This study characterized the pharmacokinetic interaction between Compound Danshen Tablets (CDT) and atorvastatin (ATV) and investigated absorption- and transporter-related mechanisms underlying this interaction. ATV pharmacokinetics were evaluated after oral and intravenous administration in normal and hyperlipidemic/diabetic rats, with or without repeated CDT treatment. Mechanistic studies included single-pass intestinal perfusion, Caco-2 bidirectional transport assays, human multidrug resistance protein 1 inside-out vesicle transport assays, ileal P-glycoprotein (P-gp) immunoblotting, an in vivo digoxin probe-substrate experiment, and molecular docking. Repeated CDT administration increased oral ATV exposure in both normal and model rats, with the area under the plasma concentration-time curve from 0 to 24 hours and maximum plasma concentration increasing by up to 85.83% and 306.53%, respectively, whereas intravenous ATV pharmacokinetics were not significantly altered. In Caco-2 monolayers, ATV exhibited marked directional efflux, with an efflux ratio of 39.32, which was reduced to 10.99 by verapamil. CDT increased the apical-to-basolateral apparent permeability coefficient of ATV from 0.41 to 1.31 × 10-6 cm/s and reduced the efflux ratio to 9.82, whereas the basolateral-to-apical apparent permeability coefficient decreased from 16.27 to 12.90 × 10-6 cm/s. In human multidrug resistance protein 1 inside-out vesicles, CDT concentration dependently reduced ATP-dependent N-methylquinidine transport, with inhibition rates ranging from 24.97% to 80.45% at 50-800 μg/mL. Together with the ileal P-gp protein data and the digoxin probe-substrate experiment, these findings indicate that CDT enhances intestinal absorption of ATV primarily by attenuating intestinal P-gp-mediated efflux through reducing both P-gp transport activity and protein abundance. These results provide mechanistic evidence that modulation of intestinal efflux transport contributes to the increased systemic exposure of ATV after repeated CDT administration. SIGNIFICANT STATEMENT: This study identifies a clinically relevant herb-drug interaction in which repeated administration of compound Danshen tablets increases oral atorvastatin exposure in normal and hyperlipidemic/diabetic rats without materially altering intravenous atorvastatin pharmacokinetics. Integrated pharmacokinetic, single-pass intestinal perfusion, Caco-2 transport, human multidrug resistance protein 1 vesicle, ileal P-glycoprotein, and digoxin probe-substrate data support attenuation of intestinal P-glycoprotein-mediated efflux, through reduced transporter activity and protein abundance, as a mechanism enhancing atorvastatin absorption.

The recent passing of Bruce D. Hammock provides a timely opportunity to examine how his contributions to soluble epoxide hydrolase (sEH) biology, inhibitor development, and translational pharmacology extended through international scientific collaboration and mentorship. This minireview examines the Brazilian scientific network associated with Hammock as a descriptive case study and combines an independent structured evidence survey of international research-capacity initiatives with curated PubMed records, information from the Brazilian Currículo Lattes repository, and documentary evidence of scientific exchange and mentorship. Temporal, title-term, geographic, documentary, and mentorship analyses were conducted using reproducible workflows. Of 500 records screened in the evidence survey, 24 bibliographically verified unique records were retained after postsearch audit; collectively, they reported associations between sustained international partnerships and researcher training, institutional strengthening, and scientific production. These records provided comparative context and were not required to involve Hammock or members of his scientific network. PubMed retrieval yielded 17 unique indexed records. Thirteen met the criteria for the core Brazilian sEH/(1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea [TPPU]) corpus, 3 were retained as contextual records, and 1 was excluded from the core corpus because it did not address sEH or epoxide hydrolase biology. The core corpus remained anchored in sEH inhibition and lipid-mediator biology, while extending into pain, intestinal injury, arthritis, periodontal inflammation, alveolar bone loss, immune regulation, eicosanoid profiling, macrophage plasticity, and osteoblast metabolism. Brazilian institutional participation in the core publication corpus was concentrated in São Paulo, with additional participation from Minas Gerais and Paraná. The broader documentary analysis identified 43 academic outputs explicitly linked to sEH, TPPU, or the defined Brazilian scientific lineage, together with evidence of scientific exchange, access to pharmacological tools, formal mentorship, and multigenerational propagation of the research trajectory. Together, these findings support the interpretation that Hammock's legacy in drug metabolism and disposition extended beyond foundational discoveries and publications to include the local adaptation of a mechanistically coherent translational research program. More broadly, this case illustrates how pharmacological innovation, accessible experimental tools, and sustained scientific mentorship may support the development of research capacity in emerging scientific settings. SIGNIFICANT STATEMENT: Using the Brazilian network associated with Bruce D. Hammock as a case study, this minireview connects advances in soluble epoxide hydrolase biology and inhibitor development with the documented temporal development of the publication corpus, diversification of experimental applications, geographic concentration of institutional participation, and multigenerational mentorship. It shows how scientific legacy in drug metabolism and disposition may extend beyond publications and citations to include the formation of durable translational research capacity.

Organic anion transporting polypeptide 2B1 (OATP2B1) is a basolateral plasma membrane uptake transporter expressed in hepatocytes. However, the cellular internalization and degradation pathways of OATP2B1 remain poorly characterized. Filling this knowledge gap is critical for improving predictions of OATP2B1 substrate disposition and OATP2B1-mediated drug interactions. This study investigated the localization and primary degradation pathway of OATP2B1 in human hepatocytes and examined whether OATP2B1 localization was affected by metabolic dysfunction-associated steatohepatitis in human liver tissue. Sandwich-cultured human hepatocytes (SCHHs) were treated with either 25 μM chloroquine (CQ) or 10 μM MG-132 to inhibit lysosomal or proteasomal degradation, respectively. Following treatment, SCHHs were fixed and stained for OATP2B1 and lysosome-associated membrane protein (LAMP) 1, followed by confocal imaging and subsequent object-based quantitative analysis. Lysosomal inhibition by CQ in SCHHs resulted in significant accumulation of intracellular OATP2B1, which displayed major colocalization with the lysosomal marker, LAMP1 (20% ± 9.38% vs 9.5% ± 5.25%; CQ vs control, respectively; P < .0001). In contrast, the OATP2B1 total object volume and intracellular localization remained unchanged after MG-132 treatment. OATP2B1 colocalization with LAMP1 was significantly higher in liver tissue from patients with metabolic dysfunction-associated steatohepatitis (10.47% ± 4.95%) compared with control liver tissue (6.00% ± 2.06%; P < .0001). Super-resolution imaging using stimulated emission depletion microscopy revealed that OATP2B1 was localized on lysosomal membranes. In conclusion, these findings demonstrate that OATP2B1 degrades primarily via lysosomes in human hepatocytes. Lysosomal dysfunction associated with some xenobiotics or disease may increase OATP2B1 localization on lysosomal membranes. SIGNIFICANCE STATEMENT: This study confirms that lysosomes are the predominant degradation pathway for the basolateral membrane protein organic anion transporting polypeptide 2B1 (OATP2B1) in human hepatocytes. Super-resolution imaging revealed that OATP2B1 is localized on lysosomal membranes. Both lysosomal inhibition by chloroquine in vitro and lysosomal dysfunction associated with metabolic dysfunction-associated steatohepatitis in human liver tissue increased OATP2B1 colocalization with the lysosomal membrane marker.

Ivermectin is commonly administered as a prophylactic or therapeutic antiparasitic treatment without specific restrictions on the reproductive period, raising concerns about potential drug exposure in reproductive tissues and semen. This study investigated the pharmacokinetic distribution of ivermectin in plasma, seminal plasma, and spermatozoa after a single subcutaneous administration of 0.2 mg/kg to Awassi rams. Blood and semen samples were collected over a 30-day period. Ivermectin concentrations were determined by high-performance liquid chromatography, and pharmacokinetic parameters were calculated using noncompartmental analysis. Based on the longest individual detection time observed among the animals, ivermectin was detectable in plasma and spermatozoa for up to 25 days and in seminal plasma for up to 20 days. The peak plasma, seminal plasma, or sperm concentration (Cmax), area under the curve to the last concentration, area under the curve extrapolated to infinity, apparent volume of distribution, apparent clearance, area under the first moment curve to the last concentration, and area under the first moment curve extrapolated to infinity values differed significantly among the plasma, seminal plasma, and spermatozoa compartments (P < .05). Plasma Cmax (25.03 ± 6.44 ng/mL) was significantly higher than in seminal plasma (0.98 ± 0.42 ng/mL) and spermatozoa (5.33 ± 3.54 ng/g). The seminal plasma/plasma Cmax and AUC ratios were approximately 3.9% and 3.5%, respectively; this result may be due to tissue-specific barriers and efflux transport mechanisms (such as P-glycoprotein) that restrict drug transfer to the reproductive organs, although ram-specific transporter expression or activity data are not available and were not directly evaluated in this study. Ivermectin concentrations in spermatozoa (ng/g) were numerically approximately 5.4-fold higher than in seminal plasma (ng/mL); although these values are expressed in different units and are not directly equivalent, this difference may reflect the affinity of this lipophilic drug for the high-lipid structures of sperm cells. The plasma pharmacokinetic parameters were consistent with previously reported values in sheep. To our knowledge, this is the first study to simultaneously characterize the pharmacokinetic profiles of ivermectin in plasma, seminal plasma, and spermatozoa in rams. These findings provide reference data for ivermectin residue and reproductive safety assessments in breeding rams and highlight the need for further investigation into its effects on spermatological parameters and fertility. SIGNIFICANT STATEMENT: Ivermectin is widely used as an antiparasitic in livestock, including in breeding rams before or during the breeding season for prophylactic or therapeutic purposes, with no specific restrictions on its use during this period. This study is the first to simultaneously examine the pharmacokinetic profiles of ivermectin in plasma, seminal plasma, and spermatozoa in rams. The significantly lower ivermectin levels in seminal plasma than in plasma suggest restricted drug transfer into reproductive tissues, likely due to tissue-specific barriers and efflux transport mechanisms, including P-glycoprotein, although these mechanisms were not directly evaluated in this study. The prolonged presence of ivermectin in seminal plasma and spermatozoa suggests that the drug remains in reproductive compartments for a significant time after a single therapeutic dose.

The absorption, distribution, metabolism, and excretion study and the absolute bioavailability study were conducted to determine the pharmacokinetics, mass balance, metabolite profile, and absolute bioavailability of naporafenib, a selective pan-rapidly accelerated fibrosarcoma (RAF) inhibitor. This was an open-label, single-center, 2-part, fixed-sequence study. In part 1, a tracer [[14]C]-naporafenib was administered intravenously with a single 200-mg oral dose of naporafenib to 8 healthy male participants to assess the absolute bioavailability and pharmacokinetics of naporafenib. After a 15-day washout, a single 200-mg/500-μCi oral dose of [[14]C]-naporafenib was administered to the same participants in part 2 to investigate the mass balance and metabolism of naporafenib. The absolute bioavailability was determined to be 39.6%. The geometric mean clearance was 7.58 L/h, volume of distribution was 162 L, and terminal half-life was 18.1 hours. A near complete recovery (94.0%) of administered radioactivity was achieved, with 78.8% of the dose excreted in feces and 15.2% of dose recovered in the urine. The metabolism of naporafenib involved multiple primary metabolic pathways that included amide hydrolysis, oxidation, glucuronidation, dealkylation, and dehydrogenation, with secondary hydrolysis, N-acetylation, N-methylation, reduction, glucuronidation, and oxidation. No metabolite exceeded 10% of total circulating drug-related radioactivity. There were no major/disproportionate or unique human metabolites. No metabolites warranted future evaluation given low abundance. SIGNIFICANCE STATEMENT: This study provides a detailed understanding of the pharmacokinetics, disposition, and metabolism of naporafenib after oral and microtracer intravenous administration in healthy participants. This knowledge will guide future nonclinical and clinical studies evaluating drug-drug interactions, organ dysfunction, and safety of metabolites.

Podophyllotoxin is an aryltetralin lignan with potent antimitotic and antiviral activities; however, its severe toxicity restricts clinical use to topical application, and systemic exposure to it can result in life-threatening poisoning. However, its metabolic profile remains largely uncharacterized, leaving a critical gap in the biomarkers available for confirming intoxication. Here, we used an integrated nontargeted screening strategy to investigate podophyllotoxin metabolism in post-mortem blood from a rare fatal case of podophyllotoxin poisoning. Two complementary nontargeted screening strategies were established using liquid chromatography coupled to high-resolution tandem mass spectrometry data acquired in both positive and negative ion modes. SyGMa was first used to generate a predicted metabolite list of podophyllotoxin, enabling target analysis against the acquired data. Subsequently, molecular networking was applied to cluster structurally related compounds across specimens based on high-resolution tandem mass spectrometry spectral similarity, enabling visualization and discovery of unknown metabolites and their structural relationships. By integrating these approaches, a total of 11 potential metabolites were identified, including 2 phase Ⅰ metabolites and 9 phase Ⅱ metabolites, of which 5 were previously unreported. Among these, the O-demethylenated and methylated products (m/z 416.1471) are proposed as potential biomarkers for assessing podophyllotoxin exposure. The integrated workflow demonstrated herein facilitates xenobiotic metabolite identification in biological samples, and the characterized metabolites may support future metabolite annotation and exposure assessment through incorporation into mass spectral libraries. SIGNIFICANCE STATEMENT: Human metabolic data for podophyllotoxin remain scarce despite the severe toxicity associated with systemic exposure. By analyzing post-mortem blood from a fatal poisoning case, this study expands knowledge of podophyllotoxin biotransformation and proposes previously unreported metabolites. The results further demonstrate the complementary value of combining in silico prediction with molecular networking for metabolite discovery and annotation, highlighting a practical strategy for characterizing xenobiotic metabolites in limited biological specimens.

Kava is a beverage prepared from the rhizomes and roots of the tropical evergreen plant Piper methysticum. Kava contains 6 major kavalactones. Legacy studies found that kavalactones did not inhibit human cytochrome P450s in vitro at relevant concentrations, and kava did not inhibit the major P450s in humans in vivo and was devoid of clinically significant drug interactions. Nevertheless, CYP2B6 was never evaluated. Recent investigation identified that (+)-dihydromethysticin, 1 of the 6 natural kavalactones, was an inhibitor of CYP2B6 in vitro. This investigation evaluated the effects of kava extract and (+)-dihydromethysticin on expressed CYP2B6 activity-both wild-type CYP2B6.1 and active allelic variant CYP2B6.4. Effects on human liver microsomal CYP2B6 were also evaluated. Two independent kava extracts inhibited expressed and human liver microsomal CYP2B6 activity, in a concentration-dependent manner, assessed using the CYP2B6 probe substrates 7-ethoxy-4-trifluoromethyIcoumarin O-deethylation and S-ketamine N-demethylation. Ki values for CYP2B6 inhibition by (+)-dihydromethysticin alone and in kava extracts were 0.01-0.05 μM. Kava and (+)-dihydromethysticin interaction with expressed CYP2B6.1 and CYP2B6.4 generated a difference spectrum consistent with formation of a metabolite-inhibitor complex. (+)-Dihydromethysticin underwent NADPH-dependent metabolism by expressed CYP2B6 and human liver microsomes. These results show that kava extracts inhibit CYP2B6, an effect attributed to (+)-dihydromethysticin, which appears to act as a mechanism-based inhibitor. Because kava consumption achieves plasma (+)-dihydromethysticin concentrations exceeding the inhibitory Ki, there is a potential risk of in vivo CYP2B6-mediated herb-drug interactions. Such potential merits clinical drug-herb interaction studies using standard CYP2B6 substrate probes. SIGNIFICANCE STATEMENT: Kava extracts are widely used as botanical supplements for managing various health conditions. Kava extracts showed concentration-dependent inhibition of CYP2B6 in both expressed systems and human liver microsomes. Inhibition was attributed to (+)-dihydromethysticin. Spectral and metabolism studies suggested that CYP2B6 catalyzed the formation of a metabolite-inhibitor complex with (+)-dihydromethysticin alone and present in the extracts. Typical kava consumption yields plasma (+)-dihydromethysticin concentrations that exceed the inhibitory Ki; thus, showing a clear potential for clinically relevant in vivo CYP2B6-mediated herb-drug interactions.

Inflammation profoundly alters hepatic drug metabolism and transport, requiring careful consideration in pharmacotherapy to minimize adverse outcomes. In this study, we compared global transcriptomic profiles of macroscopically and histologically normal liver tissue from patients undergoing tumor metastasis resection (n = 37) and critically ill patients requiring intensive care (n = 50), alongside an advanced 3-dimensional primary human liver spheroid model. Transcriptomic analysis revealed clear segregation between cohorts, identifying 6250 differentially expressed genes and a pronounced inflammatory signature in livers from critically ill patients, marked by elevated acute-phase proteins, including C-reactive protein, serum amyloid A1, and serum amyloid A2. Three-dimensional primary human liver spheroids were analyzed with increasing cellular complexity: hepatocyte monocultures, hepatocyte-nonparenchymal cell cocultures, and triple cultures incorporating liver sinusoidal endothelial cells, with or without free fatty acid supplementation to mimic metabolic stress. Incorporation of nonparenchymal cells and liver sinusoidal endothelial cells markedly upregulated C-reactive protein expression and induced transcriptomic shifts closely mirroring inflamed human liver tissue. Pathway enrichment and targeted analysis of absorption, distribution, metabolism, and excretion genes demonstrated consistent inflammation-driven downregulation of cytochrome P450 enzymes, phase II enzymes, transporters, and nuclear receptors critical for drug metabolism and biliary efflux-patterns nearly identical to those observed between inflamed liver tissue and tissue obtained by tumor resection. These findings highlight substantial phenotype changes in macroscopically and histologically normal livers and validate advanced multicellular 3-dimensional human liver spheroids as physiologically relevant models for recapitulating inflammation-induced perturbations in hepatic pharmacokinetics, offering predictive tools for improved preclinical safety assessment and personalized dosing in patients with systemic inflammation. SIGNIFICANCE STATEMENT: Inflammation profoundly alters hepatic drug metabolism and transport, requiring careful consideration in pharmacotherapy to minimize adverse outcomes. This study highlights substantial phenotype changes caused by inflammation in macroscopically and histologically normal livers and validates advanced multicellular 3-dimensional human liver spheroids as physiologically relevant models for recapitulating inflammation-induced perturbations in hepatic pharmacokinetics.

Proteolysis targeting chimeras (PROTACs) have emerged as a promising modality over the past decade, with only a few advancing to clinical trials. As "beyond rule-of-five" compounds, PROTACs pose significant challenges for absorption, distribution, metabolism, and excretion, often necessitating iterative in vitro absorption, distribution, metabolism, and excretion assay optimization or reliance on in vivo pharmacokinetic studies, particularly to understand oral absorption. In this study, in vitro-in vivo correlations from clinically advanced PROTACs were leveraged to develop a novel classification system with results from optimized Madin-Darby Canine kidney cell line type 1 transfected with human multidrug resistance 1 and colorectal adenocarcinoma cell permeability and efflux assays that prioritizes compounds based on estimated mouse oral absorption. Receiver operating characteristic analysis was applied to define data-driven thresholds for effective permeability (Peff) and efflux ratio (ER), enabling classification of compounds into mouse absorption-relevant buckets with good discriminative performance. In the Madin-Darby Canine kidney cell line type 1 transfected with human multidrug resistance 1 assay, both individual cutoffs for normalized Peff (0.36) and normalized ER (1.72) and combined continuous score of log10 normalized Peff - 1.3∗log10 normalized ER yielded reasonable classification of PROTACs with FaFg > 20%. In the colorectal adenocarcinoma assay, both individual cutoffs for normalized Peff (0.021) and normalized ER (0.22) and combined continuous score of log10 normalized Peff - 1.2∗log10 normalized ER yielded reasonable classification of PROTACs with FaFg > 20%. This approach aims to streamline in vitro workflows and minimize the need for extensive in vivo pharmacokinetic studies when evaluating novel PROTACs in preclinical discovery studies. SIGNIFICANT STATEMENT: This work establishes optimized assay conditions to assess permeability and a novel classification strategy that enables more reliable in vitro assessment of proteolysis targeting chimeras oral absorption potential. By streamlining workflows, it reduces the need for extensive in vivo pharmacokinetic studies when evaluating novel proteolysis targeting chimeras in drug discovery.

We introduce 6-methoxy-2-naphthoic acid (MONA) as a novel fluorogenic substrate for the drug-metabolizing CYP1A2 enzyme. Oxidative demethylation of MONA by human liver microsomes resulted in a red shift and a significant increase in its fluorescence. Screening 14 recombinant human cytochrome P450 (P450) enzymes revealed detectable activity in MONA demethylation by CYP1A2 (kcat = 12 ± 2 min[-1], KM = 578 ± 106 μM), CYP2A6 (kcat = 0.42 ± 0.09 min[-1], KM = 48 ± 15 μM), and CYP1A1 (kcat = 0.19 ± 0.09 min[-1], KM = 89 ± 43 μM). The high rate of CYP1A2-catalyzed reaction along with a distinct resolution of its KM value establishes MONA as a selective fluorogenic probe for CYP1A2 activity. MONA was used to investigate the effects of chronic alcohol exposure on CYP1A2 activity using a series of 23 proteomically characterized human liver microsomes from donors with various levels of alcohol consumption. Global kinetic analysis of the set of saturation profiles yielded 2 Michaelis-Menten components with KM values of 11 and 553 μM, whose combinations adequately approximate all profiles in the dataset. The amplitudes (Vmax) of both components showed a marked increase with increasing alcohol exposure. The Vmax of the minor high-affinity component was best correlated with a combination of abundances of CYP2E1, CYP2A6, and NADPH-P450 reductase, suggesting that it reflects the activity of CYP2A6 in the complex with CYP2E1. The Vmax of the predominant CYP1A2-dependent component exhibits a pronounced correlation with the content of NADPH-P450 reductase, whose increased expression in alcohol consumers appears to be the main causative factor in alcohol-induced increase in CYP1A2 activity that takes place despite an alcohol-induced decrease in CYP1A2 expression. SIGNIFICANCE STATEMENT: We introduced 6-methoxy-2-naphthoic acid as a specific fluorogenic substrate for CYP1A2. By studying its metabolism in a series of 23 proteomically characterized human liver microsome samples from donors with various levels of alcohol consumption, we demonstrated a significant increase in CYP1A2 activity with increasing alcohol exposure despite decreased CYP1A2 expression. This increase, which is best correlated with the alcohol-induced increase in the abundance of NADPH-cytochrome P450 reductase, may have a striking effect on the pharmacokinetics of CYP1A2-metabolized antidepressants and antipsychotics in alcoholics.

Tyrosine kinase inhibitors (TKIs) are a critical aspect of therapeutic strategy in non-small cell lung cancer (NSCLC), acting against oncogenic driver alterations, including epidermal growth factor receptor mutations, anaplastic lymphoma kinase gene rearrangements, and ROS proto-oncogene 1 fusion, etc. The clinical efficacy and safety of these agents are highly dependent on their pharmacokinetic properties, particularly metabolic pathways. Among the drug-metabolizing enzymes, cytochrome P450 3A4 (CYP3A4) plays a major role in the metabolism of many clinically used TKIs in NSCLC, generating both active and inactive metabolites that significantly influence therapeutic outcomes. While the active metabolite may retain pharmacological activity, the inactive metabolites facilitate drug elimination and prevent drug accumulation in the body. In addition, several TKIs modulate CYP3A4 activity and undergo drug-drug interactions with other CYP3A4 inhibitors and inducers, which can significantly alter systemic drug exposure, leading to toxicity or reduced efficacy. Furthermore, interindividual variability in CYP3A4 activity driven by patient-specific factors such as age, sex, comorbidities, and genetic polymorphisms is another clinical concern that necessitates individualized dosing approaches for these TKI agents. This review summarizes CYP3A4-mediated metabolism of TKIs used in NSCLC therapy and addresses the metabolites formed, providing structural insights into metabolic hot spots and guiding drug development, optimization, and prodrug strategies. It also discusses potential drug-drug interactions, compares the apparent in vivo CYP3A4 dependence of clinically used TKIs, and highlights the patient-related factors associated with TKIs, which are critical determinants of the individualized dosing strategy needed for an effective therapeutic response in patients with NSCLC. SIGNIFICANCE STATEMENT: CYP3A4 is a key regulator of the pharmacokinetics, efficacy, and safety of commonly used tyrosine kinase inhibitors in non-small cell lung cancer. This review discusses the predominant role of CYP3A4 in the metabolism of these agents, highlighting the active and inactive metabolites, the clinically relevant drug-drug interactions, and the impact of patient-specific factors on metabolic variability and systemic drug exposure. These provide critical insights into drug development and optimization strategies, and underscore the implementation of individualized dosing approaches to improve clinical outcomes in patients with non-small cell lung cancer.

A simple method is described that uses pharmacokinetic parameters for an unlabeled drug to predict for a [14]C-absorption, distribution, metabolism, and excretion study, the radiochemical dose consistent with the analytical instrument for quantitative profiling of circulating metabolites by scintillation counting. Analysis of mass balance studies for 19 drugs verified the predictive method and shows that many mass balance studies are conducted with radiochemical doses either substantially higher than required and inconsistent with the as low as reasonably achievable principle or inadequate to conduct profiling of plasma with the intended instrumentation. SIGNIFICANCE STATEMENT: The method to predict the radiochemical doses for [14]C-absorption distribution, metabolism, and elimination ([14]C-ADME) studies enables research teams to select the radiochemical dose at which to conduct an ADME study consistent with the analytical instrument for plasma profiling and with the as low as reasonably achievable principle. Furthermore, it can also be used to assess whether a microdose or macrodose study is more appropriate.

Kinases catalyze the activation of certain nucleoside and nucleotide analog drugs used in the treatment of various cancers and viral infections. Because these drugs require intracellular phosphorylation to their active form, the abundance of kinases within tissues is a key factor in determining both variability in response and off-target effects. In this study, we quantified and compared protein abundances of kinases in commercially available pooled S9 fractions of adult human liver (n = 50), kidney (n = 12), lung (n = 5), intestine (n = 4), and individual adult primary hepatocytes (n = 8). These data were compared with the proteomic abundance profile of red blood cells (n = 4) from healthy donors and bone marrow samples (n = 44) from patients with acute myeloid leukemia, in addition to kinase abundance in human plasma from the publicly available data base Human Protein Atlas. Kinase protein abundance was estimated using the total protein approach in pmol/mg total protein. We detected 364 kinases, including 23 drug-metabolizing kinases, with varying abundances across tissue and biofluid types. Four drug-metabolizing kinases, adenosine kinase, cytidine/uridine monophosphate kinase 1, and nucleoside diphosphate kinases 1 and 3, were detected in all sample types. In addition, by comparing tissue-to-plasma abundance ratios of kinases with those of known secreted versus nonsecreted marker proteins, we showed that kinases are primarily present in plasma due to tissue shedding, with few (phosphoglycerate kinase 1, pyruvate kinase M, adenylate kinase 1, and nucleoside diphosphate kinase 1) that may be present as part of extracellular vesicles. Lastly, we identified the presence of 24 kinases commonly targeted by inhibitors across sample types. Overall, this study advances the understanding of the presence and variability of kinases in different tissues. SIGNIFICANCE STATEMENT: This study quantified and compared the presence and abundance of kinases, including drug-metabolizing kinases, across various human sample types. In total, 364 kinases, including 23 known drug-metabolizing kinases, were detected with varying abundances across tissue and biofluid types. This study provides important quantitative data about drug-metabolizing kinases that can be used to understand variability in response and the off-target effects of nucleoside analog drugs.

Curcumin exhibits broad biological activity, including anticancer effects, but its therapeutic potential is limited because of poor stability, rapid metabolism, and nonspecific activity, prompting the development of structurally modified analogs with improved drug-like properties. However, how such modifications influence intracellular metabolism and subcellular behavior remains insufficiently understood. In this study, the intracellular fate of the reference molecule curcumin and 3 promising curcumin-based benzothiazepine analogs with enhanced selective anticancer activity was investigated in cancerous (HCT-116) and noncancerous (IPEC-J2) intestinal cell lines. An untargeted liquid chromatography with quadrupole time-of-flight mass spectrometry approach was applied to characterize degradation and metabolic products in cell lysates and extracellular medium, complemented by fluorescence-based imaging to explore their subcellular distribution. Interestingly, all compounds were detected intracellularly in both cell lines but displayed distinct structure- and cell-dependent metabolic patterns. Sulfation was predominant in HCT-116 cells, whereas methylation was more abundant in IPEC-J2 cells. In addition, phase II modifications-methylation and sulfation-occurred preferentially at phenolic moieties. Fluorescence imaging revealed mainly cytosolic, nuclear-excluded distribution for all compounds, with limited evidence for selective organelle accumulation. These findings demonstrate that curcumin-based benzothiazepine analogs undergo distinct cell-dependent metabolic processing, highlighting intracellular metabolism as a potential contributor to their previously reported selective anticancer activity. SIGNIFICANCE STATEMENT: By combining liquid chromatography with quadrupole time-of-flight mass spectrometry and subcellular fluorescence microscopy, this study provides insight into the intracellular fate of curcumin and 3 analogs, which differed both between compounds and between cancerous HCT-116 and noncancerous IPEC-J2 cells. These findings highlight the importance of structural modifications and cell-dependent metabolic processing when evaluating bioactive compounds because such mechanisms may substantially influence biological activity and selectivity.

Fentanyl is a potent synthetic opioid widely used as a recreational drug. It is primarily metabolized in the liver to the inactive compound norfentanyl, with only a small fraction excreted unchanged via the kidneys. However, it remains unclear whether repeated high-dose exposure alters its pharmacokinetics (PK) over time. This study evaluated the effects of repeated high-dose fentanyl administration on PK and assessed sex-related differences in metabolism. Male and female Sprague-Dawley rats (n = 19) received intravenous boluses of 100 μg/kg fentanyl twice weekly for 5 weeks. After the 1st, 5th, and 10th doses, plasma concentrations of fentanyl and norfentanyl were measured using ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. PK data were analyzed using noncompartmental methods, followed by covariate analysis within a population PK model and a physiologically based PK model to identify underlying physiological factors. Noncompartmental analysis showed significantly lower Cmax and longer t1/2 of norfentanyl in females than males. Population PK analysis using a two-compartment model confirmed slower fentanyl clearance and reduced norfentanyl formation in females. Repeated dosing led to decreased fentanyl clearance after the 10th exposure. In males, higher muscle volume was inversely correlated with total body clearance, whereas in females, fat volume correlated positively with total fentanyl exposure. Repeated high-dose fentanyl administration had only modest effects on PK, limited to reduced fentanyl clearance after the final exposure. Pronounced sex differences in fentanyl metabolism were observed with female animals clearing both fentanyl and norfentanyl more slowly and producing less norfentanyl than males. SIGNIFICANT STATEMENT: In a rat model of repeated fentanyl exposure mimicking illicit use, PK changes over time were minimal, suggesting limited accumulation with intermittent dosing. In contrast, marked sex differences were observed, with female rats showing slower fentanyl clearance and reduced metabolite formation, highlighting sex-specific determinants of opioid metabolism with potential implications for interpreting toxicology findings and overdose risk.