Drug Metabolism and Disposition最新文献

Targeted covalent inhibitor (TCI) represents a noncanonical class of small molecules that function via "inactivating" the target protein through the formation of drug-protein adducts. The electrophilic groups (warheads) embedded in the TCIs are essential for their activity while also being recognized as sites susceptible to metabolism by various enzymes and endogenous nucleophiles. Given the growing knowledge of gut microbiome-mediated drug metabolism and its impact on drug absorption, distribution, metabolism, and excretion, the fate of the reactive warhead-containing TCIs in the gut warrants further understanding. In this study, we selected unsubstituted terminal acrylamides (ibrutinib, sotorasib, and divarasib), β-substituted acrylamides (afatinib, neratinib, and dacomitinib), an α-substituted acrylamide (adagrasib), an alkynamide (acalabrutinib), and a salicylaldehyde (voxelotor) to investigate. An anaerobic in vitro approach was utilized using both fecal slurry and feces-outgrown bacteria from rats, mice, and humans. The results showed that double bond reduction was the major metabolism captured for terminal acrylamides, but the activity decreases significantly when α or β substitutions are present; acalabrutinib was stable; and voxelotor was efficiently reduced to a benzyl alcohol metabolite. Synthesized TCI-GSH adducts can be efficiently hydrolyzed sequentially to cysteine adducts, which are rather stable from further microbiome modifications. There were no apparent species differences between rats, mice, and humans qualitatively, while the reductase activity observed was generally higher in the human gut microbiome. This study provides insights into both enzymatic and nonenzymatic reactions of TCIs and their thiol conjugates in the gut environment, which can be translated to the understanding of their absorption, distribution, metabolism, and excretion behavior during drug development. SIGNIFICANCE STATEMENT: Understanding the gut microbiome metabolism of targeted covalent inhibitors and their thiol conjugates will help interpret absorption, distribution, metabolism, and excretion studies for new targeted covalent inhibitors in delineating that from human metabolism, predicting clearance pathways, and assessing the impact on absorption/reabsorption. The species difference information can inform proper preclinical species for better human translation in overall drug behavior. The experimental conditions developed from this work can also be adapted to study gut microbiome metabolism in general across different species.

A new rat permeability- and perfusion-limited physiologically based pharmacokinetic model, "rat PermQ," was developed with the goal of improving concentration-time (C-t) predictions of drugs. Similar to the previously published human PermQ, drugs can reversibly distribute between capillaries and interstitial fluid by fenestra or discontinuities in capillaries or by transcellular diffusion through endothelial cells. Drugs also can be partitioned into intracellular phospholipids and neutral lipids in the cytosol. For acidic drugs, active uptake transport and an empirical protein binding correction factor were considered. A shallow distribution compartment was added for basic drugs to account for early distribution. In vitro and in vivo experimental inputs were collected in-house or from the literature. C-t profiles were predicted for 7 drugs (2 acidic, 2 neutral, and 3 basic) with 3 models: Rodgers and Rowland (RR), a perfusion-limited membrane-based model, and rat PermQ. Results indicate the importance of consistent, species-specific in vitro inputs. In general, rat PermQ predicted C-t profiles at least as well as the other models. For acidic drugs, rat PermQ predictions improved with incorporation of uptake transport and the empirical protein binding factor. For neutral drugs, RR predicted digoxin C-t profiles better compared with rat PermQ, while midazolam predictions with rat PermQ were improved with the use of in-house in vitro experimental inputs. Rat PermQ predicted C-t profiles for all 3 bases better than RR and perfusion-limited membrane-based model, and addition of a shallow compartment greatly improved the predictions. Rat and human PermQ allowed several hypotheses to be simulated for putative uptake mechanisms for atenolol and glyburide. SIGNIFICANCE STATEMENT: A new physiologically based pharmacokinetic framework, rat PermQ, was developed. This model predicted plasma concentration-time profiles of the tested drugs as well as or better than published physiologically based pharmacokinetic models. PermQ allowed several hypotheses to be simulated for uptake mechanisms in rats and humans. The work highlights the importance of accurate in vitro parameters such as drug plasma protein binding and blood-to-plasma ratio. The model can aid in testing new hypotheses to explain poorly understood observations in distribution and elimination of drugs.

Transchromosomic technology using mouse artificial chromosomes (MACs) offers a promising approach for transferring gene clusters into host organisms. This study focused on the multispecific organic anion-transporting polypeptides (OATPs) in the liver, which exhibit significant species differences between mice (Oatp1a1/Slco1a1, Oatp1a4/Slco1a4, Oatp1b2/Slco1b2) and humans (OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3). We generated an OATP1-humanized transchromosomic mouse model using a MAC vector (hOATP1-MAC mice) by transferring the human OATP1 gene cluster (SLCO1C1-SLCO1B3-SLCO1B7-SLCO1B1-SLCO1A2, 700 kbp) via an MAC into Slco1a/1b cluster knockout (KO) mice (Oatp1-KO). The human OATP1 genes were expressed in a tissue-specific manner. Plasma concentrations of the OATP1B biomarkers, coproporphyrin I and III, which were 7.2- and 23.3-fold higher in Oatp1-KO mice than in wild-type mice, were decreased by 68% and 96% in hOATP1-MAC mice, respectively. A pharmacokinetics study using pitavastatin revealed greater total body clearance (168 mL/min/kg) in hOATP1-MAC mice than in Oatp1-KO mice (100 mL/min/kg) but lower clearance than in wild-type mice (484 mL/min/kg), with bioavailability ranging from 0.66 to 0.77. In addition, drug-drug interactions were investigated using rifampicin, an OATP1B inhibitor. Rifampicin (60 mg/kg orally) increased the area under the plasma concentration-time curves of orally administered pitavastatin and grazoprevir in hOATP1-MAC mice, but not of asunaprevir. These findings demonstrated the functional expression of OATP1B1 and OATP1B3 in the mouse liver and their significant role in the systemic elimination of substrates. This is the first study to introduce multiple solute carrier drug transporter genes using artificial chromosome technology, highlighting its potential to overcome species differences in drug transport. SIGNIFICANCE STATEMENT: Transchromosomic technology holds promise for addressing species differences by introducing multiple solute carrier drug transporter genes such as OATP1. Mice OATP1-humanized using a mouse artificial chromosome vector demonstrated enhanced clearance of endogenous OATP1B biomarkers and probe drugs.

Distinct differences between sexes exist in various cardiovascular diseases. Moreover, there is a significant correlation between the pathogenesis of cardiac hypertrophy (CH) and the metabolites of arachidonic acid (AA) mediated by cytochrome P450 (CYP) enzymes. The potential link between these sex differences, the levels and the activity of CYP enzymes, and their AA-mediated metabolites remains to be elucidated. Male and female Sprague Dawley rats were injected with 1 mg/kg isoproterenol for 7 days to induce CH. Echocardiography was performed before and after the induction of CH. The hypertrophic markers and CYP enzyme levels were analyzed at the gene and protein levels using real-time polymerase chain reaction and Western blot, respectively. Heart microsomal proteins were incubated with AA, and the resulting metabolites were quantified using liquid chromatography-tandem mass spectrometry. Both sexes showed a significant degree of CH, albeit to varying extents, as the echocardiograph, heart weight/tibial length, and left ventricular parameters proved. In addition, the β/α-myosin heavy chain was 2-fold higher in male compared with female rats. Albeit the 20-hydroxyeicosatetraenoic acid (20-HETE) metabolite formation showed no increase in both sexes, the mid-chain HETEs (5- and 15-HETE) were higher in male rats, which paralleled the increase in the gene and protein levels of CYP1B1. The formation rate of the epoxyeicosatrienoic acids was almost unchanged in female-treated rats, while it was significantly decreased in male-treated rats. Our results suggest sexual dimorphism in the isoproterenol-induced CH in rats, specifically on the level of CYP enzymes and their AA-mediated metabolites. SIGNIFICANCE STATEMENT: Sexual dimorphism was observed in rats following isoproterenol-induced cardiac hypertrophy, with males showing a stronger hypertrophic response. This was linked to higher CYP1B1 gene and protein expression in males, along with sex-related differences in many cytochrome P450 enzyme activities and their mediated arachidonic acid metabolites. These findings emphasized the need for targeted, sex-specific therapeutic strategies for the management and treatment of cardiac hypertrophy and other cardiovascular disorders.

Incubation of drugs with suspension hepatocytes (SH) to determine intrinsic clearance is common in drug discovery. However, the limited duration of SH assays hampers clearance assessment of metabolically stable compounds. In turn, this has driven the development of alternative in vitro approaches to generate intrinsic clearance estimates. Culturing primary hepatocytes with supportive cells as co/tricultures has been shown to maintain morphology, viability, and drug-metabolizing enzyme function for weeks, permitting extended incubations. Another assay from our laboratory is the preloaded hepatocyte assay (preload assay), which involves preloading plated monoculture hepatocytes with compounds and measuring the loss from cells in drug-free media. This approach increases analytical sensitivity compared to assays that measure bulk compound loss in the cells plus medium. We conducted a systematic evaluation of the ability of coculture, triculture, and preload assay models to predict human in vivo clearance for 50 predominantly low-clearance compounds with a range of physicochemical properties, including equal numbers of compounds following or violating Lipinski's rule of 5, across 3 hepatocyte donors. The results were compared with SH. Co/tricultures exhibited lower inter-donor differences compared to the preload and SH assays, likely due to the blunting of environmental cues after 5 days in culture prior to compound introduction. All 3 plated models significantly reduced the number of compounds with insufficient turnover to calculate CL_int,u compared to SH (SH: 40%; preload: 18%; cocultures: 8%; tricultures: 4%), exhibited strong interexperimental reproducibility and robust predictions of blood clearance (preload: 26/41; cocultures: 31/46; tricultures: 30/48 within 3-fold of observed). SIGNIFICANCE STATEMENT: Preloading plated hepatocytes with compounds and measuring the loss in drug-free media, or culturing hepatocytes with supportive cells as co/tricultures, facilitate quantitation of metabolically stable compounds in substrate depletion assays compared to suspension hepatocytes (SH). All 4 models exhibit robust estimates of CL_int,u and CL_b, but plated models allowed assessment of several compounds found to be too stable to evaluate in SH.

The SLC22A2 gene encodes organic cation transporter 2 (OCT2), which is predominantly expressed in renal proximal tubule cells. OCT2 is critical for the active renal excretion of various cationic drugs and endogenous metabolites. OCT2 expression varies across species, with higher levels in mice and monkeys compared with humans and rats. The human OCT2 protein consists of 555 amino acids and contains 12 transmembrane domains. OCT2 functions as a uniporter, facilitating the bidirectional transport of organic cations into renal tubular cells, driven by the inside-negative membrane potential. Its expression is regulated by sex hormones, contributing to potential sex differences in Oct2 activity in rodents. OCT2 has been linked to tissue toxicity, such as cisplatin-induced nephrotoxicity. Factors such as genetic variants, age, disease states, and the coadministration of drugs, including tyrosine kinase inhibitors, contribute to interindividual variability in OCT2 activity. This, in turn, impacts the systemic exposure and elimination of drugs and endogenous substances. Regulatory agencies recommend evaluating the potential of a drug to inhibit OCT2 through in vitro and clinical drug-drug interaction (DDI) studies, often using metformin as a probe substrate. Emerging tools like transporter biomarkers and physiologically based pharmacokinetic modeling hold promise in predicting OCT2-mediated DDIs. While several OCT2 biomarkers, such as N1-methylnicotinamide, have been proposed, their reliability in predicting renal DDIs remains uncertain and requires further study. Ultimately, a better understanding of the factors influencing OCT2 activity is essential for achieving precision medicine and minimizing renal and systemic toxicity. SIGNIFICANCE STATEMENT: Organic cation transporter 2 (OCT2) is essential for the active tubular secretion of xenobiotics and endogenous cationic substances in the kidneys. This article offers a comprehensive overview of the tissue distribution, interspecies differences, and factors affecting its activity-critical for evaluating tissue toxicity and systemic exposure to cationic substances. Using OCT2 biomarkers and integrating OCT2 activity and expression data into physiologically based pharmacokinetic models are valuable tools for predicting OCT2 function and its clinical implications.

The formation of antidrug antibodies (ADAs) against antibody-drug conjugates (ADCs) can trigger a humoral immune response and change drug exposure. Although the immunogenicity assessment of an ADC drug in nonclinical nonhuman primates may not directly translate to potential immunogenicity in humans, the nonclinical ADA assay facilitates understanding the pharmacokinetic profiles of biotherapeutics. The immune response against the human IgG4 monoclonal antibody-based ADC was suspected in cynomolgus monkey serum after intravenous administration at 1.5 mg/kg. However, the conventional bridging format ADA assay presented unique challenges for the ADC molecules due to the interaction of ADC-based capture and detection reagents, which generated high background noise. Solid-phase extraction with acid dissociation (SPEAD) sample treatment allowed the selective ADA transfer to a second plate for detection while avoiding the interaction between the capture and detection reagents. The signal-to-noise ratio in the ADA assay for ADCs was notably improved with SPEAD sample treatment compared with the results from the bridging assay. Importantly, the rapid drug clearance of the ADC molecules at the later time points was well correlated with the signal-to-noise ratio of the ADA assay in monkey serum, suggesting the validity of the results. Hence, we demonstrated the utility of the SPEAD sample treatment to mitigate the critical reagent interaction that triggered the unexpectedly high background in the ADA assay. SIGNIFICANCE STATEMENT: A fit-for-purpose antidrug antibody screening assay for the human IgG4 monoclonal antibody-based antibody-drug conjugate (ADC) molecule by solid-phase extraction with acid dissociation was developed to mitigate the high background noise due to the interaction of capture and detection ADCs. A positive antidrug antibody signal was observed in the monkey serum sample, which is in line with the significant decrease in the plasma concentration of ADCs at the later time points.

Cytochrome P450 plays key roles in the biotransformation of endogenous and exogenous chemicals including drugs and environmental pollutants. The inhibition and downregulation of P450s can have therapeutic effects, and/or modulate drug metabolism. P450s are largely inhibited by small molecules; however, this strategy is often hampered by intrinsic toxicity and drug-drug interactions. Furthermore, it is challenging for small molecules to exhibit high selectivity and inhibitory efficiencies. Recently, small interfering RNA (siRNA) technology has demonstrated the potential for P450 modulation. Examples of recent applications of siRNAs in P450 gene modulation, in vitro and in vivo, are highlighted in this review. The necessity of siRNA techniques and their advantages as P450 modulators are discussed, along with a review of current obstacles and a perspective on future advancements. SIGNIFICANCE STATEMENT: This article reviews studies on the application of small interfering RNA technology to cytochrome P450 gene modulation. The necessity of siRNA methods and the benefits of their use as P450 modulators have been suggested by comparison with small-molecule drugs. Additionally, the challenges that presently limit the broader implementation of this topic are examined, and a perspective for future developments is proposed.

Geranylgeranyl diphosphate synthase produces the isoprenoid geranylgeranyl diphosphate, which is used in protein geranylgeranylation. Our previous work illustrates that geranylgeranyl diphosphate synthase inhibitors (GGSIs) disrupt Rab-mediated protein trafficking in cells, inducing the unfolded protein response pathway and apoptosis. Structure-function studies of our GGSIs, which are isoprenoid triazole bisphosphonates, have revealed a complex relationship between GGSI structure and enzymatic, cellular, and in vivo activities. The dose-limiting toxicity of this family of GGSIs is hepatic, and the mechanisms underlying their hepatic uptake are unexplored. Here, we evaluate the pharmacokinetics (PK) and biodistribution of a pair of potent GGSIs that are olefin isomers (homogeranyl [HG] and homoneryl [HN]). We investigate whether these isomers, as well as their a-methylated analogs (HG-me and HN-me), are substrates for key hepatic transporters and explore the effects of these GGSIs on the expression of a panel of hepatic transporters and cytochrome P450s. The PK/biodistribution studies revealed that both systemic exposure and liver levels of HG were significantly higher than that of HN across multiple time points. Conversely, HN was present at 4-fold higher concentrations in the bile at 2 hours postinjection relative to HG. HG-me and HN-me, but not HG or HN, were determined to be substrates of hepatic transport proteins OATP1B1 and OATP1B3. While the hepatic expression of several transporters and cytochrome P450 were altered by GGSI treatment, no significant differences in expression patterns between pairs of olefin isomers were observed. Collectively, these studies reveal that GGSI structure, including olefin stereochemistry, impacts PK profile, biodistribution, and hepatic transporter affinity. SIGNIFICANCE STATEMENT: Our understanding of the in vivo structure-activity relationship of our novel geranylgeranyl diphosphate synthase inhibitors has expanded, demonstrating that isoprenoid olefin stereochemistry impacts pharmacokinetic and biodistribution patterns and that other modifications impact transporter affinity. These studies reveal the underlying complexity of the mechanisms regulating hepatic exposure to these agents. Future studies will focus on optimizing tumor-directed geranylgeranyl diphosphate synthase inhibitor delivery while minimizing hepatic uptake.

Midostaurin and its active metabolites are substrates, mixed inhibitors/inducers of cytochrome P450 (CYP)3A4. The main objective of this study was to develop/refine a physiologically based pharmacokinetic (PBPK) model that incorporated recent clinical drug-drug interaction (DDI) data with midazolam after multiple dosing, to qualify the pharmacokinetic (PK) model simulations of midostaurin and its metabolites, and to apply it to predict untested clinical DDI scenarios with potential comedications. In this study, Simcyp PBPK model of midostaurin and its 2 metabolites was refined from a previously published model associated with endogenous biomarker 4β-hydroxycholesterol data through further optimization of CYP3A4 inhibition/induction potency and was qualified to simulate midostaurin steady-state PK. The incorporation of these parameters enabled DDI predictions of high midostaurin doses on the PK of midazolam and oral contraceptives containing ethinyl estradiol. Additionally, scaling factors for in vitro breast cancer resistance protein and the organic anion transporting polypeptide (OATP1B) inhibition were applied to account for the observed single-dose DDI with rosuvastatin and further extrapolated to predict steady-state DDI with other OATP1B drug substrates. The overall prediction results showed minimal impact of midostaurin at high doses on CYP3A substrates or an effect on the exposure of OATP1B substrates. In summary, the midostaurin PBPK model was retrospectively refined, requalified, and used to simulate the steady-state perpetrator DDI of midostaurin and its metabolites. This PBPK modeling approach and the resulting model predictions were implemented into the midostaurin product label (up to 100 mg twice a day) without the need for confirmatory clinical studies. SIGNIFICANCE STATEMENT: The manuscript describes how a midostaurin PBPK model was updated, verified, and applied to untested scenarios by a predict-learn-confirm cycle as new clinical data become available. It also provides a learning experience of prospective prediction by utilizing endogenous biomarker 4β-hydroxycholesterol to evaluate a complex CYP3A4-mediated drug interaction.