A Systematic Investigation into the Influence of Net Charge on the Biological Distribution of Radiometalated Peptides Using [68Ga]Ga-DOTA-TATE Derivatives.

Recently, several radiometalated peptides have been approved for clinical imaging and/or therapy (theranostics) of several types of cancer; nonetheless, the primary challenge that most of these peptides confront is significant renal uptake and retention, which is often dose limiting and can cause nephrotoxicity. In response to this, numerous methods have been employed to reduce the uptake of radiometalated peptides in the kidneys, and among these is adding a linker to modulate polarity and/or charge. To better understand the influence of net charge on the biodistribution of radiometalated peptides, we selected the clinically popular construct DOTA-TATE (NETSPOT/LUTATHERA) as a model system. We synthesized derivatives using manual solid-phase peptide synthesis methods including mechanical and ultrasonic agitation to effectively yield the gold standard DOTA-TATE and a series of derivatives with different net charges (+2, +1, 0, -1, -2). Dynamic PET imaging from 0 to 90 min in healthy female mice (CD1) revealed high accumulation and retention of activity in the kidneys for the net-neutral (0) charged [68Ga]Ga-DOTA-TATE and even higher for positively charged derivatives, whereas negatively charged derivatives exhibited low accumulation and fast renal excretion. Ex vivo biodistribution at 2 h post injection demonstrated a significant retention of [68Ga]Ga-DOTA-TATE (∼74 %ID/g) in the kidneys, which increased as the net positive charge per molecule increased to +1 and +2 (∼272 %ID/g and ∼333 %ID/g, respectively), but the -1 and -2 net charged molecules exhibited lower renal uptake (∼15 %ID/g and 16 %ID/g, respectively). Interestingly, the net -2 charged [68Ga]Ga-DOTA-(Glu)2-PEG4-TATE was stable in blood serum but had much higher healthy organ uptake (lungs, liver, spleen) than the net -1 compound, suggesting instability in vivo. Although the [68Ga]Ga-DOTA-PEG4-TATE derivative with a net charge of 0 also showed a decrease in kidney uptake, it also showed instability in blood serum and in vivo. Despite the superior pharmacokinetics of the net -1 charged [68Ga]Ga-DOTA-Glu-PEG4-TATE in healthy mice with respect to kidney uptake and overall profile, dynamic PET images and ex vivo biodistribution in male mice (NSG) bearing AR42J (SSTR2 overexpressing) subcutaneous tumor xenografts showed significantly diminished tumor uptake when compared to the gold standard [68Ga]Ga-DOTA-TATE. Taken together, these findings indicate unambiguously that kidney uptake and retention are significantly influenced by the net charge of peptide-based radiotracers. In addition, it was illustrated that the negatively charged peptides had substantially decreased kidney uptake, but in this instantiation the tumor uptake was also impaired.

The Chemistry of Creating Chemically Programmed Antibodies (cPAbs): Site-Specific Bioconjugation of Small Molecules.

Small-molecule drugs have been employed for years as therapeutics in the pharmaceutical industry. However, small-molecule drugs typically have short in vivo half-lives which is one of the largest impediments to the success of many potentially valuable pharmacologically active small molecules. The undesirable pharmacokinetics and pharmacology associated with some small molecules have led to the development of a new class of bioconjugates known as chemically programmed antibodies (cPAbs). cPAbs are bioconjugates in which antibodies are used to augment small molecules with effector functions and prolonged pharmacokinetic profiles, where the pharmacophore of the small molecule is harnessed for target binding and therefore biological targeting. Many different small molecules can be conjugated to large proteins such as full monoclonal antibodies (IgG), fragment crystallizable regions (Fc), or fragment antigen binding regions (Fab). In order to successfully and site-specifically conjugate small molecules to any class of antibodies (IgG, Fc, or Fab), the molecules must be derivatized with a functional group for ease of conjugation without altering the pharmacology of the small molecules. In this Review, we summarize the different synthetic or biological methods that have been employed to produce cPAbs. These unique chemistries have potential to be applied to other fields of antibody modification such as antibody drug conjugates, radioimmunoconjugates, and fluorophore-tagged antibodies.

DFO-Km: A Modular Chelator as a New Chemical Tool for the Construction of Zirconium-89-Based Radiopharmaceuticals.

Zirconium-89-labeled monoclonal antibodies and other large macromolecules such as nanoparticles hold great promise as positron emission tomography imaging agents. In general, zirconium-89 is an ideal radionuclide for long-circulating vectors such as antibodies or nanoparticles. It is also a promising radionuclide for theranostic radiopharmaceuticals due to its suitable match in half-life with actinium-225, thorium-227, lutetium-177, and others. As such, demand for new and optimized bifunctional chelators for zirconium-89 continues to grow. Herein, we present the modular chelator DFO-Km, which is octadentate and features lysine as a modular amino acid linker. The modular amino acid linker can be changed to other natural or unnatural amino acids to access different bioconjugation chemistries, while the chelating portion is unchanged thus retaining identical metal ion coordination properties to DFO-Km. The epsilon-amine in the DFO-Km linker (lysine) was used to complete synthesis of a bifunctional derivative bearing a p-SCN-Ph moiety. The chelator DFO-Km includes a redesigned hydroxamic acid, which provides more flexibility for metal ion coordination relative to the monomer used in the previously published DFO-Em. Moreover, a set of comprehensive DFT calculations were performed to model and evaluate 16 geometric isomers of Zr-(DFO-Km), which suggested the complex would form the optimum cic-cis-trans-trans octadentate Zr(IV) coordination geometry with no aqua or hydroxide ligands present. The bifunctional derivative p-SCN-Ph-DFO-Km was compared directly with the commercially available p-SCN-Ph-DFO, and both underwent efficient conjugation to a nonspecific human serum antibody (IgG) to yield two model immunoconjugates. The behavior of [89Zr]Zr-DFO-Km-IgG was studied in healthy mice for 2 weeks and compared to an equivalent cohort injected with [89Zr]Zr-DFO-IgG as a clinical "gold standard" control. PET-CT and biodistribution results revealed higher stability of [89Zr]Zr-(DFO-Km)-IgG in vivo over [89Zr]Zr-DFO-IgG, as demonstrated by the significant reduction of zirconium-89 in the whole skeleton as visualized and quantified by PET-CT at 1, 3, 7, and 14 days post-injection. Using CT-gated regions of interest over these PET-CT images, the whole skeleton was selected and uptake values were measured at 14 days post-injection of 3.6 ± 0.9 (DFO) vs 1.9 ± 0.1 (DFO-Km) %ID/g (n = 4, * p = 0.02), which represents a ∼48% reduction in bone uptake with DFO-Km relative to DFO. Biodistribution experiments performed on these same mice following the 14 day imaging time point revealed bone (both tibia) uptake values of 3.7 ± 1.3 (DFO) vs 2.0 ± 0.6 (DFO-Km) %ID/g (n = 6, * p < 0.05), with the tibia uptake values in close agreement with whole-skeleton ROI PET-CT data. These results indicate that DFO-Km is an improved chelator for [89Zr]Zr4+ applications relative to DFO. The bifunctional chelator p-SCN-Ph-DFO-Km shows potential as a new chemical tool for creating bioconjugates using targeting vectors such as antibodies, peptides, and nanoparticles.

Radiochemical, Computational, and Spectroscopic Evaluation of High-Denticity Desferrioxamine Derivatives DFO2 and DFO2p toward an Ideal Zirconium-89 Chelate Platform.

Desferrioxamine (DFO) has long been considered the gold standard chelator for incorporating [89Zr]Zr4+ in radiopharmaceuticals for positron emission tomography (PET) imaging. To improve the stability of DFO with zirconium-89 and to expand its coordination sphere to enable binding of large therapeutic radiometals, we have synthesized the highest denticity DFO derivatives to date: dodecadentate DFO2 and DFO2p. In this study, we describe the synthesis and characterization of a novel DFO-based chelator, DFO2p, which is comprised of two DFO strands connected by an p-NO2-phenyl linker and therefore contains double the chelating moieties of DFO (potential coordination number up to 12 vs 6). The chelator DFO2p offers an optimized synthesis comprised of only a single reaction step and improves water solubility relative to DFO2, but the shorter linker reduces molecular flexibility. Both DFO2 and DFO2p, each with 6 potential hydroxamate ligands, are able to reach a more energetically favorable 8-coordinate environment for Zr(IV) than DFO. The zirconium(IV) coordination environment of these complexes were evaluated by a combination of density functional theory (DFT) calculations and synchrotron spectroscopy (extended X-ray absorption fine structure), which suggest the inner-coordination sphere of zirconium(IV) to be comprised of the outermost four hydroxamate ligands. These results also confirm a single Zr(IV) in each chelator, and the hydroxide ligands which complete the coordination sphere of Zr(IV)-DFO are absent from Zr(IV)-DFO2 and Zr(IV)-DFO2p. Radiochemical stability studies with zirconium-89 revealed the order of real-world stability to be DFO2 > DFO2p ≫ DFO. The zirconium-89 complexes of these new high-denticity chelators were found to be far more stable than DFO, and the decreased molecular flexibility of DFO2p, relative to DFO2, could explain its decreased stability, relative to DFO2.

Design, Synthesis, and Evaluation of DFO-Em: A Modular Chelator with Octadentate Chelation for Optimal Zirconium-89 Radiochemistry.

Zirconium-89 has quickly become a favorite radionuclide among academics and clinicians for nuclear imaging. This radiometal has a relatively long half-life, which matches the biological half-life of most antibodies, suitable decay properties for positron emission tomography (PET), and efficient and affordable cyclotron production and purification. The "gold standard" chelator for [89Zr]Zr4+ is desferrioxamine B (DFO), and although it has been used both preclinically and clinically for immunoPET with great success, it has revealed its suboptimal stability in vivo. DFO can only bind to [89Zr]Zr4+ through its six available coordination sites made up by three hydroxamic acid (HA) moieties, which is not sufficient to saturate the coordination sphere (CN 7-8). In this study, we have designed, synthesized, and characterized a new octadentate chelator we have called DFO-Em, which is an improved derivative of our previously published dodecadentate chelator DFO2. This octadentate DFO-Em chelator is smaller than DFO2 but still satisfies the coordination sphere of zirconium-89 and forms a highly stable radiometal-chelator complex. DFO-Em was synthesized by tethering a hydroxamic acid monomer to commercially available DFO using glutamic acid as a linker, providing an octadentate chelator built on a modular amino acid-based synthesis platform. Radiolabeling performance and radiochemical stability of DFO-Em were assessed in vitro by serum stability, ethylenediamine tetraacetic acid (EDTA), and hydroxyapatite challenges. Furthermore, [89Zr]Zr-(DFO-Em) and [89Zr]Zr-DFO were injected in healthy mice and measured in vivo by PET/CT imaging and ex vivo biodistribution. Additionally, the coordination of DFO-Em with Zr(IV) and its isomers was studied using density functional theory (DFT) calculations. The radiolabeling studies revealed that DFO-Em has a comparable radiolabeling profile to the gold standard chelator DFO. The in vitro stability evaluation showed that [89Zr]Zr-(DFO-Em) was significantly more stable than [89Zr]Zr-DFO, and in vivo both had similar clearance in healthy mice with a small decrease in tissue retention for [89Zr]Zr-(DFO-Em) at 24 h post injection. The DFT calculations also confirmed that Zr-(DFO-Em) can adopt highly stable 8-coordinate geometries, which along with NMR characterization suggest no fluxional behavior and the presence of a single isomer. The modular design of DFO-Em means that any natural or unnatural amino acid can be utilized as a linker to gain access to different chemistries (e.g., thiol, amine, carboxylic acid, azide) while retaining an identical coordination sphere to DFO-Em.

Ultrasonic-Assisted Solid-Phase Peptide Synthesis of DOTA-TATE and DOTA-linker-TATE Derivatives as a Simple and Low-Cost Method for the Facile Synthesis of Chelator-Peptide Conjugates.

Peptides have been widely adopted as biological targeting vectors for applications in molecular imaging and peptide-receptor radionuclide therapy (PRRT). Somatostatin (SST) analogues such as octreotate (TATE) are exogenous ligands for somatostatin receptors (SSTRs), which are highly expressed on neuroendocrine tumors (NETs). Recently, both [68Ga]Ga-DOTA-TATE (NETSPOT) and [177Lu]Lu-DOTA-TATE (LUTATHERA) received U.S. Food and Drug Administration approval for positron emission tomography (PET) imaging and PRRT of NETs, respectively. However, to the best of our knowledge a well-described synthesis of DOTA-TATE has not been reported in the literature. Herein, we report a fully reoptimized DOTA-TATE synthesis, including the application of a simple ultrasonic bath to greatly improve yields, reduce coupling times, and decrease the amount of reagents required for each coupling step by a half. The most prevalently used cyclizing agents such as iodine, thallium(III) trifluoroacetate, hydrogen peroxide, and dimethyl sulfoxide were compared. On-resin cyclizations using mechanical agitation showed higher yields (23% and 25% using I2 and Tl(III), respectively) than off-resin (1.3% and 11% using DMSO and H2O2, respectively), and the total synthesis time of DOTA-TATE was ∼540 min excluding the cyclization step, with a total synthesis yield of ∼23%. The same manual SPPS methods/reagents were reoptimized with ultrasonic (US) agitation, resulting in an immense reduction in the total synthesis time by ∼8-fold to ∼70 min for DOTA-TATE with a higher yield (∼29% yield), and ∼13-fold to 105 min for DOTA-PEG4-TATE (∼29% yield). Also, the use of US agitation reduces the need for excess molar equivalents of the reagents to a half, which is particularly important when coupling expensive or custom-synthesized groups such as bifunctional chelators and linkers. Finally, the synthesized DOTA-TATE was successfully radiolabeled with [68Ga]Ga3+ (t1/2 = 68 min) with high radiochemical yields (30 min, 95 °C). We believe this work opens the door to the facile and low-cost synthesis of many new chelator-linker-peptide conjugates that were previously cumbersome or cost-prohibitive to produce with manual SPPS.

A Systematic Evaluation of Antibody Modification and 89Zr-Radiolabeling for Optimized Immuno-PET.

Immuno-PET using desferrioxamine (DFO)-conjugated zirconium-89 ([89Zr]Zr4+)-labeled antibodies is a powerful tool used for preclinical and clinical molecular imaging. However, a comprehensive study evaluating the variables involved in DFO-conjugation and 89Zr-radiolabeling of antibodies and their impact on the in vitro and in vivo behavior of the resulting radioimmunoconjugates has not been adequately performed. Here, we synthesized different DFO-conjugates of the HER2-targeting antibody (Ab)-trastuzumab, dubbed T5, T10, T20, T60, and T200-to indicate the molar equivalents of DFO used for bioconjugation. Next we radiolabeled the immunoconjugates with ([89Zr]Zr4+) under a comprehensive set of reaction conditions including different buffers (PBS, chelexed-PBS, TRIS/HCl, HEPES; ± radioprotectants), different reaction volumes (0.1-1 mL), variable amounts of DFO-conjugated Ab (5, 25, 50 µg), and radioactivity (0.2-1.0 mCi; 7.4-37 MBq). We evaluated the effects of these variables on radiochemical yield (RCY), molar activity (Am)/specific activity (As), immunoreactive fraction, and ultimately the in vivo biodistribution profile and tumor targeting ability of the trastuzumab radioimmunoconjugates. We show that increasing the degree of DFO conjugation to trastuzumab increased the RCY (∼90%) and Am/As (∼194 MBq/nmol; 35 mCi/mg) but decreased the HER2-binding affinity (3.5×-4.6×) and the immunoreactive fraction of trastuzumab down to 50-64%, which translated to dramatically inferior in vivo performance of the radioimmunoconjugate. Cell-based immunoreactivity assays and standard binding affinity analyses using surface plasmon resonance (SPR) did not predict the poor in vivo performance of the most extreme T200 conjugate. However, SPR-based concentration free calibration analysis yielded active antibody concentration and was predictive of the in vivo trends. Positron emission tomography (PET) imaging and biodistribution studies in a HER2-positive xenograft model revealed activity concentrations of 38.7 ± 3.8 %ID/g in the tumor and 6.3 ± 4.1 %ID/g in the liver for ([89Zr]Zr4+)-T5 (∼1.4 ± 0.5 DFOs/Ab) at 120 h after injection of the radioimmunoconjugates. On the other hand, ([89Zr]Zr4+)-T200 (10.9 ± 0.7 DFOs/Ab) yielded 16.2 ± 3.2 %ID/g in the tumor versus 27.5 ± 4.1 %ID/g in the liver. Collectively, our findings suggest that synthesizing trastuzumab immunoconjugates bearing 1-3 DFOs per Ab (T5 and T10) combined with radiolabeling performed in low reaction volumes using Chelex treated PBS or HEPEs without a radioprotectant provided radioimmunoconjugates having high Am/As (97 MBq/nmol; 17.5 ± 2.2 mCi/mg), highly preserved immunoreactive fractions (86-93%), and favorable in vivo biodistribution profile with excellent tumor uptake.

DiPODS: A Reagent for Site-Specific Bioconjugation via the Irreversible Rebridging of Disulfide Linkages.

Chemoselective reactions with thiols have long held promise for the site-specific bioconjugation of antibodies and antibody fragments. Yet bifunctional probes bearing monovalent maleimides-long the "gold standard" for thiol-based ligations-are hampered by two intrinsic issues: the in vivo instability of the maleimide-thiol bond and the need to permanently disrupt disulfide linkages in order to facilitate bioconjugation. Herein, we present the synthesis, characterization, and validation of DiPODS, a novel bioconjugation reagent containing a pair of oxadiazolyl methyl sulfone moieties capable of irreversibly forming covalent bonds with two thiolate groups while simultaneously rebridging disulfide linkages. The reagent was synthesized from commercially available starting materials in 8 steps, during which rotamers were encountered and investigated both experimentally and computationally. DiPODS is designed to be modular and can thus be conjugated to any payload through a pendant terminal primary amine (DiPODS-PEG4-NH2). Subsequently, the modification of a HER2-targeting Fab with a fluorescein-conjugated variant of DiPODS (DiPODS-PEG4-FITC) reinforced the site-specificity of the reagent, illustrated its ability to rebridge disulfide linkages, and produced an immunoconjugate with in vitro properties superior to those of an analogous construct created using traditional stochastic bioconjugation techniques. Ultimately, we believe that this work has particularly important implications for the synthesis of immunoconjugates, specifically for ensuring that the attachment of cargoes to immunoglobulins is robust, irreversible, and biologically and structurally benign.

A High-Denticity Chelator Based on Desferrioxamine for Enhanced Coordination of Zirconium-89.

Herein we report a new high-denticity chelator based on the iron siderophore desferrioxamine (DFO). Our new chelator-DFO2-is acyclic and was designed and synthesized with the purpose of improving the coordination chemistry and radiolabeling performance with radioactive zirconium-89. The radionuclide zirconium-89 ([89Zr]Zr4+) has found wide use for positron emission tomography (PET) imaging when it is coupled with proteins, antibodies, and nanoparticles. DFO2 has a potential coordination number of 12, which uniquely positions this chelator for binding large, high-valent, and oxophilic metal ions. Following synthesis of the DFO2 chelator and the [natZr]Zr-(DFO2) complex we performed density functional theory calculations to study its coordination sphere, followed by zirconium-89 radiolabeling experiments for comparisons with the "gold standard" chelator DFO. DFO (CN 6) can coordinate with zirconium in a hexadentate fashion, leaving two open coordination sites where water is thought to coordinate (total CN 8). DFO2 (potential CN 12, dodecadentate) can saturate the coordination sphere of zirconium with four hydroxamate groups (CN 8), with no room left for water to directly coordinate, and only binds a single atom of zirconium per chelate. Following quantitative radiolabeling with zirconium-89, the preformed [89Zr]Zr-(DFO) and [89Zr]Zr-(DFO2) radiometal-chelate complexes were subjected to a battery of in vitro stability challenges, including human blood serum, apo-transferrin, serum albumin, iron, hydroxyapatite, and EDTA. One objective of these stability challenges was to determine if the increased denticity of DFO2 over that of DFO imparted improved complex stability, and another was to determine which of these assays is most relevant to perform with future chelators. In all of the assays DFO2 showed superior stability with zirconium-89, except for the iron challenge, where both DFO2 and DFO were identical. Substantial differences in stability were observed for human blood serum using a precipitation method of analysis, apo-transferrin, hydroxyapatite, and EDTA challenges. These results suggest that DFO2 is a promising next-generation scaffold for zirconium-89 chelators and holds promise for radiochemistry with even larger radionuclides, which we anticipate will expand the utility of DFO2 into theranostic applications.

Structural Characterization of the Solution Chemistry of Zirconium(IV) Desferrioxamine: A Coordination Sphere Completed by Hydroxides.

Positron emission tomography (PET) using radiolabeled, monoclonal antibodies has become an effective, noninvasive method for tumor detection and is a critical component of targeted radionuclide therapy. Metal ion chelator and bacterial siderophore desferrioxamine (DFO) is the gold standard compound for incorporation of zirconium-89 in radiotracers for PET imaging because it is thought to form a stable chelate with [89Zr]Zr4+. However, DFO may not bind zirconium-89 tightly in vivo, with free zirconium-89 reportedly liberated into the bones of experimental mouse models. Although high bone uptake has not been observed to date in humans, this potential instability has been proposed to be related to the unsaturated coordination sphere of [89Zr]Zr-DFO, which is thought to consist of the 3 hydroxamate groups of DFO and 1 or 2 water molecules. In this study, we have used a combination of X-ray absorption spectroscopy and density functional theory (DFT) geometry optimization calculations to further probe the coordination chemistry of this complex in solution. We find the extended X-ray absorption fine structure (EXAFS) curve fitting of an aqueous solution of Zr(IV)-DFO to be consistent with an 8-coordinate Zr with oxygen ligands. DFT calculations suggest that the most energetically favorable Zr(IV) coordination environment in DFO likely consists of the 3 hydroxamate ligands from DFO, each with bidentate coordination, and 2 hydroxide ligands. Further EXAFS curve fitting provides additional support for this model. Therefore, we propose that the coordination sphere of Zr(IV)-DFO is most likely completed by 2 hydroxide ligands rather than 2 water molecules, forming Zr(DFO)(OH)2.

89Zr-Labeled AR20.5: A MUC1-Targeting ImmunoPET Probe.

High expression levels of the tumor-associated antigen MUC1 have been correlated with tumor aggressiveness, poor response to therapy, and poor survival in several tumor types, including breast, pancreatic, and epithelial ovarian cancer. Herein, we report the synthesis, characterization, and in vivo evaluation of a novel radioimmunoconjugate for the immuno-positron emission tomography (immunoPET) imaging of MUC1 expression based on the AR20.5 antibody. To this end, we modified AR20.5 with the chelator desferrioxamine (DFO) and labeled it with the positron-emitting radiometal zirconium-89 (t1/2 ~3.3 d) to produce [89Zr]Zr-DFO-AR20.5. In subsequent in vivo experiments in athymic nude mice bearing subcutaneous MUC1-expressing ovarian cancer xenografts, [89Zr]Zr-DFO-AR20.5 clearly delineated tumor tissue, producing a tumoral activity concentration of 19.1 ± 6.4 percent injected dose per gram (%ID/g) at 120 h post-injection and a tumor-to-muscle activity concentration ratio of 42.4 ± 10.6 at the same time point. Additional PET imaging experiments in mice bearing orthotopic MUC1-expressing ovarian cancer xenografts likewise demonstrated that [89Zr]Zr-DFO-AR20.5 enables the visualization of tumor tissue-including metastatic lesions-with promising tumor-to-background contrast.

Serial Stimulated Jitter Analysis In Juvenile Myasthenia Gravis.

INTRODUCTION: Clinical and electrophysiological studies to measures disease activity in juvenile myasthenia gravis (JMG) are limited. METHODS: Retrospective review of the clinical profile, Myasthenia Gravis Foundation of America (MGFA) scores, serial stimulated jitter analysis (Stim-JA) of the orbicularis oculi muscle, grip strength, and spirometry of patients with JMG who were followed in a multidisciplinary clinic was performed. RESULTS: Thirteen patients with JMG (9 females) with mean age of 13.2 ± 4.8 years and follow-up duration of 25.3 ± 8.3 months (range, 6-39) with ≥ 2 Stim-JA recordings were included. The mean jitter, mean percentage of apparent single-fiber action potentials (%ASFAP) with increased jitter, and mean %ASFAP with blocking at baseline values (77.3 ± 54.7 µs, 64.3% ± 35.8%, 39% ± 38.6%, respectively) and at follow-up (53 ± 45.4 µs, 51.2% ± 34.5%, 17% ± 29.4%, respectively) were abnormal; however, no statistically significant interval difference was noted. The electrophysiological data correlated significantly with Myasthenia Gravis Foundation of America (MGFA) class. Grip strength and spirometry did not correlate with MGFA class. DISCUSSION: Stimulated jitter values are sensitive biomarkers in JMG. Muscle Nerve 58: 729-732, 2018.

Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT.

Hydrolytic enzymes are a large class of biological catalysts that play a vital role in a plethora of critical biochemical processes required to maintain human health. However, the expression and/or activity of these important enzymes can change in many different diseases and therefore represent exciting targets for the development of positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radiotracers. This review focuses on recently reported radiolabeled substrates, reversible inhibitors, and irreversible inhibitors investigated as PET and SPECT tracers for imaging hydrolytic enzymes. By learning from the most successful examples of tracer development for hydrolytic enzymes, it appears that an early focus on careful enzyme kinetics and cell-based studies are key factors for identifying potentially useful new molecular imaging agents.

Matching chelators to radiometals for radiopharmaceuticals.

Radiometals comprise many useful radioactive isotopes of various metallic elements. When properly harnessed, these have valuable emission properties that can be used for diagnostic imaging techniques, such as single photon emission computed tomography (SPECT, e.g.(67)Ga, (99m)Tc, (111)In, (177)Lu) and positron emission tomography (PET, e.g.(68)Ga, (64)Cu, (44)Sc, (86)Y, (89)Zr), as well as therapeutic applications (e.g.(47)Sc, (114m)In, (177)Lu, (90)Y, (212/213)Bi, (212)Pb, (225)Ac, (186/188)Re). A fundamental critical component of a radiometal-based radiopharmaceutical is the chelator, the ligand system that binds the radiometal ion in a tight stable coordination complex so that it can be properly directed to a desirable molecular target in vivo. This article is a guide for selecting the optimal match between chelator and radiometal for use in these systems. The article briefly introduces a selection of relevant and high impact radiometals, and their potential utility to the fields of radiochemistry, nuclear medicine, and molecular imaging. A description of radiometal-based radiopharmaceuticals is provided, and several key design considerations are discussed. The experimental methods by which chelators are assessed for their suitability with a variety of radiometal ions is explained, and a large selection of the most common and most promising chelators are evaluated and discussed for their potential use with a variety of radiometals. Comprehensive tables have been assembled to provide a convenient and accessible overview of the field of radiometal chelating agents.

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.

Outpatient Utilization of the RAM Cannula for Nasal Noninvasive Ventilation in Children.

Background: The RAM cannula® consists of nasal prongs that can be used to administer oxygen, continuous, and bilevel positive airway pressure therapies. Studies have reported the efficacy and utility of the RAM cannula in inpatients requiring noninvasive ventilation (NIV); however, there is limited literature on the use of the RAM cannula to provide NIV in the outpatient setting. Objectives: This study aimed to describe the clinical features and outcomes of children who used NIV via RAM cannula in the outpatient setting. Design: Retrospective review. Methods: We conducted a retrospective review of children treated with outpatient NIV via RAM cannula at our institution between January 2010 and March 2023. The analyzed data included age, diagnoses, indications for NIV, duration of RAM cannula use, complications, and outcomes at 6 months. Results: We identified 20 patients who used outpatient NIV via RAM cannula during the study period. The median age at initiation of NIV via RAM cannula was 5.8 months (IQR 2.4-9.9 months). Indications for NIV included sleep-related hypoventilation (15%), restrictive lung disease (25%), obstructive sleep apnea (45%), and chronic respiratory failure (50%), with 6 patients having ⩾2 indications for NIV. RAM cannula was utilized for inability to tolerate conventional NIV interfaces (80%), to alleviate dyspnea (60%), and to avoid tracheostomy (55%). Patients used NIV via RAM cannula for a median duration of 7.7 months (IQR 3.7-20.6 months). Patient outcomes included ongoing usage of RAM cannula (55%), changing to conventional NIV interfaces (15%) or oxygen (10%), weaning off respiratory support (5%), and death (15%). There were no complications related to using the RAM cannula. Conclusion: Our study demonstrates the utility of outpatient NIV via RAM cannula in children with a variety of diagnoses until clinical improvement or tolerance of conventional interfaces, and for avoidance of tracheostomy.

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.

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.

Strands, networks, and continents from polystyrene dewetting at the air-water interface: implications for amphiphilic block copolymer self-assembly.

We demonstrate that nanoscale aggregates similar to those formed via amphiphilic block copolymer self-assembly at the air-water interface, including strands, networks, and continents, can be generated by the simple spreading of PS homopolymer solutions on water. Two different PS homopolymers of different molecular weight (PS-405k, M(n) = 405 000 g mol(-1) and PS-33k, M(n) = 33 000 g mol(-1)) are spread at the air-water interface at various spreading concentrations ranging from 0.25 to 3.0 mg/mL. Aggregate formation is driven by PS dewetting from water as the spreading solvent evaporates. We propose that a high spreading concentration or a high molecular weight lead to chain entanglements that restrict macromolecular mobility in the solution, enabling the kinetic trapping of nanostructures associated with early and intermediate stages of PS dewetting. Comparison of PS-405k with a mainly hydrophobic PS-b-PEO block copolymer of similar molecular weight (PSEO-392k, M(n) = 392 000 g mol(-1), 2.0 wt % PEO) allows the effect of a relatively short surface active block on aggregate formation to be investigated. We show that whereas the PEO block is not a required component for the formation of strands and other nonglobular aggregates, it does increase the number of these aggregates at a given spreading concentration and decreases the minimum spreading concentration at which these aggregates are observed, along with decreasing the dimensions and polydispersity of specific surface features. The results provide supporting evidence for the role of PS dewetting in the generation of multiple PS-b-PEO aggregate morphologies at the air-water interface, as originally described in earlier paper from our group.