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]

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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]. Xiao-yun Liu, et al. Alflutinib (AST2818), primarily metabolized by CYP3A4, is a potent CYP3A4 inducer. Acta Pharmacol Sin. 2020 Oct; 41(10): 1366-1376.

Aflutinib (AST2818) is a third-generation epidermal growth factor receptor (EGFR) inhibitor that inhibits EGFR-sensitive mutations and the T790M mutation. Previous studies have shown that aflutinib exhibits nonlinear pharmacokinetic characteristics after multiple doses, with its apparent clearance increasing with time and dose, possibly due to its self-induction of cytochrome P450 (CYP) enzymes. This study investigated the CYP isoenzymes involved in aflutinib metabolism and evaluated the enzyme inhibitory and inducing potential of aflutinib and its metabolites. Results showed that aflutinib is primarily metabolized by CYP3A4 in human liver microsomes (HLM), and CYP3A4 catalyzes the formation of AST5902. Aflutinib does not inhibit CYP isoenzymes in the HLM but does induce CYP3A4 in human hepatocytes. Rifampin is a known potent CYP3A4 inducer and is recommended by the FDA as a positive control for CYP3A4 induction assays. We found that aflutinib's inducing ability was comparable to that of rifampin. In three batches of human hepatocytes, aflutinib induced CYP3A4 expression with Emax of 9.24-fold, 11.2-fold, and 10.4-fold, respectively, while rifampin (10 μM) induced it with folds of 7.22-fold, 19.4-fold, and 9.46-fold, respectively. The EC50 for aflutinib-induced CYP3A4 mRNA expression was 0.25 μM, similar to rifampin. Furthermore, AST5902 showed significantly weaker CYP3A4 induction compared to aflutinib. Given the plasma exposure levels of both aflutinib and AST5902, both may affect the pharmacokinetics of CYP3A4 substrates. Considering that aflutinib is both a CYP3A4 substrate and a potent CYP3A4 inducer, drug interactions are expected during aflutinib treatment. [1]

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Background Aflutinib is a novel, irreversible, highly selective third-generation EGFR inhibitor currently under development for the treatment of non-small cell lung cancer patients with EGFR activating mutations and EGFR T790M resistance mutations. Aflutinib is primarily metabolized by CYP3A4 to produce its active metabolite AST5902. Both aflutinib and AST5902 contribute to the pharmacological activity in vivo. This study aimed to investigate the effects of rifampin (a potent CYP3A4 inducer) on the pharmacokinetics of aflutinib and AST5902 in healthy volunteers, thereby providing important information for drug interaction assessment and clinical application. Methods: This was a single-center, open-label, single-sequence trial, divided into two phases. Volunteers received a single dose of 80 mg aflutinib on days 1/22 and continued to receive 0.6 g rifampin from days 15 to 30. Blood samples were collected on days 1-10 and days 22-31. The pharmacokinetics of aflutinib, AST5902, and the total active ingredient of aflutinib and AST5902 were analyzed with and without rifampin. Results: Concomitant use with rifampin resulted in an 86% decrease in AUC_0-∞ and a 60% decrease in Cmax for aflutinib, while the AUC_0-∞ of AST5902 decreased by 17%, and the Cmax increased by 1.09-fold. The AUC_0-∞ and Cmax of the total active ingredients (aflutinib and AST5902) decreased by 62% and 39%, respectively. Conclusion: Rifampin, as a potent CYP3A4 inducer, significantly affects the pharmacokinetics of aflutinib and its total active ingredients (aflutinib and AST5902). These results suggest that concomitant use of potent CYP3A4 inducers should be avoided during aflutinib treatment. This study has been registered on the China Drug Research website (http://www.chinadrugtrials.org.cn). August 2021; 39(4):1011-1018.

C27H29F3N8O2

554.5668

554.236

2412155-74-7

AST5902 trimesylate;2929417-90-1

146405881

Typically exists as solid at room temperature

4

3

11

11

40

836

0

OYVNKZAYJFLKCX-UHFFFAOYSA-N

InChI=1S/C27H29F3N8O2/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/h5-11,14-15,31H,1,12-13,16H2,2-4H3,(H,33,39)(H,32,34,35)

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; 2412155-74-7; 2-Propenamide, N-[5-[[4-(1-methyl-1H-indol-3-yl)-2-pyrimidinyl]amino]-2-[methyl[2-(methylamino)ethyl]amino]-6-(2,2,2-trifluoroethoxy)-3-pyridinyl]-; 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; Des-methyl-furmonertinib; QU8GLB54KX; SCHEMBL21753618; OYVNKZAYJFLKCX-UHFFFAOYSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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