A single-domain antibody for the detection of pathological Tau protein in the early stages of oligomerization.

BACKGROUND: Soluble oligomeric forms of Tau protein have emerged as crucial players in the propagation of Tau pathology in Alzheimer's disease (AD). Our objective is to introduce a single-domain antibody (sdAb) named 2C5 as a novel radiotracer for the efficient detection and longitudinal monitoring of oligomeric Tau species in the human brain. METHODS: The development and production of 2C5 involved llama immunization with the largest human Tau isoform oligomers of different maturation states. Subsequently, 2C5 underwent comprehensive in vitro characterization for affinity and specificity via Enzyme-Linked Immunosorbent Assay and immunohistochemistry on human brain slices. Technetium-99m was employed to radiolabel 2C5, followed by its administration to healthy mice for biodistribution analysis. RESULTS: 2C5 exhibited robust binding affinity towards Tau oligomers (Kd = 6.280 nM ± 0.557) and to Tau fibers (Kd = 5.024 nM ± 0.453), with relatively weaker binding observed for native Tau protein (Kd = 1791 nM ± 8.714) and amyloid peptide (Kd > 10,000 nM). Remarkably, this SdAb facilitated immuno-histological labeling of pathological forms of Tau in neurons and neuritic plaques, yielding a high-contrast outcome in AD patients, closely mirroring the performance of reference antibodies AT8 and T22. Furthermore, 2C5 SdAb was successfully radiolabeled with 99mTc, preserving stability for up to 6 h post-radiolabeling (radiochemical purity > 93%). However, following intravenous injection into healthy mice, the predominant uptake occurred in kidneys, amounting to 115.32 ± 3.67, 97.70 ± 43.14 and 168.20 ± 34.52% of injected dose per gram (% ID/g) at 5, 10 and 45 min respectively. Conversely, brain uptake remained minimal at all measured time points, registering at 0.17 ± 0.03, 0.12 ± 0.07 and 0.02 ± 0.01% ID/g at 5, 10 and 45 min post-injection respectively. CONCLUSION: 2C5 demonstrates excellent affinity and specificity for pathological Tau oligomers, particularly in their early stages of oligomerization. However, the current limitation of insufficient blood-brain barrier penetration necessitates further modifications before considering its application in nuclear medicine imaging for humans.

COB231 targets amyloid plaques in post-mortem human brain tissue and in an Alzheimer mouse model.

Previous works have shown the interest of naturally fluorescent proflavine derivatives to label Abeta deposits in vitro. This study aimed to further characterize the properties of the proflavine 3-acetylamino-6-[3-(propargylamino)propanoyl]aminoacridine (COB231) derivative as a probe. This compound was therefore evaluated on human post-mortem and mice brain slices and in vivo in 18-month-old triple transgenic mice APPswe, PS1M146V and tauP301L (3xTgAD) mice presenting the main characteristics of Alzheimer's disease (AD). COB231 labelled amyloid plaques on brain slices of AD patients, and 3xTgAD mice at 10 and 0.1 µM respectively. However, no labelling of the neurofibrillary tangle-rich areas was observed either at high concentration or in the brain of fronto-temporal dementia patients. The specificity of this mapping was attested in mice using Thioflavin S and IMPY as positive controls of amyloid deposits. After intravenous injection of COB231 in old 3xTgAD mice, fluorescent amyloid plaques were detected in the cortex and hippocampus, demonstrating COB231 blood­brain barrier permeability. We also controlled the cellular localization of COB231 on primary neuronal cultures and showed that COB231 accumulates into the cytoplasm and not into the nucleus. Finally, using a viability assay, we only detected a slight cytotoxic effect of COB231 (< 10%) for the highest concentration (100 µM).

How organic anions accumulate in hepatocytes lacking Mrp2: evidence in rat liver.

In the liver, the accumulation of hepatobiliary contrast agents is a crucial issue to understand the images of liver scintigraphy or magnetic resonance (MR) imaging. Thus, depending on the regulation of uptake and exit membrane systems in normal and injured hepatocytes, these contrast agents will accumulate differently within cells. Gadobenate dimeglumine (Gd-BOPTA) is a hepatobiliary MR contrast agent that distributes to the extracellular space and enters into rat hepatocytes through the sinusoidal transporters, organic anion-transporting polypeptides. Gd-BOPTA is not metabolized during its transport to the canalicular membrane where it is excreted into bile through multiple resistance protein-2 (Mrp2). It is not well known how Gd-BOPTA accumulates in normal livers and in livers lacking Mrp2. We perfused livers from normal rats and from rats lacking Mrp2 with (153)Gd-BOPTA at increasing concentrations and assessed the hepatic accumulation of this agent using a gamma probe placed above the livers. By use of a pharmacokinetic model that best described the amounts of Gd-BOPTA in perfusate, bile, and hepatic tissue over time, we showed how increasing concentrations and the absence of Mrp2 modify the hepatic accumulation of the contrast agent. It is noteworthy that despite the absence of Gd-BOPTA bile excretion and a similar efflux back to sinusoids in livers lacking Mrp2, the maximal hepatic accumulation of contrast agent was similar to normal rats. We also showed how hepatic accumulation relies on the concomitant entry into and exit from hepatocytes. Such information improves our understanding of liver imaging associated with the perfusion of hepatobiliary contrast agents, which was recently introduced in clinical practice.

In vivo quantification of 5-HT1A-[18F]MPPF interactions in rats using the YAP-(S)PET scanner and a beta-microprobe.

We performed full modeling analysis of 5-HT(1A)-[(18)F]MPPF interactions using the beta-microprobe (beta P) and a YAP-(S)PET scanner. Sixteen Wistar rats were used for beta P (n=5) and YAP-(S)PET (n=5) acquisitions and metabolite studies (n=6). Time-concentration curves were obtained in the hippocampus, raphe dorsalis, frontal cortex and cerebellum, using three injections of [(18)F]MPPF at different specific activities. B'(max) values were estimated from a two (2T-5k)- and three (3T-7k)-tissue-compartment model with beta P and YAP-(S)PET time-concentration curves. The simplified reference tissue model (SRTM) was used to estimate binding potential (BP(SRTM)) values from data obtained with the first injection and the cerebellum as the reference region. Overall, the 3T-7k model provided a better fit than the 2T-5k model, as evaluated from AIC criteria in all experiments. The rank order of receptor density (B'max) values was as follows: hippocampus>raphe approximately frontal cortex>cerebellum. Non-negligible specific binding was observed in the cerebellum (B'max (beta P)=1.5+/-0.9 pmol/ml). Significant correlations (p<0.001) between B'max and BP(SRTM) values were evident with both beta P (r=0.895) and YAP-(S)PET (r=0.695). The YAP-(S)PET system underestimated the [18F]MPPF binding levels in brain due to limited resolution (i.e. partial volume), but led to similar conclusions.

SPECT quantification of benzodiazepine receptor concentration using a dual-ligand approach.

UNLABELLED: The distribution of benzodiazepine receptors in the human brain has been widely studied with SPECT using (123)I-iomazenil and semiquantitative approaches, but these methods do not allow quantification of the total receptor site concentration available for binding (B'(max)) and of the apparent equilibrium dissociation constant (K(d)/V(R)). One of the major obstacles to full quantitative studies is that pharmacologic effects preclude the administration to humans of the high doses of iomazenil required to displace the labeled ligand from the receptors. In this study, we applied a dual-ligand protocol using the unlabeled ligand flumazenil, which lacks pharmacologic effects, to quantify all binding parameters of the benzodiazepine receptor-(123)I-iomazenil interactions. METHODS: (123)I-Iomazenil SPECT and MRI were acquired in 8 healthy volunteers, one of whom had participated in a (11)C-flumazenil PET experiment. The experimental protocol consisted of injections of (123)I-iomazenil and/or unlabeled flumazenil. We developed a kinetic model to integrate the different pharmacokinetics of these 2 ligands. To simplify the model, we assumed linear relationships between iomazenil and flumazenil parameters and adjusted them using a coupled fitting procedure. The resulting constrained 5-parameter model was then used to quantify the biologic parameters. RESULTS: Across regions, we obtained B'(max) values ranging from 7 to 69 pmol/mL and K(d)V(R) values for IMZ from 2.3 to 3.7 pmol/mL. There was a close correlation in the B'(max) values calculated in the same volunteer using (123)I-iomazenil SPECT and (11)C-flumazenil PET. CONCLUSION: The dual-ligand approach can be used to quantify all model parameters with acceptable SEs. This work demonstrates a theoretic framework and initial application of SPECT to quantify binding parameters.