Gregory S. Walker

Osivelotor (GBT021601) is an orally bioavailable, small molecule hemoglobin polymerization inhibitor in clinical development for the treatment of sickle cell disease (SCD). In this Phase 1 study, the disposition of osivelotor was assessed using accelerator mass spectrometry (AMS) after a single oral 200‐mg dose containing a microtracer amount of [ ¹⁴ C]‐labeled osivelotor (~74 kBq [~2 μ Ci]) was received by nine healthy volunteers. Due to a long half‐life (27–30 days), continuous and non‐continuous sample collection was needed over a nearly 7‐month study. The results indicate an average 87.6% (47.2% in urine; 40.4% in feces) recovery of the dose in excreta. Comparison of osivelotor and total radioactivity (TRA) systemic concentration versus time profiles in whole blood and plasma suggests the absence of a long‐lived metabolite. Osivelotor was the main circulating drug‐related material in plasma (68.1% of [ ¹⁴ C]). Also observed in plasma was a glucuronide metabolite (19.7% of [ ¹⁴ C]). In whole blood, the effect compartment, only osivelotor (98.3% of [ ¹⁴ C]) was present and no metabolites were observed. The single oral 200‐mg dose of osivelotor was well tolerated. The results of this unique study design provide insights into the absorption, metabolism, and excretion (ADME) of osivelotor and support its continued development for SCD. Trial Registration: ClinicalTrials.gov NCT05718687

Human radiolabeled mass balance studies are crucial for comprehensively characterizing the absorption, distribution, metabolism, and excretion (ADME) of investigational drugs, providing essential data for drug development, regulatory evaluation, and product labeling. The IQ Consortium's Human Mass Balance Radiolabeled ADME (hADME) Working Group developed this perspective to address key considerations and highlight opportunities for these studies. This is particularly relevant as regulatory guidance, such as the 2024 US Food and Drug Administration (FDA) and China's National Medical Products Administration (NMPA), have finalized dedicated guidance documents, along with recommendations from other global regulatory bodies (including European Medicines Agency (EMA) and Pharmaceuticals and Medical Devices Agency (of Japan; PMDA)); still, there are some regional differences. This review outlines current practices and emerging trends in hADME study design. It details seven clinical designs, from conventional radiolabeled approaches to microtracer studies that employ analytical techniques, such as accelerator mass spectrometry (AMS). It further highlights alternative non‐radioactive approaches, including the use of Fluorine‐19 nuclear magnetic resonance ( ¹⁹ F‐NMR) spectroscopy. The manuscript also explores the integration of hADME studies with absolute bioavailability (ABA) assessments and highlights their impact on early drug development with case studies. This collective effort by the IQ working group highlights fit‐for‐purpose study designs to ensure comprehensive ADME characterization supporting drug registration and patient well‐being.

Radiolabeled human mass balance studies are crucial for identifying circulating metabolites and understanding drug absorption, excretion, and clearance pathways. Metabolite profiling involves quantifying all drug‐related entities, including parent drug and metabolites in plasma and excreta, using extended liquid chromatography methods coupled with detection through scintillation counting, accelerator mass spectrometry, or non‐radiolabeled approaches. Given the labor‐intensive nature of sample extraction and analysis, we propose a new paradigm that maximizes gathering information through sample pooling strategies. Our proposal introduces sample pooling strategies by integrating both individual and pooled sample schemes, simplifying decisions, and consolidating existing knowledge into a cohesive document. This aligns with the low statistical power typically associated with mass balance studies that dose six to eight subjects. In metabolite profiling, it is common practice to pool samples either from the limited number of subjects participating in a human mass balance study or from different time points of sample collection. This approach improves efficiency while preserving data integrity. Pooling reduces resource constraints and enables the concentration of samples with relatively low radioactivity levels, resulting in higher quality metabolite profiles. Nevertheless, there are situations when analyzing samples from individual subjects or time points may be preferred. This proposal presents guidance and decision trees designed to facilitate informed decisions about sample pooling to maximize data quality of metabolite profiling in human mass balance studies while efficiently managing resources. These recommendations stem from discussions within the mass balance working group of the IQ Consortium.

NMR spectroscopy is considered a benchmark analytical technique in the pharmaceutical industry for the structural characterization of organic molecules and identification of target–ligand interactions. Two- and higher-dimensional NMR experiments provide especially rich data sets that demonstrate connectivity between a variety of atoms within a diversity of molecular chemotypes. However, multidimensional NMR experiments are often limited by long acquisition times and insufficient sensitivity and/or resolution. One strategy that can mitigate these limitations is non-uniform sampling (NUS). NUS acquisition achieves faster recording times through the reduction of the number of data points in the indirectly detected dimensions. The spectrum can then be mathematically reconstructed using a variety of different algorithms. This approach can reduce the acquisition time or increase the resolution to resolve overlapping resonances. A second developing approach for the reduction of acquisition times is that of supersequences or nested experiments, such as NMR by ordered acquisition using 1H-detection (NOAH) in which multiple experiments are performed either in sequence or concomitantly in a manner to maximize data acquisition per unit time. Using these pulse sequences, the saved acquisition time can be used to increase throughput, improve sensitivity, or enhance resolution. Application of NUS and NOAH in the pharmaceutical industry will be described.

Typically human absorption, distribution, metabolism, and excretion (ADME) studies are executed using radiolabeled (e.g., carbon‐14) material, the synthesis of which is a time‐consuming activity. In this study, we were able to assess the metabolism and excretion of unlabeled nirmatrelvir (PF‐07321332) within the first‐in‐human study via a novel application of quantitative fluorine (¹⁹F) nuclear magnetic resonance (NMR) spectroscopy in place of a standard radiolabel ADME study. Six healthy participants received a single 300‐mg oral dose of nirmatrelvir (in combination with ritonavir), and excreta were collected up to 10 days. Virtually all drug‐related material was recovered within 5 days, and mass balance was achieved with 84.9 ± 8.9% (range = 70.7–95.5%) of the administered dose recovered in urine and feces. The excretion of fluorine‐containing material in urine and feces was 47.0% and 33.7%, respectively. Unchanged nirmatrelvir represented 82.5% of the normalized drug‐related material with a carboxylic acid metabolite M5, derived from hydrolysis of the P2 amide bond, present at 12.1% of dose. Nirmatrelvir was the only drug‐related entity observed in plasma. Approximately 4.2% of the dose was excreted as metabolite M8 (measured by liquid chromatography–mass spectrometry), which was ¹⁹F NMR silent due to hydrolysis of the trifluoroacetamide moiety. Hydrolysis of nirmatrelvir to M5 and M8 was shown to occur in cultures of human gut microflora. This successful demonstration of quantitative ¹⁹F NMR spectroscopy to establish the mass‐balance, excretion, and metabolic profile of nirmatrelvir offers an advantageous means to execute human ADME studies for fluorine‐containing compounds early in drug development.

Enzalutamide and apalutamide are two androgen receptor inhibitors approved for the treatment of castration-resistant prostate cancer (CRPC) and nonmetastatic castration-resistant prostate cancer (nmCRPC), respectively. Apalutamide is associated with an increased incidence of skin rash above the placebo groups in the SPARTAN trial in nmCRPC and in the TITAN trial in metastatic castration-sensitive prostate cancer patients, respectively. On the contrary, the rate of skin rash across all clinical trials (including PROSPER [nmCRPC]) for enzalutamide is similar to placebo. We hypothesized that the apalutamide-associated increased skin rash in patients could be linked to a structural difference. The 2-cyanophenyl and dimethyl moieties in enzalutamide are substituted in apalutamide with 2-cyanopyridine and cyclobutyl, respectively. In our evaluations, the 2-cyanopyridine moiety of apalutamide was chemically reactive with the thiol nucleophile glutathione, resulting in rearranged thiazoline products. Radiolabeled apalutamide, but not radiolabeled enzalutamide, was shown to react with mouse and human plasma proteins. Thiol nucleophiles decreased the extent of covalent binding to the model protein bovine serum albumin, while amine and alcohol nucleophiles had no effect, suggesting reactivity with cysteine of proteins. Subcutaneous administration of apalutamide dose dependently increased lymphocyte cellularity in draining lymph nodes in a mouse drug allergy model (MDAM). Enzalutamide, and its known analogue RD162 in which the cyanophenyl was retained but the dimethyl was replaced by cyclobutyl, demonstrated substantially less covalent binding activity and negative results in the MDAM assay. Collectively, these data support the hypothesis that the 2-cyanopyridine moiety in apalutamide may react with cysteine in proteins forming haptens, which may trigger an immune response, as indicated by the activity of apalutamide in the MDAM assay, which in turn may be leading to increased potential for skin rash versus placebo in patients in the SPARTAN and TITAN clinical trials.

Replacement of hydrogen with fluorine is a useful drug design strategy when decreases in cytochrome P450 metabolic lability are needed. In this paper, a facile two-step method of inserting fluorine into metabolically labile sites of drug molecules is described that utilizes less than 1 mg of starting material and quantitative NMR spectroscopy to ascertain the structures and concentrations of products. In the first step, hydroxyl metabolites are biosynthesized using human P450 enzymes and in the second step these metabolites are subjected to deoxyfluorination using DAST (diethylaminosulfur trifluoride). The method is demonstrated using midazolam, celecoxib, ramelteon, and risperidone as examples and CYP3A5, 2C9, 1A2, and 2D6 to catalyze the hydroxylations. The drugs and their fluoro analogues were tested for metabolic lability. 9-Fluororisperidone and 4'-fluorocelecoxib were 16X and 4X more metabolically stable than risperidone and celecoxib respectively, while 2-fluororamelteon and ramelteon were metabolized at the same rate. 1’-Fluoromidazolam was metabolized at the same rate as midazolam by CYP3A4 but was more stable in CYP3A5 incubations. The P450 catalyzed sites of metabolism of the fluorine containing analogues were determined. Some of the metabolites arose via metabolism at the fluorine substituted carbon wherein the fluorine was lost to yield aldehydes. In summary, this method offers an approach whereby metabolically labile sites can be substituted with fluorine and the products can be tested to determine whether an enhancement in metabolic stability is obtained.

The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-to-plasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation.