Evaluation of Alectinib Metabolic Stability in HLMs Using Fast LC-MS/MS Method: In Silico ADME Profile, P450 Metabolic Lability, and Toxic Alerts Screening

Abstract

Alectinib, also known as Alecensa^®, is prescribed for the therapeutic treatment of individuals diagnosed with metastatic non-small cell lung cancer (NSCLC) who have a specific genetic mutation referred to as anaplastic lymphoma kinase (ALK) positivity. The Food and Drug Administration granted regular approval to alectinib, a drug developed by Hoffmann-La Roche, Inc. (Basel, Switzerland)/Genentech, Inc. (South San Francisco, CA, USA), on 6 November 2017. The screening of the metabolic stability and identification of hazardous alarms within the chemical structure of ALC was conducted using the StarDrop software package (version 6.6), which incorporates the P450 metabolic module and DEREK software (KB 2018 1.1). The primary aim of this investigation was to develop a high-throughput and accurate LC-MS/MS technique for the quantification of ALC in the metabolic matrix (human liver microsomes; HLMs). The aforementioned methodology was subsequently employed to assess the metabolic stability of ALC in HLMs through in vitro tests, with the obtained results further validated using in silico software. The calibration curve of the ALC showed a linear correlation that exists within the concentration range from 1 to 3000 ng/mL. The LC-MS/MS approach that was recommended exhibited accuracy and precision levels for both inter-day and intra-day measurements. Specifically, the accuracy values ranged from -2.56% to 3.45%, while the precision values ranged from -3.78% to 4.33%. The sensitivity of the established approach was proved by its ability to adhere to an LLOQ of 0.82 ng/mL. The half-life (t_1/2) and intrinsic clearance (Cl_int) of ALC were estimated to be 22.28 min and 36.37 mL/min/kg, correspondingly, using in vitro experiments. The ALC exhibited a moderate extraction ratio. The metabolic stability and safety properties of newly created derivatives can be enhanced by making modest adjustments to the morpholine and piperidine rings or by substituting the substituent, as per computational software. In in silico ADME prediction, ALC was shown to have poor water solubility and high gastrointestinal absorption along with inhibition of some cytochrome P450s (CYP2C19 and CYP2C9) without inhibition of others (CYP1A2, CYP3A4, and CYP2D6) and P-glycoprotein substrate. The study design that involves using both laboratory experiments and different in silico software demonstrates a novel and groundbreaking approach in the establishment and uniformization of LC-MS/MS techniques for the estimation of ALC concentrations, identifying structural alerts and the assessment of its metabolic stability. The utilization of this study strategy has the potential to be employed in the screening and optimization of prospective compounds during the drug creation process. This strategy may also facilitate the development of novel derivatives of the medicine that maintain the same biological action by targeted structural modifications, based on an understanding of the structural alerts included within the chemical structure of ALC.

Keywords: ADME profile; DEREK software; LC-MS/MS approach; P450 metabolic mode; StarDrop software; alectinib; greenness; in vitro half-life; intrinsic clearance; metabolic stability.

Figure 1

Chemical structure of alectinib and encorafenib (IS).

Figure 2

The composite site lability (CSL) value of 0.9691 indicates that ALC exhibits a moderate level of lability in terms of its metabolism. The outcomes were assessed utilizing the P450 program of StarDrop software.

Figure 3

Structural alerts of ALC using DEREK software highlighted in red (A). P450 metabolic stability of ALC CSL at 0.9691 indicating the moderate clearance characteristic of ALC (B).

Figure 4

The ADME radar chart of ALC obtained from SwissADME software indicating the good ADME characteristics of ALC that fall within the suitable range for a pharmacological drug.

Figure 5

MRM mass spectrum of ALC [M + H]⁺ showing one mass transition from m/z 483 to m/z 396 (A) and EFB [M + H]⁺ showing two mass transitions from m/z 540 to m/z 359 and from m/z 540 to m/z 116 (B). The proposed collision-induced dissociation (the collision cell) patterns are exhibited.

Figure 6

Panel (A) displays the negative-control sample consisting of HLM matrix, which exhibits no interference at the retention times of alectinib (ALC) and encorafenib (EFB). Panel (B) presents the MRM chromatogram of the positive-control sample, which contains HLMs with EFB at a concentration of 1000 ng/mL. Panel (C) showcases the overlaid MRM charts of ALC standards, including CSs and quality controls (QCs) at various concentrations ranging from 1 to 3000 ng/mL. The ALC peak is observed at 1.38 min, while the EFB peak (at a concentration of 1000 ng/mL) appears at 0.9 min, indicating a fast LC-MS/MS method.

Figure 7

(A) ALC chromatographic peak (1.39 min) at 1 ng/mL (LLOQ) indicating the sensitivity of the proposed LC-MS/MS method; (B) EFB (IS) analytical peak (0.9 min) at 1000 ng/mL. The LLOQ can be detected very easily as seen in the figure with a height of 546.72 and peak area of 10,856 that reveals the high sensitivity of the established LC-MS/MS approach.

Figure 8

The results are presented in a circular drawing that showcases a diverse spectrum of hues, ranging from dark green (indicating the maximum degree of greenness) to red (revealing a lack of greenness). The colors described above are linked to twelve separate attributes, as illustrated in the accompanying visual depiction. The GAC score was 0.76, as seen in the middle of the circle, that indicated the greenness of the current LC-MS/MS approach.

Figure 9

The metabolic stability curve of ALC in the incubation matrix of HLMs was analyzed from 0.0 to 60 min. (A). Additionally, the linear section of the logarithmic (ln) calibration curve was examined, showing the regression equation (y = −0.0311x + 4.639; R² = 0.9951) revealing a slope of −0.0311 that was utilized in calculation of t_1/2 (22.28 min) and Cl_int (36.37 mL/min/kg) (B).

Figure 10

ALC metabolic lability curve (blue) using P450 software (version 6.6) and ALC DEREK toxicity predictions (red) indicating that morpholine and piperidine rings are responsible for the metabolic instability of ALC.