Alpha-ketoglutarate (aKG) is a central intermediate of cerebral energy metabolism and a precursor for glutamate synthesis in the brain. Alterations in aKG metabolism occur in pathological contexts, including isocitrate dehydrogenase (IDH) mutant astrocytomas and oligodendrogliomas, in which mutant IDH converts aKG to the oncometabolite 2-hydroxyglutarate. Given its central role in brain metabolism, non-invasive interrogation of aKG-dependent metabolic flux is needed. Hyperpolarized (HP) 13C MR enables real-time visualization of metabolic conversion by transiently enhancing signal intensity by several orders of magnitude. Leveraging this approach, we report the first-in-human feasibility and safety study of HP [1-13C]aKG MR spectroscopy in the healthy brain (n = 3). A standard operating procedure (SOP) was developed for sterile [1-13C]aKG dose production, achieving reproducible polarization levels averaging 30.5 ± 2.2%. Following intravenous administration, time-resolved 13C spectra in healthy volunteers demonstrated the detection of HP aKG resonance and a measurable downstream glutamate signal, consistent across repeat acquisitions, with a delayed temporal profile relative to aKG observed in a representative dataset. Although performed in healthy volunteers, these results establish feasibility for HP [1-13C]aKG metabolic imaging to open a new window into normal and pathological brain cellular metabolism.

The Blood–Brain Barrier (BBB) is a selective barrier that regulates the entry of molecules including nutrients, environmental toxins, and therapeutic medications into the brain. Its function continues to evolve postnatally, through aging, and disease states. Here we present a global proteomics analysis focused on the ontogeny and aging of proteins in human brain microvessels (BMVs), predominantly composed of brain endothelial cells. Our proteomic study quantified 6,223 proteins and revealed possible age-related alterations in BBB permeability due to basement membrane component changes through the early developmental stage and age-dependent changes in transporter expression. Age dependent expression changes were observed within nutrient transporters and transporters that play critical roles in drug disposition. This research 1) provides important information on the mechanisms that drive changes in the metabolic content of the brain with age and 2) enables the creation of physiologically based pharmacokinetic models for CNS drug distribution across different life stages.

Tumor acidosis is one of the hallmarks indicating the initiation and progression of various cancers. Here, we present a protocol for preparing a hyperpolarized (HP) 13 C -bicarbonate tissue pH MRI imaging contrast agent to detect aggressive tumors. We describe the steps for the formulation and polarization of a precursor molecule 13 C -glycerol carbonate ( 13 C-GLC), the post -dissolution reaction, and converting HP 13 C-GLC to an injectable HP 13 C -bicarbonate solution. We then detail procedures for MRI data acquisition to generate tumor pH maps for assessing tumor aggressiveness. For complete details on the use and execution of this protocol, please refer to Mu et al. 1

The effect of membrane transporters on drug disposition, efficacy and safety is now well recognized. Since the initial publication from the International Transporter Consortium, significant progress has been made in understanding the roles and functions of transporters, as well as in the development of tools and models to assess and predict transporter-mediated activity, toxicity and drug–drug interactions (DDIs). Notable advances include an increased understanding of the effects of intrinsic and extrinsic factors on transporter activity, the application of physiologically based pharmacokinetic modelling in predicting transporter-mediated drug disposition, the identification of endogenous biomarkers to assess transporter-mediated DDIs and the determination of the cryogenic electron microscopy structures of SLC and ABC transporters. This article provides an overview of these key developments, highlighting unanswered questions, regulatory considerations and future directions.

Introduction:The normal heart has remarkable metabolic flexibility that permits rapid switching between mitochondrial glucose oxidation and fatty acid (FA) oxidation to generate ATP. Loss of metabolic flexibility has been implicated in the genesis of contractile dysfunction seen in cardiomyopathy. Metabolic flexibility has been imaged in experimental models, using hyperpolarized (HP) [2-13C]pyruvate MRI, which enables interrogation of metabolites that reflect tricarboxylic acid (TCA) cycle flux in cardiac myocytes. This study aimed to develop methods, demonstrate feasibility for [2-13C]pyruvate MRI in the human heart for the first time, and assess cardiac metabolic flexibility. Methods:Good Manufacturing Practice [2-13C]pyruvic acid was polarized in a 5T polarizer for 2.5-3 hours. Following dissolution, QC parameters of HP pyruvate met all safety and sterility criteria for pharmacy release, prior to administration to study subjects. Three healthy subjects each received two HP injections and MR scans, first under fasting conditions, followed by oral glucose load. A 5cm axial slab-selective spectroscopy approach was prescribed over the left ventricle and acquired at 3s intervals on a 3T clinical MRI scanner. Results:The study protocol which included HP substrate injection, MR scanning and oral glucose load, was performed safely without adverse events. Key downstream metabolites of [2-13C]pyruvate metabolism in cardiac myocytes include the glycolytic derivative [2-13C]lactate, TCA-associated metabolite [5-13C]glutamate, and [1-13C]acetylcarnitine, catalyzed by carnitine acetyltransferase (CAT). After glucose load, 13C-labeling of lactate, glutamate, and acetylcarnitine from 13C-pyruvate increased by 39.3%, 29.5%, and 114%, respectively in the three subjects, that could result from increases in lactate dehydrogenase (LDH), pyruvate dehydrogenase (PDH), and CAT enzyme activity as well as TCA cycle flux (glucose oxidation). Conclusions:HP [2-13C]pyruvate imaging is safe and permits non-invasive assessment of TCA cycle intermediates and the acetyl buffer, acetylcarnitine, which is not possible using HP [1-13C]pyruvate. Cardiac metabolite measurement in the fasting/fed states provides information on cardiac metabolic flexibility and the acetylcarnitine pool.

Background: Recent shortages of intravenous (IV) fluids have resulted in healthcare systems converting administration of many medications from IV piggyback (IVPB) to IV push (IVP). Administering medications via IVP presents numerous advantages; however, IV site reactions such as phlebitis and infiltration may occur. Objective: The objective of this analysis is to evaluate the infusion site safety of ertapenem given as peripheral IVP compared to IVPB in adult patients. Methods: This was an institutional review board-approved, single-center, retrospective study. Patients, ages 18 or older, receiving IV ertapenem were identified. The major endpoints analyzed were IV site reactions including phlebitis and infiltration. The Naranjo Nomogram was utilized to assess the causality of the reactions to determine the likelihood of whether the event was caused by the medication itself or other factors. Results: To date, 283 administrations (92 patients) in the IVP group and 319 administrations (82 patients) in the IVPB group were analyzed. There were 13 IV site reactions compared to 8 in the IVP vs IVPB group, respectively (P-value = 0.16). Ten of the events in the IVP group were deemed "possible" and 2 deemed "doubtful," while the remaining event was considered "probable" per the Naranjo Nomogram. Of the events in the IVPB group, all 8 were found to be "possible." Conclusion: The administration of IVP ertapenem showed comparable rates of infusion site reactions compared to IVPB. Implementation of IVP ertapenem appears to be associated with infusion site safety similar to IVPB and should be considered safe to administer.

The ATP-binding cassette transporter P-glycoprotein (P-gp) limits the oral bioavailability of many drugs. Although P-gp has been well studied in humans and mice, little is known about the substrate specificities of many of its species orthologs. To address this, we performed in vitro analysis of P-gp transporter function using HEK293 cells stably expressing human, ovine, porcine, canine, and feline P-gp. We also employed a human physiologically based pharmacokinetic (PBPK) model to assess variations in digoxin exposure resulting from altered P-gp function. Compared to human P-gp, sheep P-gp had significantly less digoxin efflux (2.3-fold ±0.04 vs. 1.8-fold ±0.03, p < .0001) and all species orthologs had significantly less quinidine efflux compared with human P-gp (p < .05). Human P-gp also had significantly greater efflux of talinolol compared to sheep and dog P-gp (1.9-fold ±0.04 vs. 1.6-fold ±0.06, p = .003 and 1.6-fold ±0.05, p = .0002, respectively). P-gp expression protected all lines against paclitaxel-induced toxicity, with sheep P-gp being significantly less protective. The inhibitor verapamil demonstrated dose-dependent inhibition of all P-gp orthologs. Finally, a PBPK model showed digoxin exposure was sensitive to altered P-gp activity. Overall, our study found that species differences in this major drug transporter exist and that the appropriate species ortholog of P-gp should be evaluated during veterinary drug development.

Solid tumors such as prostate cancer (PCa) commonly develop an acidic microenvironment with pH 6.5-7.2, owing to heterogeneous perfusion, high metabolic activity, and rapid cell proliferation. In preclinical prostate cancer models, disease progression is associated with a decrease in tumor extracellular pH, suggesting that pH imaging may reflect an imaging biomarker to detect aggressive and high-risk disease. Therefore, we developed a hyperpolarized carbon-13 MRI method to image the tumor extracellular pH (pHe) and prepared it for clinical translation for detection and risk stratification of PCa. This method relies on the rapid breakdown of hyperpolarized (HP) 1,2-glycerol carbonate (carbonyl-13C) via base-catalyzed hydrolysis to produce HP 13CO32-, which is neutralized and converted to HP H13CO3-. After injection, HP H13CO3- equilibrates with HP 13CO2 in vivo and enables the imaging of pHe. Using insights gleaned from mechanistic studies performed in the hyperpolarized state, we solved issues of polarization loss during preparation in a clinical polarizer system. We successfully customized a reaction apparatus suitable for clinical application, developed clinical standard operating procedures, and validated the radiofrequency pulse sequence and imaging data acquisition with a wide range of animal models. The results demonstrated that we can routinely produce a highly polarized and safe HP H13CO3- contrast agent suitable for human injection. Preclinical imaging studies validated the reliability and accuracy of measuring acidification in healthy kidney and prostate tumor tissue. These methods were used to support an Investigational New Drug application to the U.S. Food and Drug Administration. This methodology is now ready to be implemented in human trials, with the ultimate goal of improving the management of PCa.

PURPOSE:This study aimed to develop and demonstrate the in vivo feasibility of a 3D stack-of-spiral balanced steady-state free precession(3D-bSSFP) urea sequence, interleaved with a metabolite-specific gradient echo (GRE) sequence for pyruvate and metabolic products, for improving the SNR and spatial resolution of the first hyperpolarized 13 C-MRI human study with injection of co-hyperpolarized [1-13 C]pyruvate and [13 C,15 N2 ]urea. METHODS:A metabolite-specific bSSFP urea imaging sequence was designed using a urea-specific excitation pulse, optimized TR, and 3D stack-of-spiral readouts. Simulations and phantom studies were performed to validate the spectral response of the sequence. The image quality of urea data acquired by the 3D-bSSFP sequence and the 2D-GRE sequence was evaluated with 2 identical injections of co-hyperpolarized [1-13 C]pyruvate and [13 C,15 N2 ]urea formula in a rat. Subsequently, the feasibility of the acquisition strategy was validated in a prostate cancer patient. RESULTS:Simulations and phantom studies demonstrated that 3D-bSSFP sequence achieved urea-only excitation, while minimally perturbing other metabolites (<1%). An animal study demonstrated that compared to GRE, bSSFP sequence provided an ∼2.5-fold improvement in SNR without perturbing urea or pyruvate kinetics, and bSSFP approach with a shorter spiral readout reduced blurring artifacts caused by J-coupling of [13 C,15 N2 ]urea. The human study demonstrated the in vivo feasibility and data quality of the acquisition strategy. CONCLUSION:The 3D-bSSFP urea sequence with a stack-of-spiral acquisition demonstrated significantly increased SNR and image quality for [13 C,15 N2 ]urea in co-hyperpolarized [1-13 C]pyruvate and [13 C,15 N2 ]urea imaging studies. This work lays the foundation for future human studies to achieve high-quality and high-SNR metabolism and perfusion images.

Background. Constipation is frequent in critically ill adults receiving opioids. Naloxegol (N), a peripherally acting mu-receptor antagonist (PAMORA), may reduce constipation. The objective of this trial was to evaluate the efficacy and safety of N to prevent constipation in ICU adults receiving opioids. Methods and Patients. In this single-center, double-blind, randomized trial, adults admitted to a medical ICU receiving IV opioids (≥100 mcg fentanyl/day), and not having any of 17 exclusion criteria, were randomized to N (25 mg) or placebo (P) daily randomized to receive N (25mg) or placebo (P) and docusate 100 mg twice daily until ICU discharge, 10 days, or diarrhea (≥3 spontaneous bowel movement (SBM)/24 hours) or a serious adverse event related to study medication. A 4-step laxative protocol was initiated when there was no SBM ≥3 days. Results. Only 318 (20.6%) of the 1542 screened adults during the 1/17–10/19 enrolment period met all inclusion criteria. Of these, only 19/381 (4.9%) met all eligibility criteria. After 7 consent refusals, 12 patients were randomized. The study was stopped early due to enrolment futility. The N (n = 6) and P (n = 6) groups were similar. The time to first SBM (N 41.4 ± 31.7 vs. P 32.5 ± 25.4 hours, P = 0.56) was similar. The maximal daily abdominal pressure was significantly lower in the N group (N 10 ± 4 vs. P 13 ± 5, P = 0.002). The median (IQR) daily SOFA scores were higher in N (N 7 (4, 8) vs. P 4 (3, 5), P < 0.001). Laxative protocol use was similar (N 83.3% vs. P 66.6%; P = 0.51). Diarrhea prevalence was high but similar (N 66.6% vs. P 66.6%; P = 1.0). No patient experienced opioid withdrawal. Conclusions. Important recruitment challenges exist for ICU trials evaluating the use of PAMORAs for constipation prevention. Despite being underpowered, our results suggest time to first SBM with naloxegol, if different than P, may be small. The effect of naloxegol on abdominal pressure, SOFA, and the interaction between the two requires further research.

Purpose The combined hyperpolarized (HP) 13 C pyruvate and urea MRI has provided a simultaneous assessment of glycolytic metabolism and tissue perfusion for improved cancer diagnosis and therapeutic evaluation in preclinical studies. This work aims to translate this dual‐probe HP imaging technique to clinical research. Methods A co‐polarization system was developed where [1‐ 13 C]pyruvic acid (PA) and [ 13 C, 15 N 2 ]urea in water solution were homogeneously mixed and polarized on a 5T SPINlab system. Physical and chemical characterizations and toxicology studies of the combined probe were performed. Simultaneous metabolic and perfusion imaging was performed on a 3T clinical MR scanner by alternatively applying a multi‐slice 2D spiral sequence for [1‐ 13 C]pyruvate and its downstream metabolites and a 3D balanced steady‐state free precession (bSSFP) sequence for [ 13 C, 15 N 2 ]urea. Results The combined PA/urea probe has a glass‐formation ability similar to neat PA and can generate nearly 40% liquid‐state 13 C polarization for both pyruvate and urea in 3‐4 h. A standard operating procedure for routine on‐site production was developed and validated to produce 40 mL injection product of approximately 150 mM pyruvate and 35 mM urea. The toxicology study demonstrated the safety profile of the combined probe. Dynamic metabolite‐specific imaging of [1‐ 13 C]pyruvate, [1‐ 13 C]lactate, [1‐ 13 C]alanine, and [ 13 C, 15 N 2 ]urea was achieved with adequate spatial (2.6 mm × 2.6 mm) and temporal resolution (4.2 s), and urea images showed reduced off‐resonance artifacts due to the J CN coupling. Conclusion The reported technical development and translational studies will lead to the first‐in‐human dual‐agent HP MRI study and mark the clinical translation of the first HP 13 C MRI probe after pyruvate.

Hyperpolarized [1-13C]alpha-ketoglutarate (aKG) is a metabolic probe that can provide direct access to the metabolic pathway for glutamate production or 2-HG production in altered alpha-ketoglutarate metabolism via IDH mutations. With its potential to serve as a biomarker of IDH1 mutational status in glioma and a measure of aKG metabolism in normal healthy subjects, we developed a new chemistry preparation and standard operating procedure (SOP) for on-site production of hyperpolarized GMP [1-13C]alpha-ketoglutarate and characterized the probe to investigate the feasibility of this probe in clinical setting. Following process qualification trials with HP [1-13C]aKG, an FDA IND application was prepared and submitted.

Motivation: Isocitrate dehydrogenase (IDH) mutational status is crucial for accurate diagnosis and prognosis of malignant gliomas. However, the current clinical assessment of IDH mutation requires an invasive brain biopsy for pathological testing. Goal(s): We aimed to perform first in-human MR studies using hyperpolarized [1-13C]alpha-ketoglutarate as a new probe of IDH mutational status via cancer metabolic reprogramming, along with cerebral bioenergetics. Approach: We acquired 13C MRS data from healthy brain volunteers (N=6) and glioma patients (N=6) who received hyperpolarized aKG. Results: Feasibility and safety were demonstrated in these 12 studies, with signals observed from [1-13C]alpha-ketoglutarate and its metabolite glutamate in the obtained 13C MRS data. Impact: MR molecular imaging with the new probe hyperpolarized [1-13C]alpha-ketoglutarate provided novel measurements of aKG metabolism and can investigate glioma IDH mutational status by detecting glutamate or the oncometabolite, 2-hydroxyglutarate.