Tracking the fate of bacteria-derived site-specific immunomodulators by positron emission tomography.

INTRODUCTION: Site-specific immunomodulators (SSIs) are a novel class of therapeutics made from inactivated bacterial species designed to regulate the innate immune system in targeted organs. QBECO is a gut-targeted SSI that is being advanced clinically to treat and/or prevent inflammatory bowel disease, cancer, and serious infections of the gastrointestinal (GI) tract and proximal organs, and QBKPN is a lung-targeted SSI that is in clinical development for the treatment and/or prevention of chronic inflammatory lung disease, lung cancers and respiratory tract infections. While these SSIs have demonstrated both safety and proof-of-concept in preclinical and clinical studies, detailed understanding of their trafficking and biodistribution is yet to be fully characterized. METHODS: QBECO and QBKPN were radiolabeled with [89Zr] and injected subcutaneously into healthy mice. The mice underwent Positron Emission Tomography (PET) imaging every day for eight days to track biodistribution of the SSIs. Tissue from the site of injection was collected and immunohistologically probed for immune cell infiltration. RESULTS: Differential biodistribution of the two SSIs was seen, adhering to their site-specific targeting. QBKPN appeared to migrate from the site of injection (abdomen) to the cervical lymph nodes which are nearer to the respiratory tract and lungs. QBECO remained in the abdominal region, with lymphatic trafficking to the inguinal lymph nodes, which are nearer to GI-proximal tissues/organs. Immune infiltration at the site of injection comprised of neutrophils for both SSIs, and macrophages for only QBKPN. CONCLUSION: Radiolabeling of SSIs allows for longitudinal in vivo imaging of biodistribution and trafficking. PET imaging revealed differential biodistribution of the SSIs based on the organotropism of the bacteria from which the SSI is derived. Trafficking from the site of injection to the targeted site is in part mediated via the lymphatics and involves macrophages and neutrophils.

It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

Direct mapping of kidney function by DCE-MRI urography using a tetrazinanone organic radical contrast agent.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are ongoing global health burdens. Glomerular filtration rate (GFR) is the gold standard measure of kidney function, with clinical estimates providing a global assessment of kidney health without spatial information of kidney- or region-specific dysfunction. The addition of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to the anatomical imaging already performed would yield a 'one-stop-shop' for renal assessment in cases of suspected AKI and CKD. Towards urography by DCE-MRI, we evaluated a class of nitrogen-centered organic radicals known as verdazyls, which are extremely stable even in highly reducing environments. A glucose-modified verdazyl, glucoverdazyl, provided contrast limited to kidney and bladder, affording functional kidney evaluation in mouse models of unilateral ureteral obstruction (UUO) and folic acid-induced nephropathy (FAN). Imaging outcomes correlated with histology and hematology assessing kidney dysfunction, and glucoverdazyl clearance rates were found to be a reliable surrogate measure of GFR.

Mapping vitamin B6 metabolism by hydrazoCEST magnetic resonance imaging.

A new CEST-MRI contrast agent, 2-HYNIC, capable of sensing aromatic aldehydes is reported. Pyridoxal 5'-phosphate, a key Vitamin B6 metabolite necessary for >140 biotransformations was mapped by CEST-MRI in vitro and in vivo in lung cancer. 2-HYNIC provided access to this key biomarker associated with a variety of human diseases.

Synthesis of natural/13C-enriched d-tagatose from natural/13C-enriched d-fructose.

A concise, easily scalable synthesis of a rare ketohexose, d-tagatose, was developed, that is compatible with the preparation of d-[UL-13C6]tagatose. Epimerization of the widely available and inexpensive ketohexose d-fructose at the C-4 position via an oxidation/reduction (Dess-Martin periodinane/NaBH4) was a key step in the synthesis. Overall, fully protected natural d-tagatose (3.21 g) was prepared from d-fructose (9 g) on a 50 mmol scale in 23% overall yield, after five steps and two chromatographic purifications. d-[UL-13C6]Tagatose (92 mg) was prepared from d-[UL-13C6]fructose (465 mg, 2.5 mmol) in 16% overall yield after six steps and four chromatographic purifications.

Mapping aldehydic load in vivo by positron emission tomography with [18F]NA3BF3.

A new radiotracer, [18F]NA3BF3, capable of rapid, stable, and catalyst-free complexation of aldehydes in vivo is reported. [18F]NA3BF3 was shown to bind aldehydes in live subjects using locally administered aldehyde-presenting microparticles, and was then applied to mapping aldehydic load in a mouse model of sepsis. [18F]NA3BF3 may enable the direct investigation of the chemical biology of aldehydes in living subjects, and may open avenues for the adoption of endogenous aldehydic load as an imaging biomarker of inflammatory pathology.

Detection of Active Caspase-3 in Mouse Models of Stroke and Alzheimer's Disease with a Novel Dual Positron Emission Tomography/Fluorescent Tracer [68Ga]Ga-TC3-OGDOTA.

Apoptosis is a feature of stroke and Alzheimer's disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [68Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and 68Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [68Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and ß-amyloid oligomers. In vivo, PET showed accumulation of [68Ga]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [68Ga]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [68Ga]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [68Ga]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases.

Fingerprinting Biogenic Aldehydes through Pattern Recognition Analyses of Excitation-Emission Matrices.

Biogenic carbonyls, especially aldehydes, have previously demonstrated their potential to serve as early diagnostic biomarkers for disease and injury that have not been fully realized owing, in part, to the lack of a rapid and simple point-of-care method for aldehyde identification. The ability to determine which carbonyl compound is elevated and not just the total aldehydic load may provide more disease- or injury-specific diagnostic information. Toward this end, a novel fluorophore is presented that is able to form a complex with biogenic carbonyls under catalyst-free conditions so as to give a fluorescent fingerprint of the resulting hydrazone. The successful identification of bound carbonyls was accomplished with a newly described algorithm that applied principal curvature analysis of excitation-emission matrices to reduce surface features to ellipse representations, followed by a pattern-matching routine. With this algorithm, carbonyls were identified over a range of concentrations, and mixture components were successfully parsed. Overall, the results presented lay the groundwork for novel implementations of chemometrics to low-cost, rapid, and simple-to-implement point-of-care diagnostics.

Methyl 5-MeO-N-aminoanthranilate, a minimalist fluorogenic probe for sensing cellular aldehydic load.

Methyl 5-MeO-N-aminoanthranilate, a fluorogenic probe comprising a single substituted benzene ring has been applied towards the fluorescence detection of endogenous carbonyls through rapid, catalyst-free complexation of these bio-derived markers of cell stress under physiological conditions. The products formed during the reaction between the probe and aldehydic products of lipid peroxidation, including malondialdehyde and long-chain aliphatic aldehydes relevant to the oxidative decomposition of cell membranes, have been evaluated. Live cell imaging of diethyl maleate-induced oxidative stress with or without pretreatment with α-tocopherol was carried out, with the result suggesting that the presented molecule might serve as a minimalist molecular probe capable of cellular "Aldehydic Load" detection by fluorescence microscopy. This work also outlines functional constraints of the fluorogenic probe (i.e. intramolecular cyclization), providing a realistic evaluation of methyl 5-MeO-N-aminoanthranilate for fluorescence-based aldehyde detection.

Hydrazo-CEST: Hydrazone-Dependent Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Contrast Agents.

The rapid formation of hydrazones under physiological conditions was exploited for the detection of aldehydes through chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI). A metal-free, diamagnetic contrast agent derived from N-amino anthranilic acid was introduced, which selectively "turned-on" upon hydrazone formation through an effect termed Hydrazo-CEST. While the hydrazine form of the probe produced no CEST-MRI signal enhancement, the formation of the aryl hydrazone resulted in >20 % intensity decrease in the bulk water signal through the CEST effect, as measured by 300 MHz 1 H NMR, 3 T and 7 T MRI. Both the electronic contributions of the N-amino anthranilate and the aldehyde binding partner were shown to directly impact the exchange rate of the proton on the ring-proximal nitrogen, and thus the imaging signal. Additionally, the presence of the carboxylic acid moiety ortho to the hydrazine was necessary not only for contrast production, but also for rapid hydrazone formation and prolonged hydrazone product stability under physiological conditions. This work provided the first example of an MRI-based contrast agent capable of a "turn on" response upon reaction with bioactive aldehydes, and outlined both the structural and electronic requirements to expand on Hydrazo-CEST, a novel, hydrazone-dependent subtype of diamagnetic CEST-MRI.

An Aspartyl Cathepsin Targeted PET Agent: Application in an Alzheimer's Disease Mouse Model.

BACKGROUND: Early detection of Alzheimer's disease (AD) pathology is a serious challenge for both diagnosis and clinical trials. The aspartyl protease, Cathepsin D (CatD), is overexpressed in AD and could be a biomarker of disease. We have previously designed a unique contrast agent (CA) for dual-optical and magnetic resonance imaging of the activity of the CatD class of enzymes. OBJECTIVE: To compare the uptake and retention of a novel, more sensitive, and clinically-translatable 68Ga PET tracer targeting CatD activity in 5XFAD mice and non-Tg littermates. METHODS: The targeted CA consisted of an HIV-1 Tat cell penetrating peptide (CPP) conjugated to a specialized cleavage sequence targeting aspartyl cathepsins and a DOTA conjugate chelating 68Ga. PET images were acquired using a Siemens Inveon preclinical microPET in female Tg AD mice and non-Tg age matched female littermates (n = 5-8) following intravenous CA administration at 2, 6, and 9 months of age. Additionally, 18F fluorodeoxyglucose (FDG) PET imaging was performed at 10 months to measure glucose uptake. RESULTS: The Tg mice showed significantly higher relative uptake rate of the targeting CA in the forebrain relative to hindbrain at all ages compared to controls, consistent with histology. In contrast, no differences were seen in CA uptake in other organs. Additionally, the Tg mice did not show any differences in relative uptake of FDG at 10 months of age in the forebrain relative to the hindbrain compared to age matched non-Tg controls. CONCLUSIONS: Elevated aspartryl cathepsin activity was detected in vivo in the 5XFAD mouse model of AD using a novel targeted PET contrast agent.

Unusual C7- versus Normal 5'-O-Dimethoxytritylation of 6-Arylpyrrolocytidine Analogs.

Fluorescent deoxynucleosides possessing the modified bases 6-(2-benzo[b]furyl)- and 6-(2-furyl)pyrrolocytosine (BFpC and FpC) have been synthesized along with the quencher nucleosides possessing 6-{4-[(4-dimethylamino)azo]phenyl}pyrrolocytosine (DABCYLpC) and 6-(p-nitrophenyl)pyrrolocytosine (p-NO2-PhpC) nucleobase analogs. Standard treatment of BFpC, FpC, DABCYLpC, and p-NO2-PhpC with dimethoxytrityl chloride (DMT-Cl) led to the unusual substitution on the C7 of the pyrrolocytosine skeleton. The desired 5'-O-DMT-protected nucleoside analogs were synthesized from suitably protected 5'-O-DMT cytidines. Subsequent phosphitylation smoothly afforded BFpC-, FpC-, DABCYLpC-, and p-NO2-PhpC-derived monomers suitable for standard oligonucleotide synthesis.

Prolonged In Vivo Retention of a Cathepsin D Targeted Optical Contrast Agent in a Mouse Model of Alzheimer's Disease.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that is elevated early in Alzheimer's disease (AD). We have previously developed a Targeted contrast agent (CA) to detect CatD activity in vivo, consisting of a magnetic resonance imaging/fluorescent moiety linked to a cell penetrating peptide (CPP) by means of a CatD cleavage site and have demonstrated its uptake in the brain of an AD mouse model. OBJECTIVE: The purpose of this study was to characterize the in vivo retention of a near infra-red fluorescent dye labeled version of this CA. METHODS: Six adult C57Bl/6 wild-type mice and six adult 5XFAD transgenic AD mice were studied using a small animal imaging system at five and twelve months of age using our novel Targeted CA, or two different control CAs; a Non-Targeted (lacking the CatD cleavage site) and a Non-Penetrating (lacking the CPP). Following intravenous CA administration, the optical signal was recorded within the brain and uptake and washout curves were measured and fitted to a one-phase exponential decay curve. RESULTS: In all wild-type and 5XFAD mice, the washout of the Targeted CA that included a CPP domain was significantly slower than the washout of the Non-Penetrating and Non-Targeted CA. Furthermore, the washout of the CatD Targeted CA was significantly slower in the 5XFAD mice compared to the age matched wild-type controls (p <  0.05) at 5 and 12 months of age. Control CAs showed no differences in washout. CONCLUSIONS: The prolonged retention of the CatD targeted CA in 5XFAD mice suggests this agent may be useful for AD detection.

Introduction of Peripheral Carboxylates to Decrease the Charge on Tm(3+) DOTAM-Alkyl Complexes: Implications for Detection Sensitivity and in Vivo Toxicity of PARACEST MRI Contrast Agents.

A series of structurally modified Tm(3+) DOTAM-alkyl complexes as potential PARACEST MRI contrast agents has been synthesized with the aim to decrease the overall positive charge associated with these molecules and increase their biocompatibility. Two types of structural modification have been performed, an introduction of terminal carboxylate arms to the alkyl side chains and a conjugation of one of the alkyl side chains with aspartic acid. Detailed evaluation of the magnetic resonance imaging chemical exchange contrast associated with the structurally modified contrast agents has been performed. In contrast to the acutely toxic Tm(3+) DOTAM-alkyl complexes, the structurally modified compounds were found to be tolerated well during in vivo MRI studies in mice; however, only the aspartic acid modified chelates produced an amide proton-based PARACEST signal.

Synthesis and spectral characterization of environmentally responsive fluorescent deoxycytidine analogs.

Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2'-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2'-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2'-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior.

Pyrimidine-fused heterocyclic frameworks based on an N4-arylcytosine scaffold: synthesis, characterization, and PNA oligomerization of the fluorescent cytosine analogue 5,6-benzopC.

A synthesis of an intrinsically fluorescent cytosine analogue 5,6-benzopC has been developed utilizing the reductive Ni-mediated cyclization of an N4-aryl,N4-(Boc)cytosine intermediate as a key step. 5,6-BenzopC was found to possess interesting fluorescence properties (Φ = 0.79, EtOH; Stoke's shift 113 nm). Peptide nucleic acid (PNA) oligomerization of the 5,6-benzopC monomer was carried out, followed by hybridization studies with complementary deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) which showed the modification to be well tolerated in the sequence contexts examined. Initial attempts to synthesize the heterocyclic skeleton present in 5,6-benzopC resulted in the discovery of routes to the pyrimido[1,6-a]benzimidazole, pyrimido[1,6-a]quinazoline, and pyrimido[1,6-a]benzo[b]6-bora-1,3-diazine heterocyclic frameworks.

A DOTAM-based paraCEST agent favoring TSAP geometry for enhanced amide proton chemical shift dispersion and temperature sensitivity.

The Tm(3+) chelate of DOTAM [1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane] possessing sterically demanding t-butyl amide substitution favors TSAP geometry. This chelate displayed a paraCEST signal associated with the highly shifted amide proton signal at approximately -100 ppm that was beyond the frequency of macromolecule magnetization transfer. This signal also displayed high temperature dependence (0.57 ppm °C(-1)) in the range of 35-42 °C and at neutral pH.

A dual magnetic resonance imaging/fluorescent contrast agent for Cathepsin-D detection.

Currently there are no approved biomarkers for the pre-symptomatic diagnosis of Alzheimer's disease (AD). Cathepsin-D (Cat-D) is a lysosomal protease that is present at elevated levels in amyloid plaques and neurons in patients with AD and is also elevated in some cancers. We have developed a magnetic resonance imaging (MRI)/fluorescent contrast agent to detect Cat-D enzymatic activity. The purpose of this study was to investigate the cellular and tissue uptake of this MRI/fluorescent contrast agent. The agent consists of an MRI probe [DOTA-caged metal ion (Gd³âº or Tm³âº)] and a fluorescent probe coupled to a cell-penetrating-peptide sequence by a Cat-D recognition site. The relaxivity of Gd³âº-DOTA-CAT(cleaved) was measured in 10% heat-treated bovine serum albumin (BSA) phantoms to assess contrast efficacy at magnetic fields ranging from 0.24 mT to 9.4 T. In vitro, Tm³âº-DOTA-CAT was added to neuronal SN56 cells over-expressing Cat-D and live-cell confocal microscropy was performed at 30 min. Tm³âº-DOTA-CAT was also intravenously injected into APP/PS1-dE9 Alzheimer's disease mice (n = 9) and controls (n = 8). Cortical and hippocampal uptake was quantified at 30, 60 and 120 min post-injection using confocal microscopy. The liver and kidneys were also evaluated for contrast agent uptake. The relaxivity of Gd³âº-DOTA-CAT(cleaved) was 3.3 (mM s)⁻¹ in 10% BSA at 9.4 T. In vitro, cells over-expressing Cat-D preferentially took up the contrast agent in a concentration-dependent manner. In vivo, the contrast agent effectively crossed the blood-brain barrier and exhibited a distinct time course of uptake and retention in APP/PS1-dE9 transgenic mice compared with age-matched controls. At clinical and high magnetic field strengths, Gd³âº-DOTA-CAT produced greater T1 relaxivity than Gd³âº-DTPA. Tm³âº-DOTA-CAT was taken up in a dose-dependent manner in cells over-expressing Cathepsin-D and was shown to transit the blood-brain barrier in vivo. This strategy may be useful for the in vivo detection of enzyme activity and for the diagnosis of Alzheimer's disease.

Simultaneous in vivo pH and temperature mapping using a PARACEST-MRI contrast agent.

Altered tissue temperature and/or pH is a common feature in pathological conditions, where metabolic demand exceeds oxygen supply such as in tumors and following stroke. Therefore, in vivo tissue temperature and pH may become valuable biomarkers for disease detection and the monitoring of disease progression or treatment response in conditions with altered metabolic demand. In this study, pH is measured using the amide protons of a thulium (Tm(3+)) complex with a DOTAM-Glycine-Lysine (ligand: Tm(3+)-DOTAM-Gly-Lys). The pH was uniquely determined from the linewidth of the asymmetry curve of the chemical exchange saturation transfer spectrum, independent of contrast agent concentration, or temperature for a given saturation pulse. pH maps with an inter-pixel standard deviation of less than 0.1 pH units were obtained in 10 mM Tm(3+)-DOTAM-Gly-Lys solutions with pH ranging from 6.0 to 8.0 pH units at 37°C. Temperature maps were simultaneously obtained using the chemical shift of the chemical exchange saturation transfer peak. Temperature and pH maps are demonstrated in the mouse leg (N = 3), where the mean and standard deviation for pH was 7.2 ± 0.2 pH unit and temperature was 37.4 ± 0.5°C.

DOTAM-type ligands possessing arginine pendant groups for use in PARACEST MRI.

A synthetic methodology was developed for the preparation of metal-chelating ligands that possess arginine pendant groups relying on the alkylation of 1,4,7,10-tetraazacyclododecane (cyclen) with arginine-containing electrophiles. Conditions for the selective trialkylation or peralkylation of cyclen are described, the outcome being dependent on the nature of the arginine-derived electrophile and the solvent used for the reaction. Lanthanide metal complexes of the ligands prepared by the described route were evaluated for their suitability as PARACEST contrast agents for use in magnetic resonance imaging. The Dy(3+) and Tm(3+) complexes display CEST effects that are associated with the amide protons proximate to the metal center. These signals exhibit pH dependence in the range of 6.0-8.0 and thus may have the potential for pH measurement in physiological range.