Pharmacodynamic biomarkers research in mononuclear peripheral blood cells in cancer patients treated with sunitinib or nivolumab, using a kinomic approach

L'identification de biomarqueurs prédisant la réponse au traitement est essentielle en cancérologie. La recherche de tels marqueurs doit être simple et peu invasive pour le patient. Les cellules mononucléées du sang périphérique (CMSP) constituent donc une matrice de choix. L'objectif de cette thèse a été d'évaluer les CMSP comme matrice biologique pour la recherche de biomarqueurs par approche kinomique chez des patients atteints de carcinome rénal métastatique (CCRm) ou de cancer bronchique non-à petites cellules (CBNPC), traités respectivement par sunitinib ou nivolumab. Chez 21 patients atteints de CCRm, le sunitinib et son métabolite actif inhibaient ex vivo de façon variable la majorité des tyrosine kinases. Les profils kinomiques étaient associés au score pronostic de Heng et au ratio J21/J0 de lymphocytes totaux après début de traitement. Chez 28 patients atteints CBNPC, deux populations distinctes ont été identifiées, après 15 jours de traitement par nivolumab, en fonction du profil kinomique des Sérine/Thréonine kinases. Ces deux profils étaient associés au ratio lymphocytaire CD4/CD8 à J15, traduisant ainsi l'impact du nivolumab sur ces sous-types lymphocytaires. Ce travail a permis de valider l'utilisation de CMSP pour la future recherche de biomarqueurs pharmacodynamiques par approche kinomique chez des patients traités par sunitinib ou nivolumab.Biomarker identification is essential in oncology to predict treatment response. The research of such markers should be simple and none invasive for the patient. Therefore, peripheral blood mononuclear cells (PBMC) could be a good biological matrix. The objective of this thesis was to evaluate PBMC as a biological matrix for biomarker research using a kinomic approach in metastatic renal cell carcinoma (mRCC) or lung non-small cell cancer (NSCLC) patients, treated with sunitinib or nivolumab, respectively. In 21 mRCC patients, sunitinib and its active metabolite inhibited ex vivo most of tyrosine kinases with a large interindividual variability. Kinomic profiles were associated with Heng prognostic score and the D21/D0 total lymphocytes ratio after treatment start. In 28 NSCLC patients, two distinct populations were identified after 15 days of treatment with nivolumab, based on the kinomic profile of Serine / Threonine kinases. These two profiles were associated with CD4/CD8 lymphocyte ratio at D15, reflecting the nivolumab effect on lymphocyte subsets. This work suggests that PBMC can be used to seek for future pharmacodynamic biomarkers, using a kinomic approach in patients under sunitinib or nivolumab therapy

Recherche de biomarqueurs pharmacodynamiques dans des cellules mononucléées du sang périphérique, chez des patients atteints de cancers traités par sunitinib ou nivolumab : intérêt de l’approche kinomique

Biomarker identification is essential in oncology to predict treatment response. The research of such markers should be simple and none invasive for the patient. Therefore, peripheral blood mononuclear cells (PBMC) could be a good biological matrix. The objective of this thesis was to evaluate PBMC as a biological matrix for biomarker research using a kinomic approach in metastatic renal cell carcinoma (mRCC) or lung non-small cell cancer (NSCLC) patients, treated with sunitinib or nivolumab, respectively. In 21 mRCC patients, sunitinib and its active metabolite inhibited ex vivo most of tyrosine kinases with a large interindividual variability. Kinomic profiles were associated with Heng prognostic score and the D21/D0 total lymphocytes ratio after treatment start. In 28 NSCLC patients, two distinct populations were identified after 15 days of treatment with nivolumab, based on the kinomic profile of Serine / Threonine kinases. These two profiles were associated with CD4/CD8 lymphocyte ratio at D15, reflecting the nivolumab effect on lymphocyte subsets. This work suggests that PBMC can be used to seek for future pharmacodynamic biomarkers, using a kinomic approach in patients under sunitinib or nivolumab therapy.L'identification de biomarqueurs prédisant la réponse au traitement est essentielle en cancérologie. La recherche de tels marqueurs doit être simple et peu invasive pour le patient. Les cellules mononucléées du sang périphérique (CMSP) constituent donc une matrice de choix. L'objectif de cette thèse a été d'évaluer les CMSP comme matrice biologique pour la recherche de biomarqueurs par approche kinomique chez des patients atteints de carcinome rénal métastatique (CCRm) ou de cancer bronchique non-à petites cellules (CBNPC), traités respectivement par sunitinib ou nivolumab. Chez 21 patients atteints de CCRm, le sunitinib et son métabolite actif inhibaient ex vivo de façon variable la majorité des tyrosine kinases. Les profils kinomiques étaient associés au score pronostic de Heng et au ratio J21/J0 de lymphocytes totaux après début de traitement. Chez 28 patients atteints CBNPC, deux populations distinctes ont été identifiées, après 15 jours de traitement par nivolumab, en fonction du profil kinomique des Sérine/Thréonine kinases. Ces deux profils étaient associés au ratio lymphocytaire CD4/CD8 à J15, traduisant ainsi l'impact du nivolumab sur ces sous-types lymphocytaires. Ce travail a permis de valider l'utilisation de CMSP pour la future recherche de biomarqueurs pharmacodynamiques par approche kinomique chez des patients traités par sunitinib ou nivolumab

Development and validation of a fast U-HPLC-fluorescent method for the quantification of hydroxychloroquine and its metabolites in patients with lupus.

International audienceBACKGROUND:Hydroxychloroquine (HCQ) is approved for the treatment of systemic lupus erythematosus (SLE). Therapeutic drug monitoring (TDM) of HCQ is necessary to detect non-adherence and to improve treatment efficacy in SLE patients. Liquid chromatographic-tandem mass spectroscopy and HPLC-fluorescent methods are currently used to measure whole blood concentrations of hydroxychloroquine and its two main metabolites desethylhydroxychloroquine (DHCQ) and desethylchloroquine (DCQ) in SLE patients. This study reports the development and validation of an ultra-high performance liquid chromatography (UHPLC) 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 employed for blood sample preparation. Analytes were separated under isocratic elution on a U-HPLC RP18 column with a total run time of seven minutes. The mobile phase consisted of piperazine buffer (46.4 mM, pH=9.8) and acetonitrile (68:32, v/v), 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, DCQ and DHCQ, accuracies and imprecisions ranged from -7.90 to 7.85% and 1.14 to 8.78%, respectively.CONCLUSION:A sensitive, accurate and fast U-HPLC-fluorescent method was validated and successfully applied to quantify whole blood concentrations to perform TDM of HCQ in pediatric and adult lupus patients

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

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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

International audienceObjectives 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

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

International audienceBACKGROUND: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

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

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

International audiencePURPOSE: 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

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

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