purity of 99mTc-exametazime preparations, United States Pharmacopoeia 25 (USP 25) prescribes a biological test in rats for quality control purpose. In this study, we developed a suitable high-performance liquid chromatography (HPLC) method which allows to demonstrate the relative amounts of d,l- and meso-isomer in 99mTc-exametazime and so obviates the need for a biodistribution test in animals as part of the quality control. Due to the low concentrations in which 99mTc-d,l-HMPAO is obtained (typically 2-6 ng/ml), confirmation of the identity of 99mTc-d,l-HMPAO in the monograph of the European Pharmacopoeia is now performed only indirectly by TLC and assessment of its retention time on RP-HPLC. To investigate the potential of radio-LC-MS for assessment of the identity of 99mTc-exametazime, 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO prepared using a Tc-rich eluate were analyzed using a radio-LC-MS system equipped with a time-of-flight mass spectrometer with electrospray ionization. The main peak in the radiometric channel coincided with the molecular ion mass of 99mTc-d,l-HMPAO in the mass spectrometer channel and the measured accurate mass differed only by 0.26 ppm from the theoretical mass. The identity of 99mTc-meso-HMPAO was also confirmed. Thus, radio-LC-MS allowed to obtain strong evidence for the structure of 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO at nanomolar concentration. It is concluded that radio-LC-MS can become a sensitive aid in quality control of "no carrier added" radiopharmaceutical preparations.