A novel side-bridged hybrid phosphonate/acetate pendant cyclam: Synthesis,characterization, and 64Cu small animal PET imaging

Copper-64 (t_1/2 = 12.7 h; β⁺: 0.653 MeV, 17.4%; β^?: 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik–Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with ⁶⁴Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the ⁶⁴Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in ⁶⁴Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.     

N-(3-[18F]fluoropropyl)-spiperone: The preferred 18F labeled spiperone analog for positron emission tomographic studies of the dopamine receptor

The ligands currently used for PET studies of the dopamine receptor are fluorine-18-labeled spiperone (FSp) and carbon-11 or fluorine-18-labeled N-methyl-spiperone. All three of these ligands have drawbacks in either their chemical preparation or their biological behavior. We have previously prepared a series of N-fluoroalkyl-spiperone derivatives which are simple to prepare in high radiochemical yield. N-[¹⁸F]fluoropropyl-spiperone (3-F-Pr-Sp) and N-[¹⁸F]fluoroethyl-spiperone (2-F-Et-Sp) were the most promising ligands. In vitro competitive binding studies showed affinities for the dopamine receptor of 3-F-Pr-Sp > FSp > 2-F-Et-Sp. Brain extraction studies in a primate model showed that FSp, 2-F-Et-Sp, and 3-F-Pr-Sp were not completely extracted by the brain. High bone uptake and kidney clearance was observed with 3-F-Pr-Sp, while 2-F-Et-Sp cleared through the intestine in rats. This is in contrast to FSp where clearance is through the kidney. Studies to evaluate the extraction of metabolites in the brain were carried out by administering large doses (10 mCi) of FSp, 2-F-Et-Sp and 3-F-Pr-Sp to rats and reinjecting the metabolites in blood into other rats. These experiments showed that < 0.02% of the metabolites from FSp and 3-F-Pr-Sp entered the brain, while 0.5% of the metabolites from 2-F-Et-Sp entered the brain. The majority of the activity present in the cerebellum after the administration of 2-F-Et-Sp is metabolites; therefore 2-F-Et-Sp is unsuitable for PET imaging studies. PET imaging studies in baboons and in one normal human volunteer with 3-F-Pr-Sp showed a high striatum-to-cerbellum ratio, showing that 3-F-Pr-Sp can replace ligands currently in use to study dopamine receptors.     

Evaluation of positron-emitting SCH 23390 analogs as tracers for CNS dopamine D1 receptors

Two positron-emitting analogs of SCH 23390, one labelled with ⁷⁵Br (or ⁷⁶Br) and another with ¹¹C, were evaluated as potential PET tracers for central dopamine D1 receptors. In vivo studies were performed to assess the time course of the biodistribution of these tracers in mice and to determine whether dopamine receptors mediated their uptake in the brains of these animals. Results show that indeed cerebral uptake was consistent with dopamine receptor innervation, i.e. uptake and clearance was regionally consistent with the target receptors and that specific uptake was saturable. Because of the relatively rapid pharmacokinetics of this drug, ¹¹C-labelled SCH 23390 would be best suited for PET imaging although the metabolism of this compound needs to be further examined.     

[18F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments

Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [¹⁸F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [¹⁸F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [¹⁸F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [¹⁸F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm³*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [¹⁸F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models.     

TMPHPG: a lipophilic derivative of EHPG for iron,gallium-68 and indium-111 hepatobiliary clearance

TMPHPG (N,N′-trimethylenebis[2-(2-hydroxy-3,5-dimethylphenyl)glycine]) is a derivative of the ligand EHPG (ethylenebis[2-(o-hydroxyphenyl)glycine]) but is more lipophilic and had been synthesized to increase hepatobiliary clearance. ⁵⁹Fe(III), ⁶⁸Ga(III) and ¹¹¹In(III) complexes of this ligand have been investigated as potential imaging agents for MRI, PET and SPECT, respectively. Metal-dependent differences in blood and liver clearance in mature rats have been evaluated.     

Properties of multilamellar liposomes containing 99mTcO−4: Effect of distearoylphosphatidylcholine to sphingomyelin ratio

Multilamellar liposomes composed of eight different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) were prepared containing [^99mTc]pertechnetate. The in vitro permeability of the liposome preparations in buffer and serum were measured by both dialysis and direct exposure. Liposomes composed of 25–33% SM were least permeable exhibiting leakage half-times of approximately 70 h in buffer and 52 h in serum. Pertechnetate-containing liposomes of three different lipid compositions were injected into mice and the biodistribution of ^99mTc activity was determined. All preparations were taken up primarily by the lungs and liver. The half-time for clearance of ^99mTc activity ranged from 24.8 to 30.6 h for the liver and 25.3 to 33.0 h for the lungs.     

Synthesis of [11C]sodium thiocyanate for in vivo studies of anion kinetics using positron emission tomography (PET)

Sodium thiocyanate (NaSCN) was labelled with carbon-11 for in vivo studies of anion kinetics using positron emission tomography (PET). The synthesis was complete in 35 min from end of bombardment using [¹¹Qammonium cyanide as the labelled precursor. [¹¹C]NaSCN was produced by the reaction of [¹¹C]sodium cyanide with elemental sulfur and subsequently separated by semi-preparative high performance liquid chromatography (HPLC). Radiochemical yields (isolated) were of the order of 25%. The specific activity was 18 GBq/mmol and the radiochemical purity better than 99%. A PET study performed in a healthy volunteer showed distribution of [¹¹C]SCN⁻ to areas corresponding to cortical fluid spaces known to be accessible to inorganic ions such as Cl⁻. An accumulation of the tracer was observed during the 70 min investigation, indicating at least three compartments of distribution.     

Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, 18F labeled bis(zinc(II)-dipicolylamine) complex

The synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes are known to have a high specific and selective affinity to target the exposed phosphatidylserine (PS) on the surface of dead and dying cells. An ¹⁸F-labeled DPAZn2 complex (4-¹⁸F-Fluoro-benzoyl-bis(zinc(II)-dipicolylamine), ¹⁸F-FB-DPAZn2) as positron emission tomography (PET) tracer was developed and evaluated for in vivo imaging of tumor treated with a chemical agent. The in vitro cell stain studies revealed that fluorescent DPAZn2 complexes (Dansyl-DPAZn2) stained the same cells (apoptotic and necrotic cells) as fluorescein isothiocyanate (FITC) labeled Annexin V (FITC-Annexin V). The radiosynthesis of ¹⁸F-FB-DPAZn2 was achieved through the amidation the precursor bis(2,2′-dipicolylamine) derivative (DPA2) with the prosthetic group N-succinimidyl-4-[¹⁸F]-fluorobenzoate (¹⁸F-SFB) and chelation with zinc nitrate. In the biodistribution study, the fast clearance of ¹⁸F-FB-DPAZn2 from blood and kidney was observed and high uptake in liver and intestine within 90 min postinjection was also found. For the PET imaging, significantly higher tumor uptake of ¹⁸F-FB-DPAZn2 was observed in the adriamycin (ADM)-treated Hepa1-6 hepatocellular carcinoma-bearing mice than that in the untreated tumor-model mice, while a slightly decreased tumor uptake of ¹⁸F-FDG was found in the ADM-treated tumor-bearing mice. The results indicate that ¹⁸F-FB-DPAZn2 has the similar capability of apoptosis detection as FITC-Annexin V and seems to be a potential PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy. The high uptake of ¹⁸F-FB-DPAZn2 in the abdomen needs to optimize the structure for improving its pharmacokinetics characteristics in the future work.     

Preparation and evaluation of a 99mTcN–PNP complex of sanazole analogue for detecting tumor hypoxia

A sanazole derivative, having a favorable single electron reduction potential (SERP) value compared to that of misonidazole, was synthesized and radiolabeled with [^99mTcN(PNP)] precursor to evaluate its potential as a hypoxia imaging agent. The complex, which was lipophilic, could be prepared in good yields and challenging studies with cysteine showed stability of the complex against trans-chelation. However, despite being lipophilic as well as possessing favorable SERP value, biodistribution studies of this complex in fibrosarcoma tumor bearing Swiss mice showed low uptake in tumor. This observation is possibly attributed to fast clearance of the complex from blood, whereby the complex spends insufficient time in tumor to get reduced and trapped. Though uptake in tumor was low, slow clearance of activity from tumor suggests reduction and trapping of the complex in hypoxic cells. The present ^99mTc-complex demonstrated acceptable values of tumor to blood (TBR) and tumor to muscle (TMR) ratios. However, low uptake in tumor which may not be indicative of the actual hypoxic status of the tumor, limit the utility of the complex to detect tumor hypoxia.     

Imaging incorporation of circulating docosahexaenoic acid into the human brain using positron emission tomography

Docosahexaenoic acid (DHA; 22:6n-3) is a critical constituent of the brain, but its metabolism has not been measured in the human brain in vivo. In monkeys, using positron emission tomography (PET), we first showed that intravenously injected [1-¹¹C]DHA mostly entered nonbrain organs, with ∼0.5% entering the brain. Then, using PET and intravenous [1-¹¹C]DHA in 14 healthy adult humans, we quantitatively imaged regional rates of incorporation (K*) of DHA. We also imaged regional cerebral blood flow (rCBF) using PET and intravenous [¹⁵O]water. Values of K* for DHA were higher in gray than white matter regions and correlated significantly with values of rCBF in 12 of 14 subjects despite evidence that rCBF does not directly influence K*. For the entire human brain, the net DHA incorporation rate J_in, the product of K*, and the unesterified plasma DHA concentration equaled 3.8 ± 1.7 mg/day. This net rate is equivalent to the net rate of DHA consumption by brain and, considering the reported amount of DHA in brain, indicates that the half-life of DHA in the human brain approximates 2.5 years. Thus, PET with [1-¹¹C]DHA can be used to quantify regional and global human brain DHA metabolism in relation to health and disease.     

Investigation of copper-PTSM as a PET tracer for tumor blood flow

Copper-PTSM has been shown in previous studies to act as a fluid microsphere and to be useful in quantitating blood flow in brain, myocardium, and kidneys. In this study we have evaluated this agent as a PET tumor blood flow agent. ⁶⁴Cu- or ⁶⁷Cu-labeled Cu-PTSM was administered (i.v.) to Golden Syrian hamsters with colorectal carcinoma cell implants (GW39). One minute prior to sacrifice (10–60 min after Cu-PTSM was administered) ¹²⁵I-iodoantipyrine (¹²⁵I-IAP), an agent known to measure tumor blood flow, was administered intravenously by a 3-stage, 1 min ramp infusion. Following sacrifice, samples of tumor and brain were removed (within 40 s) and the tumor and brain levels of Cu-PTSM and iodoantipyrine determined. Since the brain uptake of both Cu-PTSM and IAP is perfusion rate limited, the brain was used as a reference organ to normalize tumor levels of the two tracers. The plot of Cu-PTSM versus ¹²⁵I-IAP tumor/brain ratios showed a good linear correlation (r value of 0.97), suggesting that Cu-PTSM could be used to quantify tumor blood flow. Since the mechanism of Cu-PTSM trapping is likely to be due to glutathione levels in the tissue, and because tumor tissue glutathione levels might vary, the temporal uptake of Cu-PTSM was investigated by PET imaging both the tumor-bearing hamsters and ~300 g Copenhagen rats bearing R3227 prostate tumors. The tumors were clearly visualized and the retained copper radioactivity in the tumor was constant over the 30 min imaging period.     

Pharmacokinetics and Biodistribution of a Human Monoclonal Antibody to Oxidized LDL in Cynomolgus Monkey Using PET Imaging

Purpose

Oxidized low-density lipoprotein (LDL) plays an essential role in the pathogenesis of atherosclerosis. The purpose of this study was to characterize the pharmacokinetics (PK) of a human recombinant IgG1 antibody to oxidized LDL (anti-oxLDL) in cynomolgus monkey. The tissue biodistribution of anti-oxLDL was also investigated using positron emission tomography (PET) imaging.

Methods

Anti-oxLDL was conjugated with the N-hydroxysuccinimide ester of DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) and radiolabeled by chelation of radioactive copper-64 (⁶⁴Cu) for detection by PET. Anti-oxLDL was administered as a single intravenous (IV) dose of 10 mg/kg (as a mixture of radiolabeled and non-labeled material) to two male and two female cynomolgus monkeys. Serum samples were collected over 29 days. Two ELISA methods were used to measure serum concentrations of anti-oxLDL; Assay A was a ligand binding assay that measured free anti-oxLDL (unbound and partially bound forms) and Assay B measured total anti-oxLDL. The biodistribution was observed over a 48-hour period following dose administration using PET imaging.

Results

Anti-oxLDL serum concentration-time profiles showed a biphasic elimination pattern that could be best described by a two-compartment elimination model. The serum concentrations obtained using the two ELISA methods were comparable. Clearance values ranged from 8 to 17 ml/day/kg, while beta half-life ranged from 8 to12 days. The initial volume of distribution and volume of distribution at steady state were approximately 55 mL/kg and 150 mL/kg, respectively. PET imaging showed distribution predominantly to the blood pool, visible as the heart and great vessels in the trunk and limbs, plus diffuse signals in the liver, kidney, spleen, and bone marrow.

Conclusions

The clearance of anti-oxLDL is slightly higher than typical IgG1 antibodies in cynomolgus monkeys. The biodistribution pattern appears to be consistent with an antibody that has no large, rapid antigen sink outside the blood space.     

Blood–brain barrier permeability of ginkgolide: Comparison of the behavior of PET probes 7α-[18F]fluoro- and 10-O-p-[11C]methylbenzyl ginkgolide B in monkey and rat brains

The blood–brain barrier permeability of ginkgolide B was examined using positron emission tomography (PET) probes of a ¹⁸F-incorporated ginkgolide B ([¹⁸F]-2) and a ¹¹C-incorporated methylbenzyl-substituted ginkgolide B ([¹¹C]-3). PET studies in monkeys showed low uptake of [¹⁸F]-2 into the brain, but small amounts of [¹¹C]-3 were accumulated in the parenchyma. Furthermore, when cyclosporine A was preadministered to rats, the accumulation of [¹⁸F]-2 in the rat brain did not significantly change, however, the accumulation of [¹¹C]-3 was five times higher than that in the control rat. These results provide effective approaches for investigating the drug potential of ginkgolides.     

18F-Mefway PET Imaging of Serotonin 1A Receptors in Humans: A Comparison with 18F-FCWAY

IntroductionThe purpose of this research is to evaluate the prospects for the use of 4-(trans-¹⁸F-fluoranylmethyl)-N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexane-1-carboxamide (¹⁸F-Mefway) in comparison to ¹⁸F-trans-4-fluoro-N-2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (¹⁸F-FCWAY) for the quantification of 5-HT_1A receptors in human subjects.MethodFive healthy male controls were included for two positron emission tomography (PET) studies: ¹⁸F-FCWAY PET after the pretreatment with 500 mg of disulfiram and two months later, ¹⁸F-Mefway PET without disulfiram. Regional time-activity curves (TACs) were extracted from nine cortical and subcortical regions in dynamic PET images. Using cerebellar cortex without vermis as reference tissue, in vivo kinetics for both radioligands were compared based on the distribution volume ratio (DVR) calculated by non-invasive Logan graphical analysis and area under the curve ratio of the TACs (AUC ratio).ResultAlthough the pattern of regional uptakes in the ¹⁸F-Mefway PET was similar to that of the ¹⁸F-FCWAY PET (highest in the hippocampus and lowest in the cerebellar cortex), the amount of regional uptake in ¹⁸F-Mefway PET was almost half of that in ¹⁸F-FCWAY PET. The skull uptake in ¹⁸F-Mefway PET was only 25% of that in ¹⁸F-FCWAY PET with disulfiram pretreatment. The regional DVR values and AUC ratio values for ¹⁸F-Mefway were 17—40% lower than those of ¹⁸F-FCWAY. In contrast to a small overestimation of DVR values by AUC ratio values (< 10%) in ¹⁸F-FCWAY PET, the overestimation bias of AUC ratio values was much higher (up to 21%) in ¹⁸F-Mefway PET.ConclusionAs ¹⁸F-Mefway showed lower DVR values and greater overestimation bias of AUC ratio values, ¹⁸F-Mefway may appear less favorable than ¹⁸F-FCWAY. However, in contrast to ¹⁸F-FCWAY, the resistance to in vivo defluorination of ¹⁸F-Mefway obviates the need for the use of a defluorination inhibitor. Thus, ¹⁸F-Mefway may be a good candidate PET radioligand for 5-HT_1A receptor imaging in human.     

In Vitro and In Vivo Evaluation of a 18F-Labeled High Affinity NOTA Conjugated Bombesin Antagonist as a PET Ligand for GRPR-Targeted Tumor Imaging

Expression of the gastrin-releasing peptide receptor (GRPR) in prostate cancer suggests that this receptor can be used as a potential molecular target to visualize and treat these tumors. We have previously investigated an antagonist analog of bombesin (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂, RM26) conjugated to 1,4,7-triazacyclononane-N,N'',N''''-triacetic acid (NOTA) via a diethylene glycol (PEG₂) spacer (NOTA-P2-RM26) labeled with ⁶⁸Ga and ¹¹¹In. We found that this conjugate has favorable properties for in vivo imaging of GRPR-expression. The focus of this study was to develop a ¹⁸F-labelled PET agent to visualize GRPR. NOTA-P2-RM26 was labeled with ¹⁸F using aluminum-fluoride chelation. Stability, in vitro binding specificity and cellular processing tests were performed. The inhibition efficiency (IC₅₀) of the [^natF]AlF-NOTA-P2-RM26 was compared to that of the ^natGa-loaded peptide using ¹²⁵I-Tyr⁴-BBN as the displacement radioligand. The pharmacokinetics and in vivo binding specificity of the compound were studied. NOTA-P2-RM26 was labeled with ¹⁸F within 1 h (60-65% decay corrected radiochemical yield, 55 GBq/µmol). The radiopeptide was stable in murine serum and showed high specific binding to PC-3 cells. [^natF]AlF-NOTA-P2-RM26 showed a low nanomolar inhibition efficiency (IC₅₀=4.4±0.8 nM). The internalization rate of the tracer was low. Less than 14% of the cell-bound radioactivity was internalized after 4 h. The biodistribution of [¹⁸F]AlF-NOTA-P2-RM26 demonstrated rapid blood clearance, low liver uptake and low kidney retention. The tumor uptake at 3 h p.i. was 5.5±0.7 %ID/g, and the tumor-to-blood, -muscle and -bone ratios were 87±42, 159±47, 38±16, respectively. The uptake in tumors, pancreas and other GRPR-expressing organs was significantly reduced when excess amount of non-labeled peptide was co-injected. The low uptake in bone suggests a high in vivo stability of the Al-F bond. High contrast PET image was obtained 3 h p.i. The initial biological results suggest that [¹⁸F]AlF-NOTA-P2-RM26 is a promising candidate for PET imaging of GRPR in vivo.     

In vivo evaluation of IGF1R/IR PET ligand [18F]BMS-754807 in rodents

In vivo evaluation of [¹⁸F]BMS-754807 binding in mice and rats using microPET and biodistribution methods is described herein. The radioligand shows consistent binding characteristics, in vivo, in both species. Early time frames of the microPET images and time activity curves of brain indicate poor penetration of the tracer across the blood brain barrier (BBB) in both species. However, microPET experiments in mice and rats show high binding of the radioligand outside the brain to heart, pancreas and muscle, the organs known for higher expression of IGF1R/1R. Biodistribution analysis 2 h after injection of [¹⁸F]BMS-754807 in rats show negligible [¹⁸F]defluorination as reflected by the low bone uptake and clearance from blood. Overall, the data indicate that [¹⁸F]BMS-754807 can potentially be a radiotracer for the quantification of IGF1R/IR outside the brain using PET.     

[11C]MADAM Used as a Model for Understanding the Radiometabolism of Diphenyl Sulfide Radioligands for Positron Emission Tomography (PET)

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [¹¹C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [¹¹C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [¹¹C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 μM. Urine samples after perfusion of [¹¹C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [¹¹C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [¹¹C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.     

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a ⁶⁴Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with ⁶⁴Cu or ⁸⁹Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.     

Image Derived Input Function for [18F]-FEPPA: Application to Quantify Translocator Protein (18 kDa) in the Human Brain

In [¹⁸F]-FEPPA positron emission topography (PET) imaging, automatic blood sampling system (ABSS) is currently the gold standard to obtain the blood time activity curve (TAC) required to extract the input function (IF). Here, we compare the performance of two image-based methods of IF extraction to the ABSS gold standard method for the quantification of translocator protein (TSPO) in the human brain. The IFs were obtained from a direct delineation of the internal carotid signal (CS) and a new concept of independent component analysis (ICA). PET scans were obtained from 18 healthy volunteers. The estimated total distribution volume (V_T) by CS-IF and ICA-IF were compared to the reference V_T obtained by ABSS-IF in the frontal and temporal cortex, cerebellum, striatum and thalamus regions. The V_T values estimated using ICA-IF were more reliable than CS-IF for all brain regions. Specifically, the slope regression in the frontal cortex with ICA-IF was r² = 0.91 (p<0.05), and r² = 0.71 (p<0.05) using CS-IF.     

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO₂ or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP⁺ is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O₂ to H₂O₂. Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.