Impact of Reduced Deuterium Intake on Thermoregulation.

The role of stable hydrogen isotopes in the thermoregulation and its regulation is poorly studied. We analyzed fluctuations in body temperature and changes in thermoregulation parameters in mice under conditions of reduced deuterium intake. The study was performed on male C57BL/6 mice that consumed water with a low (10 ppm) and normal (146 ppm) deuterium content. In 7 days, fluctuations of body temperature, locomotor activity, and oxygen uptake were assessed. Deuterium depletion in the body reduced the mean value of minute fluctuations of body temperature and the mean spectral density of minute fluctuations in body temperature in the 2-20-min periods. This attested to a stabilizing effect of deuterium depletion on the rhythms of body temperature fluctuations, without significant shifts in the thermogenesis parameters. Thus, drinking water with reduced deuterium content makes them less sensitive to external influences.

Deuterated stearic acid uptake and accumulation in lipid droplets of cat oocytes.

Fatty acid uptake and accumulation in lipid droplets are essential processes of lipid metabolism. Oocyte in vitro culture in media enriched with fatty acid is used to modify the lipid content and composition, aiming to study the consequences of obesity and enhance cell cryotolerance. We applied Raman spectroscopy and deuterium labeling approach to quantify stearic acid uptake and investigate its incorporation within oocytes. Our data suggest that deuterium labeling does not affect oocyte maturation rates. The efficiency of deuterated stearic acid (dSA) uptake was shown to decrease with the increase of its concentration in culture medium and the duration of in vitro culture. The molar ratio between dSA and bovine serum albumin has no significant effect on the dSA uptake for 200 µM but modifies concentration dependence of the lipid uptake. dSA accumulates in all the lipid droplets inside oocytes. Different lipid droplets within the same oocyte exhibit different concentrations of dSA. The scatter in the dSA concentration in lipid droplets decreases with the culture time. Using dSA as an example, we provide a comprehensive description of how fatty acid concentration, its molar ratio versus bovine serum albumin, and culture time affect the uptake of the fatty acids in oocytes. Raman microspectroscopy of deuterium-labeled fatty acids is a nondestructive tool providing information about fatty acid uptake and heterogeneity of their accumulation between lipid droplets within the single oocyte.

Design, synthesis and biological activity of deuterium-based FFA1 agonists with improved pharmacokinetic profiles.

The free fatty acid receptor 1 (FFA1) is considered as a promising anti-diabetic target based on its function of glucose-stimulated insulin secretion. The previously reported compound 2 is a highly potent FFA1 agonist, but it might be suffered from poor pharmacokinetic properties because the phenylpropanoic acid is vulnerable to ß-oxidation. To identify orally available analogs, we tried to block the route of ß-oxidation by incorporating deuterium at phenylpropionic acid moiety. As expected, the deuterium-based analogs 3 and 4 exhibited better pharmacokinetic properties than parent compound 2. Although the difference of potency between compound 2 and 3 is quite small, the glucose-lowering effect of deuterium analog 3 was better than that of compound 2. Meanwhile, compound 3 docked well into the same binding pocket of TAK-875, and formed almost identical interactions of TAK-875 in binding site. Different from glibenclamide, a lower risk of hypoglycemia was observed in compound 3 even at the high dose of 60 mg/kg.

Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals.

OBJECTIVE: Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine (Austedo, Teva Pharmaceutical Industries, Ltd) is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools. DATA SOURCES: A search of the MEDLINE (1946 to present) database was undertaken using the Ovid interface. The search was conducted using the heading deuterium and then limited to Administration & Dosage, Adverse Effects, Pharmacokinetics, Pharmacology, Poisoning, Therapeutic Use, and Toxicity. STUDY SELECTION AND DATA EXTRACTION: All articles were reviewed and those with human information were included. Review articles were likewise interrogated for additional published human data. CONCLUSIONS: Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.

The Design, Synthesis and Preliminary Pharmacokinetic Evaluation of d3-Poziotinib Hydrochloride.

To establish a synthetic route to d3-poziotinib hydrochloride. Treatment of 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1) with d3-methyliodide afforded the etherization product, which reacted with 3,4-dichloro-2-fluoroaniline to generate the key intermediate d3-4-(3,4-dichloro-2-fluorophenylamino)-7-methoxyquinazolin-6-yl pivalate (3). Followed the de-protection reaction, the nucleophilic substitution (SN2) reaction with tert-butyl 4-(tosyloxy)piperidine-1-carboxylate (TSP), and the de-protection reaction of t-butoxycarbonyl (Boc) group, and the amide formation reaction with acrylyl chloride, d3-poziotinib was obtained, which was converted to hydrochloride salt by treatment with concentrated hydrochloric acid (HCl). Starting from a known compound 4-chloro-7-hydroxyquinazolin-6-yl pivalate (1), after 7 steps transformation, d3-poziotinib hydrochloride was obtained with a total yield of 9.02%. The structure of d3-poziotinib hydrochloride was confirmed by 1H-NMR, 13C-NMR, and high resolution (HR)-MS. Meanwhile, the in vitro microsomal stability experiment showed that d3-poziotinib had a longer half time (t1/2 = 4.6 h) than poziotinib (t1/2 = 3.5 h).

Synthesis of deuterium-enriched sorafenib derivatives and evaluation of their biological activities.

Deuterium substitution has been widely known that can improve the pharmacokinetic profiles due to isotope effect. Herein, a series of deuterated sorafenib derivatives have been synthesized and characterized by 1H NMR, 13C NMR and MS. Their antitumor activities were evaluated in vitro against human hepatoma cell line HepG2 and human cervical carcinoma cell line HeLa. The LogP values were detected by high-performance liquid chromatography. Subsequently, the metabolic stability and pharmacokinetics study were assessed in vitro and in vivo.

Altering Metabolic Profiles of Drugs by Precision Deuteration 2: Discovery of a Deuterated Analog of Ivacaftor with Differentiated Pharmacokinetics for Clinical Development.

Ivacaftor is currently used for the treatment of cystic fibrosis as both monotherapy (Kalydeco; Vertex Pharmaceuticals, Boston, MA) and combination therapy with lumacaftor (Orkambi; Vertex Pharmaceuticals). Each therapy targets specific patient populations: Kalydeco treats patients carrying one of nine gating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, whereas Orkambi treats patients homozygous for the F508del CFTR mutation. In this study, we explored the pharmacological and metabolic effects of precision deuteration chemistry on ivacaftor by synthesizing two novel deuterated ivacaftor analogs, CTP-656 (d9-ivacaftor) and d18-ivacaftor. Ivacaftor is administered twice daily and is extensively converted in humans to major metabolites M1 and M6; therefore, the corresponding deuterated metabolites were also prepared. Both CTP-656 and d18-ivacaftor showed in vitro pharmacologic potency similar to that in ivacaftor, and the deuterated M1 and M6 metabolites showed pharmacology equivalent to that in the corresponding metabolites of ivacaftor, which is consistent with the findings of previous studies of deuterated compounds. However, CTP-656 exhibited markedly enhanced stability when tested in vitro. The deuterium isotope effects for CTP-656 metabolism (DV = 3.8, DV/K = 2.2) were notably large for a cytochrome P450-mediated oxidation. The pharmacokinetic (PK) profile of CTP-656 and d18-ivacaftor were assessed in six healthy volunteers in a single-dose crossover study, which provided the basis for advancing CTP-656 in development. The overall PK profile, including the 15.9-hour half-life for CTP-656, suggests that CTP-656 may be dosed once daily, thereby enhancing patient adherence. Together, these data continue to validate deuterium substitution as a viable approach for creating novel therapeutic agents with properties potentially differentiated from existing drugs.

Total Synthesis and Metabolic Stability of Hispidulin and Its d-Labelled Derivative.

Hispidulin is a naturally occurring flavone known to have various Central nervous system (CNS) activities. Proposed synthetic approaches to synthesizing hispidulin have proven unsatisfactory due to their low feasibility and poor overall yields. To solve these problems, this study developed a novel scheme for synthesizing hispidulin, which had an improved overall yield as well as more concise reaction steps compared to previous methods reported. Additionally, using the same synthetic strategy, d-labelled hispidulin was synthesized to investigate its metabolic stability against human liver microsome. This work may produce new chemical entities for enriching the library of hispidulin-derived compounds.

Tracer-based estimates of protein flux in cases of incomplete product renewal: evidence and implications of heterogeneity in collagen turnover.

The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [(2)H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear "inert" or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.

Mass spectrometry-based microassay of (2)H and (13)C plasma glucose labeling to quantify liver metabolic fluxes in vivo.

Mouse models designed to examine hepatic metabolism are critical to diabetes and obesity research. Thus, a microscale method to quantitatively assess hepatic glucose and intermediary metabolism in conscious, unrestrained mice was developed. [(13)C3]propionate, [(2)H2]water, and [6,6-(2)H2]glucose isotopes were delivered intravenously in short- (9 h) and long-term-fasted (19 h) C57BL/6J mice. GC-MS and mass isotopomer distribution (MID) analysis were performed on three 40-µl arterial plasma glucose samples obtained during the euglycemic isotopic steady state. Model-based regression of hepatic glucose and citric acid cycle (CAC)-related fluxes was performed using a comprehensive isotopomer model to track carbon and hydrogen atom transitions through the network and thereby simulate the MIDs of measured fragment ions. Glucose-6-phosphate production from glycogen diminished, and endogenous glucose production was exclusively gluconeogenic with prolonged fasting. Gluconeogenic flux from phosphoenolpyruvate (PEP) remained stable, whereas that from glycerol modestly increased from short- to long-term fasting. CAC flux [i.e., citrate synthase (VCS)] was reduced with long-term fasting. Interestingly, anaplerosis and cataplerosis increased with fast duration; accordingly, pyruvate carboxylation and the conversion of oxaloacetate to PEP were severalfold higher than VCS in long-term fasted mice. This method utilizes state-of-the-art in vivo methodology and comprehensive isotopomer modeling to quantify hepatic glucose and intermediary fluxes during physiological stress in mice. The small plasma requirements permit serial sampling without stress and the affirmation of steady-state glucose kinetics. Furthermore, the approach can accommodate a broad range of modeling assumptions, isotope tracers, and measurement inputs without the need to introduce ad hoc mathematical approximations.

Deuterated Drugs.

Many drugs are carbon-based, and carbon-hydrogen bonding is particularly relevant for understanding important properties of drug molecules. Deuteration refers to the selective replacement of protium hydrogen isotope atoms in small-molecule drugs with deuterium hydrogen isotope atoms. Deuteration of a drug is most likely to affect pharmacokinetic properties, such as metabolism, rather than its pharmacodynamic effects. For this reason, the metabolism of certain drugs may be favorably influenced when deuterium is substituted for protium, resulting in improved safety, tolerability, or efficacy. Examples of deuterated drugs that have been evaluated in clinical studies include paroxetine, tetrabenazine, and dextromethorphan.

Content of deuterium in biological fluids and organs: Influence of deuterium depleted water on D/H gradient and the process of adaptation.

It is found that consumption of deuterium depleted water reduces not only the content of deuterium in biological fluids but also more than 2 times reduces the D/H gradient value along the line: mixed saliva > blood plasma. The experimental data showed that a physiological solution prepared on deuterium depleted water during induced apoptosis activates the DNA repair system, significantly reducing the number of single-stranded DNA breaks, which, in general, indicates an increase in the efficiency of defensive systems of the cell.

Resolving futile glucose cycling and glycogenolytic contributions to plasma glucose levels following a glucose load.

PURPOSE: After a glucose load, futile glucose/glucose-6-phosphate (G6P) cycling (FGC) generates [2-(2) H]glucose from (2)H(2)O thereby mimicking a paradoxical glycogenolytic contribution to plasma glucose levels. Contributions of load and G6P derived from gluconeogenesis, FGC, and glycogenolysis to plasma glucose levels need resolution. A simple methodology is proposed integrating the administration of (2)H(2)O with a glucose load containing [1-(2)H, 1-(13)C]glucose and [2-(2)H, 2-(13)C]glucose. METHODS: Mice fasted for 6 (n = 7) or 24 h (n = 5) were intraperitoneally injected with 2 mg/g 10% enriched glucose in 35 µL/g (2)H(2)O. Plasma glucose enrichment was analyzed by (2)H NMR after 30 min. RESULTS: For 6-h fasted mice, 12.3 ± 1.5% of plasma glucose was pre-existing, 44.3 ± 2.7% was load derived, and 43.4 ± 1.8% G6P derived. G6P origins were 26.0 ± 2.0% gluconeogenesis, 10.9 ± 2.6% FGC, and 6.5 ± 3.4% glycogenolysis. For 24-h fasted mice, 18.2 ± 8.5% was pre-existing, 41.1 ± 5.0 % was load derived, and 40.8 ± 4.3% G6P derived. G6P origins were 27.1 ± 3.3% gluconeogenesis, 13.1 ± 2.8% FGC, and 0.6 ± 2.4% glycogenolysis. CONCLUSION: After a glucose load, glycogenolytic contribution to plasma glucose was negligible, whereas FGC was significant for both 6- and 24-h fasted mice.

2H2O-based high-density lipoprotein turnover method for the assessment of dynamic high-density lipoprotein function in mice.

OBJECTIVE: High-density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver for clearance. Reduced HDL-cholesterol (HDLc) is associated with atherosclerosis; however, as a predictor of cardiovascular disease, HDLc has limitations because it is not a direct marker of HDL functionality. Our objective was to develop a mass spectrometry-based method for the simultaneous measurement of HDLc and ApoAI kinetics in mice, using a single (2)H2O tracer, and use it to examine genetic and drug perturbations on HDL turnover in vivo. APPROACH AND RESULTS: Mice were given (2)H2O in the drinking water, and serial blood samples were collected at different time points. HDLc and ApoAI gradually incorporated (2)H, allowing experimental measurement of fractional catabolic rates and production rates for HDLc and ApoAI. ApoE(-/-) mice displayed increased fractional catabolic rates (P<0.01) and reduced production rates of both HDLc and ApoAI (P<0.05) compared with controls. In human ApoAI transgenic mice, levels and production rates of HDLc and human ApoAI were strikingly higher than in wild-type mice. Myriocin, an inhibitor of sphingolipid synthesis, significantly increased both HDL flux and macrophage-to-feces reverse cholesterol transport, indicating compatibility of this HDL turnover method with the macrophage-specific reverse cholesterol transport assay. CONCLUSIONS: (2)H2O-labeling can be used to measure HDLc and ApoAI flux in vivo, and to assess the role of genetic and pharmacological interventions on HDL turnover in mice. Safety, simplicity, and low cost of the (2)H2O-based HDL turnover approach suggest that this assay can be scaled for human use to study effects of HDL targeted therapies on dynamic HDL function.

Hematocrit and oxygenation dependence of blood (1)H(2)O T(1) at 7 Tesla.

Knowledge of blood (1)H2O T1 is critical for perfusion-based quantification experiments such as arterial spin labeling and cerebral blood volume-weighted MRI using vascular space occupancy. The dependence of blood (1)H2O T1 on hematocrit fraction (Hct) and oxygen saturation fraction (Y) was determined at 7 T using in vitro bovine blood in a circulating system under physiological conditions. Blood (1)H2O R1 values for different conditions could be readily fitted using a two-compartment (erythrocyte and plasma) model, which are described by a monoexponential longitudinal relaxation rate constant dependence. It was found that T1 = 2171 ± 39 ms for Y = 1 (arterial blood) and 2010 ± 41 ms for Y = 0.6 (venous blood), for a typical Hct of 0.42. The blood (1)H2O T1 values in the normal physiological range (Hct from 0.35 to 0.45, and Y from 0.6 to 1.0) were determined to range from 1900 to 2300 ms. The influence of oxygen partial pressure (pO2) and the effect of plasma osmolality for different anticoagulants were also investigated. It is discussed why blood (1)H2O T1 values measured in vivo for human blood may be about 10-20% larger than found in vitro for bovine blood at the same field strength.

Acetaminophen glucuronide and plasma glucose report identical estimates of gluconeogenesis and glycogenolysis for healthy and prediabetic subjects using the deuterated water method.

Plasma glucose (2) H-enrichment in positions 5 ((2) H5) and 2 ((2) H2) from deuterated water ((2) H2 O) provides a measure of the gluconeogenic contribution to endogenous glucose production. Urinary glucuronide analysis can circumvent blood sampling but it is not known if glucuronide and glucose enrichments are equal. Thirteen subjects with impaired fasting glucose/impaired glucose tolerance and 11 subjects with normal fasting glucose and normal glucose tolerance ingested (2) H2 O to ∼0.5% body water and acetaminophen. Glucose and glucuronide (2) H5 and (2) H2 were measured by (2) H NMR spectroscopy of monoacetone glucose. For normal fasting glucose/normal glucose tolerance, (2) H5 was 0.23 ± 0.02% and 0.25 ± 0.02% for glucose and glucuronide, respectively, whereas (2) H2 was 0.47 ± 0.01% and 0.49 ± 0.02%, respectively. For impaired fasting glucose/impaired glucose tolerance, (2) H5 was 0.22 ± 0.01% and 0.26 ± 0.02% for glucose and glucuronide, respectively, whereas (2) H2 was 0.46 ± 0.01% and 0.49 ± 0.02%, respectively. The gluconeogenic contribution to endogenous glucose production measured from glucose and glucuronide were identical for both normal fasting glucose/normal glucose tolerance (48 ± 4 vs. 51 ± 3%) and impaired fasting glucose/impaired glucose tolerance (48 ± 2 vs. 53 ± 3%).

Deuterium in drug discovery: progress, opportunities and challenges.

Substitution of a hydrogen atom with its heavy isotope deuterium entails the addition of one neutron to a molecule. Despite being a subtle change, this structural modification, known as deuteration, may improve the pharmacokinetic and/or toxicity profile of drugs, potentially translating into improvements in efficacy and safety compared with the non-deuterated counterparts. Initially, efforts to exploit this potential primarily led to the development of deuterated analogues of marketed drugs through a 'deuterium switch' approach, such as deutetrabenazine, which became the first deuterated drug to receive FDA approval in 2017. In the past few years, the focus has shifted to applying deuteration in novel drug discovery, and the FDA approved the pioneering de novo deuterated drug deucravacitinib in 2022. In this Review, we highlight key milestones in the field of deuteration in drug discovery and development, emphasizing recent and instructive medicinal chemistry programmes and discussing the opportunities and hurdles for drug developers, as well as the questions that remain to be addressed.

Cytosolic phosphoenolpyruvate carboxykinase does not solely control the rate of hepatic gluconeogenesis in the intact mouse liver.

When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEPCK protein content in the control of gluconeogenesis was unclear. In this study, metabolic control of liver gluconeogenesis was quantified in groups of mice with varying PEPCK protein content. Surprisingly, livers with a 90% reduction in PEPCK content showed only a approximately 40% reduction in gluconeogenic flux, indicating a lower than expected capacity for PEPCK protein content to control gluconeogenesis. However, PEPCK flux correlated tightly with TCA cycle activity, suggesting that under some conditions in mice, PEPCK expression must coordinate with hepatic energy metabolism to control gluconeogenesis.

Use of (2)H(2)O for estimating rates of gluconeogenesis: determination and correction of error due to transaldolase exchange.

The use of deuterated water as a method to measure gluconeogenesis has previously been well validated and is reflective of normal human physiology. However, there has been concern since the method was first introduced that transaldolase exchange may lead to the overestimation of gluconeogenesis. We examined the impact of transaldolase exchange on the estimation of gluconenogenesis using the deuterated water method under a variety of physiological conditions in humans by using the gluconeogenic tracer [U-(13)C]propionate, (2)H(2)O, and (2)H/(13)C nuclear magnetic resonance (NMR) spectroscopy. When [U-(13)C]propionate was used, (13)C labeling inequality occurred between the top and bottom halves of glucose in individuals fasted for 12-24 h who were weight stable (n = 18) or had lost weight via calorie restriction (n = 7), consistent with transaldolase exchange. Similar analysis of glucose standards revealed no significant difference in the total (13)C enrichment between the top and bottom halves of glucose, indicating that the differences detected were biological, not analytical, in origin. This labeling inequality was attenuated by extending the fasting period to 48 h (n = 12) as well as by dietary carbohydrate restriction (n = 7), both conditions associated with decreased glycogenolysis. These findings were consistent with a transaldolase effect; however, the resultant overestimation of gluconeogenesis in the overnight-fasted state was modest (7-12%), leading to an error of 14-24% that was easily correctable by using either a simultaneous (13)C gluconeogenic tracer or a correction nomogram generated from data in the present study.

In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days.

Neutrophils are essential effector cells of the innate immune response and are indispensable for host defense. Apart from their antimicrobial functions, neutrophils inform and shape subsequent immunity. This immune modulatory functionality might however be considered limited because of their generally accepted short lifespan (< 1 day). In contrast to the previously reported short lifespans acquired by ex vivo labeling or manipulation, we show that in vivo labeling in humans with the use of (2)H(2)O under homeostatic conditions showed an average circulatory neutrophil lifespan of 5.4 days. This lifespan is at least 10 times longer than previously reported and might lead to reappraisal of novel neutrophil functions in health and disease.