RESUMEN
BACKGROUND: Interindividual pharmacokinetic variability may influence the clinical benefit or toxicity of cabozantinib in metastatic renal cell carcinoma (mRCC). We aimed to investigate the exposure-toxicity and exposure-response relationship of cabozantinib in unselected mRCC patients treated in routine care. METHODS: This ambispective multicenter study enrolled consecutive patients receiving cabozantinib in monotherapy. Steady-state trough concentration (Cmin,ss) within the first 3 months after treatment initiation was used for the PK/PD analysis with dose-limiting toxicity (DLT) and survival outcomes. Logistic regression and Cox proportional-hazards models were used to identify the risk factors of DLT and inefficacy in patients, respectively. RESULTS: Seventy-eight mRCC patients were eligible for the statistical analysis. Fifty-two patients (67%) experienced DLT with a median onset of 2.1 months (95%CI 0.7-8.2). In multivariate analysis, Cmin,ss was identified as an independent risk factor of DLT (OR 1.46, 95%CI [1.04-2.04]; p = 0.029). PFS and OS were not statistically associated with the starting dose (p = 0.81 and p = 0.98, respectively). In the multivariate analysis of PFS, Cmin, ss > 336 ng/mL resulted in a hazard ratio of 0.28 (95%CI, 0.10-0.77, p = 0.014). By contrast, Cmin, ss > 336 ng/mL was not statistically associated with longer OS. CONCLUSION: Early plasma drug monitoring may be useful to optimise cabozantinib treatment in mRCC patients treated in monotherapy, especially in frail patients starting at a lower than standard dose.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Anilidas/efectos adversos , Piridinas/efectos adversos , Estudios RetrospectivosRESUMEN
BACKGROUND: Reducing nivolumab dose intensity could increase patients' life quality and decrease the financial burden while maintaining efficacy. The aims of this study were to develop a population PK model of nivolumab based on data from unselected metastatic cancer patients and to simulate extended-interval regimens allowing to maintain minimal effective plasma concentrations (MEPC). METHODS: Concentration-time data (992 plasma nivolumab concentrations, 364 patients) were modeled using a two-compartment model with linear elimination clearance in Monolix software. Extended-interval regimens allowing to maintain steady-state trough concentrations (Cmin,ss) above the MEPC of 2.5 mg/L or 1.5 mg/L in >90% of patients were simulated. RESULTS: Increasing 3-times the dosing interval from 240 mg every two weeks (Q2W) to Q6W and 2-times from 480 mg Q4W to Q8W resulted in Cmin,ss above 2.5 mg/L in 95.8% and 95.4% of patients, respectively. 240 mg Q8W and 480 mg Q10W resulted in Cmin,ss above 1.5 mg/L in 91.0% and 91.8% of patients, respectively. Selection of a 240 mg Q6W regimen would decrease by 3-fold the annual treatment costs compared to standard regimen of 240 mg Q2W (from 78,744 to 26,248 in France). CONCLUSIONS: Clinical trials are warranted to confirm the non-inferiority of extended-interval compared to standard regimen.
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Esquema de Medicación , Neoplasias , Nivolumab , Humanos , Nivolumab/administración & dosificación , Nivolumab/farmacocinética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Femenino , Masculino , Anciano , Persona de Mediana Edad , Simulación por Computador , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/farmacocinética , Adulto , Anciano de 80 o más Años , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/farmacocinética , Modelos BiológicosRESUMEN
BACKGROUND: Lamotrigine is an effective antiseizure medication that can be used in the management of focal and generalized epilepsies in pediatric patients. This study was conducted to quantify and compare the solubility of lamotrigine in age-specific biorelevant media that simulated the fasted and fed conditions of the gastric and intestinal environments in pediatrics and adults. Another aim was to predict how traditional, re-formulated, modified, and new oral formulations would behave in the gastric and intestinal environments across different age groups. METHODS: Solubility studies of lamotrigine were conducted in 16 different age-specific biorelevant media over the pH range and temperature specified by the current biopharmaceutical classification system-based criteria. The age-specific biorelevant media simulated the environments in the stomach and proximal gastrointestinal tract in both fasted and fed conditions of adults and pediatric sub-populations. The solubility of lamotrigine was determined using a pre-validated HPLC-UV method. RESULTS: Lamotrigine showed low solubility in the 16 age-specific biorelevant media as indicated by a dose number of > 1. There were significant age-specific variabilities in the solubility of lamotrigine in the different age-specific biorelevant media. Pediatric/adult solubility ratios of lamotrigine fell outside the 80-125% range in 6 (50.0%) and were borderline in 3 (25.0%) out of the 12 compared media. These ratios indicated that the solubility of lamotrigine showed considerable differences in 9 out of the 12 (75.0%) of the compared media. CONCLUSION: Future studies are still needed to generate more pediatric biopharmaceutical data to help understand the performances of oral dosage forms in pediatric sub-populations.
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Productos Biológicos , Estómago , Adulto , Humanos , Niño , Lamotrigina , Solubilidad , Factores de EdadRESUMEN
The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.
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Barrera Hematoencefálica , Células Endoteliales , Humanos , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Proteómica , Transportadoras de Casetes de Unión a ATP/metabolismo , PermeabilidadRESUMEN
BACKGROUND: Dosing regimens of trastuzumab administered by intracerebroventricular (icv) route to patients with HER2-positive brain localizations remain empirical. The objectives of this study were to describe pharmacokinetics (PK) of trastuzumab in human plasma and cerebrospinal fluid (CSF) after simultaneous icv and intravenous (iv) administration using a minimal physiologically-based pharmacokinetic model (mPBPK) and to perform simulations of alternative dosing regimens to achieve therapeutic concentrations in CSF. METHODS: Plasma and CSF PK data were collected in two patients with HER2-positive brain localizations. A mPBPK model for mAbs consisting of four compartments (tight and leaky tissues, plasma and lymph) was enriched by an additional compartment for ventricular CSF. The comparison between observed and model-predicted concentrations was evaluated using prediction error (PE). RESULTS: The developed mPBPK model described plasma and CSF trastuzumab concentrations reasonably well with mean PE for plasma and CSF data of 41.8% [interquartile range, IQR = -9.48; 40.6] and 18.3% [-36.7; 60.6], respectively, for patient 1 and 11.4% [-10.8; 28.7] and 22.5% [-27.7; 77.9], respectively, for patient 2. Trastuzumab showed fast clearance from CSF to plasma with Cmin,ss of 0.56 and 0.85 mg/L for 100 and 150 mg q1wk, respectively. Repeated dosing of 100 and 150 mg q3day resulted in Cmin,ss of 10.3 and 15.4 mg/L, respectively. Trastuzumab CSF target concentrations are achieved rapidly and maintained above 60 mg/L from 7 days after a continuous perfusion at 1.0 mg/h. CONCLUSION: Continuous icv infusion of trastuzumab at 1.0 mg/h could be an alternative dosing regimen to rapidly achieve intraventricular CSF therapeutic concentrations.
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Anticuerpos Monoclonales , Encéfalo , Humanos , Trastuzumab , Anticuerpos Monoclonales/farmacocinética , Administración Intravenosa , Infusiones IntravenosasRESUMEN
The generation of plausible virtual patients (VPs) is an important step in most quantitative systems pharmacology (QSP) workflows, which requires time-intensive solving of ordinary differential equations (ODEs). However, non-physiological profiles of outputs of interest (OoI) are frequently produced, and additional acceptance/rejection steps are needed for comparing and removing VPs with predicted values outside a pre-defined range. Here, a new approach is developed to accelerate the acceptance/rejection steps by leveraging patterns of parameter associations with OoI. In most models, some parameters are monotonic with respect to OoI, such that an increase in a parameter value always induces an increase or decrease in the OoI. This monotonic property can be used to replace some ODE-solving steps with appropriate monotonic parameter value comparisons to extrapolate the rejection or interpolate the acceptance of some VPs (after simulation) to others. Two algorithms were built that directly extract plausible VPs from a pre-defined initial cohort. These algorithms were first tested using a simple tumor growth inhibition model. Analyzing 200,000 VPs took 50 s with a reference method and 3 to 41 s (depending on the initial set-up) with the first algorithm. The method was then applied to an apoptosis QSP model, in which the clinical phenotypes (i.e., treatment sensitive or resistant) of 200,000 VPs were fully characterized for four different drug regimens in 12 min as compared to over 80 min with the reference approach. Extraction of each phenotype can also be performed individually in 34 s to 8 min, demonstrating the time benefit and flexibility of this approach.
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Algoritmos , Modelos Teóricos , Simulación por Computador , Estudios de CohortesRESUMEN
The interest in therapeutic monoclonal antibodies (mAbs) has continuously growing in several diseases. However, their pharmacokinetics (PK) is complex due to their target-mediated drug disposition (TMDD) profiles which can induce a non-linear PK. This point is particularly challenging during the pre-clinical and translational development of a new mAb. This article reviews and describes the existing PK modeling approaches used to translate the mAbs PK from animal to human for intravenous (IV) and subcutaneous (SC) administration routes. Several approaches are presented, from the most empirical models to full physiologically based pharmacokinetic (PBPK) models, with a focus on the population PK methods (compartmental and minimal PBPK models). They include the translational approaches for the linear part of the PK and the TMDD mechanism of mAbs. The objective of this article is to provide an up-to-date overview and future perspectives of the translational PK approaches for mAbs during a model-informed drug development (MIDD), since the field of PK modeling has gained recently significant interest for guiding mAbs drug development.
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Anticuerpos Monoclonales , Antineoplásicos Inmunológicos , Animales , Humanos , Modelos Biológicos , Distribución Tisular , Inyecciones SubcutáneasRESUMEN
Diffuse intrinsic pontine gliomas (DIPG), the first cause of cerebral pediatric cancer death, will greatly benefit from specific and non-invasive biomarkers for patient follow-up and monitoring of drug efficacy. Since biopsies are challenging for brain tumors, molecular imaging may be a technique of choice to target and follow tumor evolution. So far, MR remains the imaging technique of reference for DIPG, although it often fails to define the extent of tumors, an essential parameter for therapeutic efficacy assessment. Thanks to its high sensitivity, positron emission tomography (PET) offers a unique way to target specific biomarkers in vivo. We demonstrated in a patient-derived orthotopic xenograft (PDOX) model in the rat that the translocator protein of 18 kDa (TSPO) may be a promising biomarker for monitoring DIPG tumors. We studied the distribution of 18F-DPA-714, a TSPO radioligand, in rats inoculated with HSJD-DIPG-007 cells. The primary DIPG human cell line HSJD-DIPG-007 highly represents this pediatric tumor, displaying the most prevalent DIPG mutations, H3F3A (K27M) and ACVR1 (R206H). Kinetic modeling and parametric imaging using the brain 18F-DPA-714 PET data enabled specific delineation of the DIPG tumor area, which is crucial for radiotherapy dose management.
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Astrocitoma , Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Glioma , Niño , Animales , Humanos , Ratas , Glioma/diagnóstico por imagen , Glioma/genética , Glioma/metabolismo , Línea Celular Tumoral , Neoplasias del Tronco Encefálico/diagnóstico por imagen , Neoplasias del Tronco Encefálico/genética , Tomografía de Emisión de Positrones/métodos , Proteínas Portadoras , Modelos Animales de Enfermedad , Biomarcadores , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de GABA-ARESUMEN
Lithium (Li) is the most widely used mood stabilizer in treating patients with bipolar disorder. However, more than half of the patients do not or partially respond to Li therapy, despite serum Li concentrations in the serum therapeutic range. The exact mechanisms underlying the pharmacokinetic-pharmacodynamic (PK-PD) relationships of lithium are still poorly understood and alteration in the brain pharmacokinetics of lithium may be one of the mechanisms explaining the variability in the clinical response to Li. Brain barriers such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a crucial role in controlling blood-to-brain and brain-to-blood exchanges of various molecules including central nervous system (CNS) drugs. Recent in vivo studies by nuclear resonance spectroscopy revealed heterogenous brain distribution of Li in human that were not always correlated with serum concentrations, suggesting regional and variable transport mechanisms of Li through the brain barriers. Moreover, alteration in the functionality and integrity of brain barriers is reported in various CNS diseases, as a cause or a consequence and in this regard, Li by itself is known to modulate BBB properties such as the expression and activity of various transporters, metabolizing enzymes, and the specialized tight junction proteins on BBB. In this review, we will focus on recent knowledge into the role of the brain barriers as key-element in the Li neuropharmacokinetics which might improve the understanding of PK-PD of Li and its interindividual variability in drug response.
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Trastorno Bipolar/tratamiento farmacológico , Encéfalo/metabolismo , Fármacos del Sistema Nervioso Central/farmacocinética , Compuestos de Litio/farmacocinética , Animales , Antimaníacos/farmacocinética , Antimaníacos/farmacología , Antimaníacos/uso terapéutico , Transporte Biológico/efectos de los fármacos , Trastorno Bipolar/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Fármacos del Sistema Nervioso Central/farmacología , Fármacos del Sistema Nervioso Central/uso terapéutico , Humanos , Compuestos de Litio/farmacología , Compuestos de Litio/uso terapéuticoRESUMEN
The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.
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Neoplasias Encefálicas/irrigación sanguínea , Cannabidiol/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Microvasos/patología , Canales Catiónicos TRPV/metabolismo , Barrera Hematoencefálica/metabolismo , Calcio/metabolismo , Cannabis/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Impedancia Eléctrica , Calor , Humanos , Extractos Vegetales/farmacología , Rojo de Rutenio/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , ortoaminobenzoatos/farmacologíaRESUMEN
Variability in drug response to lithium (Li+) is poorly understood and significant, as only 40% of patients with bipolar disorder highly respond to Li+. Li+ can be transported by sodium (Na+) transporters in kidney tubules or red blood cells, but its transport has not been investigated at the blood-brain barrier (BBB). Inhibition and/or transcriptomic strategies for Na+ transporters such as NHE (SLC9), NBC (SLC4), and NKCC (SLC12) were used to assess their role on Li+ transport in human brain endothelial cells. Na+-free buffer was also used to examine Na+/Li+ countertransport (NLCT) activity. The BBB permeability of Li+ evaluated in the rat was 2% that of diazepam, a high passive diffusion lipophilic compound. Gene expression of several Na+ transporters was determined in hCMEC/D3 cells, human hematopoietic stem-cell-derived BBB models (HBLEC), and human primary brain microvascular endothelial cells (hPBMECs) and showed the following rank order with close expression profile: NHE1 > NKCC1 > NHE5 > NBCn1, while NHE2-4, NBCn2, and NBCe1-2 were barely detected. Li+ influx in hCMEC/D3 cells was increased in Na+-free buffer by 3.3-fold, while depletion of chloride or bicarbonate had no effect. DMA (NHE inhibitor), DIDS (anionic carriers inhibitor), and bumetanide (NKCC inhibitor) decreased Li+ uptake significantly in hCMEC/D3 by 52, 51, and 47%, respectively, while S0859 (NBC inhibitor) increased Li+ influx 2.3-fold. Zoniporide (NHE1 inhibitor) and siRNA against NHE1 had no effect on Li+ influx in hCMEC/D3 cells. Our study shows that NHE1 and/or NHE5, NBCn1, and NKCC1 may play a significant role in the transport of Li+ through the plasma membrane of brain endothelial cells.
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Antimaníacos/farmacología , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Compuestos de Litio/farmacología , Proteínas Transportadoras de Solutos/metabolismo , Animales , Antimaníacos/uso terapéutico , Trastorno Bipolar/tratamiento farmacológico , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Endoteliales/efectos de los fármacos , Células HEK293 , Humanos , Compuestos de Litio/uso terapéutico , Masculino , Microvasos/citología , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Proteínas Transportadoras de Solutos/antagonistas & inhibidoresRESUMEN
PURPOSE: Oral drug delivery using NPs is a current strategy for poorly absorbed molecules. It offers significant improvement in terms of bioavailability. However, the encapsulation of proteins and peptides in polymeric NPs is a challenge. Firstly, the present study focused on the double emulsion process in order to encapsulate the OXY peptide. Then the technique was challenged by a one-step simplified process, the simple emulsion. METHODS: In order to study the influence of formulation and process parameters, factorial experimental designs were carried on. The responses observed were the NP size (<200 nm in order to penetrate the intestinal mucus layer), the suspension stability (ZP < |30| mV) and the OXY loading. RESULTS: It was thus found that the amount and the nature of surfactant, the ratio between the phases, the amount of PLA-PEG polymer and OXY, the presence of a viscosifying agent, and the duration of the sonication could significantly influence the responses. Finally, OXY-loaded NPs from both processes were obtained with NP size of 195 and 226 nm and OXY loading of 4 and 3.3% for double and simple emulsions, respectively. CONCLUSION: The two processes appeared to be suitable for OXY encapsulation and comparable in term of NP size, peptide drug load and release obtained.
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Portadores de Fármacos/química , Composición de Medicamentos/métodos , Liberación de Fármacos , Oxitocina/farmacocinética , Disponibilidad Biológica , Emulsiones , Nanopartículas/química , Oxitocina/administración & dosificación , Tamaño de la Partícula , Poliésteres/química , Polietilenglicoles/química , Proyectos de InvestigaciónRESUMEN
[18F]FEPPA is a specific ligand for the translocator protein of 18 kDa (TSPO) used as a positron emission tomography (PET) biomarker for glial activation and neuroinflammation. [18F]FEPPA radiosynthesis was optimized to assess in a mouse model the cerebral inflammation induced by an intraperitoneal injection of Salmonella enterica serovar Typhimurium lipopolysaccharides (LPS; 5 mg/kg) 24 h before PET imaging. [18F]FEPPA was synthesized by nucleophilic substitution (90 °C, 10 min) with tosylated precursor, followed by improved semi-preparative HPLC purification (retention time 14 min). [18F]FEPPA radiosynthesis were carried out in 55 min (from EOB). The non-decay corrected radiochemical yield were 34 ± 2% (n = 17), and the radiochemical purity greater than 99%, with a molar activity of 198 ± 125 GBq/µmol at the end of synthesis. Western blot analysis demonstrated a 2.2-fold increase in TSPO brain expression in the LPS treated mice compared to controls. This was consistent with the significant increase of [18F]FEPPA brain total volume of distribution (VT) estimated with pharmacokinetic modelling. In conclusion, [18F]FEPPA radiosynthesis was implemented with high yields. The new purification/formulation with only class 3 solvents is more suitable for in vivo studies.
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Anilidas/farmacología , Encefalitis/diagnóstico por imagen , Radioisótopos de Flúor/farmacología , Antígenos O/administración & dosificación , Tomografía de Emisión de Positrones , Piridinas/farmacología , Anilidas/síntesis química , Anilidas/farmacocinética , Animales , Western Blotting , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Encefalitis/inducido químicamente , Radioisótopos de Flúor/farmacocinética , Ratones , Modelos Animales , Piridinas/síntesis química , Piridinas/farmacocinética , Ensayo de Unión Radioligante , Radiometría , Receptores de GABA/metabolismo , Salmonella enterica/inmunología , Distribución TisularRESUMEN
Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per µg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels. Peptide peak identities were evaluated using a spectral library and chromatographic parameters. Sprague-Dawley rat microvessels obtained on three different days were analyzed with this method complemented by an absolute quantification multiple reaction monitoring method for transporter proteins P-gp, Bcrp, and Na+ /K+ ATPase pump using stable isotope labeled peptides as internal standard. Inter-day differences in the cell markers and BSA contamination were observed. Levels of cell markers correlated positively between each other. Then, the correlation between cell marker proteins and transporter proteins was evaluated to choose the best EC marker protein for protein quantification normalization. The membrane protein Pecam-1 showed a very high correlation with the EC-specific transporter P-gp (Pearson product-moment correlation coefficient (r) > 0.89) and moderate to high with Bcrp (r ≥ 0.77), that can be found also in pericytes and astrocytes. Therefore, Pecam-1 was selected as a marker for the normalization of the quantification of the proteins of endothelial cells.
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Transporte Biológico/fisiología , Biomarcadores/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Microvasos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Animales , Encéfalo/irrigación sanguínea , Células Endoteliales/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem/métodosRESUMEN
The purpose of the present work was to study the change in morphine metabolic ratio in obese subjects before and after Roux-en-Y Gastric Bypass (RYGB) and to identify clinical and/or biological factors associated with this change. The pharmacokinetics (PK) of oral morphine (30mg), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was performed in patients before (n=25; mean BMI=43.2 (35.4-61.9)kg/m2), 7-15days (n=16) and 6 months after RYGB (n=19; mean BMI=32.3 (25.4-46.0)kg/m2). Morphine Cmax and AUC0-inf were significantly increased and morphine Tmax significantly shortened at 6 months after RYGB compared with preoperative data, indicating an important increase in the rate and extent of morphine absorption. The morphine metabolic ratio 0-inf M3G+M6G/Morphine, decreased significantly from the preoperative to 6 months postoperative period with an average of -26% (range -74%; +21%; p=0.004), but not in the immediate post-operative period. The change in morphine metabolic ratio was associated with a change in BMI, fat mass in kg, and triglyceride levels (rho=0.5, p≤0.04). The degree of change in several markers of low-grade inflammation, or the level of liver steatosis and fibrosis before surgery, was not associated with the change in morphine metabolic ratios. Our findings indicate that RYGB-induced weight loss significantly decreases morphine metabolic ratio, arguing for an effect of morbid obesity on glucuronidation. With glucuronide exposure at 6 months similar to preoperative values, a higher morphine AUC0-inf should encourage reducing morphine dosage in patients undergoing RYGB and chronically receiving immediate-release oral morphine.
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Derivados de la Morfina/metabolismo , Morfina/metabolismo , Obesidad Mórbida/metabolismo , Femenino , Derivación Gástrica , Humanos , Masculino , Obesidad Mórbida/cirugíaRESUMEN
Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high-resolution mass spectrometry (LC-HR-MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC-HR-MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full-scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 µL plasma giving a wide polynomial dynamic range (R2 > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra- and inter-day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples.
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Baclofeno/sangre , Cromatografía Líquida de Alta Presión/métodos , Monitoreo de Drogas/métodos , Agonistas de Receptores GABA-B/sangre , Relajantes Musculares Centrales/sangre , Espectrometría de Masas en Tándem/métodos , Baclofeno/metabolismo , Agonistas de Receptores GABA-B/metabolismo , Glucurónidos/sangre , Glucurónidos/metabolismo , Humanos , Límite de Detección , Relajantes Musculares Centrales/metabolismo , Oxidación-ReducciónRESUMEN
Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Xenobióticos/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Disponibilidad Biológica , Biotransformación/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Interacciones Farmacológicas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Sustitución del Gen , Humanos , Absorción Intestinal/efectos de los fármacos , Masculino , Moduladores del Transporte de Membrana/sangre , Moduladores del Transporte de Membrana/metabolismo , Moduladores del Transporte de Membrana/farmacocinética , Moduladores del Transporte de Membrana/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Distribución Tisular/efectos de los fármacos , Xenobióticos/sangre , Xenobióticos/metabolismo , Xenobióticos/farmacologíaRESUMEN
The objective of our work was to study the association between the jejunal expression levels of P-gp, MRP2, MRP3, UGT2B7, CYP3A4, the ABCB1 c.3435C > T polymorphism, and several obesity-associated biomarkers, as well as oral morphine and glucuronides pharmacokinetics in a population of morbidly obese subjects. The pharmacokinetics of oral morphine (30 mg) and its glucuronides was performed in obese patients candidate to bariatric surgery. A fragment of jejunal mucosa was preserved during surgery. Subjects were genotyped for the ABCB1 single nucleotide polymorphism (SNP) c.3435C > T. The subjects were 6 males and 23 females, with a mean body mass index of 44.8 (35.4-61.9) kg/m(2). The metabolic ratios AUC0-inf M3G/morphine and AUC0-inf M6G/morphine were highly correlated (rs = 0.8, p < 0.0001) and were 73.2 ± 24.6 (34.7-137.7) and 10.9 ± 4.1 (3.8-20.6). The pharmacokinetic parameters of morphine and its glucuronides were not associated with the jejunal contents of P-gp, CYP3A4, MRP2, and MRP3. The jejunal content of UGT2B7 was positively associated with morphine AUC0-inf (rs = 0.4, p = 0.03). Adiponectin was inversely correlated with morphine Cmax (rs = -0.44, p = 0.03). None of the factors studied was associated with morphine metabolic ratios. The interindividual variability in the jejunal content of drug transporters and metabolizing enzymes, the ABCB1 gene polymorphism, and the low-grade inflammation did not explain the variability in morphine and glucuronide exposure. High morphine metabolic ratio argued for an increased morphine glucuronidation in morbidly obese patients.
Asunto(s)
Analgésicos Opioides/farmacocinética , Biomarcadores/análisis , Glucurónidos/farmacocinética , Yeyuno/metabolismo , Morfina/farmacocinética , Obesidad Mórbida/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Administración Oral , Adolescente , Adulto , Analgésicos Opioides/administración & dosificación , Índice de Masa Corporal , Citocromo P-450 CYP3A , Femenino , Glucurónidos/administración & dosificación , Glucuronosiltransferasa/metabolismo , Humanos , Yeyuno/efectos de los fármacos , Masculino , Persona de Mediana Edad , Morfina/administración & dosificación , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Obesidad Mórbida/tratamiento farmacológico , Polimorfismo de Nucleótido Simple/genética , Distribución Tisular , Adulto JovenRESUMEN
Protein expression levels of drug-metabolizing enzymes and transporters in human jejunal tissues excised from morbidly obese subjects during gastric bypass surgery were evaluated using quantitative targeted absolute proteomics. Protein expression levels of 15 cytochrome P450 (CYP) enzymes, 10 UDP-glucuronosyltransferase (UGT) enzymes, and NADPH-P450 reductase (P450R) in microsomal fractions from 28 subjects and 49 transporters in plasma membrane fractions from 24 of the same subjects were determined using liquid chromatography-tandem mass spectrometry. Based on average values, UGT1A1, UGT2B15, UGT2B17, SGLT1, and GLUT2 exhibited high expression levels (over 10 fmol/µg protein), though UGT2B15 expression was detected at a high level in only one subject. CYP2C9, CYP2D6, CYP3A5, UGT1A6, P450R, ABCG2, GLUT5, PEPT1, MCT1, 4F2 cell-surface antigen heavy chain (4F2hc), LAT2, OSTα, and OSTß showed intermediate levels (1-10 fmol/µg protein), and CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP2C19, CYP2J2, CYP3A7, CYP4A11, CYP51A1, UGT1A3, UGT1A4, UGT1A8, UGT2B4, ABCC1, ABCC4, ABCC5, ABCC6, ABCG8, TAUT, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OCTN1, CNT2, PCFT, MCT4, GLUT4, and SLC22A18 showed low levels (less than 1 fmol/µg protein). The greatest interindividual difference (364-fold) was detected for UGT2B17. However, differences in expression levels of other quantified UGTs (except UGT2B15 and UGT2B17), CYPs (except CYP1A1 and CYP3A5), and P450R, and all quantified transporters, were within 10-fold. Expression levels of CYP1A2 and GLUT4 were significantly correlated with body-mass index. The levels of 4F2hc showed significant gender differences. Smokers showed increased levels of UGT1A1 and UGT1A3. These findings provide a basis for understanding the changes in molecular mechanisms of jejunal metabolism and transport, as well as their interindividual variability, in morbidly obese patients.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Glucuronosiltransferasa/metabolismo , Yeyuno/metabolismo , Obesidad Mórbida/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Femenino , Transportador de Glucosa de Tipo 2/metabolismo , Transportador de Glucosa de Tipo 5/metabolismo , Humanos , Técnicas In Vitro , Intestino Delgado/metabolismo , Masculino , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas de Neoplasias/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportador de Péptidos 1 , Transportador 1 de Sodio-Glucosa/metabolismo , SimportadoresRESUMEN
The expression of aryl hydrocarbon receptor (AhR) transcription factor was detected at transcript level in freshly isolated human brain microvessels and in the hCMEC/D3 human cerebral microvascular endothelial cell line. Recent studies have demonstrated that AhR pathway is able to crosstalk with other pathways such as hypoxia signaling pathway. Therefore, we used the hCMEC/D3 cell line to investigate the potential crosstalk between AhR and hypoxia signaling pathways. First, we performed two different hypoxia-like procedures in hCMEC/D3 cells; namely, exposition of cells to 150 µM deferoxamine or to glucose and oxygen deprivation for 6 h. These two procedures led to hypoxia-inducible factor (HIF)-1α and HIF-2α proteins accumulation together with a significant induction of the two well-known hypoxia-inducible genes VEGF and GLUT-1. Both HIF-1α and -2α functionally mediated hypoxia response in the hCMEC/D3 cells. Then, we observed that a 6 h exposure to 25 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin, a strong AhR ligand, up-regulated CYP1A1 and CYP1B1 expression, and that this effect was AhR dependent. Regarding AhR and hypoxia crosstalk, our experiments revealed that an asymmetric interference between these two pathways effectively occurred in hCMEC/D3 cells: hypoxia pathway interfered with AhR signaling but not the other way around. We studied the putative crosstalk of AhR and hypoxia pathways in hCMEC/D3 human cerebral microvascular endothelial cells. While hypoxia decreased the expression of the two AhR target genes CYP1A1 and CYP1B1, AhR activation results in no change in hypoxia target gene expression. This is the first sign of AhR and hypoxia pathway crosstalk in an in vitro model of the human cerebral endothelium.