The multiple roles of autophagy in uveal melanoma and the microenvironment.

PURPOSE: Uveal melanoma (UM) is the most common primary malignant intraocular tumor in adults, and effective clinical treatment strategies are still lacking. Autophagy is a lysosome-dependent degradation system that can encapsulate abnormal proteins, damaged organelles. However, dysfunctional autophagy has multiple types and plays a complex role in tumorigenicity depending on many factors, such as tumor stage, microenvironment, signaling pathway activation, and application of autophagic drugs. METHODS: A systematic review of the literature was conducted to analyze the role of autophagy in UM, as well as describing the development of autophagic drugs and the link between autophagy and the tumor microenvironment. RESULTS: In this review, we summarize current research advances regarding the types of autophagy, the mechanisms of autophagy, the application of autophagy inhibitors or agonists, autophagy and the tumor microenvironment. Finally, we also discuss the relationship between autophagy and UM. CONCLUSION: Understanding the molecular mechanisms of how autophagy differentially affects tumor progression may help to design better therapeutic regimens to prevent and treat UM.

LINC01278 Induces Autophagy to Inhibit Tumour Progression by Suppressing the mTOR Signalling Pathway.

Uveal melanoma (UM) is an aggressive intraocular malignant tumour that is closely related to autophagic dysfunction. We aimed to identify autophagy-related long noncoding RNAs (lncRNAs) to elucidate the molecular mechanism of UM. Here, we show that LINC01278 is a new potential biomarker with clinical prognostic value in UM through bioinformatics analysis. Application of an autophagy inhibitor (3-MA) and an autophagy agonist (MG-132) indicated that LINC01278 can inhibit UM cell proliferation, migration, and invasion by inducing autophagy. A xenograft nude mouse model was used to examine the tumorigenesis of UM cells in vivo. Mechanistically, LINC01278 can inhibit the mTOR signalling pathway to activate autophagy, as shown by experiments with an mTOR agonist (MHY1485) and mTOR inhibitor (rapamycin) treatment. Our findings indicate that LINC01278 functions as a tumour suppressor by inhibiting the mTOR signalling pathway to induce autophagy. Targeting the LINC01278-mTOR axis might be a novel and promising therapeutic approach for UM.

PTK6 inhibits autophagy to promote uveal melanoma tumorigenesis by binding to SOCS3 and regulating mTOR phosphorylation.

Autophagy dysfunction is one of the common causes of tumor formation and plays an important role in uveal melanoma (UM). However, little is known about the regulatory mechanisms of autophagy in UM. Here, we show that PTK6 can promote the proliferation, migration, and invasion of UM cells by inhibiting autophagy. SOCS3 can inhibit the proliferation, migration, and invasion of UM cells. Overexpression of SOCS3 can partially rescue the PTK6-induced promotion of UM cell proliferation, migration, and invasion. Mechanistically, PTK6 can bind to SOCS3, and SOCS3 can downregulate the expression of PTK6. Furthermore, PTK6 can upregulate the phosphorylation of mTOR to inhibit autophagy. Taken together, our findings demonstrate the functions of PTK6 and SOCS3 in UM cells and targeting the SOCS3-PTK6 signaling axis might be a novel and promising therapeutic strategy for patients with UM.

Potential drug-drug interaction of olverembatinib (HQP1351) using physiologically based pharmacokinetic models.

Olverembatinib (HQP1351) is a third-generation BCR-ABL tyrosine kinase inhibitor for the treatment of chronic myeloid leukemia (CML) (including T315I-mutant disease), exhibits drug-drug interaction (DDI) potential through cytochrome P450 (CYP) enzymes CYP3A4, CYP2C9, CYP2C19, CYP1A2, and CYP2B6. A physiologically-based pharmacokinetic (PBPK) model was constructed based on physicochemical and in vitro parameters, as well as clinical data to predict 1) potential DDIs between olverembatinib and CYP3A4 and CYP2C9 inhibitors or inducers 2), effects of olverembatinib on the exposure of CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 substrates, and 3) pharmacokinetics in patients with liver function injury. The PBPK model successfully described observed plasma concentrations of olverembatinib from healthy subjects and patients with CML after a single administration, and predicted olverembatinib exposure increases when co-administered with itraconazole (strong CYP3A4 inhibitor) and decreases with rifampicin (strong CYP3A4 inducer), which were validated by observed data. The predicted results suggest that 1) strong, moderate, and mild CYP3A4 inhibitors (which have some overlap with CYP2C9 inhibitors) may increase olverembatinib exposure by approximately 2.39-, 1.80- to 2.39-, and 1.08-fold, respectively; strong, and moderate CYP3A4 inducers may decrease olverembatinib exposure by approximately 0.29-, and 0.35- to 0.56-fold, respectively 2); olverembatinib, as a "perpetrator," would have no or limited impact on CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activity 3); systemic exposure of olverembatinib in liver function injury with Child-Pugh A, B, C may increase by 1.22-, 1.79-, and 2.13-fold, respectively. These simulations inform DDI risk for olverembatinib as either a "victim" or "perpetrator".

Comprehensive PBPK model to predict drug interaction potential of Zanubrutinib as a victim or perpetrator.

A physiologically based pharmacokinetic (PBPK) model was developed to evaluate and predict (1) the effect of concomitant cytochrome P450 3A (CYP3A) inhibitors or inducers on the exposures of zanubrutinib, (2) the effect of zanubrutinib on the exposures of CYP3A4, CYP2C8, and CYP2B6 substrates, and (3) the impact of gastric pH changes on the pharmacokinetics of zanubrutinib. The model was developed based on physicochemical and in vitro parameters, as well as clinical data, including pharmacokinetic data in patients with B-cell malignancies and in healthy volunteers from two clinical drug-drug interaction (DDI) studies of zanubrutinib as a victim of CYP modulators (itraconazole, rifampicin) or a perpetrator (midazolam). This PBPK model was successfully validated to describe the observed plasma concentrations and clinical DDIs of zanubrutinib. Model predictions were generally within 1.5-fold of the observed clinical data. The PBPK model was used to predict untested clinical scenarios; these simulations indicated that strong, moderate, and mild CYP3A inhibitors may increase zanubrutinib exposures by approximately four-fold, two- to three-fold, and <1.5-fold, respectively. Strong and moderate CYP3A inducers may decrease zanubrutinib exposures by two- to three-fold or greater. The PBPK simulations showed that clinically relevant concentrations of zanubrutinib, as a DDI perpetrator, would have no or limited impact on the enzyme activity of CYP2B6 and CYP2C8. Simulations indicated that zanubrutinib exposures are not impacted by acid-reducing agents. Development of a PBPK model for zanubrutinib as a DDI victim and perpetrator in parallel can increase confidence in PBPK models supporting zanubrutinib label dose recommendations.

Pharmacokinetics analysis based on target-mediated drug distribution for RC18, a novel BLyS/APRIL fusion protein to treat systemic lupus erythematosus and rheumatoid arthritis.

BACKGROUND AND PURPOSE: RC18 is a novel recombinant fusion protein targeting on B lymphocyte stimulator (BLyS). We aimed to develop and qualify a population pharmacokinetics (PopPK) model for RC18 in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients, taking into account the mechanistic target-mediated drug disposition (TMDD) process. METHODS: A TMDD model of RC18 was developed using data from two phase I clinical trial (n = 23). The TMDD structural model was developed by simultaneous fitting of the serum free RC18 and serum RC18-BLyS complex. Potential covariates were screened using stepwise method, and predictive performance was qualified using a prediction-corrected visual predictive check (pcVPC) and bootstrap. RESULTS: A two compartment TMDD model with first order absorption for subcutaneous administration was built. The final model included a significant relationship between distribution volume of the central compartment and body weight. And the baseline of immunoglobulin IgG had significant effect on the baseline of target BLyS. The plots from goodness-of-fit and pcVPC confirmed good predictive performance of this TMDDmodel. CONCLUSIONS: This mechanistic TMDD model integrated the interaction of RC18 with its target BLyS and accurately predicts both RC18 and RC18-BLyS complex profiles in RA and SLE patients. Simulated target change profiles can be used to help guide rational dose regimen selection and used as a biomarker for efficacy evaluation.

A high-performance liquid chromatography-tandem mass spectrometry method for the determination of lifrafenib, a novel RAF kinase and EGFR inhibitor, in human plasma and urine and its application in clinical pharmacokinetic study.

Lifirafenib (BGB-283), a dual inhibitor trageting BRAF kinase and EGFR, showed favorable efficacy and safety in treating patients with different cancer types harboring mutations in BRAF, KRAS and NRAS. In order to support the clinical pharmacokinetic study, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to quantify lifirafenib concentration in human plasma and urine. Plasma samples were purified using protein precipitation. Urine samples were pre-treated by adding tween 80 with the purpose of preventing non-specific adsorption, then extracted by centrifugation. Chromatographic separation was achieved on Phenomenex Luna C18 column with a gradient elution. The mass detection was performed using electrospray ionization (ESI) source under multiple reaction monitoring (MRM) in positive ionization mode. The method was fully validated, and the result of inter-assay and intra-assay precisions were less than 15% and the accuracy within the scope of ±15%. The linear range for plasma and urine covered from 10 to 10,000 ng/mL and 1 to 200 ng/mL, respectively, with correlation coefficients of 0.99. The validation for matrix effect, recovery, stability and carryover were met the acceptance criteria. The method showed robust and sensitive, it successfully fulfilled the requirement of clinical pharmacokinetic study of lifirafenib in Chinese patients with locally advanced or metastatic solid tumors.

Early metabolic characterization of brain tissues after whole body radiation based on gas chromatography-mass spectrometry in a rat model.

Radiation-induced brain injury involves acute, early delayed and late delayed damage based on the time-course and clinical manifestations. The acute symptoms are mostly transient and reversible, whereas the late delayed radiation-induced changes are progressive and irreversible. Therefore, evaluation of the organ-specific early response to ionizing radiation exposure is necessary for improving treatment strategies and minimizing possible damage at an early stage after radiation exposure. In the current study, the gas chromatography-mass spectrometry technique based on metabolomics coupled with metabolic correlation network was applied to investigate the early metabolic characterization of rat brain tissues following irradiation. Our findings showed that the metabolic response to irradiation was not just limited to the variations of individual metabolite levels, but also accompanied by alterations of network correlations among various metabolites. Metabolite clustering indicated that energy metabolism disorder and inflammation response were induced following radiation exposure. The correlation networks revealed that the strong positive correlations of differential metabolites were highly reduced and significant negative linkages were highlighted in irradiated groups even without statistical changes in metabolic levels. Our findings provided new insights into our understanding of the radiation-induced acute brain injury mechanism and clues as to the therapy target for clinical applications.

Metabolites characterization of a novel DPP-4 inhibitor, imigliptin in humans and rats using ultra-high performance liquid chromatography coupled with synapt high-resolution mass spectrometry.

Imigliptin has been reported as a novel dipeptidyl-peptidase-IV (DPP-4) inhibitor to treat type 2 Diabetes Mellitus (T2DM), and is currently being tested in clinical trials. In the first human clinical study, imigliptin was well tolerated and proved to be a potent DPP-4 inhibitor. Considering its potential therapeutic benefits and promising future, it is of great importance to study the metabolite profiles in the early stage of drug development. In the present study, a robust and reliable analytical method based on the ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method combined with MassLynx software was established to investigate the characterization of metabolites of imigliptin in human and rat plasma, urine and feces after oral administration. As a result, a total of 9 metabolites were identified in humans, including 6, 9 and 8 metabolites in human plasma, urine, and feces, respectively. A total of 11 metabolites were identified in rats, including 7, 10 and 8 metabolites in rat plasma, urine, and feces, respectively. In addition, 6 of the metabolites detected in humans and rats were phase I metabolites, including demethylation, carboxylation, hydroxylation and dehydrogenation metabolites, and 5 of the metabolites were phase II metabolites, including acetylation and glucuronidation. There was no human metabolite detected compared to those in rats. The major metabolites detected in human plasma (M1 and M2) were products resulting from acetylation, and hydroxylation followed by dehydrogenation. M1 was the major metabolite in rat plasma. M2 and the parent drug were the major drug-related substances in human urine. The parent drug was the major drug-related substances in rat urine. M2, M5 (hydroxylation product) and M6 (2 × hydroxylation and acetylation product) were the predominant metabolites in human feces. M2 and M5 were the major metabolites in rat feces. In addition, renal clearance was the major route of excretion for imigliptin.