Chronic Administration of Δ9-Tetrahydrocannabinol Alters Brain Glucose Uptake and Improves Waiting Impulsivity in the Rat.

Introduction: Δ9-Tetrahydrocannabinol (THC) acts as an agonist at cannabinoid receptors. Its chronic intake affects many behaviors, including cognitive processes. The aims of this study in rats are to assess the chronic effects of THC on impulsivity and on regional brain glucose uptake. Materials and Methods: For the determination of "waiting impulsivity," a total of 20 male Lister Hooded rats were trained to perform a reaction time task, followed by a baseline test of impulsivity and baseline glucose uptake measurements with [18F]-fluoro-2-deoxy-D-glucose and positron emission tomography (PET). Then, 10 rats each received 3 mg/kg THC or vehicle injected intraperitoneally daily for 21 days. Subsequently, a second behavioral test and PET measurements were performed, and blood THC concentrations were determined. Analyses of variance of brain regions of the impulsivity network with the parameter "standardized uptake value" regarding glucose uptake and correlation analyses of the collected parameters were carried out. Discussion: After chronic THC treatment, decreased glucose uptake (p-values <0.05) was found in cingulate cortex, hippocampus, amygdala, thalamus, and cerebellar cortex, as compared with vehicle-treated rats. The number of correct no-go responses (increased waiting time) significantly increased (p<0.05) in THC-treated rats. Furthermore, correct no-go responses correlated positively and strongly with the THC blood concentrations (Spearman's ρ=0.79, p<0.01). Conclusion: These findings reflect a specific reduction in impulsive behavior after chronic THC treatment, showing a functionally relevant influence of THC on "waiting impulsivity" with reduced selective glucose uptake at the same time.

Genetic Code Expansion for Site-Specific Labeling of Antibodies with Radioisotopes.

Due to their target specificity, antibody-drug conjugates─monoclonal antibodies conjugated to a cytotoxic moiety─are efficient therapeutics that can kill malignant cells overexpressing a target gene. Linking an antibody with radioisotopes (radioimmunoconjugates) enables powerful diagnostics and/or closely related therapeutic applications, depending on the isotope. To generate site-specific radioimmunoconjugates, we utilized genetic code expansion and subsequent conjugation by inverse electron-demand Diels-Alder cycloaddition reactions. We show that, using this approach, site-specific labeling of trastuzumab with either zirconium-89 (89Zr) for diagnostics or lutetium-177 (177Lu) for therapeutics yields efficient radioimmunoconjugates. Positron emission tomography imaging revealed a high accumulation of site-specifically 89Zr-labeled trastuzumab in tumors after 24 h and low accumulation in other organs. The corresponding 177Lu-trastuzumab radioimmunoconjugates were comparably distributed in vivo.

Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA.

BACKGROUND: RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice. RESULTS: NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland. CONCLUSIONS: The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.

Characterization of activation induced [18]F-FDG uptake in Dendritic Cells.

AIM: Activation of immune cells leads to enhanced glucose uptake that can be visualized by [18]F-Fluorodeoxyglucose ([18]F-FDG) positron emission tomography/computed tomography (PET/CT). Dendritic cells (DC) are essential for the function of the adaptive immune system. In contrast to other immune cells metabolic changes leading to an increase of [18]F-FDG uptake are poorly investigated. Here, we analysed the impact of different DC activation pathways on their [18]F-FDG uptake. This effect was then used to radiolabel DC with [18]F-FDG and track their migration in vivo. METHODS: DC were generated from bone marrow progenitors (BMDC) or isolated from spleens (SPDC) of C57BL/6 mice. After stimulation with the TLR ligands LPS and CpG or anti-CD40 antibody for up to 72 hours activation markers and glucose transporters (GLUTs) were measured by flow cytometry. Uptake of [18]F-FDG was measured by gamma-counting. DC lysates were analysed for expression of glycolysis relevant proteins by mass spectrometry (MS). [18]F-FDG-labeled DC were injected into footpads of mice to image DC migration. RESULTS: BMDC and SPDC showed strong upregulation of activation markers predominantly 24 hours after TLR stimulation followed by higher uptake of [18]F-FDG. In line with this, the expression of GLUTs was upregulated during the course of activation. Furthermore, MS analyses of DC lysates revealed differential regulation of glycolysis relevant proteins according to the stimulatory pathway. As a proof of principle, DC were labeled with [18]F-FDG upon activation to follow their migration in vivo via PET/MRI. CONCLUSION: Immune stimulation of DC leads to enhanced [18]F-FDG uptake into DC, representing the typical shift to aerobic glycolysis in immune cells after activation.

Quantification of the Cannabinoid Type 1 Receptor Availability in the Mouse Brain.

Introduction: The endocannabinoid system is involved in several diseases such as addictive disorders, schizophrenia, post-traumatic stress disorder, and eating disorders. As often mice are used as the preferred animal model in translational research, in particular when using genetically modified mice, this study aimed to provide a systematic analysis of in vivo cannabinoid type 1 (CB1) receptor ligand-binding capacity using positron emission tomography (PET) using the ligand [18F]MK-9470. We then compared the PET results with literature data from immunohistochemistry (IHC) to review the consistency between ex vivo protein expression and in vivo ligand binding. Methods: Six male C57BL/6J (6-9 weeks) mice were examined with the CB1 receptor ligand [18F]MK-9470 and small animal PET. Different brain regions were evaluated using the parameter %ID/ml. The PET results of the [18F]MK-9470 accumulation in the mouse brain were compared with immunohistochemical literature data. Results: The ligand [18F]MK-9470 was taken up into the mouse brain within 5 min after injection and exhibited slow kinetics. It accumulated highly in most parts of the brain. PET and IHC classifications were consistent for most parts of the telencephalon, while brain regions of the diencephalon, mesencephalon, and rhombencephalon were rated higher with PET than IHC. Conclusions: This preclinical [18F]MK-9470 study demonstrated the radioligand's applicability for imaging the region-specific CB1 receptor availability in the healthy adult mouse brain and thus offers the potential to study CB1 receptor availability in pathological conditions.

Modeling Vestibular Compensation: Neural Plasticity Upon Thalamic Lesion.

The present study in rats was conducted to identify brain regions affected by the interruption of vestibular transmission and to explore selected aspects of their functional connections. We analyzed, by positron emission tomography (PET), the regional cerebral glucose metabolism (rCGM) of cortical, and subcortical cerebral regions processing vestibular signals after an experimental lesion of the left laterodorsal thalamic nucleus, a relay station for vestibular input en route to the cortical circuitry. PET scans upon galvanic vestibular stimulation (GVS) were conducted in each animal prior to lesion and at post-lesion days (PLD) 1, 3, 7, and 20, and voxel-wise statistical analysis of rCGM at each PLD compared to pre-lesion status were performed. After lesion, augmented metabolic activation by GVS was detected in cerebellum, mainly contralateral, and in contralateral subcortical structures such as superior colliculus, while diminished activation was observed in ipsilateral visual, entorhinal, and somatosensory cortices, indicating compensatory processes in the non-affected sensory systems of the unlesioned side. The changes in rCGM observed after lesion resembled alterations observed in patients suffering from unilateral thalamic infarction and may be interpreted as brain plasticity mechanisms associated with vestibular compensation and substitution. The second set of experiments aimed at the connections between cortical and subcortical vestibular regions and their neurotransmitter systems. Neuronal tracers were injected in regions processing vestibular and somatosensory information. Injections into the anterior cingulate cortex (ACC) or the primary somatosensory cortex (S1) retrogradely labeled neuronal somata in ventral posteromedial (VPM), posterolateral (VPL), ventrolateral (VL), posterior (Po), and laterodorsal nucleus, dorsomedial part (LDDM), locus coeruleus, and contralateral S1 area. Injections into the parafascicular nucleus (PaF), VPM/VPL, or LDDM anterogradely labeled terminal fields in S1, ACC, insular cortex, hippocampal CA1 region, and amygdala. Immunohistochemistry showed tracer-labeled terminal fields contacting cortical neurons expressing the µ-opioid receptor. Antibodies to tyrosine hydroxylase, serotonin, substance P, or neuronal nitric oxide-synthase did not label any of the traced structures. These findings provide evidence for opioidergic transmission in thalamo-cortical transduction.

Using immuno-PET imaging to monitor kinetics of T cell-mediated inflammation and treatment efficiency in a humanized mouse model for GvHD.

PURPOSE: Hematopoietic stem cell transplantation is the only curative treatment for several hematological malignancies and immune deficiency syndromes. Nevertheless, the development of graft-versus-host disease (GvHD) after transplantation is a severe complication with high morbidity and mortality. The aim of this study was to image human T cells during GvHD development and their migration into GvHD-related organs. By using a radiolabeled anti-human CD3 monoclonal antibody (mAb), we were able to visualize GvHD progression in a humanized mouse model. METHODS: Human peripheral blood mononuclear cells (PBMC) were transferred into immunodeficient mice (initially n = 11 mice/group) to induce GvHD. One group additionally received regulatory T cells (Treg) for prevention of GvHD. T cell migration was visualized by sequential small animal PET/MRI using 89Zr-labeled anti-human CD3 mAb. Flow cytometry and immunohistochemistry were used to measure T cell frequencies in relevant organs at different time points after engraftment. RESULTS: Using radiolabeled anti-CD3 mAb, we successfully visualized human T cells in inflamed organs of mice by 89Zr-anti-CD3-PET/MRI. Upon GvHD progression, we observed increased numbers of CD3+ T cells in the liver (22.9% on day 3; 94.2% on day 10) and the spleen (4.4% on day 3; 58.8% on day 10) which correlated with clinical symptoms. The liver showed distinct spot-like lesions representing a strong focal accumulation of T cells. Administration of Treg prior GvHD induction reduced T cell accumulation in the liver from 857 ± 177 CD3+ cells/mm2 to 261 ± 82 CD3+ cells/mm2 and thus prevented GvHD. CONCLUSION: 89Zr-labeled anti-human CD3 mAb can be used as a proof of concept to detect the exact spatio-temporal distribution of GvHD-mediating T cells. In the future, radiolabeled T cell-specific mAb could be employed as a predictive early biomarker during the course of GvHD maybe even before clinical signs of the disease become evident. Furthermore, monitoring T cell migration and proliferation might improve tailored GvHD therapy.

Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging.

BACKGROUND: Pretargeted imaging allows the use of short-lived radionuclides when imaging the accumulation of slow clearing targeting agents such as antibodies. The biotin-(strept)avidin and the bispecific antibody-hapten interactions have been applied in clinical pretargeting studies; unfortunately, these systems led to immunogenic responses in patients. The inverse electron demand Diels-Alder (IEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO)-functionalized targeting vector is a promising alternative for clinical pretargeted imaging due to its fast reaction kinetics. This strategy was first applied in nuclear medicine using an 111In-labelled Tz to image TCO-functionalized antibodies in tumour-bearing mice. Since then, the IEDDA has been used extensively in pretargeted nuclear imaging and radiotherapy; however, these studies have only been performed in mice. Herein, we report the 44Sc labelling of a Tz and evaluate it in pretargeted imaging in Wistar rats. RESULTS: 44Sc was obtained from an in house 44Ti/44Sc generator. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized tetrazine was radiolabelled with 44Sc resulting in radiochemical yields of 85-95%, a radiochemical purity > 99% at an apparent molar activity of 1 GBq/mmol. The 44Sc-labelled Tz maintained stability in solution for up to 24 h. A TCO-functionalized bisphosphonate, which accumulates in skeletal tissue, was used as a targeting vector to evaluate the 44Sc-labelled Tz. Biodistribution data of the 44Sc-labelled Tz showed specific uptake (0.9 ± 0.3% ID/g) in the bones (humerus and femur) of rats pre-treated with the TCO-functionalized bisphosphonate. This uptake was not present in rats not receiving pre-treatment (< 0.03% ID/g). CONCLUSIONS: We have prepared a 44Sc-labelled Tz and used it in pretargeted PET imaging with rats treated with TCO-functionalized bisphosponates. This allowed for the evaluation of the IEDDA reaction in animals larger than a typical mouse. Non-target accumulation was low, and there was a 30-fold higher bone uptake in the pre-treated rats compared to the non-treated controls. Given its convenient half-life and the ability to perform positron emission tomography with a previously studied DOTA-functionalized Tz, scandium-44 (t1/2 = 3.97 h) proved to be a suitable radioisotope for this study.

68Ga[Ga]-, 111In[In]-oxine: a novel strategy of in situ radiolabeling of HPMA-based micelles.

Polymeric micelles are of increasing interest as drug delivery vehicles since they can accumulate in tumor tissue through EPR effect and deliver their hydrophobic cargo. The pharmacology can be visualized and quantified noninvasively by molecular imaging techniques. Here, a novel, fast and efficient technique for radiolabeling various HPMA-LMA based micellar aggregates with hydrophobic oxine-complexes of the trivalent radiometals 68Ga and 111In was investigated. The radiometal-oxine complexes resemble the hydrophobic drug 111In[In]-oxine considered for the diagnosis of infection and inflammation. Promising in vitro stability lead to in vivo evaluation in healthy mice in terms of quantitative ex vivo organ distribution. The results show that while the hydrophobic radiometal-oxine complexes were safely encapsulated in aqueous saline, they left the polymeric micelles slowly in contact with blood serum and more rapidly in vivo. Due to the similarity between the radiometal complexes and hydrophobic drugs transported in the polymeric micelles this has significant implications for further strategies on transport mechanisms of hydrophobically encapsulated drugs.

In vivo imaging of the immune response upon systemic RNA cancer vaccination by FDG-PET.

BACKGROUND: [18F]Fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-PET) is commonly used in the clinic for diagnosis of cancer and for follow-up of therapy outcome. Additional to the well-established value in tumor imaging, it bears potential to depict immune processes in modern immunotherapies. T cells enhance their glucose consumption upon activation and are crucial effectors for the success of such novel therapies. In this study, we analyzed the T cell immunity in spleen after antigen-specific stimulation of T cells via highly innovative RNA-based vaccines using FDG-PET/MRI. For this purpose, we employed systemic administration of RNA-lipoplexes encoding the endogenous antigen of Moloney murine leukemia virus (gp70) which have been previously shown to induce potent innate as well as adaptive immune mechanisms for cancer immunotherapy. Feasibility of clinical imaging of increased splenic FDG uptake was demonstrated in a melanoma patient participating in a clinical phase 1 trial of a tetravalent RNA-lipoplex cancer vaccine. RESULTS: We observed exclusive increase of glucose uptake in spleen compared to other organs thanks to liposome-mediated RNA targeting to this immune-relevant organ. In vivo and ex vivo FDG uptake analysis in the spleen of vaccinated mice correlated well with antigen-specific T cell activation. Moreover, the use of an irrelevant (antigen non-specific) RNA also resulted in enhanced FDG uptake early after vaccination through the activation of several other splenic cell populations. The glucose uptake was also dependent on the dose of RNA administered in line with the activation and frequencies of proliferating antigen-specific T cells as well as the general activation pattern of splenic cell populations. CONCLUSIONS: Our preclinical results show rapid and transient vaccination-induced increase of FDG uptake within the spleen reflecting immune activation preceding T cell proliferation. FDG-PET/CT in patients is also capable to image this immune activation resulting in a new potential application of FDG-PET/CT to image immune processes in new immunological therapies.

In vivo Evaluation of [225Ac]Ac-DOTAZOL for α-Therapy of Bone Metastases.

BACKGROUND: Conjugates of bisphosphonates with macrocyclic chelators possess high potential in bone targeted radionuclide imaging and therapy. DOTAZOL, zoledronic acid conjugated to DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), demonstrated promising results in vivo in small animals as well as in first patient applications using 68Ga for diagnosis via PET and the lowenergy ß-emitter 177Lu for therapy of painful bone metastases. In consideration of the fact that targeted α-therapy probably offers various advantages over the use of ß--emitters, the 225Ac-labelled derivative [225Ac]Ac-DOTAZOL was synthesized and evaluated in vivo. Here, we report on radiolabelling and biodistribution of [225Ac]Ac-DOTAZOL in healthy Wistar rats. METHODS: DOTAZOL was labelled with 225Ac and injected without further purification into the tail vein with activities of 404 ± 47 kBq per animal. Ex vivo biodistribution studies were performed in healthy Wistar rats at 1 hour, 24 hours, 5 days and 10 days post injection. The accumulation of [225Ac]Ac- DOTAZOL on healthy bone and soft tissue organs was determined in terms of SUV. The results were compared to those of other radiolabelled bisphosphonates such as [68Ga]Ga-DOTAZOL and [177Lu]Lu- DOTAZOL. A group of 7 animals was observed over a period of 3 month after application of 394 kBq ± 10 kBq of [225Ac]Ac-DOTAZOL for signs of toxicity. After 3 months, kidneys were microscopically analysed for signs of chronic kidney damage. RESULTS: Radiolabelling of DOTAZOL with 225Ac at 98 °C provided radiochemical yields ≥98 % within 30 minutes. [225Ac]Ac-DOTAZOL showed high femur uptake (SUVfemur = 4.99 ± 0.97, 10 d p.i.), which was comparable to that of other Me(III)-DOTAZOL derivatives. Ratios between bone uptake and blood pool activity reached levels of 5, 940, 2181 and 2409 at 1 hour, 24 hours, 5 days and 10 days post injection. During the observation period of the first two month no toxicity was observed clinically. Histopathology of kidneys after 3 month revealed significant tubular damage in most of the animals. CONCLUSION: [225Ac]Ac-DOTAZOL repeats the well-known pharmacology of DOTAZOL derivatives in preclinical evaluations. It thus may be considered for translational application together with strategies to reduce renal toxicity.

Long-term biodistribution study of HPMA-ran-LMA copolymers in vivo by means of 131I-labeling.

BACKGROUND: For the evaluation of macromolecular drug delivery systems suitable pre-clinical monitoring of potential nanocarrier systems is needed. In this regard, both short-term as well as long-term in vivo tracking is crucial to understand structure-property relationships of polymer carrier systems and their resulting pharmacokinetic profile. Based on former studies revealing favorable in vivo characteristics for 18F-labeled random (ran) copolymers consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) and lauryl methacrylate (LMA) - including prolonged plasma half-life as well as enhanced tumor accumulation - the presented work focuses on their long-term investigation in the living organism. METHODS: In this respect, four different HPMA-based polymers (homopolymers as well as random copolymers with LMA as hydrophobic segment) were synthesized and subsequent radioactive labeling was accomplished via the longer-lived radioisotope 131I. In vivo results, concentrating on the pharmacokinetics of a high molecular weight HPMA-ran-LMA copolymer, were obtained by means of biodistribution and metabolism studies in the Walker 256 mammary carcinoma model over a time-span of up to three days. Besides, a direct comparison with the 18F-radiolabeled polymer was drawn. To consider physico-chemical differences between the differently labeled polymer (18F or 131I) on the critical micelle concentration (CMC) and the size of the polymeric micelles, those properties were determined using the 19F- or 127I-functionalized polymer. Special emphasis was laid on the time-dependent correlation between blood circulation properties and corresponding tumor accumulation, particularly regarding the enhanced permeability and retention (EPR) effect. RESULTS: Studies revealed, at first, differences in the short time (2h) body distribution, despite the very similar properties (molecular structure, CMC and size of the micellar aggregates) of the non-radioactive 19F- and 127I-functionalized polymers. Long-term investigations with the 131I-labeled polymer demonstrated that, despite a polymer clearance from the blood within 72h, there was still an increase in tumor uptake observed over time. Regarding the stability of the 131I-label, ex vivo biodistribution experiments, considering the uptake in the thyroid, indicated low metabolism rates. CONCLUSION: The observed in vivo characteristics strongly underline the EPR effect. The findings illustrate the need to combine information of different labeling approaches and in vivo evaluation techniques to generate an overall pharmacokinetic picture of potential nanocarriers in the pre-clinical setting. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENTS: The in vivo behavior of the investigated HPMA-ran-LMA copolymer demonstrates great potential in terms of an effective accumulation in the tumor.

Highly Loaded Semipermeable Nanocapsules for Magnetic Resonance Imaging.

Magnetic resonance imaging has become an essential tool in medicine for the investigation of physiological processes. The key issues related to contrast agents, i.e., substances that are injected in the body for imaging, are the efficient enhancement of contrast, their low toxicity, and their defined biodistribution. Polyurea nanocapsules containing the gadolinium complex Gadobutrol as a contrast agent in high local concentration and high relaxivity up to 40 s-1 mmol-1 L are described. A high concentration of the contrast agent inside the nanocapsules can be ensured by increasing the crystallinity in the shell of the nanocapsules. Nanocapsules from aliphatic polyurea are found to display higher crystallinity and higher relaxivity at an initial Gadobutrol concentration of 0.1 m than aromatic polyurea nanocapsules. The nanocapsules and the contrast agent are clearly identified in cells. After injection, the nanocarriers containing the contrast agent are mostly found in the liver and in the spleen, which allow for a significant contrast enhancement in magnetic resonance imaging.

Toll like receptor mediated immune stimulation can be visualized in vivo by [18F]FDG-PET.

INTRODUCTION: High uptake of [18F]-2-fluorodeoxyglucose ([18F]FDG) by inflammatory cells is a frequent cause of false positive results in [18F]FDG-positron-emission tomography (PET) for cancer diagnostics. Similar to cancer cells, immune cells undergo significant increases in glucose utilization following activation, e.g., in infectious diseases or after vaccination during cancer therapy. The aim of this study was to quantify certain immune effects in vitro and in vivo by [18F]FDG-PET after stimulation with TLR ligands and specific antibodies. METHODS: In vivo [18F]FDG-PET/magnetic resonance imaging (MRI) and biodistribution was performed with C57BL/6 mice immunized with CpG or LPS. Cellular [18F]FDG-uptake assays were performed with B cells and T cells or with whole spleen cells after stimulation with CpG, LPS and anti-CD3/CD28. In vitro and in vivo activation of B and T cells was examined by concomitant FACS analysis to correlate immune cell activation with the strength of [18F]FDG accumulation. RESULTS: We could show that TLR mediated activation of B cells increases [18F]FDG uptake, and that B cells show faster kinetics and greater effect than T cells stimulated by the CD3/CD28 pathway. In the whole spleen cell population the [18F]FDG signal was triggered mainly by the activation of B cells, corresponding closely to expression of typical stimulation markers. This finding could also been seen in vivo in [18F]FDG-PET/MRI, where the spleen was clearly visible after TLR stimulation and B cells showed upregulation of CD80 and CD86. CONCLUSION: In vivo TLR stimulation can be visualized by increased [18F]FDG uptake in lymphoid organs. The signal generated in the spleen after immunization might be mainly attributed to the activation of B cells within. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Knowledge of the composition of cells that take up [18F]FDG during vaccination or in response to therapy may improve successful treatment of cancer patients in the future.

Fate of linear and branched polyether-lipids in vivo in comparison to their liposomal formulations by 18F-radiolabeling and positron emission tomography.

In this study, linear poly(ethylene glycol) (PEG) and novel linear-hyperbranched, amphiphilic polyglycerol (hbPG) polymers with cholesterol (Ch) as a lipid anchor moiety were radiolabeled with fluorine-18 via copper-catalyzed click chemistry. In vivo investigations via positron emission tomography (PET) and ex vivo biodistribution in mice were conducted. A systematic comparison to the liposomal formulations with and without the polymers with respect to their initial pharmacokinetic properties during the first hour was carried out, revealing remarkable differences. Additionally, cholesterol was directly labeled with fluorine-18 and examined likewise. Both polymers, Ch-PEG27-CH2-triazole-TEG-(18)F and Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F (TEG: triethylene glycol), showed rapid renal excretion, whereas the (18)F-cholesten displayed retention in lung, liver, and spleen. Liposomes containing Ch-PEG27-CH2-triazole-TEG-(18)F revealed a hydrodynamic radius of 46 nm, liposomal Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F showed a radius of 84 nm and conventional liposomes with (18)F-cholesten 204 nm, respectively. The results revealed fast uptake of the conventional liposomes by liver, spleen, and lung. Most importantly, the novel hbPG-polymer stabilized liposomes showed similar behavior to the PEG-shielded vesicles. Thus, an advantage of multifunctionality is achieved with retained pharmacokinetic properties. The approach expands the scope of polymer tracking in vivo and liposome tracing in mice via PET.

Selective binding to monoamine oxidase A: in vitro and in vivo evaluation of (18)F-labeled ß-carboline derivatives.

In this study we synthesized four different (18)F-labeling precursors for the visualization of the monoamino oxidase A using harmol derivatives. Whereas two are for prosthetic group labeling using [(18)F]fluoro-d2-methyl tosylate and 2-[(18)F]fluoroethyl-tosylate, the other three precursors are for direct nucleophilic (18)F-labeling. Additionally the corresponding reference compounds were synthesized. The syntheses of [(18)F]fluoro-d2-methyl-harmol and 2-[(18)F]fluoroethyl-harmol were carried out using harmol as starting material. For direct nucleophilic (18)F-labeling of the tracers carrying oligoethyled spacers (PEG), a toluenesulfonyl leaving group was employed. The radiolabeling, purification and formulation for each tracer was optimized and evaluated in vitro and in vivo. Stability tests in human serum showed that all tracers were stable over the observation period of 60 min. µPET studies using of the synthesized tracers revealed that the tracers carrying PEG spacers showed no sufficient brain uptake. Consequently, the (18)F-fuoro alkylated tracers [(18)F]fluoro-d2-methyl-harmol and 2-[(18)F]fluoroethyl-harmol were further evaluated showing SUVs in the brain of 1.0±0.2 g/mL and 3.4±0.5 g/mL after 45 min, respectively. In blockade studies the selectivity and specificity of both tracers were demonstrated. However, for [(18)F]fluoro-d2-methyl-harmol a rapid washout from the brain was also observed. In vitro binding assays revealed that 2-[(18)F]fluoroethyl-harmol (IC50=0.54±0.06 nM) has a higher affinity than the (18)F-fluoro-d2-methylated ligand (IC50=12.2±0.6 nM), making 2-[(18)F]fluoroethyl-harmol superior to the other evaluated compounds and a promising tracer for PET imaging of the MAO A.

18F-Radiolabeling, preliminary evaluation of folate-pHPMA conjugates via PET.

The synthesis of a 10.5 kDa and a 52.5 kDa polymer, based on pHPMA functionalized with tyramine for (18) F-labeling and a folate derivative as targeting moiety, is reported. FCS studies are conducted using Oregon Green-labeled conjugates. No aggregation is observed for the 10.5 kDa conjugate, but strong aggregation for the 52.5 kDa conjugate. In vivo studies are conducted using Walker-256 mammary carcinoma model to determine body distribution as function of size and especially targeting unit. These in vivo studies show a higher short time (2 h) accumulation for both conjugates in the tumor than for untargeted pHPMA, confirmed by blockade studies. The 10.5 kDa polymer accumulates with 0.46% ID g(-1) and the 52.5 kDa polymer with 0.28% ID g(-1) in the tumor after 2 h, demonstrating the potential of the folate-targeting concept.

In vitro and in vivo structure-property relationship of (68)Ga-labeled Schiff base derivatives for functional myocardial pet imaging.

PURPOSE: SPECT (e.g., with (99m)Tc-sestamibi) is routinely used for imaging myocardial damage, even though PET could offer a higher spatial resolution. Using the generator-gained isotope (68)Ga would allow a rapid supply of the tracer in the diagnostic unit. For this reason, the aim of the study was to develop (68)Ga-labeled PET tracers based on different Schiff base amines and to evaluate the cardiomyocyte uptake in vitro as well as the biodistribution of the tracers in vivo. PROCEDURES: Fifteen different Schiff bases (basing on 3 different backbones) were synthesized and labeled with (68)Ga. Lipophilicity varied between 0.87 ± 0.24 and 2.72 ± 0.14 (logD value). All tracers were positively charged and stable in plasma and apo-transferrin solution. In vitro uptake into cardiomyocytes was assessed in HL-1 cells in the absence and presence of the ionophor valinomycin. In vivo accumulation in the heart and in various organs was assessed by small animal PET imaging as well as by ex vivo biodistribution. The results were compared with (99m)Tc-sestamibi and (18)F-flurpiridaz. RESULTS: All cationic Schiff bases were taken up into cardiomyocytes but the amount varied by a factor of 10. When destroying the membrane potential, the cellular uptake was markedly reduced in most of the tracers, indicating the applicability of these tracers for identifying ischemic myocardium. PET imaging revealed that the in vivo myocardial uptake reached a constant value approximately 10 min after injection but the intracardial amount of the tracer varied profoundly (SUV 0.46 to 3.35). The most suitable tracers showed a myocardial uptake which was comparable to that of (99m)Tc-sestamibi. CONCLUSIONS: (68)Ga-based Schiff bases appear suitable for myocardial PET images with uptake comparable to (99m)Tc-sestamibi but offering higher spatial resolution. By systematical variation of the backbone and the side chains, tracers with optimal properties can be identified for further clinical evaluation.

Evaluation of P-glycoprotein (abcb1a/b) modulation of [(18)F]fallypride in MicroPET imaging studies.

[(18)F]Fallypride ([(18)F]FP) is an important and routinely used D2/D3 antagonist for quantitative imaging of dopaminergic neurotransmission in vivo. Recently it was shown that the brain uptake of the structurally related [(11)C]raclopride is modulated by P-glycoprotein (P-gp), an important efflux transporter at the blood-brain barrier. The purpose of this study was to determine whether the brain uptake of [(18)F]FP is influenced by P-gp. For examination of this possible modulation microPET studies were performed in a rat and a mouse model. Hence, [(18)F]FP was applied to Sprague Dawley rats, half of them being treated with the P-gp inhibitor cyclosporine A (CsA). In a second experimental series the tracer was applied to three different groups of FVB/N mice: wild type, P-gp double knockout (abcb1a/1b (-/-)) and CsA-treated mice. In CsA-treated Sprague Dawley rats [(18)F]FP showed an elevated standard uptake value in the striatum compared to the control animals. In FVB/N mice a similar effect was observed, showing an increasing uptake from wild type to CsA-treated and double knockout mice. Since genetically or pharmacologically induced reduction of P-gp activity increased the uptake of [(18)F]FP markedly, we conclude that [(18)F]FP is indeed a substrate of P-gp and that the efflux pump modulates its brain uptake. This effect - if true for humans - may have particular impact on clinical studies using [(18)F]FP for assessment of D2/3 receptor occupancy by antipsychotic drugs. This article is part of the Special Issue Section entitled 'Neuroimaging in Neuropharmacology'.

An allogenic site-specific rat model of bone metastases for nuclear medicine and experimental oncology.

Bone metastases are a major problem in several tumor entities affecting the therapeutic decision and the patient's prognosis. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are promising techniques for identifying bone tumors using gamma- or positron-emitting labeled radiotracers, but the same tracers if labeled with beta-emitters may also be used to apply therapeutic radionuclides for localized irradiation. For the tracer development specifically accumulating in osseous lesions, animal models of bone metastasis are needed. A technique was developed for tumor cell injection into the circulation of the hind limb of rats. For tumor implantation, the arteria epigastrica caudalis superficialis (a branch of the femoral artery) was cannulated, and 2×10(5) cells were injected. By using the allogenic Walker 256 mammary carcinoma cell line, isolated bone metastases were induced. For visualizing of the tumor growth, PET with 18F-fluoride was performed weekly on a µ-PET system. After 2-3 weeks, tumor invasion was confirmed by histology. Three weeks after tumor cell inoculation, PET images showed signs of bone metastases in 9 out of 11 animals. The tumors were located either in the proximal tibia/fibula or in the distal femur. At this time, the animals showed no restrictions in mobility. The tumors grew constantly over time. The final histological analysis showed tumors growing invasively into the bone matrix. With this model, new SPECT or PET tracers can be evaluated for their potency of accumulating in bone metastases in vivo and to determine which are therefore suitable for diagnosis and/or therapy.