Development and Evaluation of Ontogeny Functions of the Major UDP-Glucuronosyltransferase Enzymes to Underwrite Physiologically Based Pharmacokinetic Modeling in Pediatric Populations.

Uridine 5'-diphospho-glucuronosyltransferases (UGTs) demonstrate variable expression in the pediatric population. Thus, understanding of age-dependent maturation of UGTs is critical for accurate pediatric pharmacokinetics (PK) prediction of drugs that are susceptible for glucuronidation. Ontogeny functions of major UGTs have been previously developed and reported. However, those ontogeny functions are based on in vitro data (i.e., enzyme abundance, in vitro substrate activity, and so on) and therefore, may not translate to in vivo maturation of UGTs in the clinical setting. This report describes meta-analysis of the literature to develop and compare ontogeny functions for 8 primary UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B10, UGT2B15, and UGT2B17) based on published in vitro and in vivo studies. Once integrated with physiologically based pharmacokinetics modeling models, in vivo activity-based ontogeny functions demonstrated somewhat greater prediction accuracy (mean squared error, MSE: 0.05) compared to in vitro activity (MSE: 0.104) and in vitro abundance-based ontogeny functions (MSE: 0.129).

Xenografted Tumors Share Comparable Fraction Unbound and Can Be Surrogated by Mouse Lung Tissue.

Free (unbound) drug concentration at the site of action is the key determinant of biologic activity since only unbound drugs can exert pharmacological and toxicological effects. Unbound drug concentration in tumors for solid cancers is needed to understand/explain/predict pharmacokinetics, pharmacodynamics, and efficacy relations. Fraction unbound (fu ) in tumors is usually determined across several xenografted tumors derived from various cell lines in the drug discovery stage, which is time consuming and a resource burden. In this study, we determined the fu values for a set of diverse compounds (comprising acid, base, neutral, zwitterion, and covalent drugs) across five different xenografted tumors and five commercially available mouse tissues to explore the correlation of fu between tumors and the possibility of surrogate tissue(s) for tumor fu (fu,tumor) determination. The crosstumor comparison showed that fu,tumor values across tumors are largely comparable, and systematic tissue versus tumor comparison demonstrated that only lung tissue had comparable fu to all five tumors (fu values within twofold change for >80% compounds in both comparisons). These results indicated that mouse lung tissue can be used as a surrogate matrix for a fu,tumor assay. This study will increase efficiency in fu,tumor assessment and reduce animal use (adapting the replace, reduce, and refine principle) in drug discovery. SIGNIFICANCE STATEMENT: The free drug concept is a well accepted principle in drug discovery research. Currently, tumor fraction unbound (fu,tumor) is determined in several tumors derived from different cell lines to estimate free drug concentrations of a compound. The results from this study indicated that fu,tumor across xenografted tumors is comparable, and fu,tumor can be estimated using a surrogate tissue, mouse lung. The results will increase efficiency in fu,tumor assessment and reduce animal use in drug discovery.

Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers.

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Therapeutic Protein Drug Interactions: A White Paper From the International Consortium for Innovation and Quality in Pharmaceutical Development.

Typically, therapeutic proteins (TPs) have a low risk for eliciting meaningful drug interactions (DIs). However, there are select instances where TP drug interactions (TP-DIs) of clinical concern can occur. This white paper discusses the various types of TP-DIs involving mechanisms such as changes in disease state, target-mediated drug disposition, neonatal Fc receptor (FcRn), or antidrug antibodies formation. The nature of TP drug interaction being investigated should determine whether the examination is conducted as a standalone TP-DI study in healthy participants, in patients, or assessed via population pharmacokinetic analysis. DIs involving antibody-drug conjugates are discussed briefly, but the primary focus here will be DIs involving cytokine modulation. Cytokine modulation can occur directly by certain TPs, or indirectly due to moderate to severe inflammation, infection, or injury. Disease states that have been shown to result in indirect disease-DIs that are clinically meaningful have been listed (i.e., typically a twofold change in the systemic exposure of a coadministered sensitive cytochrome P450 substrate drug). Type of disease and severity of inflammation should be the primary drivers for risk assessment for disease-DIs. While more clinical inflammatory marker data needs to be collected, the use of two or more clinical inflammatory markers (such as C-reactive protein, albumin, or interleukin 6) may help broadly categorize whether the predicted magnitude of inflammatory disease-DI risk is negligible, weak, or moderate to strong. Based on current knowledge, clinical DI studies are not necessary for all TPs, and should no longer be conducted in certain disease patient populations such as psoriasis, which do not have sufficient systemic inflammation to cause a meaningful indirect disease-DI.

Absorption, metabolism and excretion of [14C]-sotorasib in healthy male subjects: characterization of metabolites and a minor albumin-sotorasib conjugate.

PURPOSE: The objectives of this study were to characterize the absorption, metabolism, and excretion of sotorasib and determine the metabolites present in plasma, urine, and feces in healthy male subjects following a single oral 720 mg dose containing approximately 1 µCi of [14C]-sotorasib. METHODS: Urine, feces, and plasma were collected post-dose and assayed for total radioactivity and profiled for sotorasib metabolites. Urine and plasma were also assayed for sotorasib pharmacokinetics. In addition, in vitro studies were performed to determine the enzymes responsible for formation of major circulating metabolites and protein adducts in human plasma. RESULTS: Sotorasib was rapidly absorbed, with a median time to peak concentration of 0.75 h. Mean t1/2,z of plasma sotorasib, whole blood total radioactivity, and plasma total radioactivity were 6.35, 174, and 128 h, respectively. The geometric mean cumulative recovery was 80.6%; the majority was excreted in feces (74.4%) with a low percentage excreted in urine (5.81%). M10, sotorasib, and M24 were present at 31.6%, 22.2%, and 13.7% of total radioactivity in plasma extracts, respectively. M10 and sotorasib were present at < 5% of administered radioactivity in urine, while only unchanged sotorasib, at 53% of administered radioactivity, was identified in feces. A sotorasib-albumin adduct was identified in plasma as a minor constituent, consistent with the observed radioactivity profile in plasma/blood. CONCLUSION: Sotorasib metabolism involves nonenzymatic glutathione conjugation, GGT-mediated hydrolysis of glutathione adduct, and direct CYP3A and CYP2C8-mediated oxidation. Elimination of sotorasib is predominantly fecal excretion, suggesting dose reduction is not necessary with renal impairment.

Targeting the Mitotic Kinesin KIF18A in Chromosomally Unstable Cancers: Hit Optimization Toward an In Vivo Chemical Probe.

Chromosomal instability (CIN) is a hallmark of cancer that results from errors in chromosome segregation during mitosis. Targeting of CIN-associated vulnerabilities is an emerging therapeutic strategy in drug development. KIF18A, a mitotic kinesin, has been shown to play a role in maintaining bipolar spindle integrity and promotes viability of CIN cancer cells. To explore the potential of KIF18A, a series of inhibitors was identified. Optimization of an initial hit led to the discovery of analogues that could be used as chemical probes to interrogate the role of KIF18A inhibition. Compounds 23 and 24 caused significant mitotic arrest in vivo, which was sustained for 24 h. This would be followed by cell death either in mitosis or in the subsequent interphase. Furthermore, photoaffinity labeling experiments reveal that this series of inhibitors binds at the interface of KIF18A and tubulin. This study represents the first disclosure of KIF18A inhibitors with in vivo activity.

Absorption, Distribution, Metabolism, and Excretion of [14C]-Sotorasib in Rats and Dogs: Interspecies Differences in Absorption, Protein Conjugation and Metabolism.

Sotorasib is a first-in-class, targeted covalent inhibitor of Kirsten rat sarcoma viral oncogene homolog (KRAS)G12C approved by the FDA to treat patients with locally advanced or metastatic non-small cell lung cancer with the KRASG12C mutation. The mass balance, excretion, and metabolism of [14C]-sotorasib was characterized in rats and dogs after a single dose of 60 or 500 mg/kg, respectively. Mean recovery was >90% for both species. Excretion of unchanged sotorasib was a minor pathway in rats, accounting for <4% of administered dose in urine and <7% of administered dose in feces. Approximately 66% of administered dose was recovered in the bile from bile duct cannulated rats as metabolites. Excretion of unchanged sotorasib was the major excretion pathway in dogs, likely caused by solubility-limited absorption. Major pathways of sotorasib biotransformation included glutathione conjugation and oxidative metabolism. In vitro experiments demonstrated that nonenzymatic conjugation (Michael addition) was the primary mechanism of the reaction with glutathione. Extended radioactivity profiles in blood and plasma were observed in rats, but not dogs, after dosing with [14C]-sotorasib. In vitro experiments demonstrated that sotorasib-protein adducts were observed with both rat hemoglobin and serum albumin, explaining the extended radioactivity profile. SIGNIFICANCE STATEMENT: This study characterized the mass balance, excretion, and metabolism of [14C]-sotorasib, a covalent Kirsten rat sarcoma viral oncogene homolog G12C inhibitor, in rats and dogs. Rapid absorption and extensive metabolism of sotorasib was observed in rats, while sotorasib was primarily excreted unchanged in dog feces, likely due to solubility-limited absorption. Protein adducts with rat hemoglobin and serum albumin were characterized, explaining observed extended blood and plasma radioactivity profiles. The primary biotransformation pathway, glutathione conjugation, was mediated through nonenzymatic conjugation.

Mercapturate pathway metabolites of sotorasib, a covalent inhibitor of KRASG12C, are associated with renal toxicity in the Sprague Dawley rat.

Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.

Strain-Dependent Variability of Early Discovery Small Molecule Pharmacokinetics in Mice: Does Strain Matter?

Drug discovery programs routinely perform pharmacokinetic (PK) studies in mice to prioritize lead compounds based on anticipated exposure-efficacy and exposure-toxicity relationships. Because of logistical and/or technical issues, the strain of mouse in early discovery PK studies may not always match the strain in toxicity or efficacy studies. This elicits the question do appreciable strain-dependent differences in PK parameters exist to an extent that would warrant conducting PK studies in a strain that matches efficacy and toxicity models? To understand the impact that strain may have on PK parameters, we selected eight marketed drugs with well characterized absorption, distribution, metabolism, and excretion properties and diverse structures to perform PK studies in three common mouse strains (Bagg Albino c, C57BL/6, and CD-1). Some statistical strain-dependent differences were observed; however, we found good general agreement of PK parameters between strains: 88%, 100%, 75%, 76%, 94%, and 88% of compounds were within twofold across strains for clearance, volume of distribution at steady state, t 1/2, C max, T max, and oral bioavailability, respectively. Overall, we recommend that an approach using a single strain of mouse is appropriate for discovery screening PK studies, provided that proper caution is exercised. SIGNIFICANCE STATEMENT: The mouse strain in discovery pharmacokinetic (PK) studies may not match the strain in efficacy and toxicology studies. Currently, there is a gap in the literature addressing whether differences in PK parameters across mouse strains exist such that multiple PK studies are warranted. The results from this study indicated that the PK properties of clinically used drugs between mouse strains are within an acceptable range such that single strain PK is appropriate.

Fraction Unbound for Liver Microsome and Hepatocyte Incubations for All Major Species Can Be Approximated Using a Single-Species Surrogate.

It is well recognized that nonspecific binding of a drug within an in vitro assay (f u) can have a large impact on in vitro to in vivo correlations of intrinsic clearance. Typically, this value is determined experimentally across multiple species in the drug-discovery stage. Herein we examine the feasibility of using a single species (rat) as a surrogate for other species using a panel of small molecules representing highly diverse structures and physiochemical classes. The study demonstrated that 86% and 92% of the tested compounds measured in the mouse, dog, monkey, and human were within 2-fold of rat values for f u in microsomes and hepatocytes, respectively. One compound, amiodarone, exhibited unique species-dependent binding where the f u was approximately 10-fold higher in human microsomes and 20-fold higher in human hepatocytes compared with the average of the other species tested. Overall, these data indicate that using a single species (rat) f u as a surrogate for other major species, including humans, is a means to increase the throughput of measuring nonspecific binding in vitro.

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.

A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry.

RATIONALE: The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein-SM complex). METHODS: A Waters SYNAPT G2 mass spectrometer (MS) was operated in MS(e) mode with appropriate collision energy conditions during the MS(2) acquisition for fragmentation of protein-small molecule adducts to generate characteristic small molecule product ions. RESULTS: Ibrutinib, an acrylamide-containing small molecule drug, was shown to form adducts with rat serum albumin in ex vivo experiments and these adducts were detected by relying solely on the CID product ions generated from ibrutinib. Additionally, ibrutinib produced three CID product ions, one of which was a selective protein-ibrutinib fragment ion not produced by the compound alone. CONCLUSIONS: Herein we describe a tag-free mass spectral detection technique for protein-small molecule conjugates that relies on the unique product ion fragmentation profile of the small molecule. This technique allows the detection of macromolecular ions containing the adducted small molecule from complex protein matrices through mass range selection for the unique product ions in the CID spectra.

Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.

Benchmarking in vitro covalent binding burden as a tool to assess potential toxicity caused by nonspecific covalent binding of covalent drugs.

Despite several advantages of covalent inhibitors (such as increased biochemical efficiency, longer duration of action on the target, and lower efficacious doses) over their reversible binding counterparts, there is a reluctance to use covalent inhibitors as a drug design strategy in pharmaceutical research. This reluctance is due to their anticipated reactions with nontargeted macromolecules. We hypothesized that there may be a threshold limit for nonspecific covalent binding, below which a covalent binding drug may be less likely to cause toxicity due to irreversible binding to off-target macromolecules. Estimation of in vivo covalent binding burden from in vitro data has previously been used as an approach to distinguish those agents more likely to cause toxicity (e.g., hepatotoxicity) via metabolic activation to reactive metabolites. We have extended this approach to nine covalent binding drugs to determine in vitro covalent binding burden. In vitro covalent binding burden was determined by incubating radiolabeled drugs with pooled human hepatocytes. These data were scaled to an estimate of in vivo covalent binding burden by combining the in vitro data with daily dose. Scaled in vivo daily covalent binding burden of marketed covalent drugs was found to be under 10 mg/day, which is in agreement with previously reported threshold value for metabolically activated reversible drugs. Covalent binding was also compared to the intrinsic reactivities of the covalent inhibitors assessed using nucleophiles glutathione and N-α-acetyl lysine. The intrinsic reactivity did not correlate with observed in vitro covalent binding, which demonstrated that the intrinsic reactivity of the electrophilic groups of covalent drugs does not exclusively account for the extent of covalent binding. The ramifications of these findings for consideration of using a covalent strategy in drug design are discussed.

Comparative study of the affinity and metabolism of type I and type II binding quinoline carboxamide analogues by cytochrome P450 3A4.

Compounds that coordinate to the heme-iron of cytochrome P450 (CYP) enzymes are assumed to increase metabolic stability. However, recently we observed that the type II binding quinoline carboxamide (QCA) compounds were metabolically less stable. To test if the higher intrinsic clearance of type II binding compounds relative to type I binding compounds is general for other metabolic transformations, we synthesized a library of QCA compounds that could undergo N-dealkylation, O-dealkylation, benzylic hydroxylation, and aromatic hydroxylation. The results demonstrated that type II binding QCA analogues were metabolically less stable (2- to 12-fold) at subsaturating concentration compared to type I binding counterparts for all the transformations. When the rates of different metabolic transformations between type I and type II binding compounds were compared, they were found to be in the order of N-demethylation > benzylic hydroxylation> O-demethylation > aromatic hydroxylation. Finally, for the QCA analogues with aza-heteroaromatic rings, we did not detect metabolism in aza-aromatic rings (pyridine, pyrazine, pyrimidine), indicating that electronegativity of the nitrogen can change regioselectivity in CYP metabolism.

Small molecule quantification by liquid chromatography-mass spectrometry for metabolites of drugs and drug candidates.

Identification and quantification of the metabolites of drugs and drug candidates are routinely performed using liquid chromatography-mass spectrometry (LC-MS). The best practice is to generate a standard curve with the metabolite versus the internal standard. However, to avoid the difficulties in metabolite synthesis, standard curves are sometimes prepared using the substrate, assuming that the signal for substrate and the metabolite will be equivalent. We have tested the errors associated with this assumption using a series of very similar compounds that undergo common metabolic reactions using both conventional flow electrospray ionization LC-MS and low-flow captive spray ionization (CSI) LC-MS. The differences in standard curves for four different types of transformations (O-demethylation, N-demethylation, aromatic hydroxylation, and benzylic hydroxylation) are presented. The results demonstrate that the signals of the substrates compared with those of the metabolites are statistically different in 18 of the 20 substrate-metabolite combinations for both methods. The ratio of the slopes of the standard curves varied up to 4-fold but was slightly less for the CSI method.

The kinetic mechanism for cytochrome P450 metabolism of type II binding compounds: evidence supporting direct reduction.

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at sub-saturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; (1) Dead-end type II binding, (2) a rapid equilibrium between type I and II binding modes before reduction, and (3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.