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Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.
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Andrógenos , Semen , Niño , Adulto , Humanos , Masculino , Animales , Ratones , Andrógenos/metabolismo , Testículo/metabolismo , Progesterona/metabolismo , Estrógenos/metabolismo , Transducción de SeñalRESUMEN
Nicardipine hydrochloride is an anti-hypertensive drug that is used off-label to treat hypertension in children. A previous oral formulation of nicardipine hydrochloride was developed using a commercial vehicle as an excipient. However, ready-to-use vehicles are prone to supply shortages, and their composition may undergo substantial modifications. The aim of this study was to propose a new oral formulation of nicardipine hydrochloride 2 mg/mL using simple excipients. The formulation included hydroxypropylmethylcellulose, simple syrup, polysorbate 80, sodium saccharin, citrate buffer, strawberry flavor and 0.2% potassium sorbate. The uniformity of content was maintained before and after agitation. Nicardipine hydrochloride concentration assessed by HPLC-MS/MS remained above 90% for 365 days before opening and for 28 days after opening. pH and osmolality were maintained throughout the study, and no microbial contamination was observed. The uniformity of mass of the delivered doses was evaluated using four different devices. A new oral formulation of nicardipine hydrochloride 2 mg/mL was developed using simple and safe excipients. Pharmacological and clinical parameters remain to be assessed and compared with those of the previous formulation.
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Background: Steroids play a key role in numerous physiological processes. Steroid determination is a useful tool to explore various endocrine diseases. Because of its specificity, mass spectrometry is considered to be a reference method for the determination of steroids in serum compared to radioimmunoassay. This technology could progress towards more automation for the optimal organization of clinical laboratories and ultimately for the benefit of patients. Methods: A fully automated ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and fully validated to determine five steroids in serum. Sample preparation was based on protein precipitation with filtration followed by online solid phase extraction. Chromatographic separation was performed using a biphenyl stationary phase. Results: The method was successfully validated according to European Medicine Agency guidelines. Coefficients of variation did not exceed, respectively, 8.4% and 8.1% for intra- and inter-assay precision. Method comparison with radioimmunoassay showed a proportional bias for all compounds, except for testosterone in men. Comparison with another LC-MS/MS method demonstrated acceptable concordance for all steroids, although a small bias was observed for androstenedione. Conclusion: The novelty of this method is that it has been fully automated. Automation provides benefits in traceability and allows significant savings in cost and time.
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BACKGROUND: Levosimendan (LVSMD) is a calcium-sensitizer inotropic and vasodilator agent whose use might have a beneficial effect on the weaning of venoarterial extracorporeal membrane oxygenation (VA-ECMO). In light of LVSMD pharmacological characteristics, we hypothesized that ECMO may induce major pharmacokinetic (PK) modifications for LVSMD and its metabolites. OBJECTIVE: The aim of this study was to investigate the PK of LVSMD and its metabolites, and to assess the effects of ECMO on PK parameters. METHODS: We conducted a multicentric, prospective study (NCT03681379). Twenty-seven infusions of LVSMD were performed, allowing for the collection of 255 blood samples. Non-linear mixed-effects modeling software (MONOLIX®) was used to develop a parent-metabolite PK model of LVSMD and its metabolites. RESULTS: Most patients received a 0.2 µg/kg/min infusion of LVSMD over 24 h. After elimination of non-reliable samples or concentrations below the limit of quantification, 166, 101 and 85 samples were considered for LVSMD, OR-1855 and OR-1896, respectively, of which 81, 53 and 41, respectively, were drawn under ECMO conditions. Parent-metabolite PK modeling revealed that a two-compartment model with first-order elimination best described LVSMD PK. Use of a transit compartment allowed for an explanation of the delayed appearance of circulating OR-1855 and OR-1896, with the latter following a first-order elimination. Patient weight influenced the central volume of distribution and elimination of LVSMD. ECMO support increased the elimination rate of LVSMD by 78%, and ECMO also slowed down the metabolite formation rate by 85% for OR-1855, which in turn is converted to the active metabolite OR-1896, 14% slower than without ECMO. Simulated data revealed that standard dosing may not be appropriate for patients under ECMO, with a decrease in the steady-state concentration of LVSMD and lower exposure to the active metabolite OR-1896. CONCLUSIONS: ECMO altered PK parameters for LVSMD and its metabolites. An infusion of LVSMD over 48 h, instead of 24 h, with a slightly higher dose may promote synthesis of the active metabolite OR-1896, which is responsible for the long-term efficacy of LVSMD. Further trials evaluating ECMO effects using a PK/pharmacodynamic approach may be of interest. REGISTRATION: ClinicalTrials.gov identifier number NCT03681379.
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Oxigenación por Membrana Extracorpórea , Recién Nacido , Humanos , Niño , Simendán , Estudios Prospectivos , Enfermedad Crítica/terapia , Antibacterianos/farmacocinéticaRESUMEN
Type 2 diabetes (T2D) and hypertension (HTN) are common risk factors of cardiovascular diseases (CVD) characterized by chronic low-grade systemic inflammation and impaired endothelial function. This study aimed to assess whether levels of non-enzymatic, lipoxygenase (LOX)- and cytochrome P450 (CYP)-derived arachidonic acid (ARA) metabolites, which are known regulators of vascular homeostasis, are affected by HTN and T2D. For this objective, 17 plasma level derivatives of ARA were quantitated by chromatography coupled with mass spectrometry in 44 patients (12 healthy, 8 HTN, 7 T2D, and 17 HTN + T2D). Effects of hyperglycemic and hyperinsulinemic clamps on ARA metabolite levels were assessed in seven healthy subjects. No significant differences in the plasma levels of ARA metabolites were observed for T2D patients compared with healthy volunteers. HTN was associated with an alteration of ARA metabolite correlation patterns with increased 20-, 19-, 15-, and 8-hydroxyeicosatrienoic acid (HETE). A decrease of 20-HETE was also observed during both hyperglycemic and hyperinsulinemic clamps. Additional experiments are needed to assess whether the modulation of HETE metabolites in HTN may be of interest. Furthermore, although not affected by T2D, it remains to investigate whether the decrease of 20-HETE observed during clamps may be related to the regulation of glucose tolerance and insulin signaling.
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In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.
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Espermatogénesis , Testículo , Animales , Barrera Hematotesticular/metabolismo , Fertilidad , Masculino , Ratas , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.
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Productos Biológicos , Vacunas , Boranos , Cromatografía Líquida de Alta Presión/métodos , Ácido Edético , Metanol , Espectrometría de Masas en Tándem/métodosRESUMEN
Intravenous (i.v.) morphine is a safe, robust, and recommended treatment for severe pain using the titration principle. Despite its high efficacy, it is impacted by organizational constraints related to venous access. Nebulized (NEB) morphine may represent an alternative for titration but pharmacokinetic (PK) properties of short nebulization using routine devices need evaluation. Twenty-seven healthy volunteers were included to receive NEB or i.v. morphine administration using increasing amounts according to Dixon's reference method. Plasma morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) were quantified. PK modeling and simulations were performed using Monolix. Dixon's method exhibited a significantly higher morphine dose regimen in the NEB group versus the i.v. group (6.2 [5.3-7.1] vs. 3.0 [2.0-4.0] mg, p < 0.001). Morphine, M3G, and M6G dose-normalized exposure were significantly lower in the NEB group versus the i.v. group: morphine (19 [13-23] vs. 1044 [702-1266] µg min/L, p < 0.001), M3G (245 [162-287] vs. 3752 [2487-5165] µg min/L, p < 0.001) and M6G (28 [21-43] vs. 466 [370-723] µg min/L, p < 0.001). The model that best fitted the data consisted in a transit compartment for morphine absorption, three compartments for morphine distribution followed by multiple transit compartments (8.2 and 57.5-min transit time for M3G and M6G, respectively) and a first order elimination for M3G and M6G. Morphine bioavailability in the NEB group was 3.5% using the i.v. group as reference. Administration route and sex significantly influenced morphine and metabolite PKs. This work aimed to evaluate the PKs of NEB morphine compared with the i.v. route. Despite a bioavailability to improve, NEB morphine administration using a routine device is suitable to plan morphine titration.
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Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacocinética , Derivados de la Morfina/metabolismo , Morfina/administración & dosificación , Morfina/farmacocinética , Administración por Inhalación , Adulto , Simulación por Computador , Relación Dosis-Respuesta a Droga , Femenino , Voluntarios Sanos , Humanos , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Modelos Biológicos , Nebulizadores y Vaporizadores , Factores SexualesRESUMEN
(1) Background: Glioblastoma is the most common malignant brain tumor in adults. Its etiology remains unknown in most cases. Glioblastoma pathogenesis consists of a progressive infiltration of the white matter by tumoral cells leading to progressive neurological deficit, epilepsy, and/or intracranial hypertension. The mean survival is between 15 to 17 months. Given this aggressive prognosis, there is an urgent need for a better understanding of the underlying mechanisms of glioblastoma to unveil new diagnostic strategies and therapeutic targets through a deeper understanding of its biology. (2) Methods: To systematically address this issue, we performed targeted and untargeted metabolomics-based investigations on both tissue and plasma samples from patients with glioblastoma. (3) Results: This study revealed 176 differentially expressed lipids and metabolites, 148 in plasma and 28 in tissue samples. Main biochemical classes include phospholipids, acylcarnitines, sphingomyelins, and triacylglycerols. Functional analyses revealed deep metabolic remodeling in glioblastoma lipids and energy substrates, which unveils the major role of lipids in tumor progression by modulating its own environment. (4) Conclusions: Overall, our study demonstrates in situ and systemic metabolic rewiring in glioblastoma that could shed light on its underlying biological plasticity and progression to inform diagnosis and/or therapeutic strategies.
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BACKGROUND: Fabry disease (FD) is an X-linked lysosomal disease due to a deficiency in the activity of the lysosomal α-galactosidase A (GalA), a key enzyme in the glycosphingolipid degradation pathway. FD is a complex disease with a poor genotype-phenotype correlation. FD could involve kidney, heart or central nervous system impairment that significantly decreases life expectancy. The advent of omics technologies offers the possibility of a global, integrated and systemic approach well-suited for the exploration of this complex disease. MATERIALS AND METHODS: Sixty-six plasmas of FD patients from the French Fabry cohort (FFABRY) and 60 control plasmas were analyzed using liquid chromatography and mass spectrometry-based targeted metabolomics (188 metabolites) along with the determination of LysoGb3 concentration and GalA enzymatic activity. Conventional univariate analyses as well as systems biology and machine learning methods were used. RESULTS: The analysis allowed for the identification of discriminating metabolic profiles that unambiguously separate FD patients from control subjects. The analysis identified 86 metabolites that are differentially expressed, including 62 Glycerophospholipids, 8 Acylcarnitines, 6 Sphingomyelins, 5 Aminoacids and 5 Biogenic Amines. Thirteen consensus metabolites were identified through network-based analysis, including 1 biogenic amine, 2 lysophosphatidylcholines and 10 glycerophospholipids. A predictive model using these metabolites showed an AUC-ROC of 0.992 (CI: 0.965-1.000). CONCLUSION: These results highlight deep metabolic remodeling in FD and confirm the potential of omics-based approaches in lysosomal diseases to reveal clinical and biological associations to generate pathophysiological hypotheses.
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Nicardipine is an antihypertensive drug that may be used off-label by oral route to treat hypertension in children. Currently commercially available tablets are inappropriate for oral use in children and manufactured hard capsules are not suitable for easy dose individualization to achieve target blood pressure. We aimed to fulfill this lack of appropriate dose forms by developing an oral liquid formulation of nicardipine. We compounded an extemporaneous 2 mg/mL nicardipine solution in InOrpha® vehicle for oral use with 1% polysorbate 80. A HPLC-MS/MS stability-indicating method was developed and validated. The stability was assessed under room temperature and refrigerated storage conditions. Nicardipine concentration remained above 95% of the initial concentration for 90 days in both storage conditions, without apparition of degradation products. Organoleptic parameters, pH, osmolality, viscosity and density were assessed and remained stable throughout storage. A uniformity of content was maintained before and after agitation of the packaging bottles. Mass uniformity of delivered doses was also ensured. Finally, the formulation met the Pharmacopoeia specifications for microbiological contaminations. In this study we report a compounded formulation of nicardipine for oral use in pediatrics. This solution, which could be easily manufactured, is being used in our hospital. Pharmacological and clinical parameters including bioavailability, pharmacokinetics, efficacy and tolerance remain to be assessed.
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Antihipertensivos , Hipertensión , Administración Oral , Niño , Cromatografía Líquida de Alta Presión , Composición de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Humanos , Hipertensión/tratamiento farmacológico , Nicardipino , Suspensiones , Espectrometría de Masas en TándemRESUMEN
Purpose/Aims: Infectious keratitis is a major cause of visual impairment and blindness worldwide. Common difficulties in treating fungal keratitis prompt new therapeutic possibilities. In this study, intrastromal voriconazole and posaconazole, and topical posaconazole were tested for their potential to obtain therapeutic cornea concentrations. Materials and Methods: Pharmacokinetics of triazole intracorneal/eye drop administration was studied in rats. Sixty-two rats were treated either by voriconazole or posaconazole. Twenty-nine and 33 rats received intrastromal injection of voriconazole solution (1 µl, 10 mg/ml) and posaconazole solution (1 µl, 18 mg/ml), respectively, administered under microscopic examination with a 32 gauge needle in the left cornea. Posaconazole (1.8% solution) eye drops were used. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS, at 30 min, 3 h, 6 h, 24 h, 48 h, 72 h, and 144 h (6 days) post-intrastromal injection. The entire rat cornea was used for chromatography analyses. Results: In anesthetized rats, single intracorneal injection resulted, after 30 min, in respectively, >300 ng/mg and >260 ng/mg cornea concentrations, dropping to low levels within hours, while staying low in plasma. The effect of hourly posaconazole eye drops resulted in >10 ng/mg cornea concentration, which was maintained with instillations every 2 and then every 4 h. Conclusion: Our results show that there is little interest of intrastromal triazole administration due to the short duration of high cornea concentrations obtained after intracorneal injection. Posaconazole eye drops maintain therapeutic cornea concentrations in rats and could be used to treat severe infectious keratitis.
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Antifúngicos/farmacocinética , Córnea/metabolismo , Triazoles/farmacocinética , Voriconazol/farmacocinética , Administración Oftálmica , Animales , Antifúngicos/administración & dosificación , Cromatografía Liquida , Sustancia Propia/efectos de los fármacos , Inyecciones Intraoculares , Masculino , Pruebas de Sensibilidad Microbiana , Soluciones Oftálmicas , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Voriconazol/administración & dosificaciónRESUMEN
Chemo-induced thrombocytopenia is a limiting toxicity among patients receiving temozolomide (TMZ) as first-line treatment for glioblastoma. We aimed to compare early platelet concentration kinetics, hematological safety profile, and impact on survival following the initiation of either the brand-name or a generic TMZ formulation. A retrospective trial was conducted in patients suffering from newly diagnosed glioblastoma. Patients were treated with TMZ at 75 mg/m2 per day during six weeks, concomitantly with radiotherapy. Platelet concentration was collected each week. Primary endpoint was to perform a linear mixed-effect model of platelet concentration kinetic over weeks. A total of 147 patients were included as follows: 96 received the brand-name TMZ, and 51 received a generic TMZ formulation. Exposition to the generic was a significant variable that negatively influenced the platelet kinetics in the radiotherapy and concomitant TMZ phase, P = 0.02. Grade ≥3 chemo-induced thrombocytopenia was more frequent in the generic group: 19.6% [95% CI 8.7-30.5%] vs 3.1% [0-6.6%], P = 0.001. Exposition to the generic formulation of TMZ led to increase early treatment discontinuation due to TMZ-induced thrombocytopenia and was a worsening independent prognostic factor on overall survival: adjusted HR 1.83 [1.21-2.8], P = 0.031. These data suggest that exposition to a generic formulation of TMZ vs the brand-name product is associated with higher early platelet decrease leading to clinically relevant impacts on treatment schedule in glioblastoma. Further prospective trials are needed to confirm these results.
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Antineoplásicos Alquilantes/efectos adversos , Plaquetas/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Medicamentos Genéricos/efectos adversos , Glioblastoma/tratamiento farmacológico , Temozolomida/efectos adversos , Trombocitopenia/inducido químicamente , Antineoplásicos Alquilantes/química , Neoplasias Encefálicas/mortalidad , Composición de Medicamentos , Medicamentos Genéricos/química , Femenino , Glioblastoma/mortalidad , Humanos , Cinética , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Temozolomida/química , Trombocitopenia/sangre , Trombocitopenia/diagnóstico , Trombocitopenia/mortalidad , Resultado del TratamientoRESUMEN
Metabolic phenotyping is poised as a powerful and promising tool for biomarker discovery in inherited metabolic diseases. However, few studies applied this approach to mcopolysaccharidoses (MPS). Thus, this innovative functional approach may unveil comprehensive impairments in MPS biology. This study explores mcopolysaccharidosis VI (MPS VI) or Maroteauxâ»Lamy syndrome (OMIM #253200) which is an autosomal recessive lysosomal storage disease caused by the deficiency of arylsulfatase B enzyme. Urine samples were collected from 16 MPS VI patients and 66 healthy control individuals. Untargeted metabolomics analysis was applied using ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry. Furthermore, dermatan sulfate, amino acids, carnitine, and acylcarnitine profiles were quantified using liquid chromatography coupled to tandem mass spectrometry. Univariate analysis and multivariate data modeling were used for integrative analysis and discriminant metabolites selection. Pathway analysis was done to unveil impaired metabolism. The study revealed significant differential biochemical patterns using multivariate data modeling. Pathway analysis revealed that several major amino acid pathways were dysregulated in MPS VI. Integrative analysis of targeted and untargeted metabolomics data with in silico results yielded arginine-proline, histidine, and glutathione metabolism being the most affected. This study is one of the first metabolic phenotyping studies of MPS VI. The findings might shed light on molecular understanding of MPS pathophysiology to develop further MPS studies to enhance diagnosis and treatments of this rare condition.
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Metaboloma , Metabolómica , Mucopolisacaridosis VI/metabolismo , Adolescente , Adulto , Anciano , Niño , Preescolar , Biología Computacional/métodos , Femenino , Humanos , Masculino , Redes y Vías Metabólicas , Metabolómica/métodos , Persona de Mediana Edad , Anotación de Secuencia Molecular , Mucopolisacaridosis VI/genética , Fenotipo , Adulto JovenRESUMEN
Stability data of toxics or drugs in gel-based or mechanical separation blood collection tubes are lacking, especially for therapeutic drug monitoring and clinical toxicology procedures. According to ISO 15189 accreditation standard, laboratories need to master the entire preanalytical process including the stability of analytes in a specific tube. Here we explored the impact of BD PST™ II and Barricor™ separator tubes on the stability of 167 therapeutic compounds and common drugs of abuse in plasma samples using LC-MS/MS. Forty drugs were significantly affected by the use of PST™ II tubes, including antidepressants (11/26), neuroleptics (9/13), cardiovascular drugs (5/26), anxiolytics and hypnotics (4/25) and some drugs of abuse (5/26). Six compounds exhibited significant reduction by the mechanical Barricor™ tubes. Ten drugs exhibited low (<85%) but non-significant recoveries due to inter-assay variability. Besides, a logPâ¯>â¯3.3 was determined as a cut-off value to predict a potential lack of stability in PST™ II gel tubes with an 86.4% sensitivity and a 61.4% specificity. As a consequence, determination of drugs with a logPâ¯>â¯3.3 should be carried out with caution in plasma samples withdrawn on PST™ II. The study showed the Barricor™ and non-gel tubes cause less drug interference and are recommended for the drugs studied.
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Ansiolíticos/sangre , Antidepresivos/sangre , Recolección de Muestras de Sangre , Fármacos Cardiovasculares/sangre , Hipnóticos y Sedantes/sangre , Drogas Ilícitas/sangre , Recolección de Muestras de Sangre/normas , Cromatografía Liquida , Evaluación Preclínica de Medicamentos , Monitoreo de Drogas , Geles/química , Humanos , Espectrometría de Masas en TándemRESUMEN
Ifosfamide (IFA) is a potent alkylating antitumoral agent, but its use is limited by neurological side effects. IFA is a racemic mixture of two enantiomeric forms, R-IFA and S-IFA with a stereoselective metabolism by CYP3A4 and CYP2B6, leading either to bioactive or to toxic pathways. In three consecutive cases of pediatric patients who exhibited IFA-induced encephalopathy (IIE), genotyping of clinically relevant single-nucleotide polymorphisms associated with decreased CYP3A4 and CYP2B6 activities was performed. Genetic investigations revealed the presence of CYP2B6 rs4803419 (C>T) in one patient while the two others carried the CYP2B6*6 allelic variant. All patients carried CYP3A4 wild-type genotype (CYP3A4*1/*1). Because CYP2B6-deficient alleles may be responsible for an increased conversion of S-IFA into neurotoxic metabolites, screening for CYP2B6 polymorphisms may help to avoid IIE and improve clinical outcomes.
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Antineoplásicos Alquilantes/efectos adversos , Neoplasias Óseas/tratamiento farmacológico , Encefalopatías/inducido químicamente , Encefalopatías/genética , Citocromo P-450 CYP2B6/genética , Ifosfamida/efectos adversos , Osteosarcoma/tratamiento farmacológico , Variantes Farmacogenómicas , Polimorfismo de Nucleótido Simple , Administración Intravenosa , Adolescente , Antineoplásicos Alquilantes/metabolismo , Encefalopatías/diagnóstico , Encefalopatías/tratamiento farmacológico , Niño , Citocromo P-450 CYP2B6/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Humanos , Ifosfamida/metabolismo , Masculino , Azul de Metileno/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Farmacogenética , Fenotipo , Factores de Riesgo , Resultado del TratamientoRESUMEN
Epoxyeicosatrienoic acids (EETs) are vasodilating lipid mediators metabolized into dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. We aimed to develop a LC-MS/MS method to quantify EETs and DHETs in human plasma and monitored their levels during vascular endothelial stimulation. Plasma samples, collected from 14 healthy and five hypertensive subjects at baseline and during radial artery endothelium-dependent flow-mediated dilatation, were spiked with internal standards. Lipids were then extracted by a modified Bligh and Dyer method and saponified to release bound EETs and DHETs. Samples were purified by a second liquid-liquid extraction and analyzed by LC-MS/MS. The assay allowed identification of (±)8(9)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-EET); (±)11(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-EET); (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET); (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DHET); (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DHET); and (±)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DHET). (±)5(6)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (5,6-EET) was virtually undetectable due to its chemical instability. The limits of quantification were 0.25 ng/mL for DHETs and 0.5 ng/mL for EETs. Intra- and inter-assay variations ranged from 1.6 to 13.2%. Heating induced a similar increase in 8,9-EET, 11,12-EET, and 14,15-EET levels and in corresponding DHET levels in healthy but not in hypertensive subjects. We validated a sensitive LC-MS/MS method for measuring simultaneously plasma EET and DHET regioisomers in human plasma and showed its interest for assessing endothelial function.
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Ácidos Araquidónicos/sangre , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Estudios de Casos y Controles , Endotelio Vascular/metabolismo , Humanos , Hipertensión/metabolismo , Límite de DetecciónRESUMEN
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
Voriconazole (VCZ) use is limited by its narrow therapeutic range and significant interpatient variability in exposure. This study aimed to assess (i) the impact of CYP2C19 genotype on VCZ exposure and (ii) the doses required to achieve the therapeutic range in adult patients with invasive fungal infections (IFIs). Therapeutic drug monitoring (TDM) of VCZ, based on trough concentration measurement, and CYP2C19 genotyping were used to guide VCZ dosing in Caucasian patients with IFIs. The two common polymorphisms in Caucasians (CYP2C19*2 and *17), associated with decreased or increased CYP2C19 activity, respectively, were correlated with the daily VCZ dose, pharmacokinetic parameters and concentration-to-dose ratio. In total, 111 trough concentration measurements from 35 genotyped patients were analysed using linear mixed-effect models. The mean VCZ doses required to achieve target concentrations were significantly higher in CYP2C19*17 carriers compared with CYP2C19*1/*1 individuals (P<0.001): 2.57±0.25mg/kg twice daily in CYP2C19*1/*1 patients versus 3.94±0.39mg/kg and 6.75±0.54mg/kg in *1/*17 and *17/*17 patients, respectively. In addition, exposure to VCZ correlated with the CYP2C19*17 variant. Indices of exposure for CYP2C19*2 carriers were in line with the functional effect of this polymorphism compared with CYP2C19*1/*1 individuals, however comparisons of doses required to achieve target concentrations were not statistically different. The CYP2C19*17 allele predicted both VCZ exposure and dose required to achieve effective and non-toxic concentrations. CYP2C19 genotyping appears useful to guide VCZ initial dosing when coupled with TDM and to explain subtherapeutic concentrations frequently observed in clinical practice.