Metabolic Stability In Human Liver Microsomes Research Articles

Ultra-fast UPLC–MS/MS approach for estimating X-376 in human liver microsomes: Evaluation of metabolic stability via in silico software and in vitro analysis

X-376 is a novel anaplastic lymphoma kinase (ALK) inhibitor that is capable of penetrating the blood brain barrier. This makes it suitable for use in patients with ALK-positive non-small cell lung cancer (NSCLC) who have metastases in the central nervous system. This study developed a highly sensitive, fast, eco-friendly, and reliable UPLC-MS/MS approach to quantify X-376 in human liver microsomes (HLMs). This approach was used to evaluate X-376's metabolic stability in HLMs in vitro. The UPLC-MS/MS analytical technique validation followed US-FDA bio-analytical method validation guidelines. StarDrop software, containing P450 metabolic and DEREK modules, was utilized to scan X-376's chemical structure for metabolic lability and hazardous warnings. X-376 and Encorafenib (ENF as internal standard) were resoluted on the Eclipse Plus C18 column utilizing an isocratic mobile phase method. The X-376 calibration curve was linear from 1 to 3000 ng/mL. The precision and accuracy of this study's UPLC-MS/MS approach were tested for intra- and inter-day measurements. Inter-day accuracy was −1.32% to 9.36% while intra-day accuracy was −1.5% to 10.00%. The intrinsic clearance (Clint) and in vitro half-life (t1/2) of X-376 were 59.77 mL/min/kg and 13.56 min. The high extraction ratio of X-376 supports the 50 mg twice-daily dose for ALK-positive NSCLC and CNS metastases patients. In silico software suggests that simple structural changes to the piperazine ring or group substitution in drug design may improve metabolic stability and safety compared to X-376.

A Sensitive, Green, and Fast LC–MS/MS Analytical Method for the Quantification of Ribociclib: Evaluation of the Metabolic Stability in HLMs

Ribociclib (Kisqali®) is a pharmacological agent that has great selectivity as a cyclin-dependent kinase 4/6 inhibitor. It has received regulatory approval for its application in the treatment of breast cancer. The objective of the current study was to develop a rapid, green, highly sensitive, validated, and specific LC–MS/MS approach for the quantification of RCB in human liver microsomes (HLMs) over the linear range of 1–3000 ng/mL (LLOQ: 0.98 ng/mL). The inter- and intraday precision and accuracy exhibited values ranging from −0.31% to 3.16% and −5.67% to 5.46% correspondingly. The eco-scale technique (AGREE program) was employed to examine the environmental impact of the existing LC–MS/MS technology. The in vitro half-life and intrinsic clearance of RCB were determined to be 23.58 min and 34.39 mL/min/kg, respectively, which indicated the intermediate extraction ratio of RCB. The in silico P450 software (version 6.6) was used to confirm and validate the practical results. The metabolism of RBC was previously studied by our research group, indicating that the piperazine ring and N-dimethyl group are responsible for the metabolic instability of RCB. Drug discovery studies can be conducted taking into account this concept, allowing the development of new drugs with an enhanced safety profile and good metabolic stability.

Design, synthesis and biological evaluation of chalcone derivatives as potent and orally active hCYP3A4 inhibitors

Human cytochrome P450 3A4 (hCYP3A4), one of the most important drug-metabolizing enzymes, catalyze the metabolic clearance of ∼50% therapeutic drugs. CYP3A4 inhibitors have been used for improving the in vivo efficacy of hCYP3A4-substrate drugs. However, most of existing hCYP3A4 inhibitors may trigger serious adverse effects or undesirable effects on endogenous metabolism. This study aimed to discover potent and orally active hCYP3A4 inhibitors from chalcone derivatives and to test their anti-hCYP3A4 effects both in vitro and in vivo. Following three rounds of screening and structural optimization, the isoquinoline chalcones were found with excellently anti-hCYP3A4 effects. SAR studies showed that introducing an isoquinoline ring on the A-ring significantly enhanced anti-CYP3A4 effect, generating A10 (IC50 = 102.10 nM) as a promising lead compound. The 2nd round of SAR studies showed that introducing a substituent group at the para position of the carbonyl group on B-ring strongly improved the anti-CYP3A4 effect. As a result, C6 was identified as the most potent hCYP3A4 inhibitor (IC50 = 43.93 nM) in human liver microsomes (HLMs). C6 also displayed potent anti-hCYP3A4 effect in living cells (IC50 = 153.00 nM), which was superior to the positive inhibitor ketoconazole (IC50 = 251.00 nM). Mechanistic studies revealed that C6 could potently inhibit CYP3A4-catalyzed N-ethyl-1,8-naphthalimide (NEN) hydroxylation in a competitive manner (Ki = 30.00 nM). Moreover, C6 exhibited suitable metabolic stability in HLMs and showed good safety profiles in mice. In vivo tests demonstrated that C6 (100 mg/kg, orally administration) significantly increased the AUC(0-inf) of midazolam by 3.63-fold, and strongly prolonged its half-life by 1.66-fold compared with the vehicle group in mice. Collectively, our findings revealed the SARs of chalcone derivatives as hCYP3A4 inhibitors and offered several potent chalcone-type hCYP3A4 inhibitors, while C6 could serve as a good lead compound for developing novel, orally active CYP3A4 inhibitors with improved druglikeness properties.

Evaluation of ADMET Predictor in Early Discovery Drug Metabolism and Pharmacokinetics Project Work.

A dataset consisting of measured values for LogD, solubility, metabolic stability in human liver microsomes (HLMs), and Caco-2 permeability was used to evaluate the prediction models for lipophilicity (S+LogD), water solubility (S+Sw_pH), metabolic stability in HLM (CYP_HLM_Clint), intestinal permeability (S+Peff), and P-glycoprotein (P-gp) substrate identification (P-gp substrate) in the software ADMET Predictor (AP) from Simulations Plus. The dataset consisted of a total of 4,794 compounds, with at least data from metabolic stability determinations in HLM, from multiple discovery projects at Medivir. Our evaluation shows that the global AP models can be used for categorization of high and low values based on predicted results for metabolic stability in HLM and intestinal permeability, and to give good predictions of LogD (R2= 0.79), guiding the synthesis of new compounds and for prioritizing in vitro ADME experiments. The model seems to overpredict solubility for the Medivir compounds, however. We also used the in-house datasets to build local models for LogD, solubility, metabolic stability, and permeability by using artificial neural network (ANN) models in the optional Modeler module of AP. Predictions of the test sets were performed with both the global and the local models, and the R2 values for linear regression for predicted versus measured HLM in vitro intrinsic clearance (CLint) based on logarithmic data were 0.72 for the in-house model and 0.53 for the AP model. The improved predictions with the local models are likely explained both by the specific chemical space of the Medivir dataset and laboratory-specific assay conditions for parameters that require biologic assay systems. SIGNIFICANCE STATEMENT: AP is useful early in projects for predicting and categorizing LogD, metabolic stability, and permeability, to guide the synthesis of new compounds, and for prioritizing in vitro ADME experiments. The building of local in-house prediction models with the optional AP Modeler Module can yield improved prediction success since these models are built on data from the same experimental setup and can also be based on compounds with similar structures.

Discovery of a Novel Small-Molecule Modulator of C-X-C Chemokine Receptor Type 7 as a Treatment for Cardiac Fibrosis.

C-X-C chemokine receptor type 7 (CXCR7) is involved in cardiac and immune pathophysiology. We report the discovery of a novel 1,4-diazepine CXCR7 modulator, demonstrating for the first time the role of pharmacological CXCR7 intervention in cardiac repair. Structure-activity-relationship (SAR) studies demonstrated that a net reduction in lipophilicity (log D) and an incorporation of saturated ring systems yielded compounds with good CXCR7 potencies and improvements in oxidative metabolic stability in human-liver microsomes (HLM). Tethering an ethylene amide further improved the selectivity profile (e.g., for compound 18, CXCR7 Ki = 13 nM, adrenergic α 1a Kb > 10 000 nM, and adrenergic β 2 Kb > 10 000 nM). The subcutaneous administration of 18 in mice led to a statistically significant increase in circulating concentrations of plasma stromal-cell-derived factor 1α (SDF-1α) of approximately 2-fold. Chronic dosing of compound 18 in a mouse model of isoproterenol-induced cardiac injury further resulted in a statistically significant reduction of cardiac fibrosis.

Absorption and metabolism characteristics of pristimerin as determined by a sensitive and reliable LC–MS/MS method

In this research, a sensitive and reliable LC–MS/MS method was developed and applied to determine the concentration of pristimerin in rat plasma, cell incubation media and metabolism incubation mixtures. The absolute oral bioavailability of pristimerin is 28.4% at a dose of 1mg·kg−1, and the bioavailability was poor. The bidirectional transport of pristimerin across Caco-2 cells was studied in vitro. A markedly higher transport of pristimerin across Caco-2 cells was observed in the basolateral-to-apical direction and was abrogated in the presence of the P-gp inhibitor, verapamil. The result indicated that P-gp might be involved in the transport of pristimerin in intestine. The phase I and phase II metabolic stability was also investigated using human liver microsomes (HLM) and S9 fractions, respectively. Pristimerin was stable in S9 fractions but metabolized in HLM with a half-life of 20.4min, which indicated that pristimerin could be mainly metabolized by phase I enzymes. In conclusion, the absolute oral bioavailability of pristimerin in plasma, transport across Caco-2 cell monolayers, and metabolic stability in HLM and S9 fractions were systematically investigated by using a sensitive and reliable LC–MS/MS method.