Nicole Bausbacher

68Ga-BPAMD: PET-imaging of bone metastases with a generator based positron emitter

PURPOSE Bone metastases are a serious aggravation for patients suffering from cancer. Therefore, early recognition of bone metastases is of great interest for further treatment of patients. Bisphosphonates are widely used for scintigraphy of bone lesions with (99m)Tc. Using the (68)Ge/(68)Ga generator together with a macroyclic bisphosphonate a comparable PET-tracer comes into focus. PROCEDURES The bisphosphonate DOTA-conjugated ligand BPAMD was labelled with (68)Ga. [(68)Ga]BPAMD was evaluated in vitro concerning binding to hydroxyapatite and stability. The tracers in vivo accumulation was determined on healthy rats and bone metastases bearing animals by μ-PET. RESULTS BPAMD was labelled efficiently with (68)Ga after 10 min at 100°C. [(68)Ga]BPAMD showed high in vitro stability within 3h and high binding to hydroxyapatite. Consequently, μ-PET experiments revealed high accumulation of [(68)Ga]BPAMD in regions of pronounced remodelling activity like bone metastases. CONCLUSIONS (68)Ga BPAMD reveals great potential for diagnosis of bone metastases via PET/CT. The straight forward (68)Ga-labelling could be transferred to a kit-preparation of a cyclotron-independent PET tracer instantaneously available in many clinical sites using the (68)Ge/(68)Ga generator.

Activation of P-glycoprotein (Pgp)-mediated drug efflux by extracellular acidosis: in vivo imaging with 68Ga-labelled PET tracer.

PurposeIn vitro it has been shown that the functional activity of P-glycoprotein (Pgp), an important drug transporter responsible for multidrug resistance, can be strongly increased by extracellular acidosis. Here mitogen-activated protein kinases (MAPK) (p38, ERK1/2) seem to play an important role for signal transduction. However, it is unclear whether these effects are also relevant in vivo.MethodsWith the newly developed PET tracer Schiff base-based 68Ga-MFL6.MZ the functional Pgp activity was visualized under acidic conditions and during inhibition of MAPKs non-invasively by means of microPET in rat tumours. Tumours were acidified either by inspiratory hypoxia (8% O2) or by injection of lactic acid. Inhibitors of the MAPK were injected intratumourally.ResultsWith increasing tumour volume the tumour pH changed from 7.0 to 6.7 and simultaneously the Pgp activity increased almost linearly. When the tumour was acidified by direct lactic acid injection the PET tracer uptake was reduced by 20% indicating a higher transport rate out of the cells. Changing the inspiratory O2 fraction to 8% dynamically led to a reduction of extracellular pH and in parallel to a decrease of tracer concentration. While inhibition of the p38 pathway reduced the Pgp transport rate, inhibition of ERK1/2 had practically no impact.ConclusionAn acidic extracellular environment significantly stimulates the Pgp activity. The p38 MAPK pathway plays an important role for Pgp regulation in vivo, whereas ERK1/2 is of minor importance. From these results new strategies for overcoming multidrug resistance (e.g. reducing tumour acidosis, inhibition of p38) may be developed.

18F-Labeling and evaluation of novel MDL 100907 derivatives as potential 5-HT2A antagonists for molecular imaging

INTRODUCTION The serotonergic system, especially the 5-HT2A receptor, is involved in various diseases and conditions. It is a very interesting target for medicinal applications. METHODS Two novel 5-HT2A tracers, namely, [(18)F]DD-1 and the enantiomeric pure (R)-[(18)F]MH.MZ, were radiolabeled by (18)F-fluoroalkylation of the corresponding desmethyl analogue. In vitro binding autoradiography on rat brain slices was performed to test the affinity and selectivity of these tracers. Moreover, first microPET experiments of (R)-[(18)F]MH.MZ were carried out in Sprague-Dawley rats. RESULTS [(18)F]DD-1 (K(i)=3.23 nM) and (R)-[(18)F]MH.MZ (K(i)=0.72 nM) were (18)F-fluoroalkylated by the secondary synthon [(18)F]FETos in a radiochemical yield (RCY) of >70%. The final formulation for both tracers took no longer than 100 min with an overall RCY of approximately 40%. It provided [(18)F]tracers with a purity >96% and a typical specific activity of 25-35 GBq/mumol. Autoradiographic images of (R)-[(18)F]MH.MZ (5) and [(18)F]DD-1 (4) showed excellent visualization and selectivity of the 5-HT2A receptor for (R)-[(18)F]MH.MZ and less specific binding for [(18)F]DD-1. The binding potential (BP) of (R)-[(18)F]MH.MZ was determined to be 2.6 in the frontal cortex and 2.2 in the cortex (n=4), whereas the cortex-to-cerebellum ratio was determined to be 3.2 at steady state (n=4). Cortex-to-cerebellum ratios of (R)-[(18)F]MH.MZ were almost twice as much as compared with the racemic [(18)F]MH.MZ. Thereby, equal levels of specific activities were used. High uptake could be demonstrated in cortex regions. CONCLUSION Labeling of both novel tracers was carried out in high RCY. Autoradiography revealed (R)-[(18)F]MH.MZ as a very selective and affine 5-HT2A tracer (K(i)=0.72 nM), whereas [(18)F]DD-1 showed no reasonable distribution pattern on autoradiographic sections. Moreover, results from microPET scans of (R)-[(18)F]MH.MZ hint on improved molecular imaging characteristics compared with those of [(18)F]MH.MZ. Therefore, (R)-[(18)F]MH.MZ appears to be a highly potent and selective serotonergic PET ligand in small animals.

Labelling of commercially available human serum albumin kits with 68Ga as surrogates for 99mTc-MAA microspheres

An alternative method of labelling different commercially available human serum albumin kits with generator produced (68)Ga as possible surrogate markers for the investigation of the biodistribution, and to exclude a possible lung shunt in patients with solid primary tumours and liver metastases by positron emission-tomography (PET) and effective substitute for (99m)Tc HSA as a PET perfusion tracer is described. The radioactive labelling with (68)Ga was based on the elution of a commercial (68)Ge/(68)Ga generator system, which uses a modified tin dioxide column to absorb (68)Ga. We obtained (68)Ga-MAA and (68)Ga-HSA in radiochemical yields of 79 ± 5% with less than 5% free (68)Ga. The whole procedure takes a total of less than 30 min from elution to the final product.

Assessing p-Glycoprotein (Pgp) Activity In Vivo Utilizing 68Ga–Schiff Base Complexes

PurposeThe p-glycoprotein (Pgp) is the most prominent member of active drug transporters leading to a multidrug-resistant phenotype. For identification of tumors functionally overexpressing Pgp in vivo, non-invasive imaging techniques are needed.ProceduresSix Schiff base compounds were synthesized and labeled with 68Ge/68Ga generator-derived 68Ga. The compounds were studied in vitro in Pgp-positive tumor cells. The property of being a Pgp substrate was tested by comparison of the tracers uptake in R-3327 Dunning prostate carcinoma AT1 cells in presence and absence of the Pgp-inhibitor verapamil. In vivo investigations were performed with tumor-bearing rats imaged with micro-positron emission tomography.ResultsAll ligands were labeled with 68Ga in yields of >92% beside one (~55%). The tracers showed different accumulation within the cells in vitro (4–60%). In blocking experiments, the ratio (blocked to unblocked) varied from 1.8 to 1.0. For in vivo experiments, 68Ga–ENBDMPI and 68Ga–MFL6.MZ were selected. The tumors showed specific uptake of the tracer. Direct intratumoral injection of verapamil increased the tracer concentration by ~25% reflecting the functional Pgp activity.ConclusionsTwo 68Ga-labeled ligands appear to be valuable for imaging non-invasively the intratumoral Pgp activity. On a long term, patients with multidrug-resistant tumors pre-therapeutically may be identified prior to treatment.

Predicting the in vivo release from a liposomal formulation by IVIVC and non-invasive positron emission tomography imaging.

This study aimed to predict the in vivo performance from the in vitro release of a low-molecular weight model compound, [(18)F]-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG), from liposomes and by means of positron emission tomography (PET). Liposomes composed of hydrogenated phosphatidylcholine (HPC) were prepared by a freeze-thaw method. Particle size distribution was measured by dynamic light scattering (DLS). In vitro release was examined with a dispersion method detecting the radioactivity of [(18)F]FDG. In vivo release of [(18)F]FDG, following i.p. injection of the liposomes in rats, was determined by using a Micro-PET scanner. Convolution was performed to predict the in vivo profiles from the in vitro data and to establish an in vitro-in vivo correlation (IVIVC). The in vivo predictions slightly underestimated the experimentally determined values. The magnitude of the prediction errors (13% and 19%) displayed a satisfactory IVIV relationship leaving yet room for further improvement. This study demonstrated for the first time the use of PET in attaining an IVIVC for a parenterally administered modified release dosage form. It is therefore possible to predict target tissue concentrations, e.g., in the brain, from in vitro release experiments. IVIVC using non-invasive PET imaging could thus be a valuable tool in drug formulation development, resulting in reduced animal testing.

Selective binding to monoamine oxidase A: In vitro and in vivo evaluation of 18F-labeled β-carboline derivatives

In this study we synthesized four different (18)F-labeling precursors for the visualization of the monoamino oxidase A using harmol derivatives. Whereas two are for prosthetic group labeling using [(18)F]fluoro-d2-methyl tosylate and 2-[(18)F]fluoroethyl-tosylate, the other three precursors are for direct nucleophilic (18)F-labeling. Additionally the corresponding reference compounds were synthesized. The syntheses of [(18)F]fluoro-d2-methyl-harmol and 2-[(18)F]fluoroethyl-harmol were carried out using harmol as starting material. For direct nucleophilic (18)F-labeling of the tracers carrying oligoethyled spacers (PEG), a toluenesulfonyl leaving group was employed. The radiolabeling, purification and formulation for each tracer was optimized and evaluated in vitro and in vivo. Stability tests in human serum showed that all tracers were stable over the observation period of 60 min. μPET studies using of the synthesized tracers revealed that the tracers carrying PEG spacers showed no sufficient brain uptake. Consequently, the (18)F-fuoro alkylated tracers [(18)F]fluoro-d2-methyl-harmol and 2-[(18)F]fluoroethyl-harmol were further evaluated showing SUVs in the brain of 1.0±0.2 g/mL and 3.4±0.5 g/mL after 45 min, respectively. In blockade studies the selectivity and specificity of both tracers were demonstrated. However, for [(18)F]fluoro-d2-methyl-harmol a rapid washout from the brain was also observed. In vitro binding assays revealed that 2-[(18)F]fluoroethyl-harmol (IC50=0.54±0.06 nM) has a higher affinity than the (18)F-fluoro-d2-methylated ligand (IC50=12.2±0.6 nM), making 2-[(18)F]fluoroethyl-harmol superior to the other evaluated compounds and a promising tracer for PET imaging of the MAO A.

18F-Radiolabeling, preliminary evaluation of folate-pHPMA conjugates via PET.

The synthesis of a 10.5 kDa and a 52.5 kDa polymer, based on pHPMA functionalized with tyramine for (18) F-labeling and a folate derivative as targeting moiety, is reported. FCS studies are conducted using Oregon Green-labeled conjugates. No aggregation is observed for the 10.5 kDa conjugate, but strong aggregation for the 52.5 kDa conjugate. In vivo studies are conducted using Walker-256 mammary carcinoma model to determine body distribution as function of size and especially targeting unit. These in vivo studies show a higher short time (2 h) accumulation for both conjugates in the tumor than for untargeted pHPMA, confirmed by blockade studies. The 10.5 kDa polymer accumulates with 0.46% ID g(-1) and the 52.5 kDa polymer with 0.28% ID g(-1) in the tumor after 2 h, demonstrating the potential of the folate-targeting concept.

Fate of linear and branched polyether-lipids in vivo in comparison to their liposomal formulations by 18F-radiolabeling and positron emission tomography.

In this study, linear poly(ethylene glycol) (PEG) and novel linear-hyperbranched, amphiphilic polyglycerol (hbPG) polymers with cholesterol (Ch) as a lipid anchor moiety were radiolabeled with fluorine-18 via copper-catalyzed click chemistry. In vivo investigations via positron emission tomography (PET) and ex vivo biodistribution in mice were conducted. A systematic comparison to the liposomal formulations with and without the polymers with respect to their initial pharmacokinetic properties during the first hour was carried out, revealing remarkable differences. Additionally, cholesterol was directly labeled with fluorine-18 and examined likewise. Both polymers, Ch-PEG27-CH2-triazole-TEG-(18)F and Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F (TEG: triethylene glycol), showed rapid renal excretion, whereas the (18)F-cholesten displayed retention in lung, liver, and spleen. Liposomes containing Ch-PEG27-CH2-triazole-TEG-(18)F revealed a hydrodynamic radius of 46 nm, liposomal Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F showed a radius of 84 nm and conventional liposomes with (18)F-cholesten 204 nm, respectively. The results revealed fast uptake of the conventional liposomes by liver, spleen, and lung. Most importantly, the novel hbPG-polymer stabilized liposomes showed similar behavior to the PEG-shielded vesicles. Thus, an advantage of multifunctionality is achieved with retained pharmacokinetic properties. The approach expands the scope of polymer tracking in vivo and liposome tracing in mice via PET.

An allogenic site-specific rat model of bone metastases for nuclear medicine and experimental oncology.

Bone metastases are a major problem in several tumor entities affecting the therapeutic decision and the patients prognosis. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are promising techniques for identifying bone tumors using gamma- or positron-emitting labeled radiotracers, but the same tracers if labeled with beta-emitters may also be used to apply therapeutic radionuclides for localized irradiation. For the tracer development specifically accumulating in osseous lesions, animal models of bone metastasis are needed. A technique was developed for tumor cell injection into the circulation of the hind limb of rats. For tumor implantation, the arteria epigastrica caudalis superficialis (a branch of the femoral artery) was cannulated, and 2×10(5) cells were injected. By using the allogenic Walker 256 mammary carcinoma cell line, isolated bone metastases were induced. For visualizing of the tumor growth, PET with 18F-fluoride was performed weekly on a μ-PET system. After 2-3 weeks, tumor invasion was confirmed by histology. Three weeks after tumor cell inoculation, PET images showed signs of bone metastases in 9 out of 11 animals. The tumors were located either in the proximal tibia/fibula or in the distal femur. At this time, the animals showed no restrictions in mobility. The tumors grew constantly over time. The final histological analysis showed tumors growing invasively into the bone matrix. With this model, new SPECT or PET tracers can be evaluated for their potency of accumulating in bone metastases in vivo and to determine which are therefore suitable for diagnosis and/or therapy.