[14C]Ivarmacitinib (SHR0302) exhibits significant species differences in metabolic and excretory processes between humans and rats. In humans, urine is the primary excretion route (60.95% of dose), with SHR161279 (mono-oxidation) as the main urinary metabolite (33.61%). In rats, fecal excretion predominates (84.1% of dose), with parent drug (54.5%) and SHR146622 (N-demethylation, 8.50%) as major fecal components. To elucidate the potential mechanisms underlying these species differences, studies were conducted to investigate the metabolism and excretion pathways of ivarmacitinib in humans and rats. Mechanistically, the distinct metabolic profiles are driven by species-specific cytochrome P450 activities. Human liver microsome preferentially produced SHR161279 via CYP2C8, whereas rat liver microsome preferentially produced SHR146622 via CYP3A. Regarding disposition and excretion, in humans, transporter studies showed that SHR161279 was excreted into bile via P-glycoprotein, whereas ivarmacitinib and SHR146622 underwent passive diffusion into blood. Subsequently, ivarmacitinib (accounting for 90.42% in area under the curve-pooled plasma) entered the kidneys probably via passive diffusion. Ivarmacitinib was metabolized in human kidney microsome to form SHR161279. In rats, ivarmacitinib and its metabolites were substrates for P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2, leading to predominant fecal excretion. Enzyme kinetics also indicated that ivarmacitinib was metabolized more slowly in human liver microsome. In contrast, in rats, ivarmacitinib was more metabolized to highly polar metabolites, which were more readily transported into bile via transporters localized on the canalicular membrane of hepatocytes, ultimately leading to species differences in excretion pathways between humans and rats. SIGNIFICANCE STATEMENT: This study elucidates the interspecies difference mechanism in the metabolic and excretory pathways of ivarmacitinib in humans and rats. Urine excretion is predominant in humans, unlike the predominant fecal excretion observed in rats. Meanwhile, SHR161279 is the primary metabolite in human urine, whereas SHR146622 is the primary metabolite in rat feces. These differences are attributed to the expression and activity of cytochrome P450 enzymes and transporters.

BACKGROUND:BPI-460372 is an orally available, covalent, irreversible small molecule inhibitor of the transcriptional enhanced associate domain (TEAD) 1/3/4, which is currently in clinical development for the treatment of cancers with Hippo pathway alterations. OBJECTIVE:This study aimed to determine the cytochrome P450 (CYP) phenotyping, metabolic stability, and in vitro and in vivo metabolic profile of BPI-460372. METHODS:The CYP phenotyping and metabolic stability were assessed by measuring the depletion of substrate. The metabolic profile in hepatocytes and rat and dog plasma was analyzed using ultra-high-performance liquid chromatography combined with Orbitrap tandem mass spectrometry (UHPLC-Orbitrap-HRMS). RESULTS:BPI-460372 was mainly metabolized by CYP2D6, CYP3A4, and CYP1A2. BPI-460372 exhibited low clearance in human, monkey, and rat hepatocytes, while moderate clearance in dog and mouse hepatocytes. A total of 10 metabolites were identified in five species of hepatocytes, and no human-unique metabolite was detected. In rat plasma and dog plasma, the primary metabolites were M407 (BPI-460430) and M423 (BPI-460456), respectively. The two metabolites were quantitatively determined in rat and dog plasma in pharmacokinetic and toxicological studies. The major metabolic site was 2-fluoro-acrylamide, and major metabolic pathways in hepatocytes, and rat and dog plasma involved oxidative defluorination, hydration, glutathione (GSH) conjugation, hydrolysis, cysteine conjugation, and N-acetyl cysteine conjugation. β-lyase pathway contributed to the metabolism of BPI-460372 in rats to a certain degree. CONCLUSION:This study elucidated the metabolism of BPI-460372 and provided a basis for pharmacokinetic and toxicological species selection, human pharmacokinetics prediction, and assessment of clinical co-administration limitations and possible metabolic pathways in humans.

Aims The primary objective of this study was to determine the pharmacokinetics, mass balance and biotransformation of [ 14 C]GT0918 in humans after the drug was administered to healthy Chinese male subjects. Methods The absorption, metabolism, and excretion (AME) of GT0918 was characterized via isotope labelling technology in six healthy Chinese male subjects after receiving a single 200 mg oral dose of [ 14 C]GT0918 (80 μCi), and the phenotype, together with the metabolic mechanism of GT0918, was confirmed in vitro. Results The medium T max of total radioactivity was 6.00 h (4.00–8.00 h) post‐dose, and the mean C max was 10.5 μg eq./mL (8.7–12.3 μg eq./mL) in plasma. Drug‐related components in the plasma were eliminated slowly, with a mean t 1/2 of 67.7 h (54.4–90.7 h), and the radioactivity of the plasma samples from some subjects was above the below the quantization limit (BQL) until 17 days post‐dose. After 19 days of dosing, the mean cumulative excreted radioactivity was 82.81% (79.07–86.07%) of the dose, including 29.47% (26.71–32.02%) in urine and 53.34% (52.01–55.62%) in faeces, indicating that the drug‐related components of GT0918 were mainly excreted by faeces. Metabolite profiling revealed that the parent drug was detected in plasma, as well as in faeces and not in urine. In plasma, the most abundant metabolite was GT0955, a mono‐oxidative metabolite of GT0918; in urine, the primary metabolite was GT0795, a metabolite of oxazole ring‐opening followed by N ‐dealkylation; in faeces, the two main metabolites were M551 and the glucuronidation of GT0955. The majority of the metabolites were formed via an important aldehyde intermediate derived from the oxazole ring‐opening, and the intermediate was trapped by methoxyamine hydrochloride in the in‐vitro study. CYP3A4 is the main enzyme involved in the metabolism of GT0918. Conclusions Overall, all the dosed subjects completed the study, and GT0918 was found to be safe, with no grade II or above adverse events reported. A total dose of 82.81% was quantified in the urine (29.47%) and faeces (53.34%) of healthy adult male subjects after a single oral administration of 200 mg (80 μCi) GT0918 ([ 14 C]GT0918). The metabolism of GT0918 is catalysed predominantly by CYP3A4, and an uncommon pathway of oxazole ring‐opening to an aldehyde intermediate has also been proposed.

SAF-189s is a promising highly selective, brain-penetrant, next-generation inhibitor of anaplasticlymphoma kinase (ALK)and oncogenic c-ros oncogene (ROS1). The pharmacokinetics, massbalance, and metabolism of SAF-189s were measured in six healthy Chinese male subjects afterreceiving a single oral dose of 160 mg [14C]SAF-189s (150 μCi). SAF-189s was rapidly absorbedwith a median Tmax of 4.0 h. The arithmetic mean half-life of total radioactivity in plasma wasapproximately 32.1 h. The ratio of mean total drug-related substance concentration in whole bloodto that in plasma (B/PAUC) was 3.06, indicating that the drug was predominantly distributed inblood cells. After 336 h of drug administration, the average cumulative excretion of radioactivityaccounted for 96.98% of the total dose, with 6.24% of the drug excreted in urine and 90.74% ofthe drug excreted in feces. In total, 14 metabolites were identified. SAF-189s was the predominantcomponent in plasma but was scarcely detected in urine and feces. Oxidative metabolism mediatedby CYP3A4 was determined to be the primary metabolic pathway for SAF-189s, with theisopropyl group being the most susceptible metabolizing site. M543 was identified as the mainoxidative metabolite of SAF-189s in humans, and its production was likely affected by bothCYP3A and intestinal microbiota. Following a single oral dose of [14C]SAF-189s, SAF-189s andits principal metabolites were primarily excreted via feces. The main metabolic pathway wasoxidation, likely catalyzed by both CYP3A and intestinal microbiota.

SAF-189s is a promising highly selective, brain-penetrant, next-generation inhibitor of anaplastic lymphoma kinase (ALK) and c-ROS proto-oncogene 1 (ROS1). The pharmacokinetics, mass balance, and metabolism of SAF-189s were measured in 6 healthy Chinese male participants after receiving a single oral dose of 160 mg [14C]SAF-189s (150 μCi). SAF-189s was rapidly absorbed with a median Tmax of 4.0 hours. The arithmetic mean half-life of total radioactivity in plasma was approximately 32.1 hours. The ratio of mean total drug-related substance concentration in whole blood to that in plasma (B/PAUC) was 3.06, indicating that the drug was predominantly distributed in blood cells. After 336 hours of drug administration, the average cumulative excretion of radioactivity accounted for 96.98% of the total dose, with 6.24% of the drug excreted in urine and 90.74% of the drug excreted in feces. In total, 14 metabolites were identified. SAF-189s was the predominant component in plasma but was scarcely detected in urine and feces. Oxidative metabolism mediated by CYP3A4 was determined to be the primary metabolic pathway for SAF-189s, with the isopropyl group being the most susceptible metabolizing site. M543 was identified as the main oxidative metabolite of SAF-189s in humans, and its production was likely affected by both CYP3A and intestinal microbiota. After a single oral dose of [14C]SAF-189s, SAF-189s and its principal metabolites were primarily excreted via feces. The main metabolic pathway was oxidation, likely catalyzed by both CYP3A and intestinal microbiota. SIGNIFICANCE STATEMENT: This study investigated the absorption and disposition of SAF-189s, a promising next-generation inhibitor of ALK/ROS1 administered for the treatment of ALK+/ROS1+ non-small cell lung cancer. The results demonstrated that SAF-189s and its metabolites were primarily excreted via feces, with metabolism likely mediated by both the cytochrome P450 system and gut microbiota. These findings provide essential pharmacokinetic and safety data, encourage further studies on drug interactions and dose adjustments, and support the involvement of gut microbiota, thereby guiding future research.

Furmonertinib demonstrated potent efficacy as a newly developed tyrosine kinase inhibitor for the treatment of patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer. In vitro research showed that furmonertinib is metabolized to its active metabolite AST5902 via the cytochrome P450 (CYP) enzyme CYP3A4. Furmonertinib is a strong CYP3A4 inducer, while the metabolite is a weaker CYP3A4 inducer. In clinical studies, nonlinear pharmacokinetics were observed during chronic dosing. The apparent clearance showed time- and dose-dependent increases. In this evaluation, a combination of in vitro data using radiolabeled compounds, clinical pharmacokinetic data, and drug-drug interaction (DDI) data of furmonertinib in oncology patients and/or in healthy subjects was used to develop a physiologically based pharmacokinetic (PBPK) model. The model was built in PK-Sim Version 11 using a total of 44 concentration-time profiles of furmonertinib and its metabolite AST5902. Suitability of the predictive model performance was demonstrated by both goodness-of-fit plots and statistical evaluation. The model predicted the observed monotherapy concentration profiles of furmonertinib well, with 32/32 predicted AUClast (area under the curve until the last concentration measurement) values and 32/32 maximum plasma concentration (Cmax) ratios being within twofold of the respective observed values. In addition, 8/8 predicted DDI AUClast and Cmax ratios with furmonertinib as a victim of CYP3A4 inhibition or induction were within twofold of their respective observed values. Potential applications of the final model include the prediction of DDIs for chronic administration of CYP3A4 perpetrators along with furmonertinib, considering auto-induction of furmonertinib and its metabolite AST5902.

Piperine has been reported to inhibit the enzyme activity of cytochrome P450 (CYP) 3A4. The aim of this study was to develop and validate a physiologically based pharmacokinetic (PBPK) model for piperine and to predict potential food-drug interactions (FDIs) between piperine and CYP3A4 substrate drugs using these models. The PBPK model for piperine was successfully developed and validated. Using this model, FDIs with ten CYP3A4 substrate drugs were simulated. The predicted area under the curve (AUC) ratios (with and without piperine, following a 7-day intake of 20 mg/day) for six drugs were found to exceed 1.25, with significant increases in AUC observed for ritonavir (31%), nifedipine (34%), cyclosporine (35%), triazolam (36%), alfentanil (39%), and simvastatin (59%) in humans. These findings suggest that caution should be exercised when consuming amounts of black pepper equivalent to a daily intake of 20 mg piperine during treatment with CYP3A4 substrate drugs, as it may significantly alter their pharmacokinetics.

P2Y12 receptor inhibitors are commonly used in clinical antiplatelet therapy, typically alongside other medications. Vicagrel, a promising P2Y12 receptor inhibitor, has submitted a new drug marketing application to the United States Food and Drug Administration. Its primary metabolites and some metabolic pathways are identical to those of clopidogrel. The aim of this study was to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine (DCMB) on the metabolism and pharmacokinetics of vicagrel. In vitro incubation with human and rat liver microsomes revealed that DCMB significantly inhibited the methylation of vicagrel's thiol metabolite M15-1. Rats were orally administered 6 mg/kg [14C]vicagrel (100 μCi/kg) 1 hour after peritoneal injection with or without DCMB (80 mg/kg). Compared with the control group, the plasma of DCMB-pretreated rats exhibited maximum plasma concentration (C max) decrease and time to reach C max (T max) delay for all vicagrel-related substances, the methylation product of the thiol metabolite (M9-2), and the derivatization product of the active thiol metabolite (MP-M15-2). However, no significant changes in area under the curve (AUC) or half-life (t 1/2) were observed. DCMB had negligible effect on the total radiological recovery of vicagrel within 72 hours, although the rate of vicagrel excretion slowed down within 48 hours. DCMB had a negligible impact on the metabolic pathway of vicagrel. Overall, the present study found that DCMB did not significantly affect the total exposure, metabolic pathways, metabolite profiles, or total excretion rates of vicagrel-related metabolites in rats, but led to C max decrease, T max delay, and slower excretion rate within 48 hours. SIGNIFICANCE STATEMENT: This study used liquid chromatography-tandem mass spectrometry combined with radiolabeling technology to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine on the absorption, metabolism, and excretion of vicagrel in rats. This work helps to better understand the in vivo metabolism of active thiol metabolites of P2Y12 inhibitors such as clopidogrel, vicagrel, etc.

Tislelizumab, an anti-programmed cell death protein 1 monoclonal antibody, in combination with chemotherapy, showed promising antitumor activity in patients with extensive-stage small-cell lung cancer (ES-SCLC) receiving first-line treatment in the phase 2 BGB-A317-206 study. Here, we present the final analysis of the randomized, double-blind, placebo-controlled, phase 3 RATIONALE-312 study (NCT04005716), which compared efficacy and safety of tislelizumab plus chemotherapy with placebo plus chemotherapy as first-line treatment in patients with ES-SCLC.

Detailed protocols for ctDNA sequencing

Bevacizumab is a humanized monoclonal antibody used in the treatment of advanced colorectal and non-small cell lung cancer. Our main aim was to establish a simple, economical, and high efficiency liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying the content of bevacizumab in various biological fluids (rat, cynomolgus monkey, and human serum). A surrogate peptide of bevacizumab, specifically FTFSLDTSK, was generated through trypsin hydrolysis, and quantified using an isotopically labeled peptide containing two amino acids, FTFSLDTSK[13C6, 15N2]ST, as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry variabilities. The pre-treatment process included denaturation, disulfide bond reduction and alkylation, trypsin hydrolysis, and termination of the reaction, with a total duration of approximately 2.5-3 h. The results of the methodological validation showed that the linear range in three different biological matrices was 0.2 µg/mL to300 µg/mL, with an LLOQ of 0.2 µg/mL. The precision and accuracy of the measurements met the required standards. The validated LC-MS/MS method was used to conduct pharmacokinetic analysis in rats administered bevacizumab at a dose of 10 mg/kg intravenously.

As third-generation tyrosine kinase inhibitors, furmonertinib and osimertinib exhibit better efficacy than first- and second-generation tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer. However, radioactive pharmacokinetics studies showed that parent-related components remain in human plasma for at least 21 days after oral administration. Similar pharmacokinetic profiles were found in pyrotinib and neratinib, which have been identified to covalently bind with human serum albumin at Lys-190, leading to low extraction recovery in protein precipitation. However, the binding mechanism of furmonertinib and osimertinib in human plasma has not been confirmed. Comprehensive techniques were used to investigate the mechanism of this binding, including ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry and online/offline radioactivity profiling. SDS-PAGE and further autoradiography were also used to detect drug-protein adducts. We found that most furmonertinib exists in the human plasma following ex vivo incubation in the form of protein-drug adducts. Only lysine-furmonertinb adducts were found in pronase digests. A standard reference of lysine-furmonertinib was synthesized and confirmed by NMR. Through peptide mapping analysis, we confirmed that furmonertinib almost exclusively binds with human serum albumin (HSA) in plasma following ex vivo incubation, via Michael addition at Lys-195 and Lys-199, instead of Lys-190. Two peptides found to bond with furmonertinib were ASSAKQR and LKCASLQK. Osimertinib was also found to bond with Lys-195 and Lys-199 of HSA via peptide mapping analysis. SIGNIFICANCE STATEMENT: Here we report that furmonertinib and osimertinib can covalently bind with human serum albumin at the site of Lys-195 and Lys-199 instead of Lys-190, potentially leading to the long duration of drug-protein adducts in the human body.

Background: Catalpol, one of the main bioactive components isolated from Rehmannia glutinosa, was developed by Suzhou Youseen for the treatment of ischemic stroke; however, preclinical information about its absorption, distribution, metabolism, and excretion (ADME) in animals is inadequate. Objective: This study aimed to illuminate the pharmacokinetics (PK), mass balance (MB), tissue distribution (TD), and metabolism of catalpol after a single intragastric administration of 30 mg/kg (300 μCi/kg) [3H]catalpol in rats. Methods: Radioactivity in plasma, urine, feces, bile, and tissues was measured by liquid scintillation counting (LSC), and metabolite profiling was characterized by UHPLC-β-ram and UHPLC-Q-Exactive plus MS. Results: The radio pharmacokinetic results showed that catalpol was rapidly absorbed by Sprague‒Dawley (SD) rats, with a median Tmax of 0.75 h and an arithmetic mean half-life (t1/2) of the total radioactivity of approximately 1.52 h in plasma. The mean recovery of the total radioactive dose was 94.82%±1.96% over 168 h postdose (57.52%±12.50% in the urine and 37.30%±12.88% in the feces). The parent drug catalpol was the predominant drugrelated substance in rat plasma and urine, while M1 and M2, two unidentified metabolites, were detected in feces. When [3H]catalpol was incubated with β-glucosidase and rat intestinal flora, we found that the same metabolites M1 and M2 were produced in both incubation systems. Conclusions: Catalpol was excreted mainly through the urine. The drug-related substances were primarily concentrated in the stomach, large intestine, bladder, and kidney. Only the parent drug was detected in the plasma and urine, and M1 and M2 were detected in the feces. We speculate that the metabolism of catalpol in rats was mainly mediated by the intestinal flora, resulting in an aglycone-containing hemiacetal hydroxyl structure.