<i>In vitro</i> Metabolic Stability of Drugs and Applications of LC-MS in Metabolite Profiling

Krishna, Marothu Vamsi; Padmalatha, K.; Madhavi, Gorrepati

doi:10.5772/intechopen.99762

Cited by 8 publications

(8 citation statements)

References 101 publications

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“…A compound's metabolic stability is a crucial element to consider during the early phases of drug research. Even if a molecule has intriguing pharmacological properties, it will never become a drug if it has low metabolic stability (Krishna et al, 2021), for this reason we used two web servers to predict the metabolic stability of the LaSOM 335: SwissADME and BioTransformer 3.0. The SwissADME server predicted that LaSOM 335 has a high gastrointestinal absorption, a medium skin permeation and does not pass the blood-brain barrier.…”

Section: Metabolic Stability Predictionmentioning

confidence: 99%

LaSOM335, active against bladder cancer cells, interferes withLet‐60(hRas) and reducesCD73 expression/activity

et al. 2023

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Bladder cancer is the fourth most common malignancy in men. It can present along the entire continuum of severity, from mild to well‐differentiated disease to extremely malignant tumors with low survival rates. Human RAS genes are the most frequently mutated oncogenes in human cancers, and the critical role of aberrant Ras protein function in carcinogenesis is well established. Therefore, considerable efforts have been devoted to the development of anti‐Ras inhibitors for cancer treatment. This study presents the biphenyl dihydropyrimidinone LaSOM 335 with high activity against T24 bladder cancer cells (IC50 = 10.73 ± 0.53 μM) and selectivity of cytotoxicity for this cancer cell line compared to two non‐cancer cell lines investigated. Furthermore, we also show that this compound reduced vulvar development in the mutant let‐60 gene of Caenorhabditis elegans. Let‐60 is a homolog of the mammalian Ras gene. In addition, we observed that LaSOM 335 inhibits the enzymatic activity of CD73 and decreases CD73 expression. Possibly, this expression decrease is due to downstream EGFR signaling via the Ras–Raf–ERK pathway, that directly regulates CD73 expression via ERK1/2. Evidence suggests that non‐immunomodulating functions of CD73 play an equally important role for cancer cell survival, progression, and migration. Regarding we also notice that LaSOM 335 was safe in the in vivo model of C. elegans. The set of these findings makes this biphenyl dihydropyrimidinone a promising candidate for further investigations in the bladder cancer field.

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Section: Metabolic Stability Predictionmentioning

confidence: 99%

LaSOM335, active against bladder cancer cells, interferes withLet‐60(hRas) and reducesCD73 expression/activity

et al. 2023

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“…So, the current analytical method (LC-MS/MS) was established and validated in HLM’s matrix for quantifying SLP using calibration standards and unknowns. The results of metabolic stability are included in the calculation of two parameters [the intrinsic clearance (Cl int ) and the in vitro half-life (t 1/2 )] of SLP [ 11 ] using an ‘in vitro t 1/2 ′ approach (‘well-stirred’ model) [ 12 , 13 ] as it is considered the most utilized model in various metabolism experiments owing to its ease. In silico software data were utilized as a clue for the in vitro incubation experiments and generated data about the rate of SLP metabolism that provided a proposed figure for SLP in vivo bioavailability [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method

et al. 2023

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Selpercatinib (SLP; brand name Retevmo®) is a selective and potent RE arranged during transfection (RET) inhibitor. On 21 September 2022, the FDA granted regular approval to SLP (Retevmo, Eli Lilly, and Company). It is considered the only and first RET inhibitor for adults with metastatic or locally advanced solid tumors with RET gene fusion. In the current experiment, a highly specific, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying SLP in human liver microsomes (HLMs) was developed and applied to the metabolic stability evaluation of SLP. The LC-MS/MS method was validated following the bioanalytical methodology validation guidelines outlined by the FDA (linearity, selectivity, matrix effect, accuracy, precision, carryover, and extraction recovery). SLP was detected by a triple quadrupole detector (TQD) using a positive ESI source and multiple reaction monitoring (MRM) mode for mass spectrometric analysis and estimation of analytes ions. The IS-normalized matrix effect and extraction recovery were acceptable according to the FDA guidelines for the bioanalysis of SLP. The SLP calibration standards were linear from 1 to 3000 ng/mL HLMs matrix, with a regression equation (y = 1.7298x + 3.62941) and coefficient of variation (r2 = 0.9949). The intra-batch and inter-batch precision and accuracy of the developed LC-MS/MS method were −6.56–5.22% and 5.08–3.15%, respectively. SLP and filgotinib (FLG) (internal standard; IS) were chromatographically separated using a Luna 3 µm PFP (2) stationary phase (150 × 4.6 mm) with an isocratic mobile phase at 23 ± 1 °C. The limit of quantification (LOQ) was 0.78 ng/mL, revealing the LC-MS/MS method sensitivity. The intrinsic clearance and in vitro t1/2 (metabolic stability) of SLP in the HLMs matrix were 34 mL/min/kg and 23.82 min, respectively, which proposed an intermediate metabolic clearance rate of SLP, confirming the great value of this type of kinetic experiment for more accurate metabolic stability predictions. The literature review approved that the established LC-MS/MS method is the first developed and reported method for quantifying SLP metabolic stability.

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“…In silico prediction of the GTB metabolic lability was performed utilizing StarDrop's software package that contains the P450 metabolism model before starting the in vitro metabolic incubation study to exhibit the worth of the current UPLC-ESI-MS/MS methodology and to spare time and resources [19]. After validation of the established UPLC-ESI-MS/MS method, the application was done in a practical assessment of the two metabolic stability factors [the in vitro half-life (t 1/2 ) and intrinsic clearance (Cl int )] of GTB [20]. In silico software (P450 metabolic model) and in vitro HLMs metabolic incubation with HLMs were utilized for the determination of GTB metabolic stability to reveal more information about the rate of GTB metabolism and to permit the determination of in vivo bioavailability.…”

Section: Introductionmentioning

confidence: 99%

An UPLC–ESI–MS/MS Bioanalytical Methodology for the Quantification of Gilteritinib in Human Liver Microsomes: Application to In Vitro and In Silico Metabolic Stability Estimation

et al. 2023

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Gilteritinib (Xospata®) is a tyrosine kinase inhibitor (TKI) that works by inhibiting numerous receptor tyrosine kinases, involving AXL and FMS-like tyrosine kinase 3 (FLT3). Gilteritinib (GTB) was approved (28 November 2018) by the USFDA for the treatment of refractory or relapsed (R/R) acute myeloid leukemia (AML) with a FLT3 mutation. In the current study, a fast, highly sensitive, and specific ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) analytical methodology was created for GTB determination in human liver microsomes (HLMs) utilizing an electrospray ionization (ESI) source. The developed methodology (UPLC–ESI–MS/MS) was utilized in the assessment of GTB metabolic stability. The UPLC–ESI–MS/MS methodology was validated following the rules of the FDA that include selectivity, linearity, accuracy, precision, matrix effect, stability, and extraction recovery. The generated data of the optimized validation parameters of the current UPLC–ESI–MS/MS methodology were acceptable as reported in the FDA guidelines. GTB parent ions were generated in the ESI source (positive mode) and GTB daughter ions (two) were quantified in the mass analyzer utilizing multiple reaction monitoring (MRM) modes. The plotted GTB calibration curve showed a wide range of linearity from 1 ng/mL to 3000 ng/mL in HLMs matrix (y = 1.7298x + 3.62941 and r2 = 0.9949). The intraday and interday precision and accuracy outcomes of the current UPLC–ESI–MS/MS methodology were 0.35–11.39% and 0.27–4.32%, respectively. GTB and encorafenib (EFB) (internal standard; IS) were resoluted utilizing a reversed stationary phase (ZORBAX Eclipse plus C18 column; 1.8 μm PS, 2.1 mm ID, and 50 mm L) at 22 ± 2 °C. The calculated lower limit of quantification (LLOQ) was 0.94 ng/mL, revealing the UPLC–ESI–MS/MS methodology sensitivity. The two metabolic stability factors including in vitro half-life (t1/2) and intrinsic clearance (Clint) of GTB were 14.32 min and 56.64 mL/min/kg, respectively, predicting the moderate extraction ratio and good bioavailability of GTB. The current UPLC–ESI–MS/MS methodology is fast, sensitive and exhibits a wider range of linearity (1 to 3000 ng/mL) compared to other reported methods and is considered the first validated methodology for the determination of GTB metabolic stability.

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In vitro Metabolic Stability of Drugs and Applications of LC-MS in Metabolite Profiling

Cited by 8 publications

References 101 publications

LaSOM335, active against bladder cancer cells, interferes withLet‐60(hRas) and reducesCD73 expression/activity

LaSOM335, active against bladder cancer cells, interferes withLet‐60(hRas) and reducesCD73 expression/activity

Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method

An UPLC–ESI–MS/MS Bioanalytical Methodology for the Quantification of Gilteritinib in Human Liver Microsomes: Application to In Vitro and In Silico Metabolic Stability Estimation

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