EGFR[1]

In this study, researchers 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. [1]
Considering the high exposure of AST5902 and the structures of alflutinib and AST5902, we also evaluated the CYP3A4 induction potential of AST5902. At low concentrations, AST5902 inhibited the mRNA transcription of CYP3A4, but the underlying mechanisms remain unclear. Compared to alflutinib and rifampin, the in vitro induction effects of AST5902 were found to be less significant. However, given the plasma exposure of AST5902, AST5902 is also likely to cause a clinical DDI with CYP3A4-sensitive substrates.[1]

In phase I/II clinical trials, the Cmax and AUC of a single-dose alflutinib were elevated in a dose-dependent manner among NSCLC patients in the dose range of 20–240 mg. After multiple doses, the increase in alflutinib exposure was less than that of a single dose. The AUC of AST5902 greatly increased, even exceeding that of alflutinib in the 240 mg dosage group. In addition, alflutinib showed a time-dependent and dose-dependent increase in clearance (CL/F) following multiple doses. CYP phenotyping studies and CYP enzyme induction indicated that alflutinib was a substrate and inducer of CYP3A4. Thus, the self-induction of alflutinib may be the reason for the phenomenon observed in clinical trials. Given the exposure of human alflutinib at an 80 mg dose, alflutinib is speculated to activate clinical pharmacokinetic DDIs when coadministered with CYP3A4-sensitive substrates, including midazolam and triazolam. Considering that the activation of pregnane X receptor (PXR) can induce CYP3A and CYP2C, further evaluation of the potential of alflutinib to induce CYP2C should be conducted.[1]

Metabolism of alflutinib in HLMs[1]
Before starting the experiments, the HLMs were thawed gently on ice. Then, 3 µM alflutinib was added to the HLMs (0.5 mg protein/mL) in 100 mM phosphate-buffered saline (PBS; pH 7.4) to a total volume of 100 μL. After incubating at 37 °C for 3 min, the reactions were initiated by the addition of 1.0 mM NADPH. Following 1 h of incubation, the reactions were terminated by mixing with ice-cold acetonitrile at the same volume. All incubations were performed in duplicate and then analyzed by UPLC-UV/Q-TOF MS.

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Incubation of HLMs with specific CYP inhibitors[1]
HLMs were used to study the effects of CYP enzyme inhibitors on the metabolism of alflutinib. The incubation mixture (100 µL) consisted of alflutinib (3 µM), HLMs (0.5 mg protein/mL), NADPH (1 mM), PBS (100 mM, pH 7.4) and a selective CYP inhibitor. The chemical inhibitors were as follows: α-naphthoflavone (2 µM) for CYP1A/2 C, quercetin (20 µM) for CYP2C8, sulfaphenazole (6 µM) for CYP2C9, ticlopidine (24 µM) for CYP2B6/2C19, quinidine (8 µM) for CYP2D6, chlormethiazole (24 µM) for CYP2E1, ketoconazole (2 µM) for CYP3A and ABT (1 mM) for all CYP enzymes. These inhibitors were preincubated with HLMs in the presence of NADPH for 10 min before adding the substrate. After that, the reactions were initiated by incubation at 37 °C for 60 min. Finally, the reactions were terminated by the addition of 100 µL of ice-cold acetonitrile. All incubations were performed in duplicate, and the formation of metabolites was evaluated in the absence or presence of inhibitors.

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The metabolism of alflutinib through recombinant human CYP isoenzyme[1]
To identify the specific isoform that participates in the metabolism of alflutinib, 3 µM alflutinib was mixed with recombinant human CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, or 3A5 (25 pmol P450/mL) in a total volume of 100 μL. The reactions were initiated and terminated by the addition of 1 mM NADPH and 100 µL of ice-cold acetonitrile, respectively. The incubation was carried at 37 °C for 60 min. All reactions were conducted in duplicate, followed by UPLC-UV/Q-TOF MS analysis.

Upon incubation of 3 μM alflutinib with human hepatocytes at 37 °C for 3 h, the primary metabolite was identified as AST5902, accounting for 52% of the remaining alflutinib concentration. Other metabolites accounted for less than 1.2% of alflutinib (unpublished data). In addition, AST5902 exerted CYP3A4 induction potential, which might contribute to the induction effect of alflutinib.[1]
Enzyme induction of alflutinib and AST5902 on the human CYP3A4 enzyme[1]
For the assessment of enzyme induction, 7 × 105 hepatocytes/mL were seeded into a collagen-coated 24-well plate and placed in a 37 °C humidified incubator with 5% CO2 for 24 h. The hepatocytes were treated with the human CYP3A4 enzyme inducer rifampin (10 μM), alflutinib or AST5902 (0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 or 5 μM) or 0.1% DMSO (control group) once daily for three consecutive days. After treatment, RNA extraction was performed with TRIzol according to the manufacturer’s protocol. cDNA synthesis was carried out using the PrimeScript RT reagent kit. Real-time PCR was conducted on a StepOnePlus real-time PCR system using the SYBR green Premix Ex Taq kit. The forward primer and reverse primer for CYP3A4 were 5′-ATCACTAGCACATCATTTGGAG-3′ and 5′-GGAATGGAAAGGTTATTGAGAG-3′, respectively. For GAPDH, the forward and reverse primers were 5′-AGAAGGCTGGGGCTCATTTG-3′ and 5′-GAGGGGCCATCCACAGTCTTC-3′, respectively. The levels of cDNA were quantitated by the comparative threshold cycle method using GAPDH as an internal standard. EC50 is the concentration of inducer at 50% maximal effect of induction, which is obtained by a nonlinear regression test carried by GraphPad Prism version 5.0.

[1]. Alflutinib (AST2818), primarily metabolized by CYP3A4, is a potent CYP3A4 inducer. Acta Pharmacol Sin. 2020 Oct; 41(10): 1366-1376.

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.[1]

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Background Alflutinib is a novel irreversible and highly selective third-generation EGFR inhibitor currently being developed for the treatment of non-small cell lung cancer patients with activating EGFR mutations and EGFR T790M drug-resistant mutation. Alflutinib is mainly metabolized via CYP3A4 to form its active metabolite AST5902. Both alflutinib and AST5902 contribute to the in vivo pharmacological activity. The aim of this study was to investigate the effects of rifampicin (a strong CYP3A4 inducer) on the pharmacokinetics of alflutinib and AST5902 in healthy volunteers, thus providing important information for drug-drug interaction evaluation and guiding clinical usage. Methods This study was designed as a single-center, open-label, and single-sequence trial over two periods. The volunteers received a single dose of 80 mg alflutinib on Day 1/22 and continuous doses of 0.6 g rifampicin on Day 15-30. Blood sampling was conducted on Day 1-10 and Day 22-31. The pharmacokinetics of alflutinib, AST5902, and the total active ingredients (alflutinib and AST5902) with or without rifampicin co-administration were respectively analyzed. Results Co-administration with rifampicin led to 86% and 60% decreases in alflutinib AUC0-∞ and Cmax, respectively, as well as 17% decrease in AST5902 AUC0-∞ and 1.09-fold increase in AST5902 Cmax. The total active ingredients (alflutinib and AST5902) exhibited 62% and 39% decreases in AUC0-∞ and Cmax, respectively. Conclusions As a strong CYP3A4 inducer, rifampicin exerted significant effects on the pharmacokinetics of alflutinib and the total active ingredients (alflutinib and AST5902). The results suggested that concomitant strong CYP3A4 inducers should be avoided during alflutinib treatment. This trial was registered at http://www.chinadrugtrials.org.cn . The registration No. is CTR20191562, and the date of registration is 2019-09-12. Source: Invest New Drugs. 2021 Aug;39(4):1011-1018. doi: 10.1007/s10637-021-01071-z.

C30H41F3N8O11S3

842.883753538132

842.2

2929417-90-1

AST5902;2412155-74-7

155971210

Yellow to orange solid powder

6

20

11

55

929

0

KRCDPFVVSHJNOV-UHFFFAOYSA-N

InChI=1S/C27H29F3N8O2.3CH4O3S/c1-5-23(39)33-20-14-21(25(40-16-27(28,29)30)36-24(20)37(3)13-12-31-2)35-26-32-11-10-19(34-26)18-15-38(4)22-9-7-6-8-17(18)22;3*1-5(2,3)4/h5-11,14-15,31H,1,12-13,16H2,2-4H3,(H,33,39)(H,32,34,35);3*1H3,(H,2,3,4)

methanesulfonic acid;N-[5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]-2-[methyl-[2-(methylamino)ethyl]amino]-6-(2,2,2-trifluoroethoxy)pyridin-3-yl]prop-2-enamide

AST5902 trimesylate; AST5902 (trimesylate); AST5902 trimesylate; 2929417-90-1; AST5902 (trimesylate);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 50 mg/mL (59.32 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.97 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.97 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)