Simultaneous quantification of four hormone therapy drugs by LC-MS/MS: Clinical applications in breast cancer patients.

INTRODUCTION: Aromatase inhibitors such as anastrozole, letrozole, exemestane and selective estrogen down-regulator (SERD) fulvestrant are used mostly to treat breast cancer estrogen receptor positive in post-menopausal women. These drugs are given either through the oral route or by intramuscular injection. They have shown great inter-individual variability with a risk of cardiometabolic disorders. Hence the importance of their therapeutic drug monitoring not only for exposure-efficacy but also exposure-toxicity. We describe here a LC-MS/MS method for the simultaneous quantification of anastrozole, letrozole, exemestane and fulvestrant in human plasma. MATERIAL AND METHODS: Plasma samples were prepared by a single-step protein precipitation. The liquid chromatography system was paired with a triple quadrupole mass spectrometer. Quantification were achieved in Multiple Reactions Monitoring mode and the electrospray ionization was in positive mode. RESULTS: The method demonstrated consistent analytical performance across various parameters, including linearity, specificity, sensitivity, matrix effect, upper and lower limits of quantification, extraction recovery, precision, accuracy, hemolysis effect, dilution integrity, and stability under different storage conditions, in accordance with established guidelines. The analysis time for each run was 4 min. Calibration curves exhibited linearity within the 1-100 ng/mL range, with correlation coefficients > 0.99 for the four analytes. Plasma concentrations from 42 patients were integrated into the selected calibration. Stability assessments indicated that the four drugs remained stable at - 20 °C for three months, 15 days under refrigeration, up to 7 days at room temperature, and after three freeze-thaw cycles. CONCLUSION: We have developed and validated this quantitative method for therapeutic drug monitoring of those four hormone therapy drugs:anastrozole, letrozole, fulvestrant and exemestane. This method can be also used for future clinical pharmacokinetics /pharmacodynamics studies.

Determination of plasma concentration of Belimumab by LC-MS/MS: Method development, validation, and clinical application.

INTRODUCTION: Belimumab is a monoclonal antibody against B cell activating factor (BLyS). This monoclonal antibody (mAb) has been shown to be effective in reducing disease activity in patients with systemic lupus erythematosus (SLE). Belimumab is available in two forms as a lyophilized powder for intravenous (IV) use, or single-dose syringe for subcutaneous (SC) use. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of belimumab in human serum. MATERIAL AND METHODS: All analyses relied on nano-surface and molecular-orientation limited (nSMOL) proteolysis coupled with LC-MS/MS. Quantifications was performed in multiple reactions monitoring (MRM) mode, and electrospray ionization was conducted in positive mode. RESULTS: Belimumab was quantified with signature peptide QAPGQGLEWMGGIPFGTAK and normalized using P14R. The total run time per assay was 10 min. Linearity was measured from 5 to 800 µg/mL (r² > 0.995). Accuracy and precision based on three quality control levels range from 11.2 - 9.51 % and 1.24 - 13.12 % respectively. The carryover was less than 7 %. In all, 87 patient samples were processed (65, IV; 22, SC). Mean concentration of belimumab was significantly higher for SC (93.0 ± 74.0 µg/mL) than for IV (67.4 ± 38.9 µg/mL) administration. CONCLUSION: We have developed the first method of belimumab quantification combining LC-MS/MS and nSMOL proteolysis. It can be used for future clinical pharmacokinetic studies of belimumab and for investigating the relationship between belimumab concentration, efficacy, and toxicity in SLE patients.

Total and Unbound Pharmacokinetics of Cefiderocol in Critically Ill Patients.

BACKGROUND: Cefiderocol is a siderophore cephalosporin antibiotic active against Gram-negative bacteria, including extended-spectrum beta-lactamase and carbapenemase-producing strains. The pharmacokinetics of cefiderocol has been studied in healthy subjects and particularly in phase II and III studies. This retrospective study investigated intravenous cefiderocol population pharmacokinetics in adult patients treated by cefiderocol. METHODS: We studied 55 consecutive patients hospitalized in an intensive care unit. Cefiderocol plasma samples were obtained on different occasions during treatment. Plasma concentration was assayed using mass spectrometry. Data analysis was performed using a non-linear mixed-effect approach via Monolix 2020R1. RESULTS: A total of 205 plasma samples were obtained from 55 patients. Eighty percent of patients received cefiderocol for ventilator-associated pneumonia due to carbapenem-resistant Pseudomonas aeruginosa infection. Cefiderocol concentration time-courses were best fit to a two-compartment open model with first-order elimination. Elimination clearance was positively related to renal function (estimated by the CKD formula). Adding albumin plasma binding in the model significantly improved the model assuming a ~40% unbound drug fraction given a ~40 g/L albuminemia. The final model included CKD plus cefiderocol plasma binding effects. Fat-free mass was better than total body weight to influence, via the allometric rule, clearance and volume terms, but this effect was negligible. The final clearance based on free circulating drug (CLU) for a typical patient, CKD = 90, was 7.38 L/h [relative standard error, RSE, 22%] with a between-subject variability of 0.47 [RSE 10%] (exponential distribution). CONCLUSION: This study showed that albumin binding and CKD effects were significant predictors of unbound and total plasma cefiderocol concentrations. Our results indicate that individual adjustment of cefiderocol can be used to reach high minimum inhibitory concentrations based on an estimation of unbound drug concentration and optimize therapeutic efficacy.

Ciprofloxacin population pharmacokinetics during long-term treatment of osteoarticular infections.

BACKGROUND: Ciprofloxacin is an antibiotic used in osteoarticular infections owing to its very good bone penetration. Very few pharmacokinetic data are available in this population. OBJECTIVES: To investigate oral ciprofloxacin population pharmacokinetics in adult patients treated for osteoarticular infections and propose guidance for more effective dosing. METHODS: A retrospective population-pharmacokinetic analysis was performed on 92 consecutive hospitalized patients in the orthopaedic department. Ciprofloxacin plasma samples were obtained on one or two occasions during treatment. Plasma concentration was measured using ultra-performance liquid chromatography system coupled with tandem mass spectrometry. Data analysis was performed using a non-linear mixed-effect approach via Monolix 2019R2. RESULTS: A total of 397 plasma samples were obtained with 11.5% and 41.6% of patients being below the therapeutic target for Gram-negative and staphylococcal infections, respectively. Ciprofloxacin pharmacokinetics were best described by a two-compartment model with a first-order absorption. Ciprofloxacin apparent plasma clearances and volumes of distribution were dependent on patients' fat-free mass according to the allometric rule. Elimination clearance was also positively related to renal function through the modification of diet in renal disease equation (MDRD) and rifampicin co-administration. When patients are co-treated with rifampicin, ciprofloxacin dosage should be increased by 50% to 60%. CONCLUSIONS: This study showed that free-fat mass was a better size predictor than total body weight for ciprofloxacin clearance and volumes terms. Moreover, both MDRD and rifampicin status were significant predictors of individual ciprofloxacin clearance. Our study suggests that individual adjustment of ciprofloxacin dose in osteoarticular infections with less-susceptible bacteria might be indicated to reach required efficacy targets.

Simple and accurate quantitative analysis of cefiderocol and ceftobiprole in human plasma using liquid chromatography-isotope dilution tandem mass spectrometry: interest for their therapeutic drug monitoring and pharmacokinetic studies.

OBJECTIVES: Cefiderocol and ceftobiprole are new generation cephalosporin antibiotics that exhibit high inter-individual plasma concentration variability that potentially impact their efficacy or toxicity. The aim of this study was to develop and validate a selective, simple, and fast UPLC-MS/MS method for simultaneous quantification of cefiderocol and ceftobiprole in human plasma to enable their therapeutic drug monitoring (TDM) and support PK and PK/PD studies, in particular in critically ill patients. METHODS: After a simple and fast single-step protein precipitation, cefiderocol and ceftobiprole were separated on a Waters Acquity UPLC BEH C18 column by linear gradient elution; with subsequent detection by Shimadzu MS 8060 triple quadrupole tandem mass spectrometer in a positive ionization mode. RESULTS: Analysis time was 5 min per run. The analytical performance of the method in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect (ME), extraction recovery (ER), limit of quantification, dilution integrity, and stability of analytes under different conditions met all criteria for a bioanalytical method for the quantification of drugs. The calibration curves were linear over the range of 1-200 mg/L for cefiderocol and 0.5-100 mg/L for ceftobiprole with a linear regression coefficient above 0.995 for both. CONCLUSIONS: A simple, fast, and selective liquid chroma-tography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of cefiderocol and ceftobiprole. This new method was successfully applied to the measurement of plasma concentration of cefiderocol and ceftobiprole in critically ill patients and showed good performance for their therapeutic monitoring and optimizing antibiotic therapy.

UPLC-MS/MS method for the simultaneous quantification of bictegravir and 13 others antiretroviral drugs plus cobicistat and ritonavir boosters in human plasma.

A sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for 14 antiretroviral drugs and 2 boosters in human plasma. Plasma (100 µL) was precipitated with a solution of acetonitrile containing labelled internal standards. The compounds were separated with a total chromatic run time of 6 min using an Acclaim TM RSLC 120 C18 column (2.1 × 100 mm, 2.2 µm). The method was fully validated according to the European Medecines Agency guidelines. Linearity of all analytes concentrations was validated up to 5000 ng/mL. Lower limits of quantification were ranged from 2.5 ng/mL to 10 ng/mL according to compounds. Intra-day and inter-day precision ranged from 0.2% to 8.9% and accuracies were below 13%. This UPLC-MS/MS method can be applied to clinical pharmacology research and therapeutic drug monitoring in patients living with HIV.

Lithium effects on serine-threonine kinases activity: High throughput kinomic profiling of lymphoblastoid cell lines from excellent-responders and non-responders bipolar patients.

Objectives: Lithium is the leading mood stabiliser for maintenance treatment in bipolar disorder (BD). However, response to lithium is heterogeneous with more than 60% of patients experiencing partial or no response. In vitro and in vivo molecular studies have reported the implication of kinases in the pathophysiology of BD.Methods: Since kinases are putative targets for lithium therapeutic action, we conducted the first pilot study using kinase array technology to evaluate the global serine/threonine kinases (STK) profiles in cell lines from BD I subtype patients classified as lithium excellent-responders (ER) and non-responder (NR) to lithium treatment.Results: We found significant differences in the basal STK profiles between ER and NR to lithium. We also tested lithium influence on the global STK profile and found no significant difference between ER vs NR cell lines.Conclusions: The results obtained in this exploratory study suggest that multiplex kinase activity profiling could provide a complementary approach in the study of biomarkers of therapeutic response in BD.

Differential Kinase Activation in Peripheral Blood Mononuclear Cells from Non-Small-Cell Lung Cancer Patients Treated with Nivolumab.

In the era of precision medicine, research of biomarkers for identification of responders to nivolumab therapy is a major challenge. Peripheral blood mononuclear cells (PBMC) could be an interesting surrogate tissue for identifying pharmacodynamic biomarkers. The aim of this exploratory study was to investigate the global serine/threonine kinase (STK) activity in PBMC from non-small-cell lung cancer (NSCLC) patients using a high throughput kinomic profiling method. PamChip® microarrays were used to explore the STK kinomic profile in PBMC from 28 NSCLC patients before nivolumab initiation (D0) and on day 14 (D14) of the first administration. Two clusters of patients (A and B) were identified at D0, median overall survival (OS) tended to be longer in cluster A than in B (402 vs. 112.5 days, respectively; p = 0.15). Interestingly, the PD-L1 tumor cell score (p = 0.045), the count of CD8+ cells (p = 0.023) and the total body weight (p = 0.038) were statistically different between the clusters. On D14, clusters C and D were identified. Greater activity of most STK, especially those of the PI3K/Akt signaling pathway, was noticed among cluster C. No significant difference between C and D was observed regarding OS. Considering the small number of patients, results from this preliminary study are not conclusive. However, the 4-fold longer median OS in cluster A paves the way to further investigate, in a larger cohort of NSCLC patients, the benefit of basal STK kinomic profile in PBMC to identify responders to nivolumab therapy.

Pharmacokinetics and Pharmacodynamics of Once-daily Prolonged-release Tacrolimus in Liver Transplant Recipients.

PURPOSE: Limited published data are available regarding the pharmacokinetic (PK) and pharmacodynamic (PD) variables of prolonged-release tacrolimus (PRT) after liver transplantations. The goal of this study was to compare the PK and PD profiles of PRT in early and stable liver transplant recipients by developing a population PK model of PRT and investigating the profile of calcineurin activity (CNA) in the peripheral blood mononuclear cells. METHODS: A conversion from BID immediate-release tacrolimus (IRT) to once-daily PRT based on a one-to-one daily dose was performed at day 7 (D7) and D90 posttransplantation in groups A (n = 12) and B (n = 12), respectively. Extensive PK samplings, including whole-blood tacrolimus (TAC) concentration, and CNA assessments were performed at D14 and D104 in groups A and B, respectively. TAC concentration-time data (N = 221) were analyzed by using nonlinear mixed effects modeling. FINDINGS: A 2-compartment model with linear elimination and a delayed first-order absorption characterized by 2 transit compartments best described the PK data. Model-predicted dose-normalized (6.0 mg/d) area under the TAC concentration-time curve over the dosing interval in groups A and B was similar (geometric mean, 235.6 ng/mL · h [95% CI, 139.6-598.7] vs 224.6 ng/mL · h [95% CI, 117.6-421.5], respectively; P = 0.94). Area under the CNA versus time curve over the dosing interval did not differ between groups (4897 [3437] and 4079 [1008] pmol/min/106 cells; P = 0.50). In group A, trough CNA at D14 posttransplantation was statistically higher than that measured just before the switch to PRT (ie, D7 posttransplantation) (198 [92] vs 124 [72] pmol/min/106cells, n = 8; P = 0.048); no statistical difference in TAC concentration was observed (P = 0.11). In group B, no statistical difference between D90 and D104 was observed in either trough CNA (149 [78] vs 172 [82] pmol/min/106 cells, n = 6; P = 0.18) or TAC (P = 0.17) concentration. No graft rejection was observed in either of the groups. IMPLICATIONS: This study suggests that one-to-one dosage conversion to once-daily PRT during the early posttransplantation period could result in significant CNA variations but without causing graft rejection. Further investigations in larger cohorts are warranted to confirm these results. ClinicalTrials.gov identifier: NCT02105155.

Development and Validation of a Fast Ultra-High Performance Liquid Chromatography-Fluorescent Method for the Quantification of Hydroxychloroquine and Its Metabolites in Patients With Lupus.

BACKGROUND: Hydroxychloroquine (HCQ) is approved for the treatment of systemic lupus erythematosus (SLE). Therapeutic drug monitoring of HCQ is necessary to detect nonadherence and to improve treatment efficacy in patients with SLE. Liquid chromatographic-tandem mass spectroscopy and high performance liquid chromatography (HPLC)-fluorescent methods are currently used to measure whole blood concentrations of HCQ and its 2 main metabolites desethylhydroxychloroquine and desethylchloroquine in patients with SLE. This study reports the development and validation of an ultra-HPLC (U-HPLC) method with fluorescence detection for the simultaneous quantification of HCQ and its metabolites in whole blood. METHODS: After adding chloroquine (internal standard) to the samples, a single-step protein precipitation and a subsequent filtration were used for blood sample preparation. Analytes were separated under isocratic elution on a U-HPLC RP18 column with a total run time of 7 minutes. The mobile phase consisted of piperazine buffer (46.4 mM, pH = 9.8) and acetonitrile (68:32, vol/vol), which was delivered at a flow rate of 0.4 mL/min. Fluorescence excitation and emission wavelengths were 335 and 390 nm, respectively. Assay performance parameters were evaluated per FDA bioanalytical guidelines. RESULTS: The calibration curve was linear from 125 to 4000 ng/mL for HCQ. The lower limit of quantification was 10 ng/mL for all analytes. For HCQ, desethylchloroquine, and desethylhydroxychloroquine, accuracies and imprecisions ranged from -7.90% to 7.85% and 1.14% to 8.78%, respectively. CONCLUSIONS: A sensitive, accurate, and fast U-HPLC-fluorescent method was validated and successfully applied to quantify whole blood concentrations to perform therapeutic drug monitoring of HCQ in pediatric and adult lupus patients.

Clinical Pharmacokinetics and Pharmacodynamics of Dabrafenib.

Dabrafenib is a potent and selective inhibitor of BRAF-mutant kinase that is approved, as monotherapy or in combination with trametinib (mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor), for unresectable or metastatic BRAF-mutated melanoma, advanced non-small cell lung cancer and anaplastic thyroid cancer harbouring the BRAFV600E mutation. The recommended dose of dabrafenib is 150 mg twice daily (bid) under fasted conditions. After single oral administration of the recommended dose, the absolute oral bioavailability (F) of dabrafenib is 95%. Dabrafenib shows a time-dependent increase in apparent clearance (CL/F) following multiple doses, which is likely due to induction of its own metabolism through cytochrome P450 (CYP) 3A4. Therefore, steady state is reached only after 14 days of daily dose administration. Moreover, the extent of this auto-induction process is dependent on the dose, which explains why dabrafenib systemic exposure at steady state increases less than dose proportionally over the dose range of 75-300 mg bid. The main elimination route of dabrafenib is the oxidative metabolism via CYP3A4/2C8 and biliary excretion. Among the three major metabolites identified, hydroxy-dabrafenib appears to contribute to the pharmacological activity. Age, sex and body weight did not have any clinically significant influence on plasma exposure to dabrafenib. No dose adjustment is needed for patients with mild renal or hepatic impairment, whereas the impacts of severe impairment on dabrafenib pharmacokinetics remain unknown. Considering that dabrafenib is a substrate of CYP3A4/2C8 and is a CYP3A4/2B6/2C inducer, drug-drug interactions are expected with dabrafenib. The relationship between clinical outcomes and plasma exposure to dabrafenib and hydroxy-dabrafenib should be investigated more deeply.

Circulating Tumor DNA Measurement by Picoliter Droplet-Based Digital PCR and Vemurafenib Plasma Concentrations in Patients with Advanced BRAF-Mutated Melanoma.

BACKGROUND: Circulating tumor DNA (ctDNA) has been reported as a prognostic marker in melanoma. In BRAF V600-mutant melanoma, a plasma under-exposure to vemurafenib could favor emerging resistance but no biological data are available to support this hypothesis. OBJECTIVE: We aimed to investigate the relationship between vemurafenib plasma concentrations and the ctDNA plasma concentration during follow-up of BRAF-mutated melanoma patients. PATIENTS AND METHODS: Eleven patients treated with single-agent vemurafenib for advanced BRAF V600-mutant melanoma were analyzed in an exploratory monocentric study. The vemurafenib plasma concentration was measured by liquid chromatography. ctDNA was extracted from plasma samples and the ctDNA concentration was evaluated using picoliter droplet-based digital PCR with Taqman® detection probes targeting the BRAF p.V600E/K mutation and wild-type BRAF sequences. RESULTS: At baseline, plasma ctDNA was detectable in 72% (n = 8/11) of patients and the ctDNA concentration decreased in 88% of these patients (n = 7/8) from day (D) 0 to D15 after vemurafenib initiation. During follow-up, an increased ctDNA concentration was detected in nine patients: in five patients, the first increase in ctDNA concentrations followed a decrease in vemurafenib concentrations. More interestingly, an inverse correlation between vemurafenib concentration and ctDNA concentrations was demonstrated (p = 0.026). The ctDNA concentration at baseline was associated with overall survival (hazard ratio = 2.61, 95% CI 1.04-6.56; p = 0.04). CONCLUSIONS: This study demonstrates the relevance of vemurafenib plasma monitoring during the follow-up of metastatic melanoma patients. Plasma drug monitoring and ctDNA concentrations could be combined to monitor tumor evolution in melanoma patients treated with anti-BRAF therapies.

A simple HPLC-UV method for quantification of enzalutamide and its active metabolite N-desmethyl enzalutamide in patients with metastatic castration-resistant prostate cancer.

Enzalutamide is currently approved for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC). To date, a single liquid chromatographic-tandem mass spectroscopy method is available to measure plasma enzalutamide concentrations in mCRPC patients. In this work, an accurate and sensitive HPLC-UV method has been developed for the simultaneous determination of enzalutamide and its active metabolite, N-desmethyl enzalutamide in plasma from mCRPC patients. Before precipitation of proteins with acetonitrile, samples were spiked with nilutamide (internal standard). Separation of analytes was achieved under isocratic elution on a C18 Kinetex column. The mobile phase consisted of a mixture of ammonium acetate buffer (pH=4.6, 20mM) and acetonitrile (60:40, v/v), and was delivered at a flow rate of 1.5mL/min throughout a 9-min run. UV detection was performed at 270nm. The method was linear over a concentration range of 0.50-50.0µg/mL for both analytes. Within- and between-day imprecision and accuracy were ≤10% at concentrations 0.75, 5.00, and 50.0µg/mL. This method has been implemented to assay steady-state trough plasma concentrations (n=30) of enzalutamide and N-desmethyl enzalutamide in 16 mCRPC patients. Overall, this HPLC-UV method is well-suited for routine application in clinical laboratories to perform therapeutic drug monitoring of enzalutamide in mCRPC patients.

Potential drug-drug interactions with abiraterone in metastatic castration-resistant prostate cancer patients: a prevalence study in France.

PURPOSE: Abiraterone acetate combined with prednisone improves survival in metastatic castration-resistant prostate cancer (mCRPC) patients. This oral anticancer agent may result in drug-drug interactions (DDI). We aimed to evaluate the prevalence of DDI with abiraterone and the possible determinants for the occurrence of these DDI. METHODS: We performed a single centre retrospective review from electronic medical records of mCRPC patients treated with abiraterone from 2011 to 2015. Potential DDI with abiraterone were identified using Micromedex and were categorized by a 4-point scale severity. RESULTS: Seventy-two out of ninety-five mCRPC pts (median age: 77 years [68-82]) had comorbidities. The median number of drugs used per patient was 7 [5-9]. 66 potential DDI with abiraterone were detected in 49 patients (52%): 39 and 61% were classified as major and moderate DDI, respectively. In the univariate analysis, pain (p < 0.0001), hypo-albuminemia (p = 0.032), and higher ECOG performance status (PS) (p = 0.013) were significantly associated with a higher risk of DDI with abiraterone. Pain (p < 0.0001) and PS (p = 0.018) remained significant in the multivariate analysis. CONCLUSIONS: Polypharmacy is an issue among mCRPC patients. In our study, half of the patients have potential DDI with abiraterone. Patients with pain and poor PS are at higher risk of DDI with abiraterone. A medication review by a pharmacist is of crucial importance to prevent DDI with abiraterone.

Development and validation of an ELISA method for the quantification of nivolumab in plasma from non-small-cell lung cancer patients.

Nivolumab, an anti PD-1 monoclonal antibody, has been approved for the treatment of previously treated advanced or metastatic non-small-cell lung cancer (NSCLC). The aim of this study was to develop and validate an ELISA method for the quantification of nivolumab in plasma from patients with NSCLC in order to perform future pharmacokinetic/pharmacodynamic (PK/PD) studies. A home-made ELISA was developed and validated according to the general recommendations for the immunoassays. Then, the ELISA method was applied to quantify plasma trough levels (Cmin) of nivolumab (3mg/kg every two weeks) in 27 NSCLC patients at days 14, 28 and 42 after start of treatment. Blood samples were collected just before the infusion on days 0 (baseline), 14, 28 and 42 after start of treatment. The dynamic calibration range for nivolumab assay was 5-100µg/mL. Within- and between-day imprecision for quality controls (5, 20 and 75µg/mL) were less than 5 and 12%, respectively. The mean (±standard deviation) nivolumab Cmin was 17.3±4.8µg/mL (coefficient of variation, CV=27.8%), 25.0±9.7µg/mL (CV=38.8%) and 33.0±12.9µg/mL (CV=39.1%) on days 14, 28 and 42, respectively. IgG (p=0.002) and ALT (p=0.041) were independently associated with plasma nivolumab Cmin at day 42. The present ELISA method for quantification of nivolumab in plasma from NSCLC patients is sensitive and accurate enough to be used for further PK/PD investigations.

Correlation between clinical response to sorafenib in hepatocellular carcinoma treatment and polymorphisms of P-glycoprotein (ABCB1) and of breast cancer resistance protein (ABCG2): monocentric study.

OBJECTIVES: We studied the relation between the polymorphism of P-glycoprotein (P-gp) and of breast cancer resistance protein (BCRP), encoded by ABCB1 and ABCG2 genes, respectively, and the pharmacokinetic variability and clinical response during the treatment with sorafenib of hepatocellular carcinoma. METHODS: At the Paul Brousse Hospital in Villejuif, France, 47 consecutive patients with advanced HCC treated with a single agent sorafenib, were enrolled. Sorafenib exposure was measured by its plasma concentration 3 h after oral administration of 400 mg (bid) by liquid chromatography. All enrolled patients were genotyped for ABCB1 (rs2032582; rs1045642) and ABCG2 (rs2231137; rs2231142; rs2622604) by blood genomic DNA extraction and Mass ARRAY genotyping. The clinical response was evaluated after 3months of treatment according to the RECIST criteria. KEY FINDINGS: Significant associations between sorafenib exposure and the studied polymorphisms were observed for ABCB1 3435C>T, ABCG2 34G>A, ABCG2 1143C>T and ABCG2 421C>A, but not for ABCB1 2677G>TA SNP. In heterozygous patients for ABCB1 3435 C>T, ABCG2 34 G>A and ABCG2 1143 C>T polymorphisms were significantly associated with the lowest sorafenib plasma levels. Those patients presented a tendency to have the best clinical evolution. CONCLUSION: Heterozygous forms of the studied polymorphisms could be associated with a better therapeutic response.

Dimethyl Fumarate Controls the NRF2/DJ-1 Axis in Cancer Cells: Therapeutic Applications.

The transcription factor NRF2 (NFE2L2), regulates important antioxidant and cytoprotective genes. It enhances cancer cell proliferation and promotes chemoresistance in several cancers. Dimethyl fumarate (DMF) is known to promote NRF2 activity in noncancer models. We combined in vitro and in vivo methods to examine the effect of DMF on cancer cell death and the activation of the NRF2 antioxidant pathway. We demonstrated that at lower concentrations (<25 µmol/L), DMF has a cytoprotective role through activation of the NRF2 antioxidant pathway. At higher concentrations, however (>25 µmol/L), DMF caused oxidative stress and subsequently cytotoxicity in several cancer cell lines. High DMF concentration decreases nuclear translocation of NRF2 and production of its downstream targets. The pro-oxidative and cytotoxic effects of high concentration of DMF were abrogated by overexpression of NRF2 in OVCAR3 cells, suggesting that DMF cytotoxicity is dependent of NRF2 depletion. High concentrations of DMF decreased the expression of DJ-1, a NRF2 protein stabilizer. Using DJ-1 siRNA and expression vector, we observed that the expression level of DJ-1 controls NRF2 activation, antioxidant defenses, and cell death in OVCAR3 cells. Finally, antitumoral effect of daily DMF (20 mg/kg) was also observed in vivo in two mice models of colon cancer. Taken together, these findings implicate the effect of DJ-1 on NRF2 in cancer development and identify DMF as a dose-dependent modulator of both NRF2 and DJ-1, which may be useful in exploiting the therapeutic potential of these endogenous antioxidants. Mol Cancer Ther; 16(3); 529-39. ©2017 AACR.

Plasma vemurafenib exposure and pre-treatment hepatocyte growth factor level are two factors contributing to the early peripheral lymphocytes depletion in BRAF-mutated melanoma patients.

The therapeutic response to vemurafenib, a BRAF serine-threonine kinase inhibitor, exhibits large variations between patients. Evaluation of factors predicting the clinical efficacy of vemurafenib may help to identify patients at high risk of non-response in the early phase of treatment. The aim of this study was to analyze the pharmacokinetics of vemurafenib by a population approach and to evaluate the relationship between plasma drug exposure and pre-treatment plasma hepatocyte growth factor (HGF) levels with clinical effects (progression-free survival (PFS), peripheral lymphocytes depletion) in patients with metastatic BRAFV600 mutated melanoma treated with single agent vemurafenib. Concentration-time data (n=332) obtained in 44 patients were analyzed using the NONMEM program. Pre-treatment plasma levels of HGF (n=36) were assayed by ELISA method. A Cox model was used to identify prognostic factors associated with progression-free survival (PFS), and a linear regression to identify factors contributing to the depletion of peripheral lymphocytes at day 15. Steady-state pharmacokinetics of vemurafenib was described by a one compartment model with first order absorption and first order elimination. None of the tested covariates explained the inter-patient variability in CL/F. A significant decrease in total lymphocytes count was observed within the first 15days (median ratio Day15/Day0=0.66, p<0.0001). Patients with Day15/Day0 ratio below 0.66 had longer PFS (14 vs 4 months, HR=0.41, CI95%=[0.15-0.77], p=0.0095). In the multivariate Cox model analysis, ECOG PS was the only parameter independently associated with PFS (grade 1 vs 0, HR=3.26, CI95%=[1.29-8.22], p=0.01 and grade ≥2 vs 0, HR=4.77, CI95%=[1.52-14.95], p=0.007). Plasma vemurafenib exposure (p=0.046) and pre-treatment HGF levels (p=0.003) were independently associated with the total lymphocyte ratio Day15/Day0. These findings show that plasma vemurafenib exposure and pre-treatment HGF levels are two factors contributing to the early peripheral lymphocytes depletion which itself is associated with PFS.

TGF-ß2 induces Grb2 to recruit PI3-K to TGF-RII that activates JNK/AP-1-signaling and augments invasiveness of Theileria-transformed macrophages.

Theileria-infected macrophages display many features of cancer cells such as heightened invasive capacity; however, the tumor-like phenotype is reversible by killing the parasite. Moreover, virulent macrophages can be attenuated by multiple in vitro passages and so provide a powerful model to elucidate mechanisms related to transformed macrophage virulence. Here, we demonstrate that in two independent Theileria-transformed macrophage cell lines Grb2 expression is down-regulated concomitant with loss of tumor virulence. Using peptidimer-c to ablate SH2 and SH3 interactions of Grb2 we identify TGF-receptor II and the p85 subunit of PI3-K, as Grb2 partners in virulent macrophages. Ablation of Grb2 interactions reduces PI3-K recruitment to TGF-RII and decreases PIP3 production, and dampens JNK phosphorylation and AP-1-driven transcriptional activity down to levels characteristic of attenuated macrophages. Loss of TGF-R>PI3-K>JNK>AP-1 signaling negatively impacts on virulence traits such as reduced JAM-L/ITG4A and Fos-B/MMP9 expression that contribute to virulent macrophage adhesion and invasiveness.