RESUMO
Real-time visualization of metabolic processes in vivo provides crucial insights into conditions like cancer and metabolic disorders. Metabolic magnetic resonance imaging (MRI), by amplifying the signal of pyruvate molecules through hyperpolarization, enables non-invasive monitoring of metabolic fluxes, aiding in understanding disease progression and treatment response. Signal Amplification By Reversible Exchange (SABRE) presents a simpler, cost-effective alternative to dissolution dynamic nuclear polarization, eliminating the need for expensive equipment and complex procedures. We present the first in vivo demonstration of metabolic sensing in a human pancreatic cancer xenograft model compared to healthy mice. A novel perfluorinated Iridium SABRE catalyst in a fluorinated solvent and methanol blend facilitated this breakthrough with a 1.2-fold increase in [1-13C]pyruvate SABRE hyperpolarization. The perfluorinated moiety allowed easy separation of the heavy-metal-containing catalyst from the hyperpolarized [1-13C]pyruvate target. The perfluorinated catalyst exhibited recyclability, maintaining SABRE-SHEATH activity through subsequent hyperpolarization cycles with minimal activity loss after the initial two cycles. Remarkably, the catalyst retained activity for at least 10â cycles, with a 3.3-fold decrease in hyperpolarization potency. This proof-of-concept study encourages wider adoption of SABRE hyperpolarized [1-13C]pyruvate MR for studying in vivo metabolism, aiding in diagnosing stages and monitoring treatment responses in cancer and other diseases.
RESUMO
An untargeted metabolomic study identified four potential lung cancer diagnostic biomarkers in human urine. One of the potential biomarkers was an unidentified feature possessing a m/z value of 561+. "561+" was isolated from human urine and tentatively identified as 27-nor-5ß-cholestane-3α,7α,12α,24,25 pentol glucuronide with unknown C24,25 stereochemistry using 1H NMR and mass spectrometry. In a prior report, the C24,25 stereochemistry of the aglycone, 27-nor-5ß-cholestane-3α,7α,12α,24,25 pentol, was found to be 24S,25R through GC analysis of the acetonide-TMS derivative. An authentic sample was prepared and found not to have the same stereochemistry as "561+". To identify the C24,25 stereochemistry, four C24,C25 diastereoisomeric alcohols of 27-nor-5ß-cholestane-3α,7α,12α,24,25 pentol were prepared from chiral amino acids. Using an LCMS method, the C24,C25 stereochemistry of the "561+" aglycone was determined to be 24R,25S. With the correct aglycone in hand, it was coupled with glucuronic acid to complete the first reported synthesis of 27-nor-5ß-cholestane-3α,7α,12α,24R,25S pentol glucuronide. Deuterium labeled 27-nor-5ß-cholestane-3α,7α,12α,24R,25S pentol was also synthesized for use as an internal standard for MS quantitation.
Assuntos
Biomarcadores Tumorais , Glucuronídeos , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/urina , Neoplasias Pulmonares/diagnóstico , Biomarcadores Tumorais/urina , Glucuronídeos/urina , Glucuronídeos/química , Deutério/química , Masculino , FemininoRESUMO
Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.
Assuntos
Irídio , Ácido Pirúvico , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética , ÁguaRESUMO
Carboxylic acid is a commonly utilized functional group for covalent surface conjugation of carbon nanoparticles that is typically generated by acid oxidation. However, acid oxidation generates additional oxygen containing groups, including epoxides, ketones, aldehydes, lactones, and alcohols. We present a method to specifically enrich the carboxylic acid content on fluorescent nanodiamond (FND) surfaces. Lithium aluminum hydride is used to reduce oxygen containing surface groups to alcohols. The alcohols are then converted to carboxylic acids through a rhodium (II) acetate catalyzed carbene insertion reaction with tert-butyl diazoacetate and subsequent ester cleavage with trifluoroacetic acid. This carboxylic acid enrichment process significantly enhanced nanodiamond homogeneity and improved the efficiency of functionalizing the FND surface. Biotin functionalized fluorescent nanodiamonds were demonstrated to be robust and stable single-molecule fluorescence and optical trapping probes.
RESUMO
Alpha-ketoglutarate (α-KG) is a key metabolite and signaling molecule in cancer cells, but the low permeability of α-KG limits the study of α-KG mediated effects in vivo. Recently, cell-permeable monoester and diester α-KG derivatives have been synthesized for use in vivo, but many of these derivatives are not compatible for use in hyperpolarized carbon-13 nuclear magnetic resonance spectroscopy (HP-13C-MRS). HP-13C-MRS is a powerful technique that has been used to noninvasively trace labeled metabolites in real time. Here, we show that using diethyl-[1-13C]-α-KG as a probe in HP-13C-MRS allows for noninvasive tracing of α-KG metabolism in vivo.
Assuntos
Membrana Celular/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Animais , Transporte Biológico , Isótopos de Carbono , Linhagem Celular Tumoral , Ácido Glutâmico/genética , Glutamina/genética , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais , PermeabilidadeRESUMO
Drastic sensitivity enhancement of dynamic nuclear polarization is becoming an increasingly critical methodology to monitor real-time metabolic and physiological information in chemistry, biochemistry, and biomedicine. However, the limited number of available hyperpolarized 13C probes, which can effectively interrogate crucial metabolic activities, remains one of the major bottlenecks in this growing field. Here, we demonstrate [1-13C] N-acetyl cysteine (NAC) as a novel probe for hyperpolarized 13C MRI to monitor glutathione redox chemistry, which plays a central part of metabolic chemistry and strongly influences various therapies. NAC forms a disulfide bond in the presence of reduced glutathione, which generates a spectroscopically detectable product that is separated from the main peak by a 1.5 ppm shift. In vivo hyperpolarized MRI in mice revealed that NAC was broadly distributed throughout the body including the brain. Its biochemical transformation in two human pancreatic tumor cells in vitro and as xenografts differed depending on the individual cellular biochemical profile and microenvironment in vivo. Hyperpolarized NAC can be a promising non-invasive biomarker to monitor in vivo redox status and can be potentially translatable to clinical diagnosis.
Assuntos
Acetilcisteína/metabolismo , Encéfalo/metabolismo , Isótopos de Carbono/análise , Glutationa/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Apoptose , Proliferação de Células , Humanos , Imageamento por Ressonância Magnética , Camundongos , Oxirredução , Neoplasias Pancreáticas/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To further explore the scope of our recently developed "fluorination on Sep-Pak" method, we prepared two well-known positron emission tomography (PET) tracers 21-[18F]fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione furanyl norprogesterone ([18F]FFNP) and 16ß-[18F]fluoro-5α-dihydrotestosterone ([18F]FDHT). Following the "fluorination on Sep-Pak" method, over 70% elution efficiency was observed with 3 mg of triflate precursor of [18F]FFNP. The overall yield of [18F]FFNP was 64-72% (decay corrected) in 40 min synthesis time with a molar activity of 37-81 GBq/µmol (1000-2200 Ci/mmol). Slightly lower elution efficiency (~55%) was observed with the triflate precursor of [18F]FDHT. Fluorine-18 labeling, reduction, and deprotection to prepare [18F]FDHT were performed on Sep-Pak cartridges (PS-HCO3 and Sep-Pak plus C-18). The overall yield of [18F]FDHT was 25-32% (decay corrected) in 70 min. The molar activity determined by using mass spectrometry was 63-148 GBq/µmol (1700-4000 Ci/mmol). Applying this quantitative measure of molar activity to in vitro assays [18F]FDHT exhibited high-affinity binding to androgen receptors (Kd~2.5 nM) providing biological validation of this method.
Assuntos
Di-Hidrotestosterona/química , Radioisótopos de Flúor/química , Norpregnenos/química , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Feminino , Halogenação , Humanos , Masculino , Espectrometria de Massas , Estrutura MolecularRESUMO
Effective means to identify the role of reactive oxygen species (ROS) mediating several diseases including cancer, ischemic heart disease, stroke, Alzheimer's and other inflammatory conditions in in vivo models would be useful. The cyclic nitrone 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is a spin trap frequently used to detect free radicals in vitro using Electron Paramagnetic Resonance (EPR) spectroscopy. In this study, we synthesized 13C-labeled DMPO for hyperpolarization by dynamic nuclear polarization, in which 13C NMR signal increases more than 10,000-fold. This allows in vivo 13C MRI to investigate the feasibility of in vivo ROS detection by the 13C-MRI. DMPO was 13C-labeled at C5 position, and deuterated to prolong the T1 relaxation time. The overall yield achieved for 5-13C-DMPO-d9 was 15%. Hyperpolarized 5-13C-DMPO-d9 provided a single peak at 76â¯ppm in the 13C-spectrum, and the T1 was 60â¯s in phosphate buffer making it optimal for in vivo 13C MRI. The buffered solution of hyperpolarized 5-13C-DMPO-d9 was injected into a mouse placed in a 3â¯T scanner, and 13C-spectra were acquired every 1â¯s. In vivo studies showed the signal of 5-13C-DMPO-d9 was detected in the mouse, and the T1 decay of 13C signal of hyperpolarized 5-13C-DMPO-d9 was 29â¯s. 13C-chemical shift imaging revealed that 5-13C-DMPO-d9 was distributed throughout the body in a minute after the intravenous injection. A strong signal of 5-13C-DMPO-d9 was detected in heart/lung and kidney, whereas the signal in liver was small compared to other organs. The results indicate hyperpolarized 5-13C-DMPO-d9 provided sufficient 13C signal to be detected in the mouse in several organs, and can be used to detect ROS in vivo.
Assuntos
Óxidos N-Cíclicos/farmacocinética , Coração/diagnóstico por imagem , Rim/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Espécies Reativas de Oxigênio/análise , Animais , Isótopos de Carbono , Óxidos N-Cíclicos/síntese química , Deutério , Feminino , Rim/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Detecção de SpinRESUMO
PURPOSE: To develop an implantable wireless coil with parametric amplification capabilities for time-domain electron paramagnetic resonance (EPR) spectroscopy operating at 300 MHz. METHODS: The wireless coil and lithium phthalocyanine (LiPc), a solid paramagnetic probe, were each embedded individually in a biocompatible polymer polydimethoxysiloxane (PDMS). EPR signals from the LiPc embedded in PDMS (LiPc/PDMS) were generated by a transmit-receive surface coil tuned to 300 MHz. Parametric amplification was configured with an external pumping coil tuned to 600 MHz and placed between the surface coil resonator and the wireless coil. RESULTS: Phantom studies showed significant enhancement in signal to noise using the pumping coil. However, no influence of the pumping coil on the oxygen-dependent EPR spectral linewidth of LiPc/PDMS was observed, suggesting the validity of parametric amplification of EPR signals for oximetry by implantation of the encapsulated wireless coil and LiPc/PDMS in deep regions of live objects. In vivo studies demonstrate the feasibility of this approach to longitudinally monitor tissue pO2 in vivo and also monitor acute changes in response to pharmacologic challenges. The encapsulated wireless coil and LiPc/PDMS engendered no host immune response when implanted for â¼3 weeks and were found to be well tolerated. CONCLUSIONS: This approach may find applications for monitoring tissue oxygenation to better understand the pathophysiology associated with wound healing, organ transplantation, and ischemic diseases.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/instrumentação , Oximetria/instrumentação , Tecnologia sem Fio/instrumentação , Animais , Desenho de Equipamento , Feminino , Camundongos , Camundongos Nus , Imagens de Fantasmas , Próteses e Implantes , CicatrizaçãoRESUMO
Traditional radiosynthetic optimization faces the challenges of high radiation exposure, cost, and inability to perform serial reactions due to tracer decay. To accelerate tracer development, we have developed a strategy to simulate radioactive 18 F-syntheses by using tracer-level (nanomolar) non-radioactive 19 F-reagents and LC-MS/MS analysis. The methodology was validated with fallypride synthesis under tracer-level 19 F-conditions, which showed reproducible and comparable results with radiosynthesis, and proved the feasibility of this process. Using this approach, the synthesis of [18 F]MDL100907 was optimized under 19 F-conditions with greatly improved yield. The best conditions were successfully transferred to radiosynthesis. A radiochemical yield of 19% to 22% was achieved with the radiochemical purity >99% and the molar activity 38.8 to 53.6 GBq/ µmol (n = 3). The tracer-level 19 F-approach provides a high-throughput and cost-effective process to optimize radiosynthesis with reduced radiation exposure. This new method allows medicinal and synthetic chemists to optimize radiolabeling conditions without the need to use radioactivity.
Assuntos
Radioisótopos de Flúor/química , Fluorbenzenos/química , Piperidinas/química , Compostos Radiofarmacêuticos/síntese química , Antagonistas da Serotonina/químicaRESUMO
Glutamine (Gln) and its analogues may serve as imaging agents for tumor diagnosis using positron emission tomography (PET), especially for tumors with negative [(18)F]FDG scan. We report the first automated synthesis of [(18)F](2S,4R)-4-fluoroglutamine ([(18)F]FGln) on a GE TRACERlab™ FX-N Pro module. [(18)F]FGln was obtained in 80±3min with a radiochemical yield of 21±3% (n=5, uncorrected). The radiochemical purity was >98%, and optical purity 90±5%. The synthesis is highly reproducible with good chemical purity, radiochemical yield, and is suitable for translation to cGMP production.
Assuntos
Radioisótopos de Flúor , Glutamina/análogos & derivados , Compostos Radiofarmacêuticos/síntese química , Automação/instrumentação , Automação/métodos , Técnicas de Química Sintética/instrumentação , Técnicas de Química Sintética/métodos , Estabilidade de Medicamentos , Glutamina/síntese química , Humanos , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Tecnologia Radiológica/instrumentação , Tecnologia Radiológica/métodosRESUMO
The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence have been extensively studied in the laboratory setting, sometimes by generating fluorophore-tagged analogs. Here, we synthesized two Pt(II) complexes containing ethane-1,2-diamine ligands linked to a BODIPY fluorophore, and compared their biological activity with previously reported Pt(II) complexes conjugated to carboxyfluorescein and carboxyfluorescein diacetate. The cytotoxicity and DNA damage capacity of Pt-fluorophore complexes was compared to cisplatin, and the Pt-BODIPY complexes were found to be more cytotoxic with reduced cytotoxicity in cisplatin-resistant cells. Microscopy revealed a predominately cytosolic localization, with nuclear distribution at higher concentrations. Spheroids grown from parent and resistant cells revealed penetration of Pt-BODIPY into spheroids, and retention of the cisplatin-resistant spheroid phenotype. While most activity profiles were retained for the Pt-BODIPY complexes, accumulation in resistant cells was only slightly affected, suggesting that some aspects of Pt-fluorophore cellular pharmacology deviate from cisplatin.
Assuntos
Cisplatino/análogos & derivados , Complexos de Coordenação/síntese química , Dano ao DNA/efeitos dos fármacos , Corantes Fluorescentes/química , Platina/química , Platina/toxicidade , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos de Boro/química , Linhagem Celular Tumoral , Cisplatino/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Microscopia ConfocalRESUMO
Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.
Assuntos
Galactoquinase/antagonistas & inibidores , Animais , Benzoxazóis/síntese química , Benzoxazóis/química , Benzoxazóis/metabolismo , Cristalografia por Raios X , Galactoquinase/genética , Galactoquinase/metabolismo , Galactosefosfatos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Camundongos , Microssomos Hepáticos/metabolismo , Conformação Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
[reaction: see text] Two formal total syntheses of the (-)-salicylihalamides, based on chiral pool approaches, are reported. D-glucose and L-rhamnose were used to prepare advanced intermediates 23 and 54, which can be converted in three or four steps, respectively, to the target compounds. The synthesis of 23 from a known D-glucose-derivative was accomplished in 12 steps and 17% overall yield, and the synthesis of 54 from a known L-rhamnose-derivative was done in nine steps and 6% overall yield. A key step in the synthesis was a ring-closing metathesis reaction to prepare the macrocyclic ring system. It was demonstrated that the phenolic protecting group was critical for inducing the preferential formation of the desired E isomer. It was further shown that the protecting group at the C13 hydroxyl group had no significant influence on the E:Z ratio during the ring-closing metathesis reaction.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Glucose , Ramnose , EstereoisomerismoRESUMO
The formal total synthesis of the (+)-salicylihalamides A and B is detailed, utilizing a chiral pool approach to generate the three stereogenic centers and a ring-closing metathesis (RCM) for the formation of the macrocyclic ring structure. Starting from a known glucose-derived alcohol, the formal total synthesis was achieved in an efficient 13-step protocol in 26% overall yield. It was found that substitution at the remote phenolic group significantly influenced the ratio of the E- and Z-double bond products in the RCM step. The introduction of phenol protecting groups provided E-isomers preferentially and also enhanced the rates of the RCM reactions.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Catálise , Ciclização , Compostos Organomercúricos/química , Fenóis/química , Rutênio/química , EstereoisomerismoRESUMO
[reaction: see text] Two formal chiral pool syntheses of the (-)-salicylihalamides A and B were achieved from commercially available 1,2,5,6-diacetone-d-glucose and l-rhamnose.