Application of Electro-Activated Dissociation Fragmentation Technique to Identifying Glucuronidation and Oxidative Metabolism Sites of Vepdegestrant by Liquid Chromatography-High Resolution Mass Spectrometry.

Drug metabolite identification is an integrated part of drug metabolism and pharmacokinetics studies in drug discovery and development. Definitive identification of metabolic modification sides of test compounds such as screening metabolic soft spots and supporting metabolite synthesis are often required. Currently, liquid chromatography-high resolution mass spectrometry is the dominant analytical platform for metabolite identification. However, the interpretation of product ion spectra generated by commonly used collision-induced disassociation (CID) and higher-energy collisional dissociation (HCD) often fails to identify locations of metabolic modifications, especially glucuronidation. Recently, a ZenoTOF 7600 mass spectrometer equipped with electron-activated dissociation (EAD-HRMS) was introduced. The primary objective of this study was to apply EAD-HRMS to identify metabolism sites of vepdegestrant (ARV-471), a model compound that consists of multiple functional groups. ARV-471 was incubated in dog liver microsomes and 12 phase I metabolites and glucuronides were detected. EAD generated unique product ions via orthogonal fragmentation, which allowed for accurately determining the metabolism sites of ARV-471, including phenol glucuronidation, piperazine N-dealkylation, glutarimide hydrolysis, piperidine oxidation, and piperidine lactam formation. In contrast, CID and HCD spectral interpretation failed to identify modification sites of three O-glucuronides and three phase I metabolites. The results demonstrated that EAD has significant advantages over CID and HCD in definitive structural elucidation of glucuronides and phase I metabolites although the utility of EAD-HRMS in identifying various types of drug metabolites remains to be further evaluated. SIGNIFICANCE STATEMENT: Definitive identification of metabolic modification sites by liquid chromatography-high resolution mass spectrometry is highly needed in drug metabolism research, such as screening metabolic soft spots and supporting metabolite synthesis. However, commonly used collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation techniques often fail to provide critical information for definitive structural elucidation. In this study, the electron-activated dissociation (EAD) was applied to identifying glucuronidation and oxidative metabolism sites of vepdegestrant, which generated significantly better results than CID and HCD.

Development and validation of a sensitive liquid chromatography-tandem mass spectrometry method for the assay of 12 substances in rat plasma and its application to rat pharmacokinetics of Epimedium and Psoraleae Fructus herb pair after oral administration.

Epimedium (EM) and Psoraleae Fructus (PF) are a traditional herb combination often used as a fixed form to treat osteoporosis disease in the clinic. However, the intricate interactions of this pair remain unknown. In our study, we undertook a comprehensive examination of their compatibility behaviors. Concurrently, a precise and sensitive quantitation method was successfully developed and validated using liquid chromatography-tandem mass spectrometry for the determination of 12 components. This method was applied in analyzing herbal extracts and biological samples (both in the portal vein and systemic plasma), which was also used to study the pharmacokinetics of the herb pair. The results indicated that the combination of EM and PF enhanced the dissolution of chemical components from PF in extracts, but it had a negligible influence on the contents of the components from EM. On the contrary, the in vivo exposure of the lowly exposed EM flavonoids significantly increased following the combination of EM and PF, whereas the highly exposed psoralen and isopsoralen were greatly reduced. These interactions might be crucial for the synergy and toxicity reduction of the herbal pair in disease treatment, which pave the way for further exploration into the clinical application and pharmacological mechanisms of EM and PF.

Metabolite characterisation of the peptide-drug conjugate LN005 in liver S9s by UHPLC-Orbitrap-HRMS.

LN005 is a peptide-drug conjugate (PDC) targeting glucose-regulated protein 78 (GRP78) to treat several types of cancer, such as breast, colon, and prostate cancer.As a new drug modality, understanding its metabolism and elimination pathways will help us to have a whole picture of it. Currently, there are no metabolic studies on LN005; therefore, this study aimed to investigate the metabolism of LN005, clarify its metabolic profile in the liver S9s of different species, and identify the major metabolic pathways and differences between species.The incubation samples were measured by ultra-high performance liquid chromatography combined with orbitrap tandem mass spectrometry (UHPLC-Orbitrap-HRMS).The results showed that LN005 was metabolised by liver S9s, and four metabolites were identified. The main metabolic pathway of LN005 in liver S9s was oxidative deamination to ketone or hydrolysis. Similar metabolic profiles were observed in mouse, rat, dog, monkey, and human liver S9s, indicating no differences between these four animal species and humans.This study provides information for the structural modification and optimisation of LN005 and affords a reference for subsequent animal experiments and human metabolism of other PDCs.

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.

Pharmacokinetics, mass balance, and metabolism of [14C]TPN171, a novel PDE5 inhibitor, in humans for the treatment of pulmonary arterial hypertension.

TPN171 is a novel phosphodiesterase-5 (PDE5) inhibitor used to treat pulmonary arterial hypertension (PAH) and erectile dysfunction (ED), which currently is undergoing phase II clinical trials in China. In this single-center, single-dose, nonrandomized, and open design study, radiolabeled [14C]TPN171 was used to investigate the metabolic mechanism, pharmacokinetic characteristics, and clearance pathways of TPN171 in 6 healthy Chinese male volunteers. Each volunteer was administered a single oral suspension of 10 mg (100 µCi) of [14C]TPN171. We found that TPN171 was absorbed rapidly in humans with a peak time (Tmax) of 0.667 h and a half-life (t1/2) of approximately 9.89 h in plasma. Excretion of radiopharmaceutical-related components was collected 216 h after administration, accounting for 95.21% of the dose (46.61% in urine and 48.60% in feces). TPN171 underwent extensive metabolism in humans. Twenty-two metabolites were detected in human plasma, urine, and feces using a radioactive detector combined with a high-resolution mass spectrometer. According to radiochromatograms, a glucuronide metabolite of O-dealkylated TPN171 exceeded 10% of the total drug-related components in human plasma. However, according to the Food and Drug Administration (FDA) guidelines, no further tests are needed to evaluate the safety of this metabolite because it is a phase II metabolite, but the compound is still worthy of attention. The main metabolic biotransformation of TPN171 was mono-oxidation (hydroxylation and N-oxidation), dehydrogenation, N-dealkylation, O-dealkylation, amide hydrolysis, glucuronidation, and acetylation. Cytochrome P450 3A4 (CYP3A4) mainly catalyzed the formation of metabolites, and CYP2E1 and CYP2D6 were involved in the oxidative metabolism of TPN171 to a lesser extent. According to the incubation data, M1 was mainly metabolized to M1G by UDP-glucuronosyltransferase 1A9 (UGT1A9), followed by UGT1A7 and UGT1A10.

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.

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.

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.

Pharmacokinetics, Mass Balance, and Metabolism of the Novel Urate Transporter 1 Inhibitor [14C]HR011303 in Humans: Metabolism Is Mediated Predominantly by UDP-Glucuronosyltransferase.

HR011303, a promising selective urate transporter 1 inhibitor, is currently being studied in a phase III clinical trial in China for the treatment of hyperuricemia and gout. In the current study, the pharmacokinetics, mass balance, and metabolism of HR011303 were examined in six healthy Chinese male subjects who received a single oral dose of 10 mg of [14C]HR011303 (80 µCi). The results showed that HR011303 was rapidly absorbed with a median time to reach C max of 1.50 hours postdose, and the arithmetic mean half-life of total radioactivity was approximately 24.2 hours in plasma. The mean blood-to-plasma radioactivity concentration ratio was 0.66, suggesting the preferential distribution of drug-related components in plasma. At 216 hours postdose, the mean cumulative excreted radioactivity was 91.75% of the dose, including 81.50% in urine and 10.26% in feces. Six metabolites were identified, and the parent drug HR011303 was the most abundant component in plasma and feces, but a minor component in urine. Glucuronidation of the carboxylic acid moiety of HR011303 was the primary metabolic pathway in humans, amounting to 69.63% of the dose (M5, 51.57% of the dose; M5/2, 18.06% of the dose) in the urine; however, it was not detected in plasma. UDP-glucuronosyltransferase (UGT) 2B7 was responsible for the formation of M5. Overall, after a single oral dose of 10 mg of [14C]HR011303 (80 µCi), HR011303 and its main metabolites were eliminated via renal excretion. The major metabolic pathway was carboxylic acid glucuronidation, which was catalyzed predominantly by UGT2B7. SIGNIFICANCE STATEMENT: This study determined the absorption and disposition of HR011303, a selective urate transporter (URAT) 1 inhibitor currently in development for the treatment of hyperuricemia and gout. This work helps to characterize the major metabolic pathways of new URAT inhibitors and identify the absorption and clearance mechanism.

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).

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.

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.

A Traceless Site-Specific Conjugation on Native Antibodies Enables Efficient One-Step Payload Assembly.

Direct chemical modification of native antibodies in a site-specific manner remains a great challenge. Ligand-directed conjugation can achieve the selective modification of antibodies, but usually requires multiple extra steps for ligand release and cargo assembly. Herein, we report a novel, traceless strategy to enable the facile and efficient one-step synthesis of site-specific antibody-drug conjugates (ADCs) by harnessing a thioester-based acyl transfer reagent. The designed reagent, consisting of an optimized Fc-targeting ligand, a thioester bridge and a toxin payload, directly assembles the toxin precisely onto the K251 position of native IgGs and simultaneously self-releases the affinity ligand in one step. With this method, we synthesized a series of K251-linked ADCs from native Trastuzumab. These ADCs demonstrated excellent homogeneity, thermal stability, and both in vitro and in vivo anti-tumor activity. This strategy is equally efficient for IgG1, IgG2, and IgG4 subtypes.

Twin drug design, synthesis and evaluation of diosgenin derivatives as multitargeted agents for the treatment of vascular dementia.

A novel series of multitargeted molecules were designed and synthesized by combining the pharmacological role of cholinesterase inhibitor and antioxidant of steroid as potential ligands for the treatment of Vascular Dementia (VD). The oxygen-glucose deprivation (OGD) model was used to evaluate these molecules, among which the most potent compound ML5 showed the highest activity. Firstly, ML5 showed appropriate inhibition of cholinesterases (ChEs) at orally 15 mg/kg in vivo. The further test revealed that ML5 promoted the nuclear translocation of Nrf2. Furthermore, ML5 has significant neuroprotective effect in vivo model of bilateral common carotid artery occlusion (BCCAO), significantly increasing the expression of Nrf2 protein in the cerebral cortex. In the molecular docking research, we predicted the ML5 combined with hAChE and Keap1. Finally, compound ML5 displayed normal oral absorption and it was nontoxic at 500 mg/kg, po, dose. We can draw the conclusion that ML5 could be considered as a new potential compound for VD treatment.

Sulfation predominates the pharmacokinetics, metabolism, and excretion of forsythin in humans: major enzymes and transporters identified.

Forsythin extracted from Forsythiae Fructus is widely used to treat fever caused by the common cold or influenza in China, Japan and Korea. The present study aimed to analyze the pharmacokinetics, metabolism and excretion routes of forsythin in humans and determine the major enzymes and transporters involved in these processes. After a single oral administration, forsythin underwent extensive metabolism via hydrolysis and further sulfation. In total, 3 of the 13 metabolites were confirmed by comparison to reference substances, i.e., aglycone M1, M1 sulfate (M2), and M1 glucuronide (M7). Hydrolysis was the initial and main metabolic pathway of the parent compound, followed by extensive sulfation to form M2 and a reduced level of glucuronidation to form M7. In addition, the plasma exposure of M2 and M7 were 86- and 4.2-fold higher than that of forsythin. Within 48 h, ~75.1% of the administered dose was found in urine, with M2 accounting for 71.6%. Further phenotyping experiments revealed that sulfotransferase 1A1 and UDP-glucuronosyltransferase 1A8 were the most active hepatic enzymes involved in the formation of M2 and M7, respectively. The in vitro kinetic study provided direct evidence that M1 showed a preference for sulfation. Sulfated conjugate M2 was identified as a specific substrate of organic anion transporter 3, which could facilitate the renal excretion of M2. Altogether, our study demonstrated that sulfation dominated the metabolism and pharmacokinetics of forsythin, while the sulfate conjugate was excreted mainly in the urine.

Species differences in the CYP3A-catalyzed metabolism of TPN729, a novel PDE5 inhibitor.

TPN729 is a novel phosphodiesterase 5 (PDE5) inhibitor used to treat erectile dysfunction in men. Our previous study shows that the plasma exposure of metabolite M3 (N-dealkylation of TPN729) in humans is much higher than that of TPN729. In this study, we compared its metabolism and pharmacokinetics in different species and explored the contribution of its main metabolite M3 to pharmacological effect. We conducted a combinatory approach of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolite identification, and examined pharmacokinetic profiles in monkeys, dogs, and rats following TPN729 administration. A remarkable species difference was observed in the relative abundance of major metabolite M3: i.e., the plasma exposure of M3 was 7.6-fold higher than that of TPN729 in humans, and 3.5-, 1.2-, 1.1-fold in monkeys, dogs, and rats, respectively. We incubated liver S9 and liver microsomes with TPN729 and CYP3A inhibitors, and demonstrated that CYP3A was responsible for TPN729 metabolism and M3 formation in humans. The inhibitory activity of M3 on PDE5 was 0.78-fold that of TPN729 (The IC50 values of TPN729 and M3 for PDE5A were 6.17 ± 0.48 and 7.94 ± 0.07 nM, respectively.). The plasma protein binding rates of TPN729 and M3 in humans were 92.7% and 98.7%, respectively. It was astonishing that the catalyzing capability of CYP3A4 in M3 formation exhibited seven-fold disparity between different species. M3 was an active metabolite, and its pharmacological contribution was equal to that of TPN729 in humans. These findings provide new insights into the limitation and selection of animal model for predicting the clinical pharmacokinetics of drug candidates metabolized by CYP3A4.

Pharmacokinetics, mass balance, and metabolism of [14C]vicagrel, a novel irreversible P2Y12 inhibitor in humans.

Vicagrel, a novel irreversible P2Y12 receptor inhibitor, is undergoing phase III trials for the treatment of acute coronary syndromes in China. In this study, we evaluated the pharmacokinetics, mass balance, and metabolism of vicagrel in six healthy male Chinese subjects after a single oral dose of 20 mg [14C]vicagrel (120 µCi). Vicagrel absorption was fast (Tmax = 0.625 h), and the mean t1/2 of vicagrel-related components was ~38.0 h in both plasma and blood. The blood-to-plasma radioactivity AUCinf ratio was 0.55, suggesting preferential distribution of drug-related material in plasma. At 168 h after oral administration, the mean cumulative excreted radioactivity was 96.71% of the dose, including 68.03% in urine and 28.67% in feces. A total of 22 metabolites were identified, and the parent vicagrel was not detected in plasma, urine, or feces. The most important metabolic spot of vicagrel was on the thiophene ring. In plasma pretreated with the derivatization reagent, M9-2, which is a methylated metabolite after thiophene ring opening, was the predominant drug-related component, accounting for 39.43% of the radioactivity in pooled AUC0-8 h plasma. M4, a mono-oxidation metabolite upon ring-opening, was the most abundant metabolite in urine, accounting for 16.25% of the dose, followed by M3-1, accounting for 12.59% of the dose. By comparison, M21 was the major metabolite in feces, accounting for 6.81% of the dose. Overall, renal elimination plays a crucial role in vicagrel disposition, and the thiophene ring is the predominant metabolic site.

Alflutinib (AST2818), primarily metabolized by CYP3A4, is a potent CYP3A4 inducer.

Alflutinib (AST2818) is a third-generation epidermal growth factor receptor (EGFR) inhibitor that inhibits both EGFR-sensitive mutations and T790M mutations. Previous study has shown that after multiple dosages, alflutinib exhibits nonlinear pharmacokinetics and displays a time- and dose-dependent increase in the apparent clearance, probably due to its self-induction of cytochrome P450 (CYP) enzyme. In this study, we investigated the CYP isozymes involved in the metabolism of alflutinib and evaluated the enzyme inhibition and induction potential of alflutinib and its metabolites. The data showed that alflutinib in human liver microsomes (HLMs) was metabolized mainly by CYP3A4, which could catalyze the formation of AST5902. Alflutinib did not inhibit CYP isozymes in HLMs but could induce CYP3A4 in human hepatocytes. Rifampin is a known strong CYP3A4 inducer and is recommended by the FDA as a positive control in the CYP3A4 induction assay. We found that the induction potential of alflutinib was comparable to that of rifampin. The Emax of CYP3A4 induction by alflutinib in three lots of human hepatocytes were 9.24-, 11.2-, and 10.4-fold, while the fold-induction of rifampin (10 µM) were 7.22-, 19.4- and 9.46-fold, respectively. The EC50 of alflutinib-induced CYP3A4 mRNA expression was 0.25 µM, which was similar to that of rifampin. In addition, AST5902 exhibited much weak CYP3A4 induction potential compared to alflutinib. Given the plasma exposure of alflutinib and AST5902, both are likely to affect the pharmacokinetics of CYP3A4 substrates. Considering that alflutinib is a CYP3A4 substrate and a potent CYP3A4 inducer, drug-drug interactions are expected during alflutinib treatment.

Pyrrolidinyl Synthetic Cathinones α-PHP and 4F-α-PVP Metabolite Profiling Using Human Hepatocyte Incubations.

For more than ten years, new synthetic cathinones (SCs) mimicking the effects of controlled cocaine-like stimulants have flooded the illegal drug market, causing numerous intoxications and fatalities. There are often no data on the pharmacokinetics of these substances when they first emerge onto the market. However, the detection of SC metabolites is often critical in order to prove consumption in clinical and forensic settings. In this research, the metabolite profile of two pyrrolidinyl SCs, α-pyrrolidinohexaphenone (α-PHP) and 4''-fluoro-α-pyrrolidinovalerophenone (4F-α-PVP), were characterized to identify optimal intake markers. Experiments were conducted using pooled human hepatocyte incubations followed by liquid chromatography-high-resolution tandem mass spectrometry and data-mining software. We suggest α-PHP dihydroxy-pyrrolidinyl, α-PHP hexanol, α-PHP 2'-keto-pyrrolidinyl-hexanol, and α-PHP 2'-keto-pyrrolidinyl as markers of α-PHP use, and 4F-α-PVP dihydroxy-pyrrolidinyl, 4F-α-PVP hexanol, 4F-α-PVP 2'-keto-pyrrolidinyl-hexanol, and 4F-α-PVP 2'-keto-pyrrolidinyl as markers of 4F-α-PVP use. These results represent the first data available on 4F-α-PVP metabolism. The metabolic fate of α-PHP was previously studied using human liver microsomes and urine samples from α-PHP users. We identified an additional major metabolite (α-PHP dihydroxy-pyrrolidinyl) that might be crucial for documenting exposure to α-PHP. Further experiments with suitable analytical standards, which are yet to be synthesized, and authentic specimens should be conducted to confirm these results.