Camizestrant is the next-generation oral selective estrogen receptor degrader and complete estrogen receptor antagonist in Phase 3 development for hormone receptor-positive breast cancer. To investigate the impact of manufacturing changes during pivotal Phase 3 studies, this open-label, randomized crossover study of 32 postmenopausal healthy volunteers determined the relative bioavailability of a tablet used in early clinical studies (Phase 1 tablet), a tablet designed for late-phase development (prototype Phase 3 tablet), and an oral solution. Absolute oral bioavailability in the fasted state (using a [14C] camizestrant intravenous microtracer) and effects of a high-fat meal on the prototype Phase 3 tablet were also determined. The geometric mean ratios (GMRs) of the prototype Phase 3/Phase 1 tablets (% [90% CI]) for Cmax and AUC, respectively were 98.7 (87.4-111.5) and 97.4 (92.6-102.5) at 75 mg (n = 15), and 96.6 (86.9-107.5) and 100.4 (96.2-104.9) at 300 mg (n = 15). GMRs of the prototype Phase 3 tablet/oral solution for Cmax and AUC were 96.2 (85.3-108.7) and 99.5 (94.6-104.6) at 75 mg (n = 15). Fed-to-fasted Cmax and AUC GMRs were 106.2 (94.3-119.7) (n = 16) and 109.8 (104.4-115.5) (n = 15) at 75 mg (n = 16), and 115.9 (104.3-128.7) and 102.3 (98.0-106.8) at 300 mg (n = 15). Absolute oral bioavailability at 75 mg (n = 6) and 300 mg (n = 6) was 42.5% (36.8%-49.0%) and 55.1% (48.5%-62.5%). The formulations showed similar exposures, supporting the planned manufacturing changes. Camizestrant exhibited moderate bioavailability; exposures were similar under fasted and high-fat meal conditions, supporting its administration with or without food.

A review covering early studies to the latest development in carbon isotope exchange and transfer reactions involving CO 2 .

Carboxylation reactions are fundamental transformations in organic chemistry, enabling the synthesis of diverse and valuable compounds, including polymers, pharmaceuticals, and agrochemicals. In recent years, carboxylation processes proceeding via carbon isotope exchange or CO2 transfer have received significant interest. These processes offer intriguing decarboxylation/carboxylation reactivity and enhance existing carboxylation pathways, in particular by delivering efficient and sustainable solutions for carbon isotope labelling. This review compares and contrasts the related fields of carbon isotope exchange and transfer reactions involving CO2. We detail early developments to the most recent advancements and discuss the advantages and limitations of each approach, in particular with regard to the efficiency of each strategy.

Abstract Computationally derived parameters have been employed to guide the rational design of an effective iridium catalyst for the C–H activation and hydrogen isotope exchange of secondary and tertiary aryl sulfonamides. By analyzing substrate binding energies and solid angles, an iridium(I) complex featuring a chelating NHC-pyridine ligand has been developed that is specifically tailored to accommodate sterically demanding sulfonamide directing groups. This catalyst has demonstrated broad applicability under mild reaction conditions, enabling efficient isotopic labeling across 23 diverse sulfonamide substrates.

The carboxylation of indoles through a CO2 transfer reaction with carboxylate salts is described. By altering the reaction conditions either N-H or C3-H carboxylation can occur. The reaction is also applicable to the carboxylation of other indole derivatives and amines. The relevance of this procedure is further demonstrated through the preparation and carbon isotope labelling of several biologically relevant carboxylated indoles/amines.

We describe a panel of flavoenzymes with the ability to catalyse stereoselective hydrogen isotope exchange (HIE) between 2H2O (D2O) and reduced nicotinamide cofactors, enabling a simple and redox-neutral route to deuterated NAD(P)H isotopomers. In screening the FMN- and FAD-dependent enzymes, which have diverse native functions, we identified catalysts (many of which are commercially available) that selectively yield the full suite of [4-2H]-NAD(P)H stereoisotopomers in a single step. In combining stereo-complementary enzymes, we also identify simple one-pot routes to dideuterated [4-2H2]-NAD(P)H. The biocatalytic methods provide near-quantitative 2H-incorporation for both NADPH and NADH under mild conditions, using readily available 2H2O as the isotope source. The observed activity indicates a reversible flavoenzyme hydride-transfer cycle, reliant on hydrogen/deuterium exchange on the transiently reduced flavin cofactors. We provide computational analysis to rationalise the stereochemical outcomes of the screened reactions. Finally, preparative-scale syntheses are described that deliver isolated deuterated cofactors in excellent yield and isotopic purity. We envisage this easily implemented procedure will simplify access to these important biochemical compounds for mechanistic studies, and may open up wider 2H2O-driven biocatalytic deuteration reactions.

Radiolabelled compound stability studies are an essential requirement to support human Absorption, Distribution, Metabolism and Excretion (hADME) clinical studies. This paper assesses the suitability of stability data extrapolation for carbon-14 labelled compounds following the principles set out in ICH Q1E guidelines. Data from seven stability studies at specific activities ranging from 8.2 to 81.0 μCi/mg have been assessed. Predictive models were employed to extrapolate stability data and determine extended retest periods, which were then subsequently verified against actual experimental results. In all cases, the predicted extended retest period aligned with the observed data. These findings demonstrate that stability extrapolations can be used to support extended retest periods for radiolabelled compounds.

Academic-Industrial collaborations have played an important role in developing new synthetic methodologies for the isotope chemistry team at AstraZeneca. In this viewpoint, we present the outcome of two collaborations and illustrate how the innovative methodologies developed through these partnerships have been effectively applied within an AstraZeneca project.

AbstractIn this study, the mass balance, pharmacokinetics (PK) and metabolism of atuliflapon, a novel 5‐lipoxygenase‐activating protein inhibitor, were investigated in healthy male subjects. A single oral dose of 200 mg [14C]atuliflapon suspension was administered to six healthy male subjects. Mass balance, PK and metabolite profiles of atuliflapon were analyzed using radioactivity monitoring and liquid chromatography with mass spectrometry analysis. The safety of atuliflapon was assessed during the study. Atuliflapon was rapidly absorbed with a median tmax of 1.5 h, followed by a biphasic decline in plasma exposure rendering a terminal half‐life of ~20 h. Unchanged atuliflapon was the predominant radioactive component in plasma, accounting for 40.1% of the total drug‐related exposure (DRE), while a direct N‐glucuronide was the only metabolite exceeding 10% of DRE, accounting for 20.9%. Renal excretion of intact atuliflapon accounted for <1% of the administered dose. In total 85.2% of administered radioactivity was recovered over 312 h with 79.3% and 5.9% in feces and urine, respectively. Parent atuliflapon contributed to approximately 40% of the recovered dose in excreta, while metabolites resulting from phase 1 oxidative pathways accounted for more than 30% of the excreted dose. Overall, a single oral dose of 200 mg [14C]atuliflapon suspension was well tolerated in healthy male subjects. The human metabolism and disposition data obtained will support future development and submissions of atuliflapon as a potential candidate drug for the treatment of cardiovascular, cardiorenal, and respiratory indications.

As part of a medicinal chemistry program aimed at discovering a mineralocorticoid receptor modulator for treatment of kidney and cardiovascular indications, multiple labeled versions of the lead compound, balcinrenone (AZD9977), were prepared. Four stable isotope labeled versions of the compound were prepared for clinical bioanalysis and biological investigations. Three of these stable isotope labeled compounds were tritiated as well as the parent for biology applications and DMPK investigations. They were prepared using a standard iodination-tritiodehalogentation approach. Finally, AZD9977 was prepared in carbon-14 labeled form for preclinical and clinical applications.

Pharmaceutical-aligned research endeavors continue to diversify, including via the installation of new chemical functionality and non-classical bioisosteres within drug design. With this, an equally high demand emerges for the direct installation of isotopic substituents into these scaffolds within drug discovery programmes, as isotopologues are essential for the elucidation of the biological efficacy and metabolic fate of the active pharmaceutical ingredient (API). The sulfoximine functional group has recently become established as a high-value unit in this context; however, general and effective methods for the synthesis of deuterium (2H, D) and tritium (3H, T) labelled analogues have remained elusive. Herein, we disclose the design and development of the first iridium-catalyzed sulfoximine-directed hydrogen isotope exchange (HIE) systems that permit the site-selective integration of a distinguishing atomic label at aromatic C(sp2)-H and more challenging C(sp3)-H moieties. Moreover, we exemplify the broad applicability of these methods within a spectrum of molecular settings, as well as in the late-stage generation of isotopically-enriched complex bioactive architectures.

An open-label, single-center, phase I study was conducted to determine the absolute bioavailability and absorption, distribution, metabolism, and excretion of capivasertib-a potent, selective AKT serine/threonine kinase inhibitor-in healthy males. In part 1, six participants received a single oral dose of capivasertib (400 mg; tablets) followed by a [14C]-radiolabeled intravenous microdose of capivasertib (100 μg). After a 14-day washout, five of the participants proceeded to part 2 and received a single oral dose of [14C]capivasertib (400 mg; solution). In part 1, median time of maximum observed concentration for capivasertib was 1.7 hours, geometric mean terminal elimination half-life was 12.9 hours, and absolute bioavailability was estimated at 28.6% (90% confidence interval, 23.9 to 34.2). In part 2, a high proportion of the administered radioactivity was recovered over the 168-hour sampling period [mean recovery: 95.1% (feces, 50.4%; urine, 44.7%)]. Unchanged capivasertib in urine accounted for 7.4% of the total dose and 21.1% of the systemically available drug. Geometric mean renal clearance was 8.3 L/h, suggesting active tubular secretion. Twelve metabolites were identified in plasma. M11 (AZ14102143)-the glucuronide conjugate of capivasertib, inactive as an AKT serine/threonine kinase inhibitor-was the most abundant, accounting for a mean 78.4% of the plasma drug-related area under the curve. Of 22 metabolites identified in excreta, M11 was the most abundant (mean 28.2% of administered dose), indicating direct glucuronidation as one of the major routes of metabolism. No new safety concerns were identified. SIGNIFICANCE STATEMENT: This study provides characterization of the pharmacokinetics of capivasertib-a potent, selective AKT serine/threonine kinase (AKT) inhibitor-including absolute bioavailability, mass balance, and metabolic fate in humans; the findings are being used to inform further clinical development. Absolute bioavailability was estimated at 28.6%, and mean recovery of the administered dose in excreta over 168 hours was 95.1%. M11 (AZ14102143)-the glucuronide conjugate, inactive as an AKT inhibitor-was the most abundant identified metabolite in plasma and excreta.

This study evaluated the mass balance and disposition of AZD4831, a novel myeloperoxidase inhibitor, in six healthy participants using a C-14-labeled microtracer coupled with analysis by accelerator mass spectrometry (AMS). A single oral dose of 10 mg C-14-AZD4831 (14.8 kBq) was administered as a solution, and C-14 levels were quantified by AMS in blood, urine, and feces over 336 hours postdose. AZD4831 was rapidly absorbed, and AZD4831 plasma concentrations declined in a biphasic manner, with a long half-life of 52 hours. AZD4831 was eliminated via metabolism and renal excretion. An N-carbamoyl glucuronide metabolite of AZD4831 (M7), formed primarily via UGT1A1, was the predominant circulating metabolite. Presumably, M7 contributed to the long half-life of AZD4831 via biliary elimination and hydrolysis/enterohepatic recirculation of AZD4831. On average, similar to 84% of administered C-14-AZD4831 was recovered by 336 hours postdose (urine, 51.2%; feces, 32.4%). Between 32%-44% of the dose was excreted as unchanged AZD4831 in urine, indicating renal elimination as the major excretory route. Only 9.7% of overall fecal recovery was recorded in the first 48 hours, with the remainder excreted over 48%-336 hours, suggesting that most fecal recovery was due to biliary elimination. Furthermore, only 6% of unchanged AZD4831 was recovered in feces. Overall, the fraction of the administered AZD4831 dose absorbed was high. C-14-AZD4831 was well tolerated. These findings contribute to increasing evidence that human absorption, distribution, metabolism, and excretion studies can be performed with acceptable mass balance recovery at therapeutically relevant doses and low radiolabel-specific activity using an AMS-C-14 microtracer approach. SIGNIFICANCE STATEMENT In this study, the human absorption, distribution, metabolism, and excretion (hADME) of the novel myeloperoxidase inhibitor AZD4831 was assessed following oral administration. This included investigation of the disposition of M7, the N-carbamoyl glucuronide metabolite. Resolution of challenges highlighted in this study contributes to increasing evidence that hADME objectives can be achieved in a single study for compounds with therapeutically relevant doses and low radiolabel-specific activity by using an AMS-C-14 microtracer approach, thus reducing the need for preclinical radiolabeled studies.

An absorption, distribution, metabolism, and excretion study was performed to determine the basic pharmacokinetic parameters, mass balance, and metabolite profiles of balcinrenone, a mineralocorticoid receptor modulator, in humans. This open-label, single-center, nonrandomized study had a two-period design. In period 1, eight healthy male subjects were dosed with a microtracer intravenous infusion of [14C]balcinrenone shortly after receiving an oral dose of unlabeled balcinrenone in a capsule. Following a 7-day washout, the same group of subjects subsequently received an oral dose of [14C]balcinrenone as a suspension in period 2. Clearance and absolute bioavailability of balcinrenone were determined to be 14.2 l/h and 52%, respectively. Renal clearance was determined to be 5.4 l/h (>fu • glomerular filtration rate), indicating elimination via active tubular secretion, which was potentially mediated by P-glycoprotein 1 and/or organic anion transporter 3, according to in vitro transporter data. In total, 94.1% of the oral dose was recovered: 45.2% in the urine and 48.9% in the feces. Balcinrenone was primarily metabolized via oxidation, and in vitro data suggest that cytochrome P450 3A4 was the main enzyme responsible. Intact [14C]balcinrenone accounted for 55% of drug-related material in the plasma; four metabolites were identified, each representing <6% of the total plasma radioactivity. In conclusion, this two-period study has determined the basic pharmacokinetic parameters of balcinrenone in humans, including absolute bioavailability and disposition. No metabolites warranted further evaluation on account of their low representation, and any contribution to the pharmacodynamic response or potential drug-drug interactions was deemed negligible. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the pharmacokinetics, disposition, and metabolism of balcinrenone following oral and microtracer intravenous administration in humans. In vitro phenotyping and transporter data granted mechanistic insights into the absorption, distribution, metabolism, and excretion properties of balcinrenone. This knowledge will guide future nonclinical and clinical studies evaluating drug-drug interactions, organ dysfunction, and safety of metabolites.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Diabetes is a global health problem that was estimated to be the 7th leading cause of death worldwide in 2016. Type 2 diabetes mellitus (T2DM) is classically associated with genetic and environmental factors, however recent studies have demonstrated that the gut microbiome, which is altered in T2DM patients, is also likely to play a significant role in disease development. Despite this, the identity of microbiome-derived metabolites that influence T2DM onset and/or progression remain elusive. Here we demonstrate that a serum biomarker for T2DM, previously of unknown structure and origin, is actually two microbiome-derived metabolites, 3-methyl-4-(trimethylammonio)butanoate (3M-4-TMAB) and 4-(trimethylammonio)pentanoate (4-TMAP). These metabolites are produced by the Lachnospiraceae family of bacteria, which are highly prevalent in the gut microbiome of T2DM patients and are associated with high dietary fat intake. Treatment of human liver cells with 3M-4-TMAB and 4-TMAP results in a distinct change in the acylcarnitine profile in these cells and significantly reduced their insulin sensitivity; both indicators of T2DM. These results provide evidence of a mechanistic link between gut microbiome derived metabolites and T2DM.### Competing Interest StatementThe authors have declared no competing interest.

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.

Herein, we report the rational, computationally-guided design of an iridium(I) catalyst system capable of enabling directed hydrogen isotope exchange (HIE) with the challenging sulfone directing group. Substrate binding energy was used as a parameter to guide rational ligand design via an in silico catalyst screen, resulting in a lead series of chelated iridium(I) NHC-phosphine complexes. Subsequent preparative studies show that the optimal catalyst system displays high levels of activity in HIE, and we demonstrate the labeling of a broad scope of substituted aryl sulfones. We also show that the activity of the catalyst is maintained at low pressures of deuterium gas and apply these conditions to tritium radiolabeling, including the expedient synthesis of a tritium-labeled drug molecule.

The microbiome-gut-brain (MGB) axis is a bi-directional route of communication that exists between the brain and the microbes that reside in the gut. The MGB axis is becoming of increasing importance as significant alterations in the gut microbiota are now linked to numerous neurological conditions, however, little is currently known about the microbiome derived mediators of communication. Here we used mass spectrometry imaging (MSI), a label free imaging technique, to identify bacterial products that cross the blood brain barrier in specific pathogen free (SPF) mice. We identified two bacterial molecules abundant in white matter regions of the murine that were absent in the brain and gut in germ free (GF) mice. We have identified the primary gut microbial producers of these metabolites to be members of the Lachnospiraceae family. Both molecules were found to be structurally similar to carnitine and localise with carnitine in the SPF mouse brain. Using a primary murine cell culture model of the central nervous system white matter we show that these molecules are capable of significantly impairing mitochondrial basal respiration. Given their systemic presence in the mouse and their presence in human biological samples, these metabolites may have significant implications for diseases associated with mitochondrial dysfunction and an altered gut microbiota. These results are the first to describe a direct molecular inter-kingdom communication between prokaryotes and the mammalian brain that can facilitate functional inhibition in mammalian brain cells.