Facile synthesis and 18F-radiolabeling of α4ß1-specific LLP2A-aryltrifluoroborate peptidomimetic conjugates.

The peptidomimetic, LLP2A, is a specific, high-affinity ligand for α4ß1 integrin receptors. Previously, several PEGylated LLP2A conjugates were evaluated in vivo as imaging agents for the detection of lymphoma, leukemia, multiple myeloma and melanoma tumours via NIR-fluorescence, 111In-SPECT, and 64Cu- and 68Ga-PET imaging. Despite these successes, to date there is no report of an 18F-labeled LLP2A conjugate. Notably, fluorine-18 is a preferred radionuclide for PET imaging, yet its short half-life and general inactivity under aqueous conditions present challenges for peptide labeling. A simple method for labeling complex biomolecules can be achieved with arylboronic acids that readily capture aqueous [18F]-fluoride ion resulting in an 18F-labeled aryltrifluoroborate ([18F]-ArBF3-) radioprosthetic group. Herein, we present the first radiosynthesis of an 18F-labeled LLP2A conjugate by both one-step 18F-labeling and one-pot two-step 18F-labeling post-'click' conjugation of the 18F-alkynyl-ArBF3- prosthetic. Competition with a fluorescent conjugate of LLP2A demonstrated specific binding of the non-radioactive isotopolog ArBF3--PEG2-LLP2A to α4ß1 integrin-expressing MOLT-4 leukemia cells, as evidenced and confirmed by fluorescence microscopy. This work provides a key first step in the development of an expanding library of [18F]-R-BF3--LLP2A radiotracers for PET imaging.

H2CHXdedpa and H4CHXoctapa-chiral acyclic chelating ligands for (67/68)Ga and (111)In radiopharmaceuticals.

The chiral acyclic ligands H2CHXdedpa (N4O2), H2CHXdedpa-bb (N4O2), and H4CHXoctapa (N4O4) (CHX = cyclohexyl/cyclohexane, H2dedpa = 1,2-[[6-carboxy-pyridin-2-yl]-methylamino]ethane, bb = N,N'-dibenzylated, H4octapa = N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid) were synthesized, complexed with Ga(III) and/or In(III), and evaluated for their potential as chelating agents in radiopharmaceutical applications. The ligands were compared to the previously studied hexadentate H2dedpa and octadentate H4octapa ligands to determine the effect adding a chiral 1R,2R-trans-cyclohexane to replace the ethylenediamine backbone would have on metal complex stability and radiolabeling kinetics. It was found that [Ga(CHXdedpa)](+) showed very similar properties to those of [Ga(dedpa)](+), with only one isomer in solution observed by NMR spectroscopy, and minimal structural changes in the solid-state X-ray structure. Like [Ga(dedpa)](+), [Ga(CHXdedpa)](+) exhibited exceptionally high thermodynamic stability constants (log KML = 28.11(8)), and the chelate retained the ability to label (67)Ga quantitatively in 10 min at room temperature at ligand concentrations of 1 × 10(-5) M. In vitro kinetic inertness assays demonstrated the [(67)Ga(CHXdedpa)](+) complex to be more stable than [(67)Ga(dedpa)](+) in a human serum competition, with 90.5% and 77.8% of (67)Ga remaining chelate-bound after 2 h, respectively. Preliminary coordination studies of H4CHXoctapa with In(III) demonstrated [In(CHXoctapa)](-) to have an equivalently high thermodynamically stable constant as [In(octapa)](-), with log KML values of 27.16(9) and 26.76(14), respectively. The [(111)In(CHXoctapa)](-) complex showed exceptionally high in vitro kinetic inertness over 120 h in human serum, comparing well with previously reported [(111)In(octapa)](-) values, and an improved stability compared to the current industry "gold standards" 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and diethylenetriaminepentaacetic acid (DTPA). Initial investigations reveal that the chiral acyclic hexadentate H2CHXdedpa and octadentate H4CHXoctapa ligands are ideal candidates for radiopharmaceutical elaboration of gallium or indium isotopes, respectively.

Nitroimidazole-Containing H2dedpa and H2CHXdedpa Derivatives as Potential PET Imaging Agents of Hypoxia with (68)Ga.

(68)Ga is an attractive radiometal for use in positron emission tomography (PET) imaging. The success of (68)Ga-based agents is dependent on a chelator that exhibits rapid radiometal incorporation, and strong kinetic inertness to prevent transchelation of (68)Ga in vivo. The linear chelating agents H2dedpa (1,2-[[6-carboxypyridin-2-yl]methylamino]ethane) and H2CHXdedpa (CHX = cyclohexyl/cyclohexane) (N4O2) have recently been developed that bind Ga(3+) quickly and under mild conditions, ideal properties to be incorporated into a (68)Ga PET imaging agent. Herein, nitroimidazole (NI) derivatives of H2dedpa and H2CHXdedpa to investigate specific targeting of hypoxic tumor cells are investigated, given that NI can be reduced and retained exclusively in hypoxic cells. Nine N,N'-bis-alkylated derivatives of H2dedpa and H2CHXdedpa have been synthesized; they have been screened for their ability to bind gallium, and cyclic voltammetry of nonradioactive complexes was performed to probe the redox cycling mechanism of NI. The compounds were radiolabeled with (67)Ga and (68)Ga and show promising radiolabeling efficiencies (>99%) when labeled at 10(-5) M for 10 min at room temperature. Moreover, stability studies (via apo-transferrin challenge, 37 °C) show that the (67)Ga complexes exhibit exceptional stability (86-99% intact) after 2 h. In vitro uptake studies under hypoxic (0.5% O2) and normoxic (21% O2) conditions in three cancerous cell lines [HT-29 (colon), LCC6(HER-2) (breast), and CHO (Chinese hamster ovarian)] were performed. Of the four H2dedpa or H2CHXdedpa NI derivatives tested, all showed preferential uptake in hypoxic cells compared to normoxic cells with hypoxic/normoxic ratios as high as 7.9 ± 2.7 after 120 min. The results suggest that these novel bis-alkylated NI-containing H2dedpa and H2CHXdedpa ligands would be ideal candidates for further testing in vivo for PET imaging of hypoxia with (68)Ga.

What a difference a carbon makes: H4octapa vs H4C3octapa, ligands for In-111 and Lu-177 radiochemistry.

The acyclic ligands H4C3octapa and p-SCN-Bn-H4C3octapa were synthesized for the first time, using nosyl protection chemistry. These new ligands were compared to the previously studied ligands H4octapa and p-SCN-Bn-H4octapa to determine the extent to which the addition of a single carbon atom to the backbone of the ligand would affect metal coordination, complex stability, and, ultimately, utility for in vivo radiopharmaceutical applications. Although only a single carbon atom was added to H4C3octapa and the metal donor atoms and denticity were not changed, the solution chemistry and radiochemistry properties were drastically altered, highlighting the importance of careful ligand design and radiometal-ligand matching. It was found that [In(C3octapa)](-) and [Lu(C3octapa)](-) were substantially different from the analogous H4octapa complexes, exhibiting fluxional isomerization and a higher number of isomers, as observed by (1)H NMR, VT-NMR, and 2D COSY/HSQC-NMR experiments. Past evaluation of the DFT structures of [In(octapa)](-) and [Lu(octapa)](-) revealed very symmetric complexes; in contrast, the [In(C3octapa)](-) and [Lu(C3octapa)](-) complexes were much less symmetric, suggesting lower symmetry and less rigidity than that of the analogous H4octapa complexes. Potentiometric titrations revealed the formation constants (log K(ML), pM) were ~2 units lower for the In(3+) and Lu(3+) complexes of H4C3octapa when compared to that of the more favorable H4octapa ligand (~2 orders of magnitude less thermodynamically stable). The bifunctional ligands p-SCN-Bn-H4C3octapa and p-SCN-Bn-H4octapa were conjugated to the antibody trastuzumab and radiolabeled with (111)In and (177)Lu. Over a 5 day stability challenge experiment in blood serum, (111)In-octapa- and (111)In-C3octapa-trastuzumab immunoconjugates were determined to be ~91 and ~24% stable, respectively, and (177)Lu-octapa- and (177)Lu-C3octapa-trastuzumab, ~89% and ~4% stable, respectively. This work suggests that 5-membered chelate rings are superior to 6-membered chelate rings for large metal ions like In(3+) and Lu(3+), which is a crucial consideration for the design of bifunctional chelates for bioconjugation to targeting vectors for in vivo work.

H(4)octapa-trastuzumab: versatile acyclic chelate system for 111In and 177Lu imaging and therapy.

A bifunctional derivative of the versatile acyclic chelator H4octapa, p-SCN-Bn-H4octapa, has been synthesized for the first time. The chelator was conjugated to the HER2/neu-targeting antibody trastuzumab and labeled in high radiochemical purity and specific activity with the radioisotopes (111)In and (177)Lu. The in vivo behavior of the resulting radioimmunoconjugates was investigated in mice bearing ovarian cancer xenografts and compared to analogous radioimmunoconjugates employing the ubiquitous chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The H4octapa-trastuzumab conjugates displayed faster radiolabeling kinetics with more reproducible yields under milder conditions (15 min, RT, ~94-95%) than those based on DOTA-trastuzumab (60 min, 37 °C, ~50-88%). Further, antibody integrity was better preserved in the (111)In- and (177)Lu-octapa-trastuzumab constructs, with immunoreactive fractions of 0.99 for each compared to 0.93-0.95 for (111)In- and (177)Lu-DOTA-trastuzumab. These results translated to improved in vivo biodistribution profiles and SPECT imaging results for (111)In- and (177)Lu-octapa-trastuzumab compared to (111)In- and (177)Lu-DOTA-trastuzumab, with increased tumor uptake and higher tumor-to-tissue activity ratios.

Imaging of enzyme replacement therapy using PET.

Direct enzyme replacement therapy (ERT) has been introduced as a means to treat a number of rare, complex genetic conditions associated with lysosomal dysfunction. Gaucher disease was the first for which this therapy was applied and remains the prototypical example. Although ERT using recombinant lysosomal enzymes has been shown to be effective in altering the clinical course of Gaucher disease, Fabry disease, Hurler syndrome, Hunter syndrome, Maroteaux-Lamy syndrome, and Pompe disease, the recalcitrance of certain disease manifestations underscores important unanswered questions related to dosing regimes, tissue half-life of the recombinant enzyme and the ability of intravenously administered enzyme to reach critical sites of known disease pathology. We have developed an innovative method for tagging acid beta-glucocerebrosidase (GCase), the recombinant enzyme formulated in Cerezyme(R) used to treat Gaucher disease, using an (18)F-labeled substrate analogue that becomes trapped within the active site of the enzyme. Using micro-PET we show that the tissue distribution of injected enzyme can be imaged in a murine model and that the PET data correlate with tissue (18)F counts. Further we show that PET imaging readily monitors pharmacokinetic changes effected by receptor blocking. The ability to (18)F-label GCase to monitor the enzyme distribution and tissue half-life in vivo by PET provides a powerful research tool with an immediate clinical application to Gaucher disease and a clear path for application to other ERTs.

H4octapa: an acyclic chelator for 111In radiopharmaceuticals.

This preliminary investigation of the octadentate acyclic chelator H(4)octapa (N(4)O(4)) with (111)In/(115)In(3+) has demonstrated it to be an improvement on the shortcomings of the current industry "gold standards" DOTA (N(4)O(4)) and DTPA (N(3)O(5)). The ability of H(4)octapa to radiolabel quantitatively (111)InCl(3) at ambient temperature in 10 min with specific activities as high as 2.3 mCi/nmol (97.5% radiochemical yield) is presented. In vitro mouse serum stability assays have demonstrated the (111)In complex of H(4)octapa to have improved stability when compared to DOTA and DTPA over 24 h. Mouse biodistribution studies have shown that the radiometal complex [(111)In(octapa)](-) has exceptionally high in vivo stability over 24 h with improved clearance and stability compared to [(111)In(DOTA)](-), demonstrated by lower uptake in the kidneys, liver, and spleen at 24 h. (1)H/(13)C NMR studies of the [In(octapa)](-) complex revealed a 7-coordinate solution structure, which forms a single isomer and exhibits no observable fluxional behavior at ambient temperature, an improvement to the multiple isomers formed by [In(DTPA)](2-) and [In(DOTA)](-) under the same conditions. Potentiometric titrations have determined the thermodynamic formation constant of the [In(octapa)](-) complex to be log K(ML) = 26.8(1). Through the same set of analyses, the [(111/115)In(decapa)](2-) complex was found to have nonoptimal stability, with H(5)decapa (N(5)O(5)) being more suitable for larger metal ions due to its higher potential denticity (e.g., lanthanides and actinides). Our initial investigations have revealed the acyclic chelator H(4)octapa to be a valuable alternative to the macrocycle DOTA for use with (111)In, and a significant improvement to the acyclic chelator DTPA.

Evaluation of the H2)dedpa scaffold and its cRGDyK conjugates for labeling with 64Cu.

Studies of the acyclic ligand scaffold H(2)dedpa and its derivatives with the peptide cRGDyK for application in copper radiopharmaceuticals are described. Previously shown to be a superb ligand for (67/68)Ga, the chelate is now shown to coordinate (64)Cu in its derivatized and nonderivatized forms rapidly under mild reaction conditions (10 min, RT, pH 5.5 10 mM sodium acetate buffered solution). The hexadentate, distorted octahedral coordination of H(2)dedpa is confirmed in the corresponding solid state X-ray crystal structure of [Cu(dedpa)]. Cyclic voltammetry determined the reduction potential of [Cu(dedpa)] to be below values found for common bioreductants. Reduction and reoxidation were irreversible but reproducible, indicating a potential change of coordination mode upon reduction of Cu(II) to Cu(I). The thermodynamic stability constant log K(CuL) was determined to be 19.16(5), comparable to other frequently used (64)Cu chelates. Serum stability of the (64)Cu labeled chelate revealed only 3% transchelation/association to serum proteins after 2 h, while the conjugates reveal 10% ([Cu(RGD1)]) and 6% ([Cu(RGD2)]) transchelation at the same time point.

H(2)azapa: a versatile acyclic multifunctional chelator for (67)Ga, (64)Cu, (111)In, and (177)Lu.

Preliminary experiments with the novel acyclic triazole-containing bifunctional chelator H2azapa and the radiometals (64)Cu, (67)Ga, (111)In, and (177)Lu have established its significant versatile potential as an alternative to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for metal-based radiopharmaceuticals. Unlike DOTA, H2azapa radiolabels quantitatively with (64)Cu, (67)Ga, (111)In, and (177)Lu in 10 min at room temperature. In vitro competition experiments with human blood serum show that (64)Cu remained predominantly chelate-bound, with only 2% transchelated to serum proteins after 20 h. Biodistribution experiments with [(64)Cu(azapa)] in mice reveal uptake in various organs, particularly in the liver, lungs, heart, intestines, and kidneys. When compared to [(64)Cu(DOTA)](2-), the lipophilic neutral [(64)Cu(azapa)] was cleared through the gastrointestinal tract and accumulated in the liver, which is common for lipophilic compounds or free (64)Cu. The chelator H2azapa is a model complex for a click-based bifunctional chelating agent, and the lipophilic benzyl "place-holders" will be replaced by hydrophilic peptides to modulate the pharmacokinetics and direct activity away from the liver and gut. The solid-state molecular structure of [In(azapa)(H2O)][ClO4] reveals a very rare eight-coordinate distorted square antiprismatic geometry with one triazole arm bound, and the structure of [(64)Cu(azapa)] shows a distorted octahedral geometry. The present study demonstrates significant potential for bioconjugates of H2azapa as alternatives to DOTA in copper-based radiopharmaceuticals, with the highly modular and "clickable" molecular scaffold of H2azapa easily modified into a variety of bioconjugates. H2azapa is a versatile addition to the "pa" family, joining the previously published H2dedpa ((67/68)Ga and (64)Cu), H4octapa ((111)In, (177)Lu, and (90)Y), and H5decapa ((225)Ac) to cover a wide range of important nuclides.

Novel use of F-DOPA PET/CT imaging in a child with paraganglioma/pheochromocytoma syndrome.

We report the use of F-DOPA PET/CT imaging in the evaluation of a teenager with marked hypertension and right pararenal, left adrenal and left para-aortic mass lesions. The use of the modality for this clinical application has not been described previously within the pediatric imaging literature. The value of this technique relative to conventional imaging modalities is discussed and warrants consideration of its use, if available, for evaluating children with suspected paragangliomas/pheochromocytomas.

Acyclic chelate with ideal properties for (68)Ga PET imaging agent elaboration.

We have investigated novel bifunctional chelate alternatives to the aminocarboxylate macrocycles NOTA (N(3)O(3)) or DOTA (N(4)O(4)) for application of radioisotopes of Ga to diagnostic nuclear medicine and have found that the linear N(4)O(2) chelate H(2)dedpa coordinates (67)Ga quantitatively to form [(67)Ga(dedpa)](+) after 10 min at RT. Concentration-dependent coordination to H(2)dedpa of either (68)Ga or (67)Ga showed quantitative conversion to the desired products with ligand concentrations as low as 10(-7) M. With (68)Ga, specific activities as high as 9.8 mCi nmol(-1) were obtained without purification. In a 2 h competition experiment against human apo-transferrin, [(67)Ga(dedpa)](+) showed no decomposition. Two bifunctional versions of H(2)dedpa are also described, and these both coordinate to (67)Ga at RT within 10 min. Complete syntheses, characterizations, labeling studies, and biodistribution profiles of the (67)Ga complexes are presented for the new platform chelates. The stability of these platform chelates is higher than that of DOTA.

Effect of electroconvulsive therapy on brain 5-HT(2) receptors in major depression.

BACKGROUND: Brain serotonin(2) (5-hydroxytryptamine(2); 5-HT(2)) receptors were considered potential targets for therapeutic efficacy of electroconvulsive therapy (ECT), but pre-clinical studies showed that electroconvulsive shock up-regulates 5-HT(2) receptors in contrast to antidepressant medications, which down-regulate brain 5-HT(2) receptors. Positron emission tomography (PET) studies in individuals with depression confirmed that antidepressant medications reduce brain 5-HT(2) receptors, but the effects of ECT on these receptors in individuals with depression are unknown. AIMS: To determine if a course of ECT alters brain 5-HT(2) receptors in individuals with depression and whether such changes correlate with improvement in symptoms. METHOD: Fifteen people with major depression, refractory to antidepressant therapy and referred for a course of ECT, had an [18F]setoperone scan during baseline drug-free washout period and another after a course of ECT. We assessed changes in brain 5-HT(2) receptors with ECT and their relationship to therapeutic outcome. RESULTS: Widespread reduction in brain 5-HT(2) receptors was observed in all cortical areas with changes slightly more prominent in the right hemisphere. There was a trend for correlation between reduction in brain 5-HT(2) receptors in right parahippocampal gyrus, right lingual gyrus and right medial frontal gyrus, and improvement in depressive symptoms. CONCLUSIONS: Unlike in rodents, and similar to antidepressants, ECT reduces brain 5-HT(2) receptors in individuals with depression. The ability of ECT to further down-regulate brain 5-HT(2) receptors in antidepressant non-responsive individuals may explain its efficacy in those people with antidepressant refractory depression.

Design, synthesis, and imaging of small amphiphilic rhenium and (99m)technetium tricarbonyl complexes.

The design, synthesis, radiolabeling, evaluation of stability, and in vivo investigation are presented of three variably charged, novel, short C(8) chain derivatized chelators for the [M(CO)(3)](+) core (M = (99m)Tc or Re). Labeling with [(99m)Tc(CO)(3)](+) showed complex formation in under 1 h reaction time and high stability toward 24 h histidine and cysteine challenges, as well as distinctive log P values for each complex. Distinct localization of small amphiphilic molecules in in vivo systems depends on the charge of the polar moiety and was studied via biodistribution (4 h) and imaging (15 min, 1, 2, and 4 h) in female C57Bl/6 mice.

Irinophore C, a novel nanoformulation of irinotecan, alters tumor vascular function and enhances the distribution of 5-fluorouracil and doxorubicin.

PURPOSE: To examine the antitumor effects of Irinophore C, a nanopharmaceutical formulation of irinotecan, on the tissue morphology and function of tumor vasculature in HT-29 human colorectal tumors. EXPERIMENTAL DESIGN: Fluorescence microscopy was used to map and quantify changes in tissue density, tumor vasculature, hypoxia, and the distribution of Hoechst 33342, a perfusion marker, and the anticancer drug, doxorubicin. Noninvasive magnetic resonance imaging was used to quantify Ktrans, the volume transfer constant of a solute between the blood vessels and extracellular tissue compartment of the tumor, as a measure of vascular function. Following treatment with Irinophore C, 19F magnetic resonance spectroscopy was used to monitor the delivery of 5-fluorouracil (5-FU) to the tumor tissue, whereas scintigraphy was used to quantify the presence of bound [14C]5-FU. RESULTS: Irinophore C decreased cell density (P = 8.42 x 10(-5)), the overall number of endothelial cells in the entire section (P = 0.014), tumor hypoxia (P = 5.32 x 10(-9)), and K(trans) (P = 0.050). However, treatment increased the ratio of endothelial cells to cell density (P = 0.00024) and the accumulation of Hoechst 33342 (P = 0.022), doxorubicin (P = 0.243 x 10(-5)), and 5-FU (P = 0.0002) in the tumor. Vascular endothelial growth factor and interleukin-8, two proangiogenic factors, were down-regulated, whereas the antiangiogenic factor TIMP-1 was up-regulated in Irinophore C-treated tumors. CONCLUSIONS: Irinophore C treatment improves the vascular function of the tumor, thereby reducing tumor hypoxia and increasing the delivery and accumulation of a second drug. Reducing hypoxia would enhance radiotherapy, whereas improving delivery of a second drug to the tumor should result in higher cell kill.

Toward [18F]-labeled aryltrifluoroborate radiotracers: in vivo positron emission tomography imaging of stable aryltrifluoroborate clearance in mice.

The use of a boronic ester as a captor of aqueous [(18)F]-fluoride has been previously suggested as a means of labeling biomolecules in one step for positron emission tomography (PET) imaging. For this approach to be seriously considered, the [(18)F]-labeled trifluoroborate should be humorally stable such that it neither leaches free [(18)F]-fluoride to the bone nor accumulates therein. Herein, we have synthesized a biotinylated boronic ester that is converted to the corresponding trifluoroborate salt in the presence of aqueous [(18)F]-fluoride. In keeping with its in vitro aqueous kinetic stability at pH 7.5, the trifluoroborate appears to clear in vivo quite rapidly to the bladder as the stable trifluoroborate salt with no detectable leaching of free [(18)F]-fluoride to the bone. When this labeled biotin is preincubated with avidin, the pharmacokinetic clearance of the resulting complex is visibly altered. This work validates initial claims that boronic esters are potentially useful as readily labeled precursors to [(18)F]-PET reagents.

The effect of LRRK2 mutations on the cholinergic system in manifest and premanifest stages of Parkinson's disease: a cross-sectional PET study.

BACKGROUND: Markers of neuroinflammation are increased in some patients with LRRK2 Parkinson's disease compared with individuals with idiopathic Parkinson's disease, suggesting possible differences in disease pathogenesis. Previous PET studies have suggested amplified dopamine turnover and preserved serotonergic innervation in LRRK2 mutation carriers. We postulated that patients with LRRK2 mutations might show abnormalities of central cholinergic activity, even before the diagnosis of Parkinson's disease. METHODS: Between June, 2009, and December, 2015, we recruited participants from four movement disorder clinics in Canada, Norway, and the USA. Patients with Parkinson's disease were diagnosed by movement disorder neurologists on the basis of the UK Parkinson's Disease Society Brain Bank criteria. LRRK2 carrier status was confirmed by bidirectional Sanger sequencing. We used the PET tracer N-11C-methyl-piperidin-4-yl propionate to scan for acetylcholinesterase activity. The primary outcome measure was rate of acetylcholinesterase hydrolysis, calculated using the striatal input method. We compared acetylcholinesterase hydrolysis rates between groups using ANCOVA, with adjustment for age based on the results of linear regression analysis. FINDINGS: We recruited 14 patients with LRRK2 Parkinson's disease, 16 LRRK2 mutation carriers without Parkinson's disease, eight patients with idiopathic Parkinson's disease, and 11 healthy controls. We noted significant between-group differences in rates of acetylcholinesterase hydrolysis in cortical regions (average cortex p=0·009, default mode network-related regions p=0·006, limbic network-related regions p=0·020) and the thalamus (p=0·008). LRRK2 mutation carriers without Parkinson's disease had increased acetylcholinesterase hydrolysis rates compared with healthy controls in the cortex (average cortex, p=0·046). Patients with LRRK2 Parkinson's disease had significantly higher acetylcholinesterase activity in some cortical regions (average cortex p=0·043, default mode network-related regions p=0·021) and the thalamus (thalamus p=0·004) compared with individuals with idiopathic disease. Acetylcholinesterase hydrolysis rates in healthy controls were correlated inversely with age. INTERPRETATION: LRRK2 mutations are associated with significantly increased cholinergic activity in the brain in mutation carriers without Parkinson's disease compared with healthy controls and in LRRK2 mutation carriers with Parkinson's disease compared with individuals with idiopathic disease. Changes in cholinergic activity might represent early and sustained attempts to compensate for LRRK2-related dysfunction, or alteration of acetylcholinesterase in non-neuronal cells. FUNDING: Michael J Fox Foundation, National Institutes of Health, and Pacific Alzheimer Research Foundation.

55Cobalt complexes with pendant carbohydrates as potential PET imaging agents.

Bis-ligand cobalt(II) complexes of four 3-hydroxy-4-pyridinone ligands with pendant carbohydrates were synthesized and examined for their potential as radiopharmaceuticals. Non-radioactive complexes were prepared on the macroscopic scale and characterized by elemental analysis, mass spectrometry, IR and UV/visible spectroscopy. Facile radiolabeling produced the 55Co complexes in high radiochemical yields (>95%). Identification of the radiolabeled compounds was accomplished by HPLC comparison with the corresponding non-radioactive complexes.

Novel "bi-modal" H2dedpa derivatives for radio- and fluorescence imaging.

A novel pyridyl functionalized analog of the promising hexadentate 68Ga3+ chelate H2dedpa (N4O2, 1,2-[[6-carboxy-pyridin-2-yl]-methylamine]ethane) was successfully synthesized and characterized. This new bifunctional chelate (BFC) was used to prepare the first proof-of-principle bi-modal H2dedpa derivative for fluorescence and nuclear imaging. Two bi-modal H2dedpa derivatives were prepared: H2dedpa-propylpyr-FITC and H2dedpa-propylpyr-FITC-(N,N'-propyl-2-NI) (FITC=fluorescein, pyr=pyridyl functionalized, NI=nitroimidazole). The ligands possess the strong gallium-coordinating atoms contained within dedpa2- that are ideal for radiolabeling with 68Ga3+ for positron-emission tomography (PET) imaging, and two fluorophores for optical imaging. In addition, one analog contains two NI moieties for specific entrapment of the tracer in hypoxic cells. These new bi-modal analogs were compared to the native unfunctionalized H2dedpa scaffold to determine the extent to which the addition of pyridyl functionalization would affect metal coordination, and complex stability. The non-radioactive gallium complexes were tested in a 3D tumor spheroid model. The novel pyridyl bis-functionalized H2dedpa ligand, H2dedpa-propylpyr-NH2, was quantitatively radiolabeled with 67Ga (RCY>99%) under reaction conditions commensurate with unfunctionalized H2dedpa (10min at room temperature) at ligand concentrations as low as 10-5M. The resultant 67Ga-complex withstood transchelation to the in vivo metal-binding competitor apo-transferrin (2h at 37°C, 93% intact), signifying that [Ga(dedpa-propylpyr-NH2)]+ is a kinetically inert complex suitable for in vivo use, but exhibited slightly reduced stability compared to the native [67Ga(dedpa)] scaffold (>99% intact). Finally, bi-model fluorescent Ga-dedpa compounds were successfully imaged in a 3D tumor spheroid model. The Ga-dedpa-FITC-NI derivative was specifically localized in the central hypoxic core of the spheroid.

Evaluation of H2CHXdedpa, H2dedpa- and H2CHXdedpa-N,N'-propyl-2-NI ligands for (64)Cu(ii) radiopharmaceuticals.

The chiral acyclic "pa" ligand (pa = picolinic acid) H2CHXdedpa (N4O2) and two NI-containing dedpa analogues (H2CHXdedpa-N,N'-propyl-2-NI, H2dedpa-N,N'-propyl-2-NI, NI = nitroimidazole) were studied as chelators for copper radiopharmaceuticals (CHX = cyclohexyl, H2dedpa = 1,2-[[carboxypyridin-2-yl]methylamino]ethane). The hexadentate ligand H2CHXdedpa was previously established as a superb system for (67/68)Ga radiochemistry. The solid state X-ray crystal structures of [Cu(CHXdedpa-N,N'-propyl-2-NI)] and [Cu(dedpa-N,N'-propyl-2-NI)] reveal the predicted hexadentate, distorted octahedral binding of the copper(ii) ion. Cyclic voltammetry of [Cu(dedpa-N,N'-propyl-2-NI)] shows that there is one reversible couple associated with the NI redox, and one irreversible but reproducible couple attributed to the Cu(ii)/Cu(i) redox cycle. Quantitative radiolabeling (>99%) of CHXdedpa(2-) and (dedpa-N,N'-propyl-2-NI)(2-) with (64)Cu was achieved under fast and efficient labeling conditions (10 min, RT, 0.5 M sodium acetate buffer, pH 5.5) at ligand concentrations as low as 10(-6) M. In vitro kinetic inertness studies of the (64)Cu labelled complexes were studied in human serum at 37 °C over 24 hours; [(64)Cu(CHXdedpa)] was found to be 98% stable compared to previously investigated [(64)Cu(dedpa)] which was only 72% intact after 24 hours.