Preclinical Characterization of the 177Lu-Labeled Prostate Stem Cell Antigen (PSCA)-Specific Monoclonal Antibody 7F5.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The resulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images revealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Molecular Response to Combined Molecular- and External Radiotherapy in Head and Neck Squamous Cell Carcinoma (HNSCC).

Combination treatment of molecular targeted and external radiotherapy is a promising strategy and was shown to improve local tumor control in a HNSCC xenograft model. To enhance the therapeutic value of this approach, this study investigated the underlying molecular response. Subcutaneous HNSCC FaDuDD xenografts were treated with single or combination therapy (X-ray: 0, 2, 4 Gy; anti-EGFR antibody (Cetuximab) (un-)labeled with Yttrium-90 (90Y)). Tumors were excised 24 h post respective treatment. Residual DNA double strand breaks (DSB), mRNA expression of DNA damage response related genes, immunoblotting, tumor histology, and immunohistological staining were analyzed. An increase in number and complexity of residual DNA DSB was observed in FaDuDD tumors exposed to the combination treatment of external irradiation and 90Y-Cetuximab relative to controls. The increase was observed in a low oxygenated area, suggesting the expansion of DNA DSB damages. Upregulation of genes encoding p21cip1/waf1 (CDKN1A) and GADD45α (GADD45A) was determined in the combination treatment group, and immunoblotting as well as immunohistochemistry confirmed the upregulation of p21cip1/waf1. The increase in residual γH2AX foci leads to the blockage of cell cycle transition and subsequently to cell death, which could be observed in the upregulation of p21cip1/waf1 expression and an elevated number of cleaved caspase-3 positive cells. Overall, a complex interplay between DNA damage repair and programmed cell death accounts for the potential benefit of the combination therapy using 90Y-Cetuximab and external radiotherapy.

Radiotherapy enhances uptake and efficacy of 90Y-cetuximab: A preclinical trial.

BACKGROUND AND PURPOSE: Systemic molecular radiotherapy utilizes internal irradiation by radionuclide-labeled tumor-targeting agents with the potential to destroy (micro-)metastases. However, doses that are applicable in solid tumors do not reach the levels nessecary for tumor control. Thus, the combination of molecular and external radiotherapy is a promising treatment strategy, as enhanced tumor doses can be delivered with and without minor overlapping toxicities. Here, we combined a 90Y-labeled anti-EGFR antibody (Cetuximab) with clinically relevant fractionated radiotherapy in a preclinical trial using head and neck squamous cell carcinoma xenograft tumors. MATERIALS AND METHODS: To model 90Y-Cetuximab uptake for treatment schedule optimization, FaDu-bearing mice were injected with near-infrared-labeled-Cetuximab at different time points during radiotherapy with differing doses. Cetuximab uptake was longitudinally followed by in vivo-optical imaging. Tumor control probability experiments with fractionated radiotherapy (30 fx, 6 weeks, 8 dose groups/ arm) in combination with 90Y-Cetuximab were performed to test the curative potential. RESULTS: Imaging of near-infrared-labeled-Cetuximab uptake revealed that low to moderate external beam doses can enhance antibody uptake. Using the optimized schedule, combination of molecular and external radiotherapy using 90Y-Cetuximab at a dose that did not result in permanent tumor inactivation in previous experiments, led to substantially increased tumor control compared to radiotherapy alone. CONCLUSION: Our results indicate that combination of radiolabeled therapeutics with clinically relevant fractionated radiotherapy has a remarkable potential to improve curative treatment outcome. Application of some radiation dose prior to injection may improve drug uptake and enable patient stratification and treatment personalization via a corresponding PET-tracer during therapy.

Radiochemical and radiopharmacological characterization of a 64 Cu-labeled α-MSH analog conjugated with different chelators.

Radiolabeled α-melanocyte-stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a ß-Ala linker (ε-Ahx-ß-Ala-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 ) was conjugated to different chelators: either to NOTA (p-SCN-Bn-1,4,7-triazacyclononane-1,4,7-triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl-9-(((4-nitrophenoxy)carbonyl)oxy)-2,4-di(pyridin-2-yl)-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate), or to DMPTACN (p-SCN-Ph-bis(2-pyridyl-methyl)-1,4,7-triaza-cyclononane), labeled with 64 Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three 64 Cu-labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The Bmax values of [64 Cu]Cu-bispidine-NAP-NS1 ([64 Cu]Cu-2) and [64 Cu]Cu-DMPTACN-NAP-NS1 ([64 Cu]Cu-3) were higher than those of [64 Cu]Cu-NOTA-NAP-NS1 ([64 Cu]Cu-1), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [64 Cu]Cu-1 and [64 Cu]Cu-3 to be renal, while that of [64 Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 minutes. All three 64 Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.

Exploring pitfalls of 64Cu-labeled EGFR-targeting peptide GE11 as a potential PET tracer.

The epidermal growth factor receptor (EGFR) represents an important molecular target for both radiotracer-based diagnostic imaging and radionuclide therapy of various cancer entities. For the delivery of radionuclides to the tumor, peptides hold great potential as a transport vehicle. With respect to EGFR, the peptide YHWYGYTPQNVI (GE11) has been reported to bind the receptor with high specificity and affinity. In the present study, GE11 with ß-alanine (ß-Ala-GE11) was conjugated to the chelating agent p-SCN-Bn-NOTA and radiolabeled with 64Cu for the first radio pharmacological evaluation as a potential probe for positron emission tomography (PET)-based cancer imaging. For better water solubility, an ethylene glycol-based linker was introduced between the peptide's N terminus and the radionuclide chelator. The stability of the 64Cu-labeled peptide conjugate and its binding to EGFR-expressing tumor cells was investigated in vitro and in vivo, and then compared with the 64Cu-labeled EGFR-targeting antibody conjugate NOTA-cetuximab. The GE11 peptide conjugate [64Cu]Cu-NOTA-linker-ß-Ala-GE11 ([64Cu]Cu-1) was stable in a buffer solution for at least 24 h but only 50% of the original compound was detected after 24 h of incubation in human serum. Stability could be improved by amidation of the peptide's C terminus (ß-Ala-GE11-NH2 (2)). Binding assays with both conjugates, [64Cu]Cu-1 and [64Cu]Cu-2, using the EGFR-expressing tumor cell lines A431 and FaDu showed no specific binding. A pilot small animal PET investigation in FaDu tumor-bearing mice revealed only low tumor uptake (standard uptake value (SUV) < 0.2) for both conjugates. The best tumor-to-muscle ratio determined was 3.75 for [64Cu]Cu-1, at 1 h post injection. In conclusion, the GE11 conjugates in its present form are not suitable for further biological investigations, since they presumably form aggregates.

Synthesis, Characterization, and Initial Biological Evaluation of [99m Tc]Tc-Tricarbonyl-labeled DPA-α-MSH Peptide Derivatives for Potential Melanoma Imaging.

α-Melanocyte stimulating hormone (α-MSH) derivatives target the melanocortin-1 receptor (MC1R) specifically and selectively. In this study, the α-MSH-derived peptide NAP-NS1 (Nle-Asp-His-d-Phe-Arg-Trp-Gly-NH2 ) with and without linkers was conjugated with 5-(bis(pyridin-2-ylmethyl)amino)pentanoic acid (DPA-COOH) and labeled with [99m Tc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pot method, while obtaining similarly high yields. Negligible trans-chelation and high stability in physiological solutions was determined for the [99m Tc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylene glycol (EG)-based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [99m Tc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the 99m Tc-labeled peptides for in vivo imaging.

Synthesis and Pharmacological Evaluation of Identified and Putative Metabolites of the A1 Adenosine Receptor Antagonist 8-Cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX).

The A1 adenosine receptor (A1 AR) antagonist [18 F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([18 F]CPFPX), used in imaging human brain A1 ARs by positron emission tomography (PET), is stable in the brain, but rapidly undergoes transformation into one major (3-(3-fluoropropyl)-8-(3-oxocyclopenten-1-yl)-1-propylxanthine, M1) and several minor metabolites in blood. This report describes the synthesis of putative metabolites of CPFPX as standards for the identification of those metabolites. Analysis by (radio)HPLC revealed that extracts of human liver microsomes incubated with no-carrier-added (n.c.a.)[18 F]CPFPX contain the major metabolite, M1, as well as radioactive metabolites corresponding to derivatives functionalized at the cyclopentyl moiety, but no N1-despropyl species or metabolites resulting from functionalization of the N3-fluoropropyl chain. The putative metabolites were found to displace the binding of [3 H]CPFPX to the A1 AR in pig brain cortex at Ki values between 1.9 and 380 nm and the binding of [3 H]ZM241385 to the A2A AR in pig striatum at Ki values >180 nm. One metabolite, a derivative functionalized at the ω-position of the N1-propyl chain, showed high affinity (Ki 2 nm) to and very good selectivity (>9000) for the A1 AR.

Novel Tumor Pretargeting System Based on Complementary l-Configured Oligonucleotides.

Unnatural mirror image l-configured oligonucleotides (L-ONs) are a convenient substance class for the application as complementary in vivo recognition system between a tumor specific antibody and a smaller radiolabeled effector molecule in pretargeting approaches. The high hybridization velocity and defined melting conditions are excellent preconditions of the L-ON based methodology. Their high metabolic stability and negligible unspecific binding to endogenous targets are superior characteristics in comparison to their d-configured analogs. In this study, a radiopharmacological evaluation of a new l-ONs based pretargeting system using the epidermal growth factor receptor (EGFR) specific antibody cetuximab (C225) as target-seeking component is presented. An optimized PEGylated 17mer-L-DNA was conjugated with p-SCN-Bn-NOTA (NOTA') to permit radiolabeling with the radionuclide 64Cu. C225 was modified with the complementary 17mer-L-DNA (c-L-DNA) strand as well as with NOTA' for radiolabeling and use for positron emission tomography (PET). Two C225 conjugates were coupled with 1.5 and 5.0 c-L-DNA molecules, respectively. In vitro characterization was done with respect to hybridization studies, competition and saturation binding assays in EGFR expressing squamous cell carcinoma cell lines A431 and FaDu. The modified C225 derivatives exhibited high binding affinities in the low nanomolar range to the EGFR. PET and biodistribution experiments on FaDu tumor bearing mice with directly 64Cu-labeled NOTA'3-C225-(c-L-DNA)1.5 conjugate revealed that a pretargeting interval of 24 h might be a good compromise between tumor accumulation, internalization, blood background, and liver uptake of the antibody. Despite internalization of the antibody in vivo pretargeting experiments showed an adequate hybridization of 64Cu-radiolabeled NOTA'-L-DNA to the tumor located antibody and a good tumor-to-muscle ratio of about 11 resulting in a clearly visible image of the tumor after 24 h up to 72 h. Furthermore, low accumulation of radioactivity in organs responsible for metabolism and excretion was determined. The presented results indicate a high potential of complementary L-ONs for the pretargeting approach which can also be applied to therapeutic radionuclides such as 177Lu, 90Y, 186Re, or 188Re.

Melanoma targeting with [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analogs: Effects of cyclization on the radiopharmaceutical properties.

The purpose of this study was to evaluate the effect of cyclization on the biological profile of a [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analog. A lactam bridge-cyclized H-Cys-Ahx-ßAla3-c[Lys4-Glu-His-D-Phe-Arg-Trp-Glu10]-Arg11-Pro-Val-NH2 (NAP-NS2) and the corresponding linear H-Cys-Ahx-ßAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) peptide were synthetized, characterized by ESI-MS spectroscopy and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16/F10 melanoma cells. The consistent [99mTc(N)(PNP3)]-labeled compounds were readily obtained in high specific activity and their stability and biological properties were assessed. As an example, the chemical identity of [99mTc(N)(NAP-NS1)(PNP3)]+ was confirmed by carrier added experiments supported by radio/UV HPLC analysis combined with ESI(+)-MS. Compared with the linear peptide, cyclization negatively affected the biological properties of NAP-NS2 peptide by reducing its binding affinity for MC1R and by decreasing the overall excretion rate of the corresponding [99mTc(N)(PNP3)]-labeled peptide from the body as well as its in vivo stability. [99mTc(N)(NAP-NS1)(PNP3)]+ was evaluated for its potential as melanoma imaging probe in murine melanoma model. Data from in vitro and in vivo studies on B16/F10 melanoma model of [99mTc(N)(NAP-NS1)(PNP3)]+ clearly evidenced that the radiolabeled linear peptide keeps its biological properties up on the conjugation to the [99mTc(N)(PNP3)]-building block. The progressive increase of the tumor-to-nontarget ratios over the time indicates a quite stable interaction between the radio-complex and the MC1R.

Radiopharmacological characterization of 64Cu-labeled α-MSH analogs for potential use in imaging of malignant melanoma.

The melanocortin-1 receptor (MC1R) plays an important role in melanoma growth, angiogenesis and metastasis, and is overexpressed in melanoma cells. α-Melanocyte stimulating hormone (α-MSH) and derivatives are known to bind with high affinity at this receptor that provides the potential for selective targeting of melanoma. In this study, one linear α-MSH-derived peptide Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) without linker and with εAhx-ß-Ala linker, and a cyclic α-MSH derivative, [Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2 (NAP-NS2) with εAhx-ß-Ala linker were conjugated with p-SCN-Bn-NOTA and labeled with (64)Cu. Radiochemical and radiopharmacological investigations were performed with regard to transchelation, stability, lipophilicity and in vitro binding assays as well as biodistribution in healthy rats. No transchelation reactions, but high metabolic stability and water solubility were demonstrated. The linear derivatives showed higher affinity than the cyclic one. [(64)Cu]Cu-NOTA-εAhx-ß-Ala-NAP-NS1 ([(64)Cu]Cu-2) displayed rapid cellular association and dissociation in murine B16F10 cell homogenate. All [(64)Cu]Cu-labeled conjugates exhibited affinities in the low nanomolar range in B16F10. [(64)Cu]Cu-2 showed also high affinity in human MeWo and TXM13 cell homogenate. In vivo studies suggested that [(64)Cu]Cu-2 was stable, with about 85 % of intact peptide in rat plasma at 2 h p.i. Biodistribution confirmed the renal pathway as the major elimination route. The uptake of [(64)Cu]Cu-2 in the kidney was 5.9 % ID/g at 5 min p.i. and decreased to 2.0 % ID/g at 60 min p.i. Due to the prospective radiochemical and radiopharmacological properties of the linear α-MSH derivative [(64)Cu]Cu-2, this conjugate is a promising candidate for tracer development in human melanoma imaging.

Synthesis and first evaluation of [¹8F]fluorocyano- and [¹8F]fluoronitro-quinoxalinedione as putative AMPA receptor antagonists.

Derivatives of quinoxalinedione (QX) were chosen as chemical lead for the development of new radioligands of the AMPA receptor, since there are several examples of QX-derivatives with high affinity. The radiosyntheses of the new compounds 6-[(18)F]fluoro-7-nitro-QX([(18)F]FNQX) and 7-[(18)F]fluoro-6-cyano-QX ([(18)F]FCQX) with radiochemical yields of 8±2 and 3± 2%, respectively, as well as the evaluation of their binding properties to the AMPA-receptor were performed. A comparison of the Ki-values of the new QX-derivatives FCQX and FNQX with mono-substituted cyanoand nitro-QX shows negligibly small differences of affinity (within the range of 1.4 to 5 µM), but exhibits a tenfold lower affinity than derivatives with two electron withdrawing groups like the 7-cyano-6-nitro-compound CNQX and the 6,7- dinitro compound DNQX. Thus, with respect to the low affinity and a high non-specific binding with in vitro and ex vivo autoradiographic studies, the new compounds do not lend themselves for in vivo imaging.

Radiolabeled Cetuximab Conjugates for EGFR Targeted Cancer Diagnostics and Therapy.

The epidermal growth factor receptor (EGFR) has evolved over years into a main molecular target for the treatment of different cancer entities. In this regard, the anti-EGFR antibody cetuximab has been approved alone or in combination with: (a) chemotherapy for treatment of colorectal and head and neck squamous cell carcinoma and (b) with external radiotherapy for treatment of head and neck squamous cell carcinoma. The conjugation of radionuclides to cetuximab in combination with the specific targeting properties of this antibody might increase its therapeutic efficiency. This review article gives an overview of the preclinical studies that have been performed with radiolabeled cetuximab for imaging and/or treatment of different tumor models. A particularly promising approach seems to be the treatment with therapeutic radionuclide-labeled cetuximab in combination with external radiotherapy. Present data support an important impact of the tumor micromilieu on treatment response that needs to be further validated in patients. Another important challenge is the reduction of nonspecific uptake of the radioactive substance in metabolic organs like liver and radiosensitive organs like bone marrow and kidneys. Overall, the integration of diagnosis, treatment and monitoring as a theranostic approach appears to be a promising strategy for improvement of individualized cancer treatment.

Evaluation of 18F-labeled benzodioxine piperazine-based dopamine D4 receptor ligands: lipophilicity as a determinate of nonspecific binding.

Derivatization of the putative neuroleptic 1-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-4-(4-fluorobenzyl)piperazine (3a) led to a series of new dopamine receptor D4 ligands displaying high affinity (Ki=1.1-15 nM) and D2/D4 subtype selectivities of about 800-6700. These ligands were labeled with the short-lived positron emitter fluorine-18 and analyzed for their potential application for imaging studies by positron emission tomography (PET). In vitro autoradiography was used to determine their nonspecific binding behavior as a result of their structural and thus physicochemical properties. The biodistribution, in vivo stability, and brain uptake of the most promising D4 radioligand candidate were determined. This proved to be 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-((6-fluoropyridin-3-yl)methyl)piperazine ([18F]3d), which revealed an excellent binding pattern with a high selectivity and limited nonspecific binding in vitro. This analogue also exhibited a high stability and an extremely high brain uptake in vivo with specific binding in hippocampus, cortex, colliculus, and cerebellum as determined by ex vivo autoradiography. Thus, [18F]3d appears as a suitable D4 radioligand for in vivo imaging, encouraging continued evaluation by PET studies.

Synthesis, radiofluorination and first evaluation of [18F]fluorophenylsulfonyl- and [18F]fluorophenylsulfinyl-piperidines as serotonin 5-HT2A receptor antagonists for PET.

In psychiatric disorders, 5-HT(2A) receptors play an important role. In order to study these receptors in vivo by positron emission tomography (PET), there is an increasing interest for subtype selective and high affinity radioligands. Up to now, no optimal radiotracer is available. Thus, 1-(2,4-difluorophenethyl)-4-(4-fluorophenylsulfonyl)piperidine (9), possessing high affinity and sufficient subtype selectivity for 5-HT(2A) receptors, and 1-(2,4-difluorophenethyl)-4-(4-fluorophenylsulfinyl)piperidine (15) have been (18)F-labelled by a nucleophilic one-step reaction. Both radiotracers could be prepared and isolated within 45 min, [(18)F]9 in a radiochemical yield (RCY) of 34.5+/-8% and [(18)F]15 of 9.5+/-2.5%. The K(i) values of 9 and 15 at 5-HT(2A) receptors towards [(3)H]ketanserin were determined to be 1.9+/-0.6 and 198+/-8 nM, respectively. Autoradiography with [(18)F]9 and [(18)F]15 on rat brain sections showed a very high nonspecific binding of >80% for [(18)F]9 and 30% to 40% nonspecific binding for [(18)F]15; however, it is still too high in order to compensate for its lower affinity. Even though the affinity of 9 is more promising compared with 15, the high nonspecific binding of both radiofluorinated tracers in rat brain does not recommend those as an in vivo PET imaging agent for serotonin 5-HT(2A) receptors in humans.

Autoradiographic comparison of in vitro binding characteristics of various tritiated adenosine A2A receptor ligands in rat, mouse and pig brain and first ex vivo results.

The adenosine A(2A) receptor in the basal ganglia is involved in the control of movement and plays a role in movement disorders such as Parkinsonism. Developing ligands to evaluate that receptor by noninvasive methods such as positron emission tomography has a high priority. In vitro radioligand binding guides the selection of ligands for in vivo application. This study measured the binding of the adenosine A(2A) receptor antagonist [(3)H]MSX-2 (3-(3-hydroxypropyl)-8-m-methoxystyryl)-7-methyl-1-propargylxanthine) to rat, mouse and pig brain by autoradiography. Other studies measured binding to membranes from PC12 pheochromocytoma cells. Those binding parameters were compared to those of the adenosine A(2A) receptor antagonist [(3)H]ZM241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino)ethyl)phenol), the adenosine A(2A) receptor agonist [(3)H]CGS21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine) and the unselective adenosine receptor agonist [(3)H]NECA (5'N-ethylcarboxamido)adenosine). The potency order (K(d)) in the three species was [(3)H]ZM241385<[(3)H]MSX-2<[(3)H]NECA<[(3)H]CGS21680. The density of [(3)H]MSX-2 binding sites was greater in the striatum than in the cortex. Preliminary ex vivo experiments showed that by 10min after iv injection, [(3)H]MSX-2 and [(3)H]CGS21680 crossed the blood-brain barrier to the extent of almost 1% ID/g brain tissue, but [(3)H]NECA and [(3)H]ZM241385 to only 0.2% ID/g. The prior administration of unlabeled ZM241385 significantly lowered brain uptake of [(3)H]MSX-2. In conclusion, [(3)H]MSX-2 has a high affinity and sufficient selectivity for the adenosine A(2A) receptor. It penetrates the blood-brain barrier. Sensitivity to photoisomerization is a limitation. Further investigations assess its suitability as a ligand for imaging the brain adenosine A(2A) receptor.

New 2alpha-tropane amides as potential PET ligands for the dopamine transporter.

Positron emission tomography (PET) imaging of dopamine transporter (DAT) density in the brain is a potentially valuable tool for studying the etiopathology of degenerative brain disorders. The present study evaluated five new potential competitive inhibitors of DAT as ligands for PET. The evaluation of the new compounds measured their ability to compete with the binding of the reference ligand 2beta-carbomethoxy-3beta-(4-[(131)I]iodophenyl)tropane [(131)I]beta-CIT to striatal and cortical membranes from rat and pig brain. Four of the new compounds structurally related to cocaine were synthesized in their 2alpha,3beta configuration; the most potent one, 3beta-(4-iodo-phenyl)-8-methyl-8-aza-bicyclo[3.2.1]octane-2alpha-carboxylic acid (2-fluoro-ethyl)-amide, was synthesized also in the 2beta,3beta configuration. For comparative studies in rat brain and new evaluation in pig brain homogenate, the established compounds beta-CIT, FP-CIT, PE2I and FETT were also synthesized and evaluated. Contrary to expectation, the 2alpha,3beta and 2beta,3beta isomers of 3-(4-iodo-phenyl)-8-methyl-8-aza-bicyclo[3.2.1]octane-2-carboxylic acid (2-fluoro-ethyl)-amide showed the same affinity constant for rat striatum (K(i)=200 nM+/-34), but in pig striatum and rat and pig cortex the 2alpha,3beta form even had a higher affinity than the 2beta,3beta form.

Synthesis and evaluation of 7-amino-2-(2(3)-furyl)-5-phenylethylamino-oxazolo[5,4-d]pyrimidines as potential A2A adenosine receptor antagonists for positron emission tomography (PET).

The brain A2A adenosine receptor (A2AAR) participates with the dopamine D2 receptor in the control of movement and also might influence behavior. Because PET is an important tool for studying the roles of receptors in disease, a ligand for imaging the brain A2AAR is desirable. This report describes the synthesis and A2AAR antagonist activities of a panel of phenyl-substituted 7-amino-2-(2-furyl)-5-phenylethylamino-oxazolo[5,4-d]pyrimidines, 11aa-af, and their 3-furyl congeners, 11ba-bd. In competitive binding studies all compounds displaced [3H]CGS21680 from the A2AAR with Ki values of 14-33 nM with selectivity for the A2AAR over the A1AR of 5- to 94-fold. Autoradiography of brain sections showed a high level of unspecific binding that obscured specific binding. Thus, these compounds are not promising PET ligands.

Derivatives of 4,6-diamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-2H-1-one: potential antagonist ligands for imaging the A2A adenosine receptor by positron emission tomography (PET).

The importance of the brain A2A adenosine receptor (A(2A)AR) in movement disorders urges the development of radiolabeled ligands for imaging those receptors by positron emission tomography (PET). This study evaluated one class of A(2A)AR antagonists, derivatives of 4-amino-6-benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-2H-1-one, 10a, as agents for imaging brain A(2A)ARs by PET.. Modifications of a literature synthesis of 10a efficiently generated analogs 10b-s for pharmacological evaluation. Radioligand binding experiments showed affinities for the rat brain A(2A)AR in the low nanomolar range but similar affinities for the A1AR and substantial unspecific binding. Autoradiography employing [3H]10a, showing that high unspecific binding obscured specific binding to both the A1AR and A(2A)AR. Thus, compounds 10b-s are unsuitable as ligands for imaging brain A(2A)ARs by PET.

Synthesis and evaluation of no-carrier-added 8-cyclopentyl-3-(3-[(18)F]fluoropropyl)-1-propylxanthine ([(18)F]CPFPX): a potent and selective A(1)-adenosine receptor antagonist for in vivo imaging.

This report describes the precursor synthesis and the no-carrier-added (nca) radiosynthesis of the new A(1) adenosine receptor (A(1)AR) antagonist [(18)F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX), 3, with fluorine-18 (half-life = 109.6 min). Nucleophilic radiofluorination of the precursor tosylate 8-cyclopentyl-3-(3-tosyloxypropyl)-7-pivaloyloxymethyl-1-propylxanthine, 2, with nca [(18)F]KF under aminopolyether-mediated conditions (Kryptofix 2.2.2/K(2)CO(3)) followed by deprotection was straightforward and, after formulation, gave the radioligand ready for injection with a radiochemical yield of 45 +/- 7%, a radiochemical purity of >98% and a specific radioactivity of >270 GBq/micromol (>7.2 Ci/micromol). Preparation time averaged 55 min. The synthesis proved reliable for high batch yields ( approximately 7.5 GBq) in routine production (n = 120 runs). The radiotracer was pharmacologically evaluated in vitro and in vivo and its pharmacokinetics in rodents determined in detail. After iv injection a high accumulation of radioactivity occurred in several regions of mouse brain including thalamus, striatum, cortex, and cerebellum. Antagonism by the specific A(1)AR antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N(6)-cyclopentyl-9-methyladenine (N-0840), but not with the A(2)AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), indicated specific and reversible binding of the radioligand to A(1)AR in cortical and subcortical regions of interest. In mouse blood at least two polar metabolites formed rapidly (50% at 5 min after tracer application). However, chromatographic analyses of brain homogenate extracts taken 60 min pi showed that >98% of radioactivity was unchanged radioligand. Chromatographic isolation and reinjection of peripherally formed radioactive metabolites revealed no accumulation of radioactivity in mouse brain, probably due to the polarity of the metabolites. These preliminary results suggest that nca [(18)F]CPFPX is a useful radioligand for the noninvasive imaging of the brain A(1)AR.