Marco Fellner

PET/CT imaging of osteoblastic bone metastases with 68Ga-bisphosphonates: first human study

Bisphosphonates are well established ligands for Tc for planar and SPECT/CT imaging of osteoblastic metastases. A novelDOTA-based bisphosphonate, (4-{[bis-(phosphonomethyl)) carbamoyl]methyl}-7,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid (BPAMD) [1, 2], was labelled using the Ge/Ga generator-derived positron emitter Ga [3], resulting in the PET tracer [Ga]BPAMD. [Ga]BPAMD was injected i.v. into a patient with known extensive bone metastases of prostate cancer. [Ga]BPAMD [maximum intensity projection (MIP) 50 min post-injection (p.i.), 462 MBq] revealed intense accumulation in multiple osteoblastic lesions in the central skeleton, ribs and proximal extremities: a = coronal PET, b = sagittal PET/CT. For comparison, c shows F-fluoride PET (sagittal, MIP 90 min p.i., 270 MBq). Metastases were detected in the whole skeleton with a maximal standardized uptake value (SUVmax) of 77.1 and 62.1 in the 10th thoracic and L2 vertebra vs 39.1 and 39.2 for F-fluoride, respectively. The advantages of this new bone imaging PET tracer are the very high target to soft tissue ratios and fast clearance, its ease of use and the generator-based availability of Ga which becomes especially important in these days of Tc shortage. While F-fluoride is adsorbed on bone surface and is related to blood flow, the bisphosphonate [Ga]BPAMD is taken up also by osteoclasts reflecting the farnesyl diphosphate synthase enzyme dynamics in the HMG-CoA reductase pathway. Since this pathway is mainly responsible for the osteoclastic bone destruction, [Ga]BPAMD may be superior in osteoclastic lesions. Finally, [Ga]BPAMD seems to be an ideal PET/CT tracer to plan and monitor bisphosphonate therapy in several bone disorders like osteoporosis, osteitis deformans, bone metastases, multiple myeloma, osteogenesis imperfecta, etc. and also to monitor radionuclide therapy for palliation of bone pain.

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.

Comparison of different phosphorus-containing ligands complexing 68Ga for PET-imaging of bone metabolism

Abstract 99mTc-phosphonate structures are well established tracers for bone tumour imaging. Our objective was to investigate different 68Ga-labelled phosphonate ligands concerning labelling kinetics, binding to hydroxyapatite and bone imaging using μ-PET. Seven macrocyclic phosphorus-containing ligands and EDTMP were labelled in nanomolar scale with n.c.a. 68Ga in Na-HEPES buffer at pH∼4. Except for DOTP, all ligands were labelled with >92% yield. Binding of the 68Ga-ligand complexes on hydroxyapatite was analysed to evaluate the effect of the number of the phosphorus acid groups on adsorption parameters. Adsorption of 68Ga-EDTMP and 68Ga-DOTP was >83%. For the 68Ga-NOTA-phosphonates an increasing binding with increasing number of phosphonate groups was observed but was still lower than 68Ga-DOTP and 68Ga-EDTMP. μ-PET studies in vivo were performed with 68Ga-EDTMP and 68Ga-DOTP with Wistar rats. While 68Ga-EDTMP-PET showed uptake on bone structures, an excess amount of the ligand (>1.5 mg EDTMP/kg body weight) had to be used, otherwise the 68Ga3+ is released from the complex and forms gallium hydroxide or it is transchelated to 68Ga-transferrin. As a result, the main focus of further phosphonate structures has to be on complex formation in high radiochemical yields with macrocyclic ligands with phosphonate groups that are not required for complexing 68Ga.

In vivo comparison of DOTA based 68Ga-labelled bisphosphonates for bone imaging in non-tumour models

Bone metastases are a class of cancerous metastases that result from the invasion of a tumor into bone. The solid mass which forms inside the bone is often associated with a constant dull ache and severe spikes in pain, which greatly reduce the quality of life of the patient. Numerous (99m)Tc-labeled bisphosphonate functionalised complexes are well established tracers for bone metastases imaging. The objective of this research was to evaluate the pharmacokinetics and behaviour of three DOTA based bisphosphonate functionalised ligands (BPAMD, BPAPD and BPPED), using both (68)Ga μ-PET in vivo imaging and ex vivo biodistribution studies in healthy Wistar rats. The compounds were labelled with (68)Ga in high yields using an ammonium acetate buffer, and subsequently purified using a cation exchange resin. High bone uptake values were observed for all (68)Ga-labelled bisphosphonates at 60 minutes p.i. The highest uptake was observed for [(68)Ga]BPPED (2.6 ± 0.3% ID/g) which compares favourably with that of [(99m)Tc]MDP (2.7 ± 0.1 ID/g) and [(18)F]fluoride (2.4 ± 0.2% ID/g). The (68)Ga-labelled DOTA-bisphosphonates showed rapid clearance from the blood and renal system, as well as low binding to soft tissue, resulting in a high bone to blood ratio (9.9 at 60 minutes p.i. for [(68)Ga]BPPED, for example). Although further studies are required to assess their performance in tumor models, the results obtained suggest that these ligands could be useful both in imaging ((68)Ga) and therapeutic treatment ((177)Lu) of bone metastases.

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.

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.

PET Imaging of the Impact of Extracellular pH and MAP Kinases on the p-Glycoprotein (Pgp) Activity

The functional activity of p-glycoprotein (Pgp) can be increased in vitro by an extracellular acidosis via activation of MAP kinases (p38, ERK1/2). In order to study these effects in vivo a new (68)Ga-labeled PET tracer was developed which serves as a substrate of the Pgp and therefore indirectly mirrors the Pgp activity. For in vivo studies, experimental tumors were imaged under acidic conditions (inspiratory hypoxia, injection of lactic acid) and during inhibition of MAP kinases in a μ-PET system. In vitro, [(68)Ga]MFL6.MZ showed an accumulation within the cells of about 20% which was increased to 30% by Pgp inhibition. In solid tumors a marked tracer uptake was observed showing spatial heterogeneity. When the tumors were acidified, the PET tracer accumulation was reduced by 20-30%. Changing the inspiratory O(2)-fraction to 8% led dynamically to a decrease in pH and in parallel to a reduced tracer concentration. Inhibition of the p38 pathway reduced the Pgp transport rate. The new (68)Ga-labeled tracer is suitable for PET imaging of the tissue Pgp activity. In vivo imaging reveals that an acidosis activates the Pgp markedly, a mechanism in which the p38-MAPK pathway seems to play an important role.

Measurement of Protein Synthesis: In Vitro Comparison of 68 Ga-DOTA-Puromycin, [ 3 H]Tyrosine, and 2-Fluoro-[ 3 H]tyrosine

AIM Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. METHODS DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68)Ga-DOTA-Pur. RESULTS No significant differences in the behavior of [(3)H]tyrosine and 2-fluoro-[(3)H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45-55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, (68)Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. (68)Ga-DOTA-Pur μPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors. SUMMARY Whereas the metabolic pathway of (68)Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [(3)H]tyrosine nor 2-fluoro-[(3)H]tyrosine can be considered useful for determination of protein synthesis.