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.
Aldehyde oxidase is a cytosolic drug-metabolizing enzyme. Idelalisib is a phosphoinositide-3-kinase-delta inhibitor that undergoes extensive biotransformation, mainly via 8-oxidation, which is catalyzed predominantly by human aldehyde oxidase (AOX1) and, to a lesser extent, cytochromes P450 (P450). In the present study, the primary objective was to test the hypothesis that idelalisib 8-oxidation is an enzyme-selective catalytic marker of AOX1 in HepG2 human hepatocellular carcinoma cells that express AOX1 but has little or no CYP3A functionality, and this approach is effective in identifying AOX1 inactivators. Enzyme kinetic analysis indicated comparable values of kcat, Km, and ratio of kcat/Km (catalytic efficiency) for idelalisib 8-oxidation catalyzed by HepG2 cells and human liver cytosol, whereas Vmax was greater for liver cytosol-catalyzed idelalisib 8-oxidation. Human recombinant AOX1, CYP3A4, and CYP3A5 were major catalysts of idelalisib 8-oxidation. However, experiments with a panel of enzyme-selective chemical inhibitors indicated that AOX1, but not P450 or xanthine oxidase, was responsible for idelalisib 8-oxidation in HepG2 cells and human liver cytosol. The HepG2 cell-catalyzed idelalisib 8-oxidation assay correctly identified time-dependent AOX1 inactivators (erlotinib, O-desmethylerlotinib, O-didesmethylerlotinib, hydralazine) and non-inactivators (gefitinib, O-desmethylgefitinib, O-desmorpholinopropylgefitinib, raloxifene). The data from the experiment to address our secondary objective indicated that erlotinib dosing increased the accumulation of idelalisib and 8-oxo-idelalisib in mouse plasma, liver, and kidney. In conclusion, 1) idelalisib 8-oxidation is an AOX1-selective catalytic marker in HepG2 cells that can be utilized to study AOX1 catalytic function; and 2) the in vivo mouse pharmacokinetic approach is not effective in identifying erlotinib as an inhibitor of idelalisib 8-oxidation.
Podophyllotoxin is an aryltetralin lignan with potent antimitotic and antiviral activities; however, its severe toxicity restricts clinical use to topical application, and systemic exposure 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 non-targeted screening strategy to investigate podophyllotoxin metabolism in postmortem blood from a rare fatal case of podophyllotoxin poisoning. Two complementary non-targeted 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 HRMS/MS 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.
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 (EGFR) mutations, anaplastic lymphoma kinase (ALK) gene rearrangements, ROS proto-oncogene (ROS1) 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 non-small cell lung cancer (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 (DDIs) 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 DDIs, 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 NSCLC patients.
Kava is a beverage prepared from the rhizomes and roots of the tropical evergreen plant Piper methysticum. Kava contains six major kavalactones. Legacy studies found that kavalactones did not inhibit human CYPs in vitro at relevant concentrations, and kava did not inhibit the major CYPs in humans in vivo and was devoid of clinically significant drug interactions. Nevertheless, CYP2B6 was never evaluated. Recent investigation identified that (+)-dihydromethysticin, one of the six natural kavalactones, was an inhibitor of CYP2B6 in vitro. This investigation evaluated kava extract and (+)-dihydromethysticin effects on expressed CYP2B6 activity- both wild-type CYP2B6.1 and the 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.
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.
The absorption, distribution, metabolism, excretion (ADME) study and the absolute bioavailability study were conducted to determine the pharmacokinetics (PK), 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 [14C]-naporafenib was administered intravenously (IV) with a single 200 mg oral dose of naporafenib to 8 healthy male participants to assess the absolute bioavailability and PK of naporafenib. Following a 15-day washout, a single 200 mg/500 μCi oral dose of [14C]-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.
Erythropoietic protoporphyria (EPP) is an inherited disorder caused by loss-of-function mutations in the ferrochelatase (FECH) gene. FECH deficiency leads to the accumulation of its substrate protoporphyrin IX (PPIX), resulting in cholestatic liver injury in patients with EPP. Because the liver is the primary organ responsible for drug metabolism and disposition, liver injury can alter these processes and compromise drug safety. However, safety-oriented precision medicine strategies for EPP remain poorly defined. Moreover, marked interindividual variability in EPP-associated liver injury has been observed, but the underlying risk factors remain unclear. To address these gaps, we performed RNA sequencing of liver tissue from an EPP mouse model carrying a Fech mutation. Compared with wild-type mice, more than 1700 genes were upregulated and more than 900 genes were downregulated in the liver of EPP mice. Pathway enrichment analysis revealed that upregulated pathways include immune activation, inflammatory responses, and extracellular matrix remodeling, whereas downregulated pathways include cholesterol biosynthesis, fatty acid β-oxidation, bile acid biosynthesis, and xenobiotic metabolism. Focusing on drug metabolism and disposition, we found broad downregulation of genes encoding phase I and phase II drug-metabolizing enzymes and drug transporters. Mechanistically, suppression of xenobiotic metabolism-related genes was associated with PPIX-induced liver injury and activation of inflammatory responses. Together, these findings demonstrate that PPIX-induced liver injury broadly disrupts hepatic signaling pathways, particularly those governing liver pathophysiology and xenobiotic metabolism. SIGNIFICANCE STATEMENT: Erythropoietic protoporphyria (EPP) can cause cholestatic liver injury with poorly defined implications for drug safety. Using an EPP mouse model, the study demonstrated that EPP-associated liver damage broadly suppresses hepatic drug-metabolizing enzymes and transporters while activating inflammatory signaling pathways. These findings can potentially be used to guide absorption, distribution, metabolism, and excretion-based precision medicine in patients with EPP to improve therapeutic safety.
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 (MASH) in human liver tissue. Sandwich-cultured human hepatocytes (SCHH) were treated with either 25 μM chloroquine or 10 μM MG132 to inhibit lysosomal or proteasomal degradation, respectively. Following treatment, SCHH were fixed and stained for OATP2B1 and lysosomal-associated membrane protein 1 (LAMP1) followed by confocal imaging and subsequent object-based quantitative analysis. Lysosomal inhibition by chloroquine in SCHH resulted in significant accumulation of intracellular OATP2B1, which displayed major colocalization with the lysosomal marker, LAMP1 (20 ±9.38% vs. 9.5 ±5.25%; chloroquine vs. control, respectively; p<0.0001). In contrast, the OATP2B1 total object volume and intracellular localization remained unchanged following MG132 treatment. OATP2B1 colocalization with LAMP1 was significantly higher in liver tissue from patients with MASH (10.47 ±4.95 %) compared to control liver tissue (6.00 ±2.06 %; p<0.0001). Lastly, 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. The lysosomal dysfunction associated with some xenobiotics or disease may increase OATP2B1 localization on lysosomal membranes.
Polycomb repressive complexes (PRCs) 1 and 2 are epigenetic regulators that suppress gene transcription through histone H3 lysine K27 trimethylation. This modification, catalyzed by enhancer of zeste homolog 2 (EZH2) within PRC2, is recognized by chromobox (CBX) proteins within PRC1, leading to chromatin condensation and transcriptional repression. Although EZH2 and CBX inhibitors have been developed for cancer therapy, the role of PRC1/2 inhibition in the regulation of drug metabolism-related genes remains unclear. In this study, we investigated the regulatory mechanisms of CYP3A4 expression by PRC1/2. Treatment with GSK126, an EZH2 inhibitor, significantly increased CYP3A4 mRNA and protein levels in HepaSH cells and ShP51 cells. Among CBX family members, CBX4 knockdown increased CYP3A4 mRNA levels, whereas its overexpression suppressed them in ShP51 cells. Consistently, CBX4 knockdown enhanced rifampicin-induced CYP3A4 expression, whereas its overexpression attenuated this induction. A coimmunoprecipitation assay revealed that CBX4 interacts with pregnane X receptor (PXR) regardless of rifampicin treatment. Chromatin immunoprecipitation analysis demonstrated that both CBX4 and PXR bind to the promoter and enhancer regions of CYP3A4, and that CBX4 knockdown increases PXR binding. Formaldehyde-assisted isolation of regulatory elements assays showed that CBX4 knockdown relaxed chromatin structure at the CYP3A4 locus, which is further enhanced by rifampicin. Collectively, these findings indicate that CBX4 interacts with PXR and likely represses CYP3A4 transcription through recognition of EZH2-mediated trimethylation of lysine 27 on histone H3 and chromatin condensation, highlighting a potential risk of drug-drug interactions associated with CBX4 inhibition. SIGNIFICANCE STATEMENT: This study provides, to our knowledge, the first evidence of epigenetic mechanisms underlying CYP3A4 suppression mediated by PRC1/2. The findings demonstrate that CBX4 interacts with PXR and binds to the CYP3A4 regulatory regions, where it recognizes enhancer of zeste homolog 2-mediated trimethylation of lysine 27 on histone H3 and represses CYP3A4 transcription via chromatin condensation. Given the increasing clinical interest in CBX inhibitors for cancer therapy, these findings highlight a potential risk of drug-drug interactions associated with CBX4 inhibition.
Organic anion transporting polypeptide (OATP) 1B1 is an influx transporter mediating the hepatic uptake of many compounds through the cell membrane. OATP1B1 activity fluctuates due to various factors; hence, evaluating OATP1B1 activity is important for pharmacokinetic studies and precision dosing of substrate drugs. This study aimed to discover novel biomarker candidates using nontargeted metabolomics. Blood samples of 432 individuals selected from the Japanese general population and 264 patients were analyzed. The samples were pretreated by more vigorous extraction methods than those conventionally used in metabolomics, and metabolites were measured comprehensively using ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. These participants were classified by a major OATP1B1 polymorphism, the c.521T>C variant, into the OATP1B1 normal function group (c.521TT) and the decreased function group (c.521TC and c.521CC). Specific biomarker candidates were extracted from metabolome profiles by multivariate analyses in the general population and patients separately. Four novel candidate compounds were identified to be probably 13'-hydroxy-gamma-tocopherol, 6-deoxodolichosterone, (20R or 20S)-24-hydroxygeminivitamin D3, and glycochenodeoxycholate-3-O-glucuronide (GCDCA-3G). The intensities of the 4 compounds were compared between the OATP1B1 normal function group and the decreased function group and among 3 groups classified by OATP1B1 c.521T>C polymorphism. GCDCA-3G, 13'-hydroxy-gamma-tocopherol, and (20R or 20S)-24-hydroxygeminivitamin D3 clearly discriminated against differences among 3 c.521T>C genotype groups, whereas 6-deoxodolichosterone discriminated between the OATP1B1 normal function group and the decreased function group. These results confirm the robustness and specificity of GCDCA-3G as an OATP1B1 biomarker and propose 13'-hydroxy-gamma-tocopherol, 6-deoxodolichosterone, and (20R or 20S)-24-hydroxygeminivitamin D3 as potentially useful novel OATP1B1 functional biomarkers. SIGNIFICANCE STATEMENT: Endogenous biomarkers for drug transporters are valuable tools for predicting adverse events caused by substrate drugs. This study aimed to explore novel biomarker candidates for organic anion transporting polypeptide (OATP) 1B1 activity using the nontargeted metabolomics approach with more vigorous pretreatment using solid-phase extraction and liquid-liquid extraction. Three novel and one conventional OATP1B1 biomarker candidates were detected by our nontargeted metabolomics. These findings contribute to the advancement of biomarker research for OATP1B1 and support further exploration of novel biomarkers.
Hepatic sexual dimorphism is critical for maintaining sex-specific metabolic pathways and xenobiotic clearance. We have demonstrated that in fibroblast growth factor 15 (Fgf15) transgenic (Tg) mice, FGF15 overexpression significantly reduced bile acid (BA) levels and induced hepatic feminization in males, marked by a male-to-female shift in expression of the drug metabolizing enzymes. This feminization is linked to the disruption of pulsatile growth hormone (GH) secretion pattern and signaling. However, it remained unclear whether FGF15 overexpression broadly disrupts hepatic sexual dimorphism or specifically drives a directional feminization program independent of biological sex. In this study, we profiled the hepatic transcriptome of female Fgf15 Tg mice and replenished male Fgf15 Tg mice with exogenous, pulsatile GH to test to what degree changes in the GH axis maintain liver dimorphism under low BA conditions. Beyond suppressing BA synthesis and altering steroid metabolism, female Tg mice altered expression of genes in xenobiotic metabolism and detoxification pathways, suggesting that the FGF15/BA axis regulates xenobiotic processing in females as well. Notably, FGF15 overexpression further amplified the baseline female expression profile of drug-metabolizing enzymes, characterized by the upregulation of female-predominant genes (Cyp2b9, Cyp3a41, Sult1e1, and Ugt1a1) and the suppression of male-predominant genes (Cyp7b1, Cyp2d9, Hsd3b5, and Igf1). Furthermore, pulsatile GH replenishment in male Tg mice partially restored male-predominant gene expression patterns. Taken together, these findings demonstrate that overexpression of intestine-derived FGF15 drives the feminization of sexually dimorphic hepatic gene expression independent of biological sex, a process that is at least partially mediated by impaired GH signaling. SIGNIFICANCE STATEMENT: Fibroblast growth factor 15 (Fgf15) overexpression intensified hepatic feminization of drug-metabolizing enzyme expression in female Fgf15 transgenic mice, similar to male Fgf15 transgenic mice. Restoring growth hormone signaling partially rescued the male-predominant gene expression pattern in male Fgf15 transgenic mice. This indicates that disruptions to growth hormone pulsatility, alongside sustained hepatic signal transducer and activator of transcription 5 activation, contribute to FGF15-induced hepatic gene feminization.
Cytochrome P450 (P450) enzymes play an important role in the biotransformation of both exogenous and endogenous compounds. Variability in P450 expression and activity impacts drug-metabolizing capacity and contributes to interindividual differences in drug exposure and therapeutic outcomes. Exogenous probe substrates have traditionally been used to assess the in vivo P450 enzyme activity. However, their application can be limited in vulnerable populations such as pregnancy, transplantation, pediatrics, and elderly. Endogenous biomarkers have emerged as minimally invasive potential alternatives that can reflect the P450 enzyme activity under different physiologic and clinical conditions. In this minireview, we critically examine various endogenous biomarkers that have been proposed to assess P450 activity, with specific emphasis on CYP3A4 and also discuss emerging endogenous biomarkers of CYP2D6 activity. We summarize mechanistic and translational applicability and regulatory guidelines in predicting drug-drug interactions and use of mathematical modeling and simulation approaches to validate the biomarkers. Additionally, we discuss the potential role of metabolomics in endogenous biomarker discovery and outline current limitations and future directions underscoring the utility of endogenous biomarkers in drug metabolism and drug disposition in patients. SIGNIFICANCE STATEMENT: Endogenous biomarkers offer a minimally invasive mechanistic approach for assessing in vivo P450 activity and complement traditional drug probe-based phenotyping strategies. This minireview evaluates the currently used endogenous biomarkers of CYP3A4 and emerging candidates for CYP2D6, highlighting their translational advances, limitations, and potential integration with metabolomics and model-informed drug development for drug disposition and interaction assessment.
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 (ADME), often necessitating iterative in vitro ADME assay optimization or reliance on in vivo pharmacokinetic (PK) studies, particularly to understand oral absorption. In this study, in vitro–in vivo correlations (IVIVC) from clinically advanced PROTACs were leveraged to develop a novel classification system with results from optimized MDCKI-MDR1 and Caco-2 permeability and efflux assays that prioritizes compounds based on estimated mouse oral absorption. Receiver operating characteristic (ROC) 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 MDCKI-MDR1 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 Caco-2 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 PK studies when evaluating novel PROTACs in preclinical discovery studies.
Curcumin exhibits broad biological activity, including anticancer effects, but its therapeutic potential is limited due to poor stability, rapid metabolism, and non-specific activity, prompting the development of structurally modified analogues with improved drug-like properties. However, how such modifications influence intracellular metabolism and subcellular behaviour remains insufficiently understood. In this study, the intracellular fate of the reference molecule curcumin and three promising curcumin-based benzothiazepane analogues with enhanced selective anticancer activity was investigated in a cancerous (HCT-116) and non-cancerous (IPEC-J2) intestinal cell line. An untargeted LC-QTOF-MS approach was applied to characterise 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. Sulphation was predominant in HCT-116 cells, whereas methylation was more abundant in IPEC-J2 cells. In addition, phase II modifications – methylation and sulphation – 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 benzothiazepane analogues undergo distinct cell-dependent metabolic processing, highlighting intracellular metabolism as a potential contributor to their previously reported selective anticancer activity.
