Zhi-Ping Zhuang

Preclinical Properties of 18F-AV-45: A PET Agent for Aβ Plaques in the Brain

β-amyloid plaques (Aβ plaques) in the brain, containing predominantly fibrillary Aβ peptide aggregates, represent a defining pathologic feature of Alzheimer disease (AD). Imaging agents targeting the Aβ plaques in the living human brain are potentially valuable as biomarkers of pathogenesis processes in AD. (E)-4-(2-(6-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)pyridin-3-yl)vinyl)-N-methyl benzenamine (18F-AV-45) is such as an agent currently in phase III clinical studies for PET of Aβ plaques in the brain. Methods: In vitro binding of 18F-AV-45 to Aβ plaques in the postmortem AD brain tissue was evaluated by in vitro binding assay and autoradiography. In vivo biodistribution of 18F-AV-45 in mice and ex vivo autoradiography of AD transgenic mice (APPswe/PSEN1) with Aβ aggregates in the brain were performed. Small-animal PET of a monkey brain after an intravenous injection of 18F-AV-45 was evaluated. Results: 18F-AV-45 displayed a high binding affinity and specificity to Aβ plaques (Kd, 3.72 ± 0.30 nM). In vitro autoradiography of postmortem human brain sections showed substantial plaque labeling in AD brains and not in the control brains. Initial high brain uptake and rapid washout from the brain of healthy mice and monkey were observed. Metabolites produced in the blood of healthy mice after an intravenous injection were identified. 18F-AV-45 displayed excellent binding affinity to Aβ plaques in the AD brain by ex vivo autoradiography in transgenic AD model mice. The results lend support that 18F-AV-45 may be a useful PET agent for detecting Aβ plaques in the living human brain.

Subcellular Localization of Sigma-2 Receptors in Breast Cancer Cells Using Two-Photon and Confocal Microscopy

Sigma-2 receptor agonists have been shown to induce cell death via caspase-dependent and caspase-independent pathways. Unfortunately, there is little information regarding the molecular function of sigma-2 receptors that can explain these results. In this study, two fluorescent probes, SW107 and K05-138, were used to study the subcellular localization of sigma-2 receptors by two-photon and confocal microscopy. The results indicate that sigma-2 receptors colocalize with fluorescent markers of mitochondria, lysosomes, endoplasmic reticulum, and the plasma membrane in both EMT-6 mouse and MDA-MB-435 human breast cancer cells. The fluorescent probe, K05-138, was internalized rapidly, reaching a plateau of fluorescent intensity at 5 min. The internalization of K05-138 was reduced approximately 40% by phenylarsine oxide, an inhibitor of endocytosis. These data suggest that sigma-2 ligands are internalized, in part, by an endocytotic pathway. The localization of sigma-2 receptors in several organelles known to have a role in both caspase-dependent and caspase-independent pathways of cell death supports the conclusions of previous studies suggesting that sigma-2 receptor ligands should be evaluated as potential cancer chemotherapeutic agents.

P-[18F]-MPPF: a potential radioligand for PET studies of 5-HT1A receptors in humans.

The purpose of this study was to develop a radiopharmaceutical that could be used to selectively image 5‐HT1A receptors with positron emission tomography (PET) No‐carrier‐added 4‐(2′‐methoxyphenyl)‐1‐[2′‐(N‐2′‐pyridinyl)‐p‐[18F]fluorobenzamido]ethylpiperazine (p‐[18F]‐MPPF, 2) was synthesized by the nucleophilic substitution of the corresponding nitro precursor 1 with K[18F]/Kryptofix 2.2.2. in dimethyl sulfoxide (DMSO) at 140°C for 20 min followed by purification with high‐performance liquid chromatography (HPLC) in 10% yield in a synthesis time of 90 min from end of bombardment (EOB). Specific activity was 1–4 Ci/μM.

Characterization of IMPY as a potential imaging agent for β-amyloid plaques in double transgenic PSAPP mice

Deposition of β-amyloid (Aβ) plaques in the brain is likely linked to the pathogenesis of Alzheimer’s disease (AD). Developing specific Aβ aggregate-binding ligands as in vivo imaging agents may be useful for diagnosis and monitoring the progression of AD. We have prepared a thioflavin derivative, 6-iodo-2-(4’-dimethylamino-)phenyl-imidazo[1,2-a]pyridine, IMPY, which is readily radiolabeled with 125I/123I for binding or single-photon emission computerized tomography (SPECT) imaging studies. Characterization of [125I]IMPY binding to plaque-like structures was evaluated in double transgenic PSAPP mice. [125I]IMPY labeled Aβ plaques in transgenic mouse brain sections, and the labeling was consistent with fluorescent staining and Aβ-specific antibody labeling. Significant amounts of Aβ plaques present in the cortical, hippocampal, and entorhinal regions of the transgenic mouse brain were clearly detected with [125I]IMPY via ex vivo autoradiography. In contrast, [125I]IMPY showed little labeling in the age-matched control mouse brain. Tissue homogenate binding further corroborated the Aβ plaque-specific distribution in various brain regions of transgenic mouse, and correlated well with the known density of Aβ deposition. Using a tissue dissection technique, [125I]IMPY showed a moderate increase in the cortical region of transgenic mice as compared to the age-matched controls. In vitro blocking of [125I]IMPY by “carrier” observed via autoradiography in mouse brain sections was not replicated by an in vivo blocking experiment in living TT mouse brain. The failure was most likely due to a significant carrier effect, which slows down the tracer in vivo metabolism, leading to an increased brain uptake. Taken together, these data indicate that [123I]IMPY is a potentially useful SPECT imaging agent for in vivo labeling of Aβ plaques in the living brain.

A comparison of amyloid fibrillogenesis using the novel fluorescent compound K114

Proteinaceous inclusions with amyloidogenic properties are a common link between many neurodegenerative diseases, including Alzheimers disease and Parkinsons disease. Histological and in vitro studies of amyloid fibrils have advanced the understanding of protein aggregation, and provided important insights into pathogenic mechanisms of these neurodegenerative brain amyloidoses. The classical amyloid dyes Congo Red (CR) and thioflavin T and S, have been used extensively to detect amyloid inclusions in situ. These dyes have also been utilized to monitor the maturation of amyloid fibrils assembled from monomer subunits in vitro. Recently, the compound (trans,trans)‐1‐bromo‐2,5‐bis‐(3‐ hydroxycarbonyl‐4‐hydroxy)styrylbenzene (BSB), derived from the structure of CR, was shown to bind to a wide range of amyloid inclusions in situ. More importantly it was also used to label brain amyloids in live animals. Herein, we show that an analogue of BSB, (trans,trans)‐1‐bromo‐2,5‐bis‐(4‐hydroxy)styrylbenzene (K114), recognizes amyloid lesions, and has distinctive properties which allowed the quantitative monitoring of the formation of amyloid fibrils assembled from the amyloid‐β peptide, α‐synuclein, and tau.

The Fluorescent Congo Red Derivative, (Trans, Trans)−1-Bromo-2,5-Bis-(3-Hydroxycarbonyl-4-Hydroxy)Styrylbenzene (BSB), Labels Diverse β-Pleated Sheet Structures in Postmortem Human Neurodegenerative Disease Brains

A novel Congo red-derived fluorescent probe (trans, trans),-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) that binds to amyloid plaques of postmortem Alzheimers disease brains and in transgenic mouse brains in vivo was designed as a prototype imaging agent for Alzheimers disease. In the current study, we used BSB to probe postmortem tissues from patients with various neurodegenerative diseases with diagnostic lesions characterized by fibrillar intra- or extracellular lesions and compared these results with standard histochemical dyes such as thioflavin S and immunohistochemical stains specific for the same lesions. These data show that BSB binds not only to extracellular amyloid beta protein, but also many intracellular lesions composed of abnormal tau and synuclein proteins and suggests that radioiodinated BSB derivatives or related ligands may be useful imaging agents to monitor diverse amyloids in vivo.

Florbetapir F-18: A Histopathologically Validated Beta-Amyloid Positron Emission Tomography Imaging Agent

Florbetapir F-18 is a molecular imaging agent combining high affinity for β-amyloid, pharmacokinetic properties that allow positron emission tomography (PET) imaging within a convenient time after dose administration, and the wide availability of the radionuclide fluorine-18. Florbetapir F-18 is prepared by nucleophilic radiofluorination in approximately 60 minutes with a decay-corrected yield of 20%-40% and with a specific activity typically exceeding 100 Ci/mmol. The florbetapir F-18 dissociation constant (K(d)) for binding to β-amyloid in brain tissue from Alzheimers disease (AD) patients was 3.7 ± 0.3 nmol/L, and the maximum binding capacity (B(max)) was 8800 ± 1600 fmol/mg protein. Autoradiography studies have shown that florbetapir F-18 selectively binds to β-amyloid aggregates in AD patient brain tissue, and the binding intensity is correlated with the density of β-amyloid quantified by standard neuropathologic techniques. Studies in animals revealed no safety concerns and rapid and transient normal brain uptake (6.8% injected dose/g at 2 minutes and 1.9% injected dose/g at 60 minutes in the mouse). Florbetapir F-18 has been well-tolerated in studies of more than 2000 human subjects. Biodistribution studies in humans revealed predominantly hepatobiliary excretion. The whole body effective dose was 7 mSv from a dose of 370 MBq. The pharmacokinetic of florbetapir F-18 make it possible to obtain a PET image with a brief (10 minutes) acquisition time within a convenient time window of 30-90 minutes after dose administration. Clinical studies have demonstrated a clear correlation between in vivo PET imaging with florbetapir F-18 and postmortem histopathologic quantitation of β-amyloid in the brain.

Short Communication New 5‐HT1A receptor antagonist: [3H]p‐MPPF

A new 5‐HT1A receptor antagonist ligand, [3H]p‐MPPF, 4‐(2′‐methoxy‐)‐phenyl‐1‐[2′‐(N‐2′‐pyridyl)‐p‐fluorobenzamido]ethyl‐piperazine, was prepared and characterized. It demonstrated high affinity and selectivity toward 5‐HT1A receptors (Kd = 0.34 ± 0.12 nM and Bmax = 145 ± 35 fmol/mg protein in rat hippocampal membrane homogenates). The binding is not sensitive to 100 μM Gpp(NH)p. Initial autoradiography studies of rat brain sections exhibit regional localization consistent with the known 5‐HT1A receptor distribution. This potential 5‐HT1A antagonist ligand may provide a powerful tool for 5‐HT1A receptor pharmacology studies in the central nervous system.

Detection of amyloid plaques by radioligands for Aβ40 and Aβ42

Alzheimer’s disease (AD) is linked to increased brain deposition of amyloid-β (Aβ) peptides in senile plaques (SPs), and recent therapeutic efforts have focused on inhibiting the production or enhancing the clearance of Aβ in brain. However, it has not been possible to measure the burden of SPs or assess the effect of potential therapies on brain Aβ levels in patients. Toward that end, we have developed a novel radioligand, [125I]TZDM, which binds Aβ fibrils with high affinity, crosses the blood-brain barrier (BBB), and labels amyloid plaques in vivo. Compared to a styrylbenzene probe, [125I]IMSB, [125I]TZDM showed a 10-fold greater brain penetration and labeled plaques with higher sensitivity for in vivo imaging. However, this ligand also labels white matter, which contributes to undesirable high background regions of the brain. Interestingly, parallel to their differential binding characteristics onto fibrils composed of 40 (Aβ40)- or 42 (Aβ42)-amino-acid-long forms of Aβ peptides, these radioligands displayed differential labeling of SPs in AD brain sections under our experimental conditions. It was observed that [125I]IMSB labeled SPs containing Aβ40, amyloid angiopathy (AA), and neurofibrillary tangles, whereas [125I]TZDM detected only SPs and Aβ42-positive AA. Since increased production and deposition of Aβ42 relative to Aβ40 may be crucial for the generation of SPs, [125I]TZDM and related derivatives may be more attractive probes for in vivo plaque labeling. Further structural modifications of TZDM to lower the background labeling will be needed to optimize the plaque-labeling property.

The 5-HT1A receptor antagonist p-MPPI blocks responses mediated by postsynaptic and presynaptic 5-HT1A receptors

The present experiments examined the ability of the novel 5-HT1A receptor antagonist to block responses mediated by postsynaptic and presynaptic 5-HT1A receptors in vivo. Pretreatment with p-MPPI reduced or blocked the effect of the 5-HT1A receptor agonist 8-OH-DPAT on two responses mediated by postsynaptic 5-HT1A receptors, reduction of body temperature and the 5-HT behavioral syndrome. Administration of p-MPPI alone did not alter body temperature or produce symptoms of the 5-HT syndrome. Pretreatment with p-MPPI also blocked the ability of 8-OH-DPAT to reduce extracellular 5-HT in the striatum, a response mediated by presynaptic 5-HT1A receptors in the dorsal raphe nucleus, but did not alter striatal 5-HT when administered alone. These results indicate that p-MPPI is an effective 5-HT1A receptor antagonist in vivo with no intrinsic activity. p-MPPI may prove to be a useful pharmacological tool for studying 5-HT1A receptors and their involvement in anxiety and affective disorders.