Translocator Protein Targeting Research Articles

Translocator protein PET imaging in temporal lobe epilepsy: A reliable test-retest study using asymmetry index.

Translocator protein (TSPO) targeting positron emission tomography (PET) imaging radioligands have potential utility in epilepsy to assess the efficacy of novel therapeutics for targeting neuroinflammation. However, previous studies in healthy volunteers have indicated limited test-retest reliability of TSPO ligands. Here, we examine test-retest measures using TSPO PET imaging in subjects with epilepsy and healthy controls, to explore whether this biomarker can be used as an endpoint in clinical trials for epilepsy. Five subjects with epilepsy and confirmed mesial temporal lobe sclerosis (mean age 36 years, 3 men) were scanned twice-on average 8 weeks apart-using a second generation TSPO targeting radioligand, [11C]PBR28. We evaluated the test-retest reliability of the volume of distribution and derived hemispheric asymmetry index of [11C]PBR28 binding in these subjects and compared the results with 8 (mean age 45, 6 men) previously studied healthy volunteers. The mean (± SD) of the volume of distribution (VT), of all subjects, in patients living with epilepsy for both test and retest scans on all regions of interest (ROI) is 4.49 ± 1.54 vs. 5.89 ± 1.23 in healthy volunteers. The bias between test and retest in an asymmetry index as a percentage was small (-1.5%), and reliability is demonstrated here with Bland-Altman Plots (test mean 1.062, retest mean 2.56). In subjects with epilepsy, VT of [11C]PBR28 is higher in the (ipsilateral) hippocampal region where sclerosis is present than in the contralateral region. When using TSPO PET in patients with epilepsy with hippocampal sclerosis (HS), an inter-hemispheric asymmetry index in the hippocampus is a measure with good test-retest reliability. We provide estimates of test-retest variability that may be useful for estimating power where group change in VT represents the clinical outcome.

Preclinical Evaluation of a Novel 99mTc-Labeled CB86 for Rheumatoid Arthritis Imaging.

Early diagnosis and therapy are crucial to control disease progression optimally and achieve a good prognosis in rheumatoid arthritis (RA). Previous study showed that a technetium-99m (99mTc)-labeled TSPO ligand (99mTc-CB256 [2-(8-(2-(bis(pyridin-2-yl)methyl)amino)acetamido)-2-(4-chlorophenyl)H-imidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide] composed of a translocator protein (TSPO) ligand CB86 [[2-(4-chlorophenyl)-8-amino-imidazo[1,2-a]-pyridin-3-yl]-N,N-di-n-propylacetamide] and di-(2-picolyl)amine, a bifunctional chelate agent, was used to image a TSPO-rich cancer cell in vitro; however, few 99mTc-CB256 in vivo evaluation has been reported so far probably due to the cytotoxicity of CB256 (ca. 75 times more than analogous CB86). Herein, we describe a novel TSPO targeting radiopharmaceutical consisting of CB86 and diethylenetriaminepentaacetic acid (DTPA), a conventional bifunctional chelating ligand in clinical trials used to prepare 99mTc-labeled CB86, and its evaluation as a 99mTc-single-photon emission computed tomography (SPECT) probe. The radiosynthesis and characterization of 99mTc-DPTA-CB86 including hydrophilicity and stability tests were determined. Additionally, the binding affinity and specificity of 99mTc-DTPA-CB86 to TSPO were evaluated using RAW264.7 macrophage cells. Biodistribution and 99mTc-SPECT studies were conducted on rheumatoid arthritis (RA) rat models after the injection of 99mTc-DTPA-CB86 with or without co-injection of unlabeled DTPA-CB86. The radiosynthesis of 99mTc-DTPA-CB86 was completed successfully with the labeling yields and radiochemical purity of 95.86 ± 2.45 and 97.45 ± 0.69%, respectively. The probe displayed good stability in vitro and binding specificity to RAW264.7 macrophage cells. In the biodistribution studies, 99mTc-DTPA-CB86 exhibited rapid inflammatory ankle accumulation. At 180 min after administration, 99mTc-DTPA-CB86 uptakes of the left inflammatory ankle were 2.35 ± 0.10 percentage of the injected radioactivity per gram of tissue (% ID/g), significantly higher than those of the normal tissues. 99mTc-SPECT imaging studies revealed that 99mTc-DTPA-CB86 could clearly identify the left inflammatory ankle with good contrast at 30–180 min after injection. Therefore, 99mTc-DTPA-CB86 may be a promising probe for arthritis 99mTc-SPECT imaging.

Design, Synthesis, and In Silico Evaluation of Methyl 2-(2-(5-Bromo/chloro-2-oxobenzoxazol-3(2H)-yl)-acetamido)-3-phenylpropanoate for TSPO Targeting

The high expression of the translocator protein (TSPO) makes it an ideal target for imaging and therapy. The present study is aimed at optimizing acetamidobenzoxazolone-based TSPO ligands by structural modification to overcome the limitations of TSPO ligands of the first two generations such as nonspecificity and polymorphism. Three different TSPO proteins, 2MGY, 4RYQ, and 4UC1, in the native and mutated form were chosen. Acetamidobenzoxazolones modified with phenylalanine methyl ester (methyl 2-(2-(5-bromo/chloro-2-oxobenzooxazol-3(2H)-yl)acetamido)-3-phenylpropanoate, ABPO-Br, ABPO-Cl) through better or comparable docking scores than the known TSPO ligands such as MBMP, FEBMP, FPBMP, and PK11195 are identified as potential TSPO ligands. ABPO-Cl and ABPO-Br were synthesized using phenylalanine methyl ester as a moiety for incorporation of the desired pharmacophoric feature. All the intermediates and final compounds were purified using column chromatography and analytical HPLC (purity > 97%). The purified compounds were characterized by 1H, 13C NMR and mass spectroscopy. Drug likeliness and comparative bioactivity analysis were performed using QikProp through prediction of various properties. Both analogs follow all the five Lipinski rules and three Jogersen’s rules predicting their drug likeliness. The other important aspects related to TSPO ligands such as blood-brain barrier penetration and better contrast have been predicted through lipophilicity (QP log P = 2.76 and 2.74 for ABPO-Br and ABPO-Cl, respectively) and serum binding (QP log Khsa = −0.18 and −0.25 for ABPO-Br and ABPO-Cl, respectively). The selectivity and distribution of these TSPO ligands were confirmed by 99mTc-ABPO-Br dynamic image in New Zealand rabbit. These results have shown that the ABPO analogs have the potential to act as better ligands as compared to known acetamidobenzoxazolone derivatives and would be of interest as a promising starting point for designing compounds for TSPO targeting.

Pro-inflammatory activation of primary microglia and macrophages increases 18 kDa translocator protein expression in rodents but not humans

The 18kDa Translocator Protein (TSPO) is the most commonly used tissue-specific marker of inflammation in positron emission tomography (PET) studies. It is expressed in myeloid cells such as microglia and macrophages, and in rodent myeloid cells expression increases with cellular activation. We assessed the effect of myeloid cell activation on TSPO gene expression in both primary human and rodent microglia and macrophages in vitro, and also measured TSPO radioligand binding with 3H-PBR28 in primary human macrophages. As observed previously, we found that TSPO expression increases (∼9-fold) in rodent-derived macrophages and microglia upon pro-inflammatory stimulation. However, TSPO expression does not increase with classical pro-inflammatory activation in primary human microglia (fold change 0.85 [95% CI 0.58–1.12], p = 0.47). In contrast, pro-inflammatory activation of human monocyte-derived macrophages is associated with a reduction of both TSPO gene expression (fold change 0.60 [95% CI 0.45–0.74], p = 0.02) and TSPO binding site abundance (fold change 0.61 [95% CI 0.49–0.73], p < 0.0001). These findings have important implications for understanding the biology of TSPO in activated macrophages and microglia in humans. They are also clinically relevant for the interpretation of PET studies using TSPO targeting radioligands, as they suggest changes in TSPO expression may reflect microglial and macrophage density rather than activation phenotype.

A bispecific antibody (ScBsAbAgn-2/TSPO) target for Ang-2 and TSPO resulted in therapeutic effects against glioblastomas

Antibody-based targeted therapy of cancers requires the antibody targeting of specific molecules inducing tumor cells apoptosis or death. Angiopoietin-2 (Agn-2) and translocator protein (TSPO) are identified as potential target molecules for glioblastoma therapy. The single chain anti-Agn-2 antibody (Anag-2) and anti-TSPO antibody (ATSPO) were obtained by monoclonal antibody screening. In the present study, for specific targeting and killing, we generated a recombinant bispecific antibody comprising a single-chain Fragment variable (ScFv) of anti-human Agn-2 and anti-human TSPO (ScBsAbAgn-2/TSPO), which is the mediator for mitochondrial apoptosis and tumor angiogenesis. In vitro, ScBsAbAgn-2/TSPO simultaneously bounded to both targets with a high antigen-binding affinity to Anag-2 and TSPO compared to the individual antibody. The higher expression of Ang-2 and TSPO was observed in bevacizumab-treated glioblastoma compared to normal rat brain endothelium. We also observed apoptosis-mediated cytotoxicity was improved, which resulted in the elimination of up to 90% of the target cells within 72 h. ScBsAbAgn-2/TSPO inhibited tumor growth, decreased vascular permeability, led to extended survival, improved pericyte coverage, depletion of tumor-associated macrophages, and increased numbers of intratumoral T lymphocytes infiltration in a murine bevacizumab-treated glioblastoma model. These findings were also confirmed ex vivo using glioblastoma cells from bevacizumab-treated rats with glioblastoma. We conclude that ScBsAbAgn-2/TSPO targeting of glioblastoma cell lines can be achieved in vitro and in vivo that the efficient elimination of glioblastoma cells supports the potential of ScBsAbAgn-2/TSPO as a potent, novel immunotherapeutic agent.

The impact of the rs6971 polymorphism in TSPO for quantification and study design

Second-generation translocator protein (TSPO) radioligands were developed to circumvent the technical short comings of 11C-PK11195, the first TSPO targeting tracer. However, in early clinical positron emission tomography (PET) studies they displayed greater inter- and intra-subject variability than was expected given the promising characteristics they showed in preclinical and in vitro studies. A great deal of this variability, although not all, can be explained by the rs6971 polymorphism in the gene encoding TSPO. This polymorphism causes a single amino acid substitution in the TSPO which, for all second-generation tracers tested in man hitherto, reduces binding affinity in mutants relative to wild type. This has obvious implications for interpretation of data, because inter-subject comparisons in PET studies are predicated on the assumption that binding affinity is consistent across all subjects. In this paper, we discuss the implications of the rs6971 polymorphism on study design, analysis and interpretation of data for clinical PET studies using second-generation TSPO radioligands.

Determination of [11C]PBR28 Binding Potential in vivo: A First Human TSPO Blocking Study

Positron emission tomography (PET) targeting the 18 kDa translocator protein (TSPO) is used to quantify neuroinflammation. Translocator protein is expressed throughout the brain, and therefore a classical reference region approach cannot be used to estimate binding potential ( BP ND). Here, we used blockade of the TSPO radioligand [11C]PBR28 with the TSPO ligand XBD173, to determine the non-displaceable volume of distribution ( V ND), and hence estimate the BP ND. A total of 26 healthy volunteers, 16 high-affinity binders (HABs) and 10 mixed affinity binders (MABs) underwent a [11C]PBR28 PET scan with arterial sampling. Six of the HABs received oral XBD173 (10 to 90 mg), 2 hours before a repeat scan. In XBD173-dosed subjects, V ND was estimated via the occupancy plot. Values of BP ND for all subjects were calculated using this V ND estimate. Total volume of distribution ( V T) of MABs (2.94 ± 0.31) was lower than V T of HABs (4.33 ± 0.29) ( P&lt;0.005). There was dose-dependent occupancy of TSPO by XBD173 (ED50 = 0.34 ± 0.13 mg/kg). The occupancy plot provided a V ND estimate of 1.98 (1.69, 2.26). Based on these V ND estimates, BP ND for HABs is approximately twice that of MABs, consistent with predictions from in vitro data. Our estimates of [11C]PBR28 V ND and hence BP ND in the healthy human brain are consistent with in vitro predictions. XBD173 blockade provides a practical means of estimating V ND for TSPO targeting radioligands.

A self-internalizing mitochondrial TSPO targeting imaging probe for fluorescence, MRI and EM

Advances in probes for cellular imaging have driven discoveries in biology and medicine. Primarily, antibodies and small molecules have been made for contrast enhancement of specific proteins. The development of new dendrimer-based tools offers opportunities to tune cellular internalization and targeting, image multiple modalities in the same molecule and explore therapeutics. The translocator protein (TSPO) offers an ideal target to develop dendrimer tools because it is well characterized and implicated in a number of disease states. The TSPO-targeted dendrimers reported here, primarily ClPhIQ-PAMAM-Gd-Liss, are cell membrane permeable nanoparticles that enable labeling of TSPO and provide contrast in fluorescence, electron microscopy and magnetic resonance imaging. The molecular binding affinity for TSPO was found to be 0.51 μM, 3 times greater than the monomeric agents previously demonstrated in our laboratory. The relaxivity per Gd3+ of the ClPhIQ23-PAMAM-Gd18 dendrimer was 7.7 and 8.0 mM-1 s-1 for r 1 and r 2 respectively, approximately double that of the clinically used monomeric Gd3+ chelates. In vitro studies confirmed molecular selectively for labeling TSPO in the mitochondria of C6 rat glioma and MDA-MB-231 cell lines. Fluorescence co-registration with Mitotracker Green® and increased contrast of osmium-staining in electron microscopy confirmed mitochondrial labeling of these TSPO-targeted agents. Taken collectively these experiments demonstrate the versatility of conjugation of our PAMAM dendrimeric chemistry to allow multi-modality agents to be prepared. These agents target organelles and use complementary imaging modalities in vitro, potentially allowing disease mechanism studies with high sensitivity and high resolution techniques.

Longitudinal PET Imaging of Muscular Inflammation Using 18F-DPA-714 and 18F-Alfatide II and Differentiation with Tumors

Aim: 18F-DPA-714 is a PET tracer that recognizes macrophage translocator protein (TSPO), and 18F-Alfatide II (18F-AlF-NOTA-E[PEG4-c(RGDfk)]2) is specific for integrin αvβ3. This study aims to apply these two tracers for longitudinal PET imaging of muscular inflammation, and evaluate the value of 18F-DPA-714 in differentiating inflammation from tumor.Methods: RAW264.7 mouse macrophage cells were used for cell uptake analysis of 18F-DPA-714. A mouse hind limb muscular inflammation model was established by intramuscular injection of turpentine oil. For the inflammation model, PET imaging was performed at different days using 18F-DPA-714 and 18F-Alfatide II. The specificity of the imaging probes was tested by co- or pre-injection of PK11195 or unlabeled RGD (Arg-Gly-Asp) peptide. PET imaging using 18F-DPA-714 was performed in A549, HT29, U87MG, INS-1, and 4T1 xenograft models. Immunofluorescence staining was performed to evaluate infiltrated macrophages and angiogenesis in inflammation and/or tumors.Results: Uptake of 18F-DPA-714 in RAW264.7 cells was 45.5% at 1 h after incubation, and could be blocked by PK11195. PET imaging showed increased 18F-DPA-714 and 18F-Alfatide II uptake at inflammatory muscles. Peak uptake of 18F-DPA-714 was seen on day 6 (4.02 ± 0.64 %ID/g), and peak uptake of 18F-Alfatide II was shown on day 12 (1.87 ± 0.35 %ID/g) at 1 h p.i.. Tracer uptakes could be inhibited by PK11195 for 18F-DPA-714 or cold RGD for 18F-Alfatide II. Moreover, macrophage depletion with liposomal clodronate also reduced the local accumulation of both tracers. A549, HT29, U87MG, INS-1, and 4T1 tumor uptakes of 18F-DPA-714 (0.46 ± 0.28, 0.91 ± 0.08, 1.69 ± 0.67, 1.13 ± 0.33, 1.22 ± 0.55 %ID/g at 1 h p.i., respectively) were significantly lower than inflammation uptake (All P < 0.05).Conclusion: PET imaging using 18F-DPA-714 as a TSPO targeting tracer could evaluate the dynamics of macrophage activation and infiltration in different stages of inflammatory diseases. The concomitant longitudinal PET imaging with both 18F-DPA-714 and 18F-Alfatide II matched the causal relationship between macrophage infiltration and angiogenesis. Moreover, we found 18F-DPA-714 uptake in several types of tumors is significantly lower than that in inflammatory muscles, suggesting 18F-DPA-714 PET has the potential for better differentiation of tumor and non-tumor inflammation.