Effects of the Thiol Methyltransferase Inhibitor (±)-2,3-Dichloro-α-Methylbenzylamine (DCMB) on the Pharmacokinetics and Metabolism of Vicagrel in Rats.

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.

Absorption, Distribution, Metabolism, and Excretion of [14C]BS1801, a Selenium-Containing Drug Candidate, in Rats.

BS1801 is a selenium-containing drug candidate with potential for treating liver and lung fibrosis. To fully elucidate the biotransformation of BS1801 in animals and provide sufficient preclinical drug metabolism data for human mass balance study, the metabolism of BS1801 in rats was investigated. We used radiolabeling techniques to investigate the mass balance, tissue distribution, and metabolite identification of BS1801 in Sprague-Dawley/Long-Evans rats after a single oral dose of 100 mg/kg (100 µCi/kg) [14C]BS1801: 1. The mean recovery of radioactive substances in urine and feces was 93.39% within 168 h postdose, and feces were the main excretion route. 2. Additionally, less than 1.00% of the dose was recovered from either urine or bile. 3. BS1801-related components were widely distributed throughout the body. 4. Fifteen metabolites were identified in rat plasma, urine, feces, and bile, and BS1801 was detected only in feces. 5. BS1801-M484, the methylation product obtained via a N-Se bond reduction in BS1801, was the most abundant drug-related component in plasma. The main metabolic pathways of BS1801 were reduction, amide hydrolysis, oxidation, and methylation. Overall, BS1801 was distributed throughout the body, and excreted mainly as an intact BS1801 form through feces. No differences were observed between male and female rats in distribution, metabolism, and excretion of BS1801.

Conference report for the 10th China Bioanalysis Forum annual meeting.

The 10th China Bioanalysis Forum annual conference was held in Suzhou between 9 and 11 June 2023. This year a full range of bioanalytical topics were discussed such as new technology and bioanalytical approaches for biotherapeutics and biomarkers, particularly in the areas of gene and cell therapy. Another research area covered extensively at the conference was drug metabolism, including new drug metabolism and pharmacokinetic methods; absorption, distribution, metabolism and excretion of new modality drugs, recent regulatory guidance such as human mass balance study and preclinical study of antibody-drug conjugates and case studies of drug metabolism support to newly approved drugs.

Safety, tolerability and pharmacokinetics of forsythin in healthy subjects: a double-blinded, placebo-controlled single-dose and multiple-dose escalation and food effect study.

BACKGROUND: Forsythin, an active compound from Forsythiae Fructus, has the potential to treat the common cold and influenza through its antipyretic-analgesic, anti-inflammatory and antiviral effects. The safety, tolerability and pharmacokinetic (PK) profile of forsythin were evaluated in healthy Chinese subjects. METHODS: This phase 1a study included three parts: double-blind, randomized, placebo-controlled single-ascending-dose (SAD) (50, 100, 200, 400, 600 or 800 mg), food effect investigation (100 mg) and multiple-ascending-dose (MAD) (50, 100 or 200 mg TID for 5 days). RESULTS: Forsythin is safe and tolerable in healthy Chinese subjects. The rates of adverse events (AEs) in the forsythin cohort were similar to those in the placebo cohort. Forsythin is well-absorbed after single or multiple doses and is extensively metabolized. The primary metabolites were aglycone M1, M1 sulphate (M2) and M1 glucuronide (M7). Exposure to forsythin (100 mg) was higher after food intake by approximately 1.4-fold, whereas M2 and M7 did not change. The steady state was reached around three days in the MAD study. Forsythin, M2 and M7 accumulation on day 5 was 1, 3 and 2, respectively. CONCLUSIONS: The safety and PK profiles of forsythin support further evaluation of its efficacy in individuals with the common cold or influenza.

A validated LC-MS/MS method for the quantification of bevacizumab in rat, cynomolgus monkey, and human serum.

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.

Pharmacokinetics, Mass Balance, Tissue Distribution, and Metabolism of [3H]Catalpol in Rats: the Main Bioactive Component of Rehmannia glutinosa for the Treatment of Ischemic Stroke.

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-ß-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 ß-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.

A mass balance study of [14C]SHR6390 (dalpiciclib), a selective and potent CDK4/6 inhibitor in humans.

SHR6390 (dalpiciclib) is a selective and effective cyclin-dependent kinase (CDK) 4/6 inhibitor and an effective cancer therapeutic agent. On 31 December 2021, the new drug application was approved by National Medical Product Administration (NMPA). The metabolism, mass balance, and pharmacokinetics of SHR6390 in 6 healthy Chinese male subjects after a single oral dose of 150 mg [14C]SHR6390 (150 µCi) in this research. The Tmax of SHR6390 was 3.00 h. In plasma, the t 1/2 of SHR6390 and its relative components was approximately 17.50 h. The radioactivity B/P (blood-to-plasma) AUC0-t ratio was 1.81, indicating the preferential distribution of drug-related substances in blood cells. At 312 h after administration, the average cumulative excretion of radioactivity was 94.63% of the dose, including 22.69% in urine and 71.93% in stool. Thirteen metabolites were identified. In plasma, because of the low level of radioactivity, only SHR6390 was detected in pooled AUC0-24 h plasma. Stool SHR6390 was the main component in urine and stool. Five metabolites were identified in urine, and 12 metabolites were identified in stool. Overall, faecal clearance is the main method of excretion.

Characterization of in-vivo human metabolites of the oral nucleoside anti-COVID-19 drug VV116 using UHPLC-Orbitrap-MS.

VV116 is an oral nucleoside anti-COVID-19 drug undergoing clinical trials in China. We aimed to characterize its metabolites in plasma, urine, and feces of healthy Chinese male subjects after a single oral administration of 400 mg VV116, by using UHPLC-UV-Orbitrap-MS. After oral administration, VV116 was almost completely converted into the metabolite 116-N1. Seventeen other metabolites produced by the subsequent metabolism of 116-N1 were also detected, including 6 phase I metabolites and 11 phase II metabolites resulting from hydrolysis, oxidative deamination, oxidation, and CN-group removal and conjugations. The results were exploratory. The major metabolite of VV116 in human plasma and urine was 116-N1, the main metabolites in feces were M2 and 116-N1. We then synthesized a reference M2 standard and confirmed its structure by MS and NMR.

Mass balance study of [14C]SHR0302, a selective and potent JAK1 inhibitor in humans.

SHR0302, a selective JAK1 inhibitor developed by Jiangsu Hengrui Pharmaceutical Co., was intended for the treatment of rheumatoid arthritis. In this study, we evaluated the pharmacokinetics, mass balance, and metabolism of SHR0302 in six healthy Chinese male subjects after a single 8 mg (80 µCi) oral dose of [14C]SHR0302.SHR0302 was absorbed rapidly (Tmax = 0.505 h), and the average t1/2 of the SHR0302-related components in plasma was approximately 9.18 h. After an oral dose was administered, the average cumulative excretion of the radioactive components was 100.56% ± 1.51%, including 60.95% ± 11.62% in urine and 39.61% ± 10.52% in faeces.A total of 16 metabolites were identified. In plasma, the parent drug SHR0302 accounted for 90.42% of the total plasma radioactivity. In urine, SHR161279 was the main metabolite, accounting for 33.61% of the dose, whereas the parent drug SHR0302 only accounted for 5.1% of the dose. In faeces, the parent drug SHR0302 accounted for 23.73% of the dose, and SHR161279 was the significant metabolite, accounting for 5.67% of the dose. In conclusion, SHR0302-related radioactivity was mainly excreted through urine (60.95%) and secondarily through faeces (39.61%).The metabolic reaction of SHR0302 in the human body is mainly through mono-oxidation and glucuronidation. The main metabolic location of SHR0302 in the human body is the pyrrolopyrimidine ring.

Covalent Binding Mechanism of Furmonertinib and Osimertinib With Human Serum Albumin.

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.

Simultaneous quantification of 3',4'-dimethoxy flavonol-3-O-glucoside and its major metabolite in human plasma by LC-MS/MS and its application to a clinical pharmacokinetic study.

Hyperlipidemia is a disease characterized by abnormal blood lipid levels and is the leading risk factor for cardiovascular disease. 3',4'-Dimethoxy flavonol-3-O-glucoside (abbreviated DF3G) is a new lipid-lowering drug created as a flavonoid structural analog. The principal metabolite of DF3G in human plasma is the aglycone glucuronide conjugate M2. The purpose of this study is to use liquid chromatography-tandem mass spectrometry to develop and validate a quantitative analysis method for DF3G and its metabolite M2 in human plasma, and to use the method to investigate the pharmacokinetics of DF3G and M2 in a clinical trial. This method employed DF3G-d6 as the internal standard, and plasma samples were processed by protein precipitation. Isocratic separation could accurately differentiate DF3G, M2, and DF3G-d6 from endogenous components in the matrix or other components in the samples, and endogenous components in the matrix had little impact on ionization efficiency. Positive electrospray ionization with multiple reaction monitoring (MRM) transitions of m/z 461.2 → 299.0 for DF3G, m/z 475.1 → 299.1 for M2 and m/z 467.1 → 305.1 for DF3G-d6 was used for quantification. The DF3G and M2 linear range for plasma were in the range of 4.00/4.00 ng/mL to 4000/4000 ng/mL. Both the analytes and the internal standard were stable regardless of whether they were in solution or plasma samples. The accuracy of the average concentration of the quality control samples was within 15% of the theoretical value, and the RSD was less than 15%. The method is rapid, accurate, straightforward, and precise. It is appropriate for the determination of DF3G and M2 concentrations in human plasma and has been successfully applied to determine the pharmacokinetic analysis in phase I clinical trials.

Investigating the Metabolic Mechanisms of Butaselen, An Ebselen Analog.

BACKGROUND: Butaselen is an ebselen analog that is under clinical trials for treating hepatic and pulmonary fibrosis. Our previous studies showed that butaselen is mainly present in human plasma in the form of M2, a free Se-methylated metabolite. OBJECTIVE: This study aimed to investigate the metabolic mechanisms of butaselen. METHODS AND RESULTS: Butaselen was incubated with human plasma. Butaselen immediately disappeared, and the butaselen-HSA (human serum albumin) adduct was detected by HPLC-HRMS, showing that butaselen covalently binds to HSA. The butaselen-HSA adduct was precipitated using acetonitrile and then incubated with PBS, Cys, and GSH for 1 hour. The product was M1, a reduced form of butaselen. The results indicated that HSA, Cys, and GSH can reduce the butaselen-HSA covalent bond. The binding site for butaselen could be the cysteine-34 residue of HSA through pronase and trypsin hydrolysis. Incubating butaselen with cysteine, butaselen-Cys, butaselen-2Cys, and M1 were generated, indicating the covalent binding and reduction of butaselen by cysteine. We incubated liver microsomes and cytosol with butaselen, 6.22 and 246 nM M2 were generated, respectively. The results demonstrated that cytosolic enzymes are mainly involved in M2 production. The amount of M2 in the liver cytosol decreased from 246 nM to 2.21 nM when 10 mM m-anisic acid (a specific TPMT enzyme inhibitor) was added, showing that TPMT is responsible for M2 formation. CONCLUSION: Butaselen was covalently bound to HSA, and the binding site was the cysteine-34 residue of HSA. The butaselen-HSA adduct was reduced by free thiol compounds to generate M1. M1 was further metabolized to M2 by cytosolic TPMT. This study provides a basis for studying the pharmacokinetics of selenium-containing drugs.

Absorption, metabolism, and excretion of [14C]YY-20394, a highly selective PI3K-Delta inhibitor in humans.

YY-20394, a highly selective PI3Kδ inhibitor, is under NDA submission for treating follicular lymphoma in China. The absorption, metabolism, and excretion of YY-20394 were evaluated in healthy Chinese male subjects following a single oral dose of 80 mg [14C]YY-20394 (100 µCi).Within 264 h post-dose, 92.1% of the administered dose was recovered, with 58.1% from urine and 34.0% from faeces. YY-20394 was rapidly absorbed in humans, and the peak plasma concentrations occurred at 1.0 h. The absorbed drug fraction was at least 58.1% according to urine recovery.In addition to the parent drug, nine metabolites were identified in plasma, urine, and faeces. Unchanged YY-20394 was the predominant drug-related component in plasma (accounting for 68.4% of the total radioactivity), urine (accounting for 90.0% of the urinary radioactivity) and faeces (accounting for 41.7% of the faecal radioactivity). In humans, the major metabolic sites were the morphine ring and side chains of piperidine rings. The major metabolic pathways involved N-dealkylation, O-dealkylation, glucuronidation and acetylation.Overall, renal elimination played a significant role in the disposition of YY-20394, and the morphine ring and the side chain of the piperidine ring was the predominant metabolic sites.

Quantification of the major circulating metabolite of BS1801, an ebselen analog, in human plasma.

BS1801 contains two selenium atoms in its structure, which is a specific inhibitor of thioredoxin reductase intended to treat fibrotic interstitial pneumonia (control pulmonary fibrosis) and liver fibrosis. It is currently in phase I clinical trial. However, there was no report about the metabolic transformation and pharmacokinetics of BS1801. In this study, BS1801 metabolites were characterized in the hepatocytes of different species (monkey, dog, mouse, rat, and human) and plasma specimens using the ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS) method. After incubation, BS1801 could not be detected in the hepatocytes of different species and human plasma. Five metabolites were identified based on the characteristic peak clusters of selenium atoms in the mass spectrum, combined with the product ions obtained by MS-MS through collision-induced-dissociation (CID), including M1 (reduction metabolite), M2 (reduction and Se-methylation metabolite), M4 (M2 further oxidized metabolite) and M5 (Se-methylation and Se-glucuronidation conjugation metabolite), of which the amount of M2 was the highest. By comparing the LC-MS information with the synthesized reference substance, the structure of M2 was confirmed. The principal BS1801 metabolic pathways were identified as reduction and Se-methylation in humans. Subsequently, an accurate and fast LC-MS/MS method was established to verify the major metabolite M2 in human plasma. Acetonitrile-induced protein precipitation was employed to extract M2 from human plasma. The metabolite was separated through XDB-C18 (4.6 × 50 mm, 1.8 µm) under isocratic elution with ammonium acetate (5 mM) containing 0.1% formic acid solution (A) and acetonitrile (B) as the mobile phases. A deuterated internal standard for M2 was prepared to overcome the influence of matrix effects during the detection. The bioanalytical method was shown to be precise, specific, accurate, and good linearity over the range of 3.00-3000 ng/mL, and was implemented to assess the pharmacokinetic profiles of M2 in healthy volunteers following a single oral administration of 450 mg BS1801. This is the first-ever study to identify and quantify the major circulating metabolite of ebselen analogs in human plasma.

Metabolite profiles and mass balance of fuzuloparib, a novel poly (ADP-ribose) polymerase inhibitor, in subjects with advanced solid cancers.

AIM: This trial (NCT04013048) investigated the metabolite profiles, mass balance and pharmacokinetics of fuzuloparib, a novel poly (ADP-ribose) polymerase inhibitor, in subjects with advanced solid cancers. METHODS: A single dose of 150 mg [14 C]fuzuloparib was administered to five subjects with advanced solid cancers. Blood, urine and faecal samples were collected, analysed for radioactivity and unchanged fuzuloparib, and profiled for metabolites. The safety of the medicine was assessed during the study. RESULTS: The maximum concentrations (Cmax ) of the total radioactivity (TRA) and unchanged fuzuloparib in plasma were 5.39 µg eq./mL and 4.19 µg/mL, respectively, at approximately 4 hours post dose. The exposure (AUC0-t ) of fuzuloparib accounted for 70.7% of the TRA in plasma, and no single metabolite was observed accounting for more than 10% of the plasma TRA. The recovery of TRA in excreta was 103.3 ± 3.8% in 288 hours, including 59.1 ± 9.9% in urine and 44.2 ± 10.8% in faeces. Sixteen metabolites of fuzuloparib were identified, including mono-oxidation (M1), hydrogenation (M2), di-oxidation (M3), trioxidation (M4), glucuronidation (M5, M7, M8) and de-ethylation (M6) products, and there was no specific binding between these metabolites and blood cells. Aliphatic hydroxylated fuzuloparib (M1-1) was the primary metabolite in the excreta, accounting for more than 40% of the dose for subjects. There were no serious adverse events observed in the study. CONCLUSION: Fuzuloparib was widely metabolized and excreted completely through urine and faeces in subjects with advanced solid cancer. Unchanged fuzuloparib was indicated to be the primary drug-related compound in circulation. [14 C]fuzuloparib was well-tolerated at the study dose.

Absorption, distribution, metabolism, and excretion of [14C]TPN729 after oral administration to rats.

TPN729, a novel phosphodiesterase type 5 (PDE5) inhibitor for the treatment of erectile dysfunction (ED), is in phase II clinical trials in China. Previous studies suggested that TPN729 possesses promising therapeutic value. In previous non-radiolabeled rat excretion studies, the recovery of TPN729 and its major metabolites accounted for approximately 8.58% of the administration dose in urine and faeces by 48 h post-dose.To solve this problem and further study the metabolism of TPN729 in rats, we used the radio-isotopic tracing technique for the first time. In this study, the mass balance, tissue distribution, and metabolism of TPN729 were evaluated in rats after a single oral dose of 25 mg/kg [14C]TPN729 (150 µCi/kg).At 168 h post-dose, the mean total radioactivity recovery of the dose was 92.13%. Faeces was the major excretion route, accounting for 74.63% of the dose, and urine excretion accounted for 17.50%. After oral administration of [14C]TPN729, radioactivity was widely distributed in all examined tissues, and a higher radioactivity concentration was observed in the stomach, large intestine, lung, liver, small intestine, and eyes. The concentration of drug-related materials were similar in plasma and blood cells. A total of 51 metabolites were identified in rat plasma, urine, faeces, and bile, and the predominant metabolically susceptible position of TPN729 was the pyrrolidine moiety. The main metabolic pathways were N-dealkylation, oxidation, and dehydrogenation.In summary, we solved the previous problem of low drug recovery, elucidated the major excretion pathway, determined the tissue distribution patterns, and investigated the metabolism of TPN729 in rats by using a radioisotopic tracing technique.

Metabolic disposition of the EGFR covalent inhibitor furmonertinib in humans.

Furmonertinib was designed for the treatment of non-small cell lung cancer (NSCLC) patients with EGFR T790M mutation. In this study, we investigated the metabolic disposition and mass balance in humans and tissue distribution in rats. After a single oral administration of 97.9 µCi/81.5 mg [14C]-furmonertinib mesylate to six healthy male volunteers, the absorption process of furmonertinib was fast with a tmax of total plasma radioactivity at 0.75 h. Afterward, furmonertinib was extensively metabolized, with the parent drug and active metabolite AST5902 accounting for 1.68% and 0.97% of total radioactivity in plasma. The terminal t1/2 of total radioactivity in plasma was as long as 333 h, suggesting that the covalent binding of drug-related substances to plasma proteins was irreversible to a great extent. The most abundant metabolites identified in feces were desmethyl metabolite (AST5902), cysteine conjugate (M19), and parent drug (M0), which accounted for 6.28%, 5.52%, and 1.38% of the dose, respectively. After intragastric administration of 124 µCi/9.93 mg/kg [14C]-furmonertinib to rats, drug-related substances were widely and rapidly distributed in tissues within 4 h. The concentration of total radioactivity in the lung was 100-fold higher than that in rat plasma, which could be beneficial to the treatment of lung cancer. Mass balance in humans was achieved with 77.8% of the administered dose recovered in excretions within 35 days after administration, including 6.63% and 71.2% in urine and feces, respectively. In conclusion, [14C]-furmonertinib is completely absorbed and rapidly distributed into lung tissue, extensively metabolized in humans, presented mostly as covalent conjugates in plasma, and slowly eliminated mostly via fecal route.

Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes.

Furmonertinib (Alflutinib, AST2818), as a third-generation epidermal growth factor receptor inhibitor with an advanced efficacy and a relatively wide safety window, has been commercially launched in China recently. However, previous clinical studies demonstrated its time- and dose-dependent clearance in a multiple-dose regimen. In vitro drug metabolism and pharmacokinetic studies have suggested that furmonertinib is mainly metabolized by cytochrome P450 3A4 (CYP3A4) and can induce these enzymes via an increased mRNA expression. This study investigated two important evaluation criteria of CYP3A4 induction by furmonertinib through quantitative proteomics and probe metabolite formation: simultaneous (1) protein expression and (2) enzyme activity with sandwich-cultured primary human hepatocytes in the same well of cell culture plates. Results confirmed that furmonertinib was a potent CYP3A4 inducer comparable with rifampin and could be used as a positive model drug in in vitro studies to evaluate the induction potential of other drug candidates in preclinical studies. In addition, inconsistencies were observed between the protein expression and enzyme activities of CYP3A4 in cells induced by rifampin but not in groups treated with furmonertinib. As such, furmonertinib could be an ideal positive control in the evaluation of CYP3A4 induction. The cells treated with 10 µM rifampin expressed 20.16 ± 5.78 pmol/mg total protein, whereas the cells induced with 0.5 µM furmonertinib expressed 4.8 ± 0.66 pmol/mg protein compared with the vehicle (0.1% dimethyl sulfoxide), which contained 0.65 ± 0.45 pmol/mg protein. The fold change in the CYP3A4 enzyme activity in the cells treated with rifampin was 5.22 ± 1.13, which was similar to that of 0.5 µM furmonertinib (3.79 ± 0.52).

Mechanistic Study on the Species Differences in Excretion Pathway of HR011303 in Humans and Rats.

Excretion of [14C]HR011303-derived radioactivity showed significant species difference. Urine (81.50% of dose) was the main excretion route in healthy male subjects, whereas feces (87.16% of dose) was the main excretion route in rats. To further elucidate the underlying cause for excretion species differences of HR011303, studies were conducted to uncover its metabolism and excretion mechanism. M5, a glucuronide metabolite of HR011303, is the main metabolite in humans and rats. Results of a rat microsome incubation study suggested that HR011303 was metabolized to M5 in the rat liver. According to previous studies, M5 is produced in both human liver and kidney microsomes. We found that M5 in the human liver can be transported to the blood by multidrug resistance-associated protein (MRP) 3, and then the majority of M5 can be hydrolyzed to HR011303. HR011303 enters the human kidney or liver through passive diffusion, whereas M5 is taken up through organic anion transporter (OAT) 3, organic anion-transporting polypeptide (OATP) 1B1, and OATP1B3. When HR011303 alone is present, it can be metabolized to M5 in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured human hepatocytes (SCHH) and excreted into bile as M5 in SCRH. Using transporter inhibitors in sandwich-cultured model and membrane vesicles expressing MRP2 or Mrp2, we found that M5 was a substance of MRP2/Mrp2, and the bile efflux of M5 was mainly mediated by MRP2/Mrp2. Considering the significant role of MRP3/Mrp3 and MRP2/Mrp2 in the excretion of glucuronides, the competition between them for M5 was possibly the determinant for the different excretion routes in humans and rats. SIGNIFICANCE STATEMENT: Animal experiments are necessary to predict dosage and safety of candidate drugs prior to clinical trials. However, extrapolation results often differ from the actual situation. For HR011303, excretory pathways exhibited a complete reversal, through urine in humans and feces in rats. Such phenomena have been observed in several drugs, but no in-depth studies have been conducted to date. In the present study, the excretion species differences of HR011303 can be explained by the competition for M5 between MRP2/Mrp2 and MRP3/Mrp3.