Recommendations on the Use of Multiple Labels in Human Mass Balance Studies.

The administration of radiolabeled drug candidates is considered the gold standard in absorption, distribution, metabolism, and excretion studies for small-molecule drugs since it allows facile and accurate quantification of parent drug, metabolites, and total drug-related material independent of the compound structure. The choice of the position of the radiolabel, typically 14C or 3H, is critical to obtain relevant information. Sometimes, a biotransformation reaction may lead to cleavage of a part of the molecule. As a result, only the radiolabeled portion can be followed, and information on the fate of the nonlabeled metabolite may be lost. Synthesis and administration of two or more radiolabeled versions of the parent drug as a mixture or in separate studies may resolve this issue but comes with additional challenges. In this paper, we address the questions that may be considered to help make the right choice whether to use a single or multiple radiolabel approach and discuss the pros and cons of different multiple-labeling strategies that can be taken as well as alternative methods that allow the nonlabeled part of the molecule to be followed. SIGNIFICANCE STATEMENT: Radiolabeled studies are the gold standard in drug metabolism research, but molecules can undergo cleavage with loss of the label. This often results in discussions around potential use of multiple labels, which seem to be occurring with increased frequency since an increasing proportion of the small-molecule drugs are tending towards larger molecular weights. This review provides insight and decision criteria in considering a multiple-label approach as well as pros and cons of different strategies that can be followed.

Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group.

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development Metabolite Bioanalysis Working Group on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from investigational new drug (IND) enabling studies, and phase I, phase II, and phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development.

Non-Labeled, Stable Labeled, or Radiolabelled Approaches for Provision of Intravenous Pharmacokinetics in Humans: A Discussion Piece.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (i.v. PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as i.v. PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade.

Targeted Small-Molecule Identification Using Heartcutting Liquid Chromatography-Infrared Ion Spectroscopy.

Infrared ion spectroscopy (IRIS) can be used to identify molecular structures detected in mass spectrometry (MS) experiments and has potential applications in a wide range of analytical fields. However, MS-based approaches are often combined with orthogonal separation techniques, in many cases liquid chromatography (LC). The direct coupling of LC and IRIS is challenging due to the mismatching timescales of the two technologies: an IRIS experiment typically takes several minutes, whereas an LC fraction typically elutes in several seconds. To resolve this discrepancy, we present a heartcutting LC-IRIS approach using a setup consisting of two switching valves and two sample loops as an alternative to direct online LC-IRIS coupling. We show that this automated setup enables us to record multiple IR spectra for two LC-features from a single injection without degrading the LC-separation performance. We demonstrate the setup for application in drug metabolism research by recording six m/z-selective IR spectra for two drug metabolites from a single 2 µL sample of cell incubation extract. Additionally, we measure the IR spectra of two closely eluting diastereomeric biomarkers for the inborn error of metabolism pyridoxine-dependent epilepsy (PDE-ALDH7A1), which shows that the heartcutting LC-IRIS setup has good sensitivity (requiring ∼µL injections of ∼µM samples) and that the separation between closely eluting isomers is maintained. We envision applications in a range of research fields, where the identification of molecular structures detected by LC-MS is required.

Considerations for Human ADME Strategy and Design Paradigm Shift(s) - An Industry White Paper.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of 14 C-labeled drug. Significant changes have already been made in the design of the hADME study for many companies, but opportunity exists to continue to re-think both the design and timing of the hADME study in light of the potential offered by newer technologies, that enable flexibility in particular to reducing the magnitude of the radioactive dose used. This paper provides considerations on the variety of current strategies that exist across a number of pharmaceutical companies and on some of the ongoing debates around a potential move to the so called "human first/human only" approach, already adopted by at least one company. The paper also provides a framework for continuing the discussion in the application of further shifts in the paradigm.

Evaluation of table-top lasers for routine infrared ion spectroscopy in the analytical laboratory.

Infrared ion spectroscopy is increasingly recognized as a method to identify mass spectrometry-detected analytes in many (bio)chemical areas and its integration in analytical laboratories is now on the horizon. Commercially available quadrupole ion trap mass spectrometers are attractive ion spectroscopy platforms but operate at relatively high pressures. This promotes collisional deactivation which directly interferes with the multiple-photon excitation process required for ion spectroscopy. To overcome this, infrared lasers having a high instantaneous power are required and therefore a majority of analytical studies have been performed at infrared free electron laser facilities. Proliferation of the technique to routine use in analytical laboratories requires table-top infrared lasers and optical parametric oscillators (OPOs) are the most suitable candidates, offering both relatively high intensities and reasonable spectral tuning ranges. Here, we explore the potential of a range of commercially available high-power OPOs for ion spectroscopy, comparing systems with repetition rates of 10 Hz, 20 kHz, 80 MHz and a continuous-wave (cw) system. We compare the performance for various molecular ions and show that the kHz and MHz repetition-rate systems outperform cw and 10 Hz systems in photodissociation efficiency and offer several advantages in terms of cost-effectiveness and practical implementation in an analytical laboratory not specialized in laser spectroscopy.

Multimodal biomarker discovery for active Onchocerca volvulus infection.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.

Quantitative Mass Spectrometry Imaging to Study Drug Distribution in the Intestine Following Oral Dosing.

Local delivery to the lower gut to treat diseases of the colon has become a topic of special attention. Tissue exposure of locally acting agents is not represented by plasma concentrations. Therefore, reliable methods to measure tissue uptake at the primary site of action (e.g., epithelial layer or lamina propria) are vital. This work investigates the suitability of mass spectrometry imaging (MSI) in quantitatively visualizing intestinal transmural drug distribution. Tofacitinib (Tofa), a drug approved for the treatment of several autoimmune diseases, including ulcerative colitis, was selected as a tool compound for feasibility studies. One- and 7-h postdose sections of the ileum, proximal- and distal-colon from rats that received an oral solution of Tofa were subjected to matrix-assisted laser desorption ionization (MALDI)-MSI. A dilution series of individual concentrations sprayed over an entire tissue section allowed for tissue type-specific quantitation. At 1 h (systemic Tmax), the signal was highest in the ileum, whereas at 7 h, the signal was highest in the colon, when the unabsorbed fraction of the compound reached the colon. A combination of three-dimensional (3D) intensity plots and hematoxylin and eosin (H&E) stains showed a visually observable gradual decrease in Tofa concentration from the lumen toward the muscular layer of the proximal colon. The high luminal concentration of Tofa indicated that flushing of the intestines with saline does not result in complete removal of the drug material from the lumen. This could cause an overestimation of drug concentration in gut tissue homogenates by conventional liquid chromatography-mass spectrometry (LC-MS) methods. This study demonstrates the utility of MSI to differentiate between the lumen and intestinal wall layers and enables proper interpretation of tissue distribution data.

Metabolism and disposition in rats, dogs, and humans of erdafitinib, an orally administered potent pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor.

This article describes in vivo biotransformation and disposition of erdafitinib following single oral dose of 3H-erdafitinib and 14C-erdafitinib to intact and bile duct-cannulated (BC) rats (4 mg/kg), 3H-erdafitinib to intact dogs (0.25 mg/kg), and 14C-erdafitinib to humans (12 mg; NCT02692677). Peak plasma concentrations of total radioactivity were achieved rapidly (Tmax: animals, 1 h; humans, 2-3 h). Recovery of drug-derived radioactivity was significantly slower in humans (87%, 384 h) versus animals (rats: 91-98%, 48 h; dogs: 81%, 72 h). Faeces was the primary route of elimination in intact rats (95%), dogs (76%), and humans (69%); and bile in BC rats (48%). Renal elimination of radioactivity was relatively low in animals (2-12%) versus humans (19%). Unchanged erdafitinib was major component in human excreta (faeces, 17%; urine, 11%) relative to animals. M6 (O-desmethyl) was the major faecal metabolite in humans (24%) and rats (intact, 46%; BC, 11%), and M2 (O-glucuronide of M6) was the prevalent biliary metabolite in rats (14%). In dogs, besides M6, majority of radioactive dose in faeces was composed of multiple minor metabolites. In humans, unchanged erdafitinib was the major circulating entity. O-demethylation of erdafitinib was the major metabolic pathway in humans and animals.

Evaluation of micropillar array columns for chromatographic separation of phosphorothioated oligonucleotides and their diastereomers.

Chromatographic analysis of therapeutic oligonucleotides is challenging due to the presence of closely related impurities, degradants or metabolites and due to the presence of phosphorothioate bonds, which introduce chiral centers. In the present study, ion pair reversed phase chromatography of oligonucleotides on micropillar array columns was investigated. Two commonly used mobile phase conditions were included. With 16.3 mM triethylamine and 400 mM hexafluoroisopropanol, the separation of 16-mer oligonucleotides differing in the number and positions of phosphorothioate linkages as well as some n-1 and n-2 truncations demonstrated complete suppression of diastereoselectivity. Although the positional phosphorothioate isomers evaluated could not be resolved, an increase in phosphorothioate bonds resulted in more retention. A therapeutic 19-mer RNA sequence with 2'-fluor and 2'-O-methyl modifications showed partial separation of some very close impurities. When using 15 mM triethyl ammonium acetate in the mobile phase, diastereomer selectivity was clearly observed for all analytes. The best result was obtained for the 19-mer RNA therapeutic mimic with four phosphorothioate bonds, since all 16 theoretical diastereomers were clearly observed under the conditions tested. A limited benchmark exercise demonstrated the improved capability of the new micropillar array columns. Therefore, these columns can be positioned as a valuable alternative when challenging oligonucleotide separations are expected.

Toward simplified oral lipid-based drug delivery using mono-/di-glycerides as single component excipients.

OBJECTIVE: This study aimed to systematically explore compositional effects for a series of lipid systems, on the in vitro drug solubilization and in vivo bioavailability of three poorly water-soluble drugs with different physico-chemical properties. SIGNIFICANCE: While many lipid-based drug products have successfully reached the market, there is still a level of uncertainty on the design guidelines for such drug products with limited understanding on the influence of composition on in vitro and in vivo performance. METHODS AND RESULTS: Lipid-based drug delivery systems were prepared using either single excipient systems based on partially digested triglycerides (i.e. mono- and/or di-glycerides) or increasingly complex systems by incorporating surfactants and/or triglycerides. These lipid systems were evaluated for both in vitro and in vivo behavior. Results indicated that simple single component long chain lipid systems are more beneficial for the absorption of the weak acid celecoxib and the weak base cinnarizine compared to equivalent single component medium chain lipid systems. Similarly, a two-component system produced by incorporating small amount of hydrophilic surfactant yields similar overall pharmacokinetic effects. The lipid drug delivery systems based on medium chain lipid excipients improved the in vivo exposure of the neutral drug JNJ-2A. The higher in vivo bioavailability of long chain lipid systems compared to medium chain lipid systems was in agreement with in vitro dilution and dispersion studies for celecoxib and cinnarizine. CONCLUSIONS: The present study demonstrated the benefits of using mono-/di-glycerides as single component excipients in LBDDS to streamline formulation screening and improve oral bioavailability for the three tested poorly water-soluble drugs.

2-Methyl-pentanoyl-carnitine (2-MPC): a urine biomarker for patent Ascaris lumbricoides infection.

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p < 0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

Mass spectrometry-based identification of ortho-, meta- and para-isomers using infrared ion spectroscopy.

Distinguishing positional isomers, such as compounds having different substitution patterns on an aromatic ring, presents a significant challenge for mass spectrometric analyses and is a frequently encountered difficulty in, for example, drug metabolism research. In contrast to mass spectrometry, IR spectroscopy is a well-known and powerful tool in the distinction of ortho-, meta- and para-isomers, but is not applicable to low-abundance compounds in complex mixtures such as often targeted in bioanalytical studies. Here, we demonstrate the use of infrared ion spectroscopy (IRIS) as a novel method that facilitates the differentiation between positional isomers of disubstituted phenyl-containing compounds and that can be applied in mass spectrometry-based complex mixture analysis. By analyzing different substitution patterns over several sets of isomeric compounds, we show that IRIS is able to consistently probe the diagnostic CH out-of-plane vibrations that are sensitive to positional isomerism. We show that these modes are largely independent of the chemical functionality contained in the ring substituents and of the type of ionization. We also show that IRIS spectra often identify the positional isomer directly, even in the absence of reference spectra obtained from physical standards or from computational prediction. We foresee that this method will be generally applicable to the identification of disubstituted phenyl-containing compounds.

Infrared ion spectroscopy: New opportunities for small-molecule identification in mass spectrometry - A tutorial perspective.

Combining the individual analytical strengths of mass spectrometry and infrared spectroscopy, infrared ion spectroscopy is increasingly recognized as a powerful tool for small-molecule identification in a wide range of analytical applications. Mass spectrometry is itself a leading analytical technique for small-molecule identification on the merit of its outstanding sensitivity, selectivity and versatility. The foremost shortcoming of the technique, however, is its limited ability to directly probe molecular structure, especially when contrasted against spectroscopic techniques. In infrared ion spectroscopy, infrared vibrational spectra are recorded for mass-isolated ions and provide a signature that can be matched to reference spectra, either measured from standards or predicted using quantum-chemical calculations. Here we present an overview of the potential for this technique to develop into a versatile analytical method for identifying molecular structures in mass spectrometry-based analytical workflows. In this tutorial perspective, we introduce the reader to the technique of infrared ion spectroscopy and highlight a selection of recent experimental advances and applications in current analytical challenges, in particular in the field of untargeted metabolomics. We report on the coupling of infrared ion spectroscopy with liquid chromatography and present experiments that serve as proof-of-principle examples of strategies to address outstanding challenges.

Strategies and analytical workflows to extend the dynamic range in quantitative LC-MS/MS analysis.

Aim: To evaluate alternative analytical strategies to extend the dynamic range in quantitative LC-MS/MS. Methods & results: Two approaches based on prior or no prior knowledge of expected exposure levels were evaluated. These approaches make use of two analytical strategies, which include the use of more than one injection volume or dilution of sample extract with solvents or solvent mixtures. A total of 16 compounds with varying logP values were classified into polar and nonpolar groups and used in this evaluation. From the two analytical strategies, three workflows were derived. Conclusion: All three workflows were successfully evaluated and resulted in good accuracy (80-120%) for all the compound groups.

Selective drug metabolite trace analysis by very high-volume injections and heartcut two-dimensional (2D)-ultrahigh performance liquid chromatography (UHPLC).

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

Apalutamide Absorption, Metabolism, and Excretion in Healthy Men, and Enzyme Reaction in Human Hepatocytes.

In this phase 1 study, the absolute bioavailability and absorption, metabolism, and excretion (AME) of apalutamide, a competitive inhibitor of the androgen receptor, were evaluated in 12 healthy men. Subjects received 240 mg of apalutamide orally plus a 15-minute intravenous infusion of 100 µg of apalutamide containing 9.25 kBq (250 nCi) of 14C-apalutamide (2 hours postdose) for absolute bioavailability assessment or plus one 400-µg capsule containing 37 kBq (1000 nCi) of 14C-apalutamide for AME assessment. Content of 14C and metabolite profiling for whole blood, plasma, urine, feces, and expired air samples were analyzed using accelerator mass spectrometry. Apalutamide absolute oral bioavailability was ≈100%. After oral administration, apalutamide, its N-desmethyl metabolite (M3), and an inactive carboxylic acid metabolite (M4) accounted for most 14C in plasma (45%, 44%, and 3%, respectively). Apalutamide elimination was slow, with a mean plasma half-life of 151-178 hours. The mean cumulative recovery of total 14C over 70 days postdose was 64.6% in urine and 24.3% in feces. The urinary excretion of apalutamide, M3, and M4 was 1.2%, 2.7%, and 31.1% of dose, respectively. Fecal excretion of apalutamide, M3, and M4 was 1.5%, 2.0%, and 2.4% of dose, respectively. Seventeen apalutamide metabolites and six main metabolic clearance pathways were identified. In vitro studies confirmed CYP2C8 and CYP3A4 roles in apalutamide metabolism.

Adduct ion formation as a tool for the molecular structure assessment of ten isomers in traveling wave and trapped ion mobility spectrometry.

RATIONALE: The separation of isomeric compounds with major differences in their physiochemical and pharmacokinetic properties is of particular importance in pharmaceutical R&D. However, the structural assessment and separation of these compounds with current analytical techniques and methods are still a challenge. In this study, we describe strategies to separate the various structural and stereo-isomers. METHODS: The separation of ten structural and stereo-isomers was investigated using Trapped and Travelling Wave ion mobility spectrometry (TIMS and TWIMS). Different strategies including adduct ion formation with Na, Li, Ag and Cs as well as fragmentation before and after the ion mobility cell were applied to separate the isomeric compounds. RESULTS: All the counter ions (in particular Na) strongly coordinated with the test analytes in all the IMS systems. The highest resolving power was achieved for the sodium and lithium adducts using TIMS-time-of-flight (TOF). However, some separation was attained on a Synapt HDMS system with its unique potential to monitor the ion mobility of the product ions. The elution order of the adduct ions was the same in all instruments, in which, unexpectedly, the para-substituted isomer of the [M + Na]+ species had the lowest collision cross section followed by the meta- and ortho-isomers. CONCLUSIONS: The formation of adduct ions could facilitate the separation of structural and even stereo-isomers by generating different molecular conformations. In addition, fragmenting isomers before or after the ion mobility cell is a valuable strategy to separate and also to assess the structures of adducts and different conformers.

Optimization of flow splitting and make-up flow conditions in liquid chromatography/electrospray ionization mass spectrometry.

RATIONALE: In liquid chromatography/mass spectrometry (LC/MS) the LC flow is often split prior to the mass spectrometer, for instance, when collecting fractions of the separated sample for other purposes or when less sensitive parallel detection is applied. The aim of this study is to optimize the actual split ratio and make-up flow composition. METHODS: Different types of splitters were evaluated in combination with a make-up flow. A home-made 1/10 T-piece splitter and commercial 1/10, 1/100 and 1/250 splitters were evaluated by continuous and accurate measurements of the actual split ratio throughout the LC gradient. The make-up flow composition was optimized for maximum electrospray ionization (ESI)-MS sensitivity in the positive mode using ESI efficiency measurements. RESULTS: Altogether 22 different solvent conditions were tested on 20 pharmaceutical compounds with a wide variety of functional groups and physicochemical properties (molecular weight, logP, and pKa ). Methanol/10 mM formic acid in water (90/10) provided on average the best results. CONCLUSIONS: Methanol/10 mM formic acid in water (90/10) proved to be the best make-up flow composition in relation to the average sensitivity obtained. Stronger acidic conditions using oxalic acid or higher formic acid concentrations had a clear positive effect on the sensitivity of compounds with low ionization efficiency. The tested split ratios were relatively stable over the main part of the gradient but showed some variation at very low and very high organic conditions. Differences were larger with methanol compared with acetonitrile containing solvent compositions and when applied without a column or with very long connecting tubing.