Yttrium-86-labelled human serum albumin microspheres: relation of surface structure with in vivo stability.

INTRODUCTION: Radiolabelled particles are an attractive tool in the therapy of malignancies of the liver. We consider particles manufactured from denatured human serum albumin (HSA) as useful carriers of therapeutic radionuclides. Covalent attachment of suitable chelators onto the surface of the spheres promises an easy access to radiolabelled HSA microspheres. METHODS: We synthesized 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) bearing smooth, medium-rough and rough surfaced HSA microspheres (mean diameter: 25 microm). In vitro stability of 86 Y-labelled particles was determined after incubation in human plasma and in a DTPA challenge experiment. In vivo stability of 86 Y DOTA-HSA microspheres was determined after single intravenous application in rats. Subsequently, the particles were completely trapped in the lung microvasculature. Thus, the lung serves in our experiments as target organ. RESULTS: DOTA-HSA microspheres were 86 Y labelled in reproducible high yields (>95%). No differences between smooth and rough surfaced spheres were found for both DOTA coupling and 86 Y labelling. Labelled microspheres showed high in vitro stability in human plasma and in DTPA solution with only 8+/-1% and 2+/-0% loss of radioactivity from the surface, respectively, 48 h postinjection (pi). The three batches (smooth, medium-rough and rough surfaced microspheres) differed considerably in their radioactivity recovery in the lungs of rats 48 h pi. Smooth particles showed the highest in vivo stability of the radiolabel on the surface of the spheres, presumably because of slower proteolytic degradation. CONCLUSION: We found that for the preparation of HSA-derived microspheres for radiotherapeutic application, smooth surfaced spheres are superior to rough spheres due to their higher in vivo stability of the radionuclide fixation.

3-O-methyl-6-18F-fluoro-L-dopa, a new tumor imaging agent: investigation of transport mechanism in vitro.

UNLABELLED: (18)F-Labeled amino acids represent a promising class of imaging agents in tumors, particularly brain tumors. However, the determination of their potential to image peripheral tumors, possibly depending on individual transport characteristics, still remains an area of investigation. The present study investigated the transport mechanism for 3-O-methyl-6-(18)F-fluoro-L-dopa (OMFD), a novel (18)F-labeled phenylalanine derivative, into tumor cells. METHODS: OMFD has routinely and reliably been prepared for clinical use in 20%-25% radiochemical yield (decay corrected, related to (18)F-F(2)) using 6-(18)F-fluoro-L-3,4-dihydroxyphenylalanine preparation devices with minor modifications. In vitro uptake assays with HT-29 (human colon adenocarcinoma) cells, FaDu (squamous cell carcinoma) cells, and RBE4 (immortalized rat brain endothelial) cells were performed with OMFD under physiologic amino acid concentrations without and with the competitive transport inhibitors 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid and alpha-(methylamino)isobutyric acid plus serine and without or with Na(+). RESULTS: Transport inhibition experiments using specific competitive inhibitors demonstrated that uptake of OMFD in all cell lines tested was mediated mainly by the sodium-independent high-capacity amino acid transport systems. The highest OMFD uptake was in FaDu cells. CONCLUSION: OMFD seems to be a promising PET tracer for imaging of amino acid transport in tumors.

Autoradiographic imaging of the serotonin transporter in the brain of rats and pigs using S-([18F]fluoromethyl)-(+)-McN5652.

The [18F]fluoromethyl analogue of (+)-McN5652 ([18F]FMe-McN) has recently been developed as a radioligand for imaging the neuronal serotonin transporter (SERT) with positron emission tomography (PET). We describe here the autoradiographic evaluation of [18F]FMe-McN in the brain of rats and pigs. Autoradiographic studies of [18F]FMe-McN performed on rat and pig brain in vitro showed a high accumulation of radioactivity in the regions rich in SERT, such as amygdala, hypothalamus, superficial gray layer of the superior colliculus, various nuclei of thalamus and substantia nigra. The binding of [18F]FMe-McN was reduced by citalopram, a highly selective inhibitor for SERT. Similar regional specific binding densities of [18F]FMe-McN were observed in both species. The regional distribution and specific binding of this radiotracer correlates well with the distribution and regional brain binding of [3H]citalopram. Region-to-cerebellum ratios of [18F]FMe-McN in vitro reached a maximum value of 20.6 in the rat and 14.5 in the pig. In addition, ex vivo autoradiography of the rat brain was performed 90 min after i.v. administration of [18F]FMe-McN. The highest regional uptake of [18F]FMe-McN was observed in the hypothalamic area, substantia nigra and amygdaloid area. There is a high correlation between the in vitro and in vivo binding. The region-to-cerebellum ratio in vivo reached a maximum value of 5.1 in the substantia nigra, the highest yet reported for an 18F-labelled SERT tracer in vivo in this region. Furthermore, the distribution volume of [18F]FMe-McN calculated from the PET data in various regions of the porcine brain is highly correlated with the SERT density as determined by in vitro autoradiography with [3H]citalopram. Thus, [18F]FMe-McN has a clear potential as a radiotracer for studies of the SERT distribution in man with PET.

Positron emission tomography imaging of the serotonin transporter in the pig brain using [11C](+)-McN5652 and S-([18F]fluoromethyl)-(+)-McN5652.

S-([(18)F]fluoromethyl)-(+)-McN5652 ([(18)F](+)-FMe-McN5652) has recently been synthesized as a new potential radiotracer for positron emission tomography (PET) imaging of the 5-HT transporter. It is an analog of [(11)C](+)McN5652, which has been used in clinical PET studies for 5-HT transporter imaging. This article describes the comparison of these two radiotracers in pigs with respect to their in vivo binding characteristics. PET images revealed that the highest accumulation of both radiotracers was found in the ventral midbrain, thalamus, olfactory lobe, and pons which is consistent with the known density of 5-HT transporters. The specific binding was determined by subtracting the values of the inactive (-) enantiomers or of the occipital cortex from those obtained with [(11)C](+)McN5652 or [(18)F](+)-FMe-McN5652 in the time period between 75 and 115 min after radiotracer injection. The specific binding of the (18)F-labeled derivative was about 40% higher than that of the (11)C-labeled derivative. A strong inhibition of the specific binding was observed for both radiotracers after pretreatment with the selective 5-HT uptake inhibitor citalopram. [(18)F](+)-FMe-McN5652 showed faster kinetics than [(11)C](+)McN5652. It reached the binding equilibrium during a study length of 120 min, which was not the case for [(11)C](+)McN5652. It is concluded that [(18)F](+)-FMe-McN5652 is suitable for 5-HT transporter imaging with PET.

Effect of hypoxia/hypercapnia on metabolism of 6-[(18)F]fluoro-L-DOPA in newborn piglets.

There is evidence that the dopaminergic system is sensitive to altered p(O(2)) in the immature brain. However, the respective enzyme activities have not been measured in the living neonatal brain together with brain oxidative metabolism. Therefore 18F-labelled 6-fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) together with positron emission tomography (PET) was used to estimate the activity of the aromatic amino acid decarboxylase (AADC) in the brain of fifteen newborn piglets (2-5 days old). Two PET scans were performed in each piglet. Eleven animals underwent a period of normoxia and moderate hypoxia/hypercapnia (H/H). The remaining four animals were used as an untreated control group. Simultaneously, the brain tissue p(O(2)) was recorded, the regional cerebral blood flow (CBF) was measured with colored microspheres and the cerebral metabolic rate of oxygen (CMRO(2)) was determined. In addition, in four untreated and six H/H treated piglets the relative amounts of fluorodopamine and the respective metabolites were determined in brain tissue samples using HPLC analysis. H/H conditions were induced by lowering the inspired fraction of oxygen from 0.35 to 0.10 and adding CO(2) to the inspired gas resulting in an arterial p(CO(2)) between 74 and 79 mmHg. H/H elicited a more than 3-fold increase of the CBF (P<0.05) so that the CMRO(2) remained unchanged throughout the H/H period. Despite this, the brain tissue p(O(2)) was reduced from 19+/-4 to 6+/-3 mmHg (P<0.05). The permeability-surface area product of FDOPA (PS(FDOPA)) was unchanged. However, the transfer rate of FDOPA (k(3)(FDOPA)) of the nigrostriatal dopaminergic system and the relative amounts of fluorodopamine and the respective metabolites were significantly increased (P<0.05). It is suggested that H/H induces an increase of AADC activity. However, an H/H-induced CBF increase maintains bulk O(2) delivery and preserves CMRO(2).

Development of technetium-99m-based CNS receptor ligands: have there been any advances?

By virtue of its ideal nuclear physical characteristics for routine nuclear medicine diagnostics and its ready availability, technetium-99m is of outstanding interest in the development of novel radiopharmaceuticals. The potential for the development of 99mTc-based radioligands for the study the receptor function in the central nervous system (CNS) is also well recognised despite the difficulties to be overcome. A fundamental challenge is the pharmacologically acceptable integration of the transition metal technetium, with its specific coordination chemistry, into the molecular entity of CNS receptor ligands. Conceptually, the ligand molecule can be assembled by three building blocks: a small neutral chelate unit, an organic linker that may also serve as a pharmacological modifier and a receptor-binding region derived from selective receptor antagonists. The recent introduction of novel technetium chelate units, particularly mixed-ligand complexes and low-valency organometallic compounds of technetium, provides an impetus for the further development of CNS receptor ligands. Moreover, progress in receptor pharmacology and the experience gained with positron emission tomography radiotracers have facilitated the design of numerous 99mTc-based CNS receptor ligands. The formidable challenge of developing 99mTc probes as single-photon emission tomography imaging agents targeting CNS receptors can be viewed with optimism given the successful development of [99mTc]TRODAT-1 as a 99mTc complex for imaging dopamine transporters in the brain, although there are a number of receptor-specific imaging agents that have so far resisted all efforts to develop them. This review presents recent advances and discusses the remaining hurdles in the design of 99mTc-based CNS receptor imaging agents.

A novel technetium-99m radioligand for the 5-HT(1A) receptor derived from desmethyl-WAY-100635 (DWAY).

This paper reports the synthesis, biological evaluation and in vitro autoradiography of a new technetium-99m radioligand with high affinity for the 5-HT(1A) receptor. The neutral complex combines an N(2)S(2) diamine dithiol (DADT) ligand as complexing moiety for oxotechnetium(V) and a 2-(1-piperazino)phenol via a 6-carbon alkyl chain, derived from desmethyl-WAY 100635 (DWAY). The complex displays an IC(50) value for the 5-HT(1A) receptor of 1.29 n M against the selective 5-HT(1A) agonist [(3)H]8-OH-DPAT, a moderate selectivity towards the alpha(1)-adrenergic receptor (IC(50) of 8.1 n M against [(3)H]prazosin) and a good selectivity for the D(2) receptor (IC(50) of 192 n M against [(3)H]spiperone) and the 5-HT(2A)receptor (IC(50) of 922 n M against [(3)H]ketanserin). Biodistribution studies in rats show an initial brain uptake of 0.56%+/-0.07% ID 2.5 min p.i. In vitro autoradiographic studies of the (99m)Tc complex in rat brains indicate a strong specific accumulation of the radioactivity in 5-HT(1A) receptor-rich brain regions.

Biodistribution and catabolism of (18)F-labeled neurotensin(8-13) analogs.

4-([(18)F]fluoro)benzoyl-neurotensin(8-13) ((18)FB-Arg(8)-Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 1) and two analogs stabilized in one and two positions ((18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 2, (18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)-Tle(12)-Leu(13)-OH, 3) were synthesized in a radiochemical yield of 25-36% and a specific activity of 5-15 GBq/mmol. The peptides were evaluated in vitro and in vivo for their potential to image tumors overexpressing neurotensin receptor 1 (NTR1) by positron emission tomography (PET). All analogs exhibited in vitro binding affinity in the low nanomolar range to NTR1-expressing human tumors, measured by quantitative receptor autoradiography, HT-29 and WiDr cells, and to sections of tumors derived from these cell lines in mice. The radiotracers were internalized in the cells in vitro, and the fluorinated peptides were able to mobilize intracellular Ca(2+) of WiDr cells. In in vivo studies in rats and in mice bearing HT-29 cell tumors, only a moderate uptake of the radioligands into the studied tumors was observed, presumed to be due to degradation in vivo and fast elimination by the kidneys. In comparison with the other analogs, the specific tumor uptake expressed as tumor-to-muscle relation was highest for the radioligand 3. The blood clearance of 3 was reduced by co-injection of peptidase inhibitors. The catabolic pathways of the radiofluorinated peptides were elucidated. The results suggest that the high binding affinity to NTR1 and the stabilization against proteolytic degradation are not yet sufficient for tumor imaging by PET.