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Introduction: Endocrine disrupting chemicals (EDCs) are known to interfere with endocrine homeostasis. Their impact on the adrenal cortex and steroidogenesis has not yet been sufficiently elucidated. This applies in particular to the ubiquitously available bisphenols A (BPA), F (BPF), and S (BPS). Methods: NCI-H295R adrenocortical cells were exposed to different concentrations (1nM-1mM) of BPA, BPF, BPS, and an equimolar mixture of them (BPmix). After 72 hours, 15 endogenous steroids were measured using LC-MS/MS. Ratios of substrate and product of CYP-regulated steps were calculated to identify most influenced steps of steroidogenesis. mRNA expression of steroidogenic enzymes was determined by real-time PCR. Results: Cell viability remained unaffected at bisphenol concentrations lower than 250 µM. All tested bisphenols and their combination led to extensive alterations in the quantified steroid levels. The most profound fold changes (FC) in steroid concentrations after exposure to BPA (>10µM) were seen for androstenedione, e.g. a 0.37±0.11-fold decrease at 25µM (p≤0.0001) compared to vehicle-treated controls. For BPF, levels of 17-hydroxyprogesterone were significantly increased by 25µM (FC 2.57±0.49, p≤0.001) and 50µM (FC 2.65±0.61, p≤0.0001). BPS treatment led to a dose-dependent decrease of 11-deoxycorticosterone at >1µM (e.g. FC 0.24±0.14, p≤0.0001 at 10µM). However, when combining all three bisphenols, additive effects were detected: e.g. 11-deoxycortisosterone was decreased at doses >10µM (FC 0.27±0.04, p≤0.0001, at 25µM), whereas 21-deoxycortisol was increased by 2.92±0.20 (p≤0.01) at 10µM, and by 3.21±0.45 (p≤0.001) at 50µM. While every measured androgen (DHEA, DHEAS, androstenedione, testosterone, DHT) was lowered in all experiments, estradiol levels were significantly increased by BPA, BPF, BPS, and BPmix (e.g. FC 3.60±0.54, p≤0.0001 at 100µM BPF). Calculated substrate-product ratios indicated an inhibition of CYP17A1-, and CYP21A2 mediated conversions, whereas CYP11B1 and CYP19A1 showed higher activity in the presence of bisphenols. Based on these findings, most relevant mRNA expression of CYP genes were analysed. mRNA levels of StAR, CYP11B1, and CYP17A1 were significantly increased by BPF, BPS, and BPmix. Discussion: In cell culture, bisphenols interfere with steroidogenesis at non-cytotoxic levels, leading to compound-specific patterns of significantly altered hormone levels. These results justify and call for additional in-vivo studies to evaluate effects of EDCs on adrenal gland functionality.
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Córtex Suprarrenal , Compostos Benzidrílicos , Disruptores Endócrinos , Fenóis , Plastificantes , Fenóis/toxicidade , Compostos Benzidrílicos/toxicidade , Humanos , Disruptores Endócrinos/toxicidade , Córtex Suprarrenal/efeitos dos fármacos , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/citologia , Plastificantes/toxicidade , Esteroides/biossíntese , Sulfonas/farmacologia , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: The escalating prescription of psychopharmacological medications to women of reproductive age underscores the growing significance of sex-specific variations in pharmacotherapy. Despite this, clinical trials have largely overlooked these differences. Preliminary data indicate sex-specific variations in the neurobiology of affective disorders and in the metabolism, pharmacodynamics, and kinetics of therapeutic drugs. This underscores the imperative for a more nuanced exploration of menstrual cycle-dependent fluctuations in psychotropic drugs. This pilot study aimed to investigate drug and hormone fluctuations in female patients with affective disorders, aiming to enhance comprehension of the interplay between cycle-related hormone fluctuations and pharmacokinetics. The ultimate goal is to facilitate more effective and safer pharmacological therapy in the future. METHODS: Blood samples were collected from 27 patients and 27 age-matched control participants at 3 distinct time points (early follicular phase, ovulation, and late luteal phase) during each menstrual cycle. Depressive and manic symptoms were assessed, and hormone concentrations were measured in the entire sample, while drug concentrations were assessed solely in the affective disorder sample using mass spectrometry. RESULTS: Significant variations in drug concentration were observed throughout the menstrual cycle for bupropion, with a trend toward altered concentration for venlafaxine. Moreover, notable differences in hormone concentrations were identified between patients and controls, even after accounting for the impact of contraceptive use, diagnoses, and medication. CONCLUSIONS: This pilot study reinforces previously reported data, underscoring the significance of sex-specific pharmacological therapy approaches. It provides further evidence supporting the interaction among sex hormones, drugs, and symptoms of affective disorders.
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Ciclo Menstrual , Progesterona , Masculino , Feminino , Humanos , Projetos Piloto , Fase Luteal , Psicotrópicos/uso terapêuticoRESUMO
Endocrine-disrupting chemicals (EDCs) are exogenous substances known to interfere with endocrine homeostasis and promote adverse health outcomes. Their impact on the adrenal cortex, corticosteroids and their physiological role in the organism has not yet been sufficiently elucidated. In this review, we collect experimental and epidemiological evidence on adrenal disruption by relevant endocrine disruptors. In vitro data suggest significant alterations of gene expression, cell signalling, steroid production, steroid distribution, and action. Additionally, morphological studies revealed disturbances in tissue organization and development, local inflammation, and zone-specific hyperplasia. Finally, endocrine circuits, such as the hypothalamic-pituitary-adrenal axis, might be affected by EDCs. Many questions regarding the detection of steroidogenesis disruption and the effects of combined toxicity remain unanswered. Not only due to the diverse mode of action of adrenal steroids and their implication in many common diseases, there is no doubt that further research on endocrine disruption of the adrenocortical system is needed.
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Córtex Suprarrenal , Disruptores Endócrinos , Córtex Suprarrenal/metabolismo , Corticosteroides/metabolismo , Disruptores Endócrinos/toxicidade , Sistema Hipotálamo-Hipofisário , Sistema Hipófise-Suprarrenal , Esteroides/metabolismoRESUMO
OBJECTIVES: Primary aldosteronism is the most common cause of endocrine hypertension and is associated with significant cardiovascular morbidities. The diagnostic workup depends on determinations of plasma aldosterone and renin which are highly variable and associated with false-positive and false-negative results. Quantification of aldosterone in 24â¯h urine may provide more reliable results, but the methodology is not well established. We aimed to establish an assay for urinary aldosterone and related steroids with suitability for clinical routine implementation. METHODS: Here, we report on the development and validation of a quantitative LC-MS/MS method for six urinary steroids: aldosterone, cortisol, 18-hydroxycorticosterone, 18-hydroxycortisol, 18-oxocortisol, tetrahydroaldosterone. After enzymatic deconjugation, total steroids were extracted using SepPak tC18 plates and quantified in positive electrospray ionization mode on a QTRAP 6500+ mass spectrometer. RESULTS: Excellent linearity was demonstrated with R2>0.998 for all analytes. Extraction recoveries were 89.8-98.4â¯% and intra- and inter-day coefficients of variations were <6.4 and <9.0â¯%, establishing superb precision. Patients with primary aldosteronism (n=10) had higher mean 24â¯h excretions of aldosterone-related metabolites than normotensive volunteers (n=20): 3.91 (95â¯% CI 2.27-5.55) vs. 1.92 (1.16-2.68)⯵mol/mol for aldosterone/creatinine, 2.57 (1.49-3.66) vs. 0.79 (0.48-1.10)⯵mol/mol for 18-hydroxycorticosterone/creatinine, 37.4 (13.59-61.2) vs. 11.61 (10.24-12.98)⯵mol/mol for 18-hydroxycortisol/creatinine, 1.56 (0.34-2.78) vs. 0.13 (0.09-0.17)⯵mol/mol for 18-oxocortisol/creatinine, and 21.5 (13.4-29.6) vs. 7.21 (4.88-9.54)⯵mol/mol for tetrahydroaldosterone/creatinine. CONCLUSIONS: The reported assay is robust and suitable for routine clinical use. First results in patient samples, though promising, require clinical validation in a larger sample set.
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Aldosterona , Hiperaldosteronismo , Humanos , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida/métodos , Creatinina , Espectrometria de Massas em Tandem/métodos , Hiperaldosteronismo/diagnósticoRESUMO
OBJECTIVE: This study aims to identify susceptibility markers for adrenal crises (AC) in educated patients with chronic adrenal insufficiency (AI). DESIGN: A case-control study involving 66 patients with AI analyzing the impact of glucocorticoid and mineralocorticoid exposure, adrenomedullary function, inflammatory parameters, and educational status on AC frequency. Patients were categorized into low (n = 32) and high (n = 34) AC frequency groups based on AC occurrence (below or 2 times above the average of the reported AC frequency of 8.3 AC/100 patient-years in a previous prospective study). METHODS: Parameters, including cortisol plasma profile and urinary steroid excretion after administration of the morning glucocorticoid dose, 24-h urinary steroid profiling, salivary cortisol profiling, and hair cortisol, estimated cortisol exposure. Polymorphisms (single nucleotide polymorphism [SNP]) of the glucocorticoid receptor (NR3C1) and mineralocorticoid receptor (NR3C2) associated with individual steroid sensitivity were assessed together with SNPs for 11ß-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11ß-hydroxysteroid dehydrogenase 2 (HSD11B2). Mineralocorticoid replacement was evaluated by serum and urinary electrolytes and osmolality, plasma-renin concentration, and ambulatory blood pressure levels. We additionally measured plasma and urinary catecholamines, serum levels of IL6 and hsCRP, and SNPs of IL6 and TNF-alpha. Patient knowledge of AC prevention was assessed by questionnaires. RESULTS: Frequent AC patients had higher daily glucocorticoid doses and hair cortisol levels, with no significant differences in other parameters investigated. AC frequency is inversely correlated with the frequency of self-reported adjustments of the glucocorticoid replacement. CONCLUSION: Higher glucocorticoid dosages in high-risk patients, despite unaffected cortisol metabolism, may be linked to decreased cortisol sensitivity or impaired glucocorticoid absorption. Proactive dose adjustments show a protective effect against AC, regardless of biological vulnerability.
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Doença de Addison , Insuficiência Adrenal , Humanos , Hidrocortisona/metabolismo , Glucocorticoides/uso terapêutico , Mineralocorticoides , Estudos de Casos e Controles , Monitorização Ambulatorial da Pressão Arterial , Interleucina-6 , Insuficiência Adrenal/epidemiologia , Insuficiência Adrenal/tratamento farmacológico , Doença de Addison/epidemiologia , Doença de Addison/genética , 11-beta-Hidroxiesteroide Desidrogenases/uso terapêutico , CausalidadeRESUMO
OBJECTIVES: Preoperative identification of malignant adrenal tumors is challenging. 24-h urinary steroid profiling by LC-MS/MS and machine learning has demonstrated high diagnostic power, but the unavailability of bioinformatic models for public use has limited its routine application. We here aimed to increase usability with a novel classification model for the differentiation of adrenocortical adenoma (ACA) and adrenocortical carcinoma (ACC). METHODS: Eleven steroids (5-pregnenetriol, dehydroepiandrosterone, cortisone, cortisol, α-cortolone, tetrahydro-11-deoxycortisol, etiocholanolone, pregnenolone, pregnanetriol, pregnanediol, and 5-pregnenediol) were quantified by LC-MS/MS in 24-h urine samples from 352 patients with adrenal tumor (281 ACA, 71 ACC). Random forest modelling and decision tree algorithms were applied in training (n = 188) and test sets (n = 80) and independently validated in 84 patients with paired 24-h and spot urine. RESULTS: After examining different models, a decision tree using excretions of only 5-pregnenetriol and tetrahydro-11-deoxycortisol classified three groups with low, intermediate, and high risk for malignancy. 148/217 ACA were classified as being at low, 67 intermediate, and 2 high risk of malignancy. Conversely, none of the ACC demonstrated a low-risk profile leading to a negative predictive value of 100% for malignancy. In the independent validation cohort, the negative predictive value was again 100% in both 24-h urine and spot urine with a positive predictive value of 87.5% and 86.7%, respectively. CONCLUSIONS: This simplified LC-MS/MS-based classification model using 24-h-urine provided excellent results for exclusion of ACC and can help to avoid unnecessary surgeries. Analysis of spot urine led to similarly satisfactory results suggesting that cumbersome 24-h urine collection might be dispensable after future validation.
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Neoplasias do Córtex Suprarrenal , Neoplasias das Glândulas Suprarrenais , Adenoma Adrenocortical , Carcinoma Adrenocortical , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/urina , Carcinoma Adrenocortical/diagnóstico , Carcinoma Adrenocortical/urina , Adenoma Adrenocortical/diagnóstico , Adenoma Adrenocortical/patologia , Adenoma Adrenocortical/urina , EsteroidesRESUMO
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a high risk of relapse and metastatic spread. The actin-bundling protein fascin (FSCN1) is overexpressed in aggressive ACC and represents a reliable prognostic indicator. FSCN1 has been shown to synergize with VAV2, a guanine nucleotide exchange factor for the Rho/Rac GTPase family, to enhance the invasion properties of ACC cancer cells. Based on those results, we investigated the effects of FSCN1 inactivation by CRISPR/Cas9 or pharmacological blockade on the invasive properties of ACC cells, both in vitro and in an in vivo metastatic ACC zebrafish model. Here, we showed that FSCN1 is a transcriptional target for ß-catenin in H295R ACC cells and that its inactivation resulted in defects in cell attachment and proliferation. FSCN1 knock-out modulated the expression of genes involved in cytoskeleton dynamics and cell adhesion. When Steroidogenic Factor-1 (SF-1) dosage was upregulated in H295R cells, activating their invasive capacities, FSCN1 knock-out reduced the number of filopodia, lamellipodia/ruffles and focal adhesions, while decreasing cell invasion in Matrigel. Similar effects were produced by the FSCN1 inhibitor G2-044, which also diminished the invasion of other ACC cell lines expressing lower levels of FSCN1 than H295R. In the zebrafish model, metastases formation was significantly reduced in FSCN1 knock-out cells and G2-044 significantly reduced the number of metastases formed by ACC cells. Our results indicate that FSCN1 is a new druggable target for ACC and provide the rationale for future clinical trials with FSCN1 inhibitors in patients with ACC.
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Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Animais , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Peixe-ZebraRESUMO
Volumetric absorptive microsampling (VAMS) has emerged as a minimally invasive alternative to conventional sampling. However, the applicability of VAMS must be investigated clinically. Therefore, the feasibility of at-home sampling was investigated for the kinase inhibitors nilotinib, cabozantinib, dabrafenib, trametinib and ruxolitinib and evaluated regarding the acceptance of at-home microsampling, sample quality of at-home VAMS and incurred sample stability. In addition, clinical validation including three different approaches for serum level predictions was performed. For this purpose, VAMS and reference serum samples were collected simultaneously. Conversion of VAMS to serum concentration was based either on a linear regression model, a hematocrit-dependent formula, or using a correction factor. During the study period 591 VAMS were collected from a total of 59 patients. The percentage of patients who agreed to perform VAMS at home ranged from 50.0 % to 84.6 % depending on the compound. 93.1 % of at-home VAMS were collected correctly. Regarding the drug stability in dried capillary blood, no stability issues were detected between on-site and at-home VAMS. Linear regression showed a strong correlation between VAMS and reference serum concentrations for nilotinib, cabozantinib, dabrafenib and ruxolitinib (r 0.9427 - 0.9674) and a moderate correlation for trametinib (r 0.5811). For clinical validation, the acceptance criteria were met for all three approaches for three of the five kinase inhibitors. Predictive performance was not improved by using individual hematocrit instead of population hematocrit and was largely independent of conversion model. In conclusion, VAMS at-home has been shown to be feasible for use in routine clinical care and serum values could be predicted based on the measured VAMS concentration for nilotinib, cabozantinib, and dabrafenib.
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Coleta de Amostras Sanguíneas , Teste em Amostras de Sangue Seco , Humanos , Estudos de ViabilidadeRESUMO
Although bariatric surgery is known to change the metabolome, it is unclear if this is specific for the intervention or a consequence of the induced bodyweight loss. As the weight loss after Roux-en-Y Gastric Bypass (RYGB) can hardly be mimicked with an evenly effective diet in humans, translational research efforts might be helpful. A group of 188 plasma metabolites of 46 patients from the randomized controlled Würzburg Adipositas Study (WAS) and from RYGB-treated rats (n = 6) as well as body-weight-matched controls (n = 7) were measured using liquid chromatography tandem mass spectrometry. WAS participants were randomized into intensive lifestyle modification (LS, n = 24) or RYGB (OP, n = 22). In patients in the WAS cohort, only bariatric surgery achieved a sustained weight loss (BMI -34.3% (OP) vs. -1.2% (LS), p ≤ 0.01). An explicit shift in the metabolomic profile was found in 57 metabolites in the human cohort and in 62 metabolites in the rodent model. Significantly higher levels of sphingolipids and lecithins were detected in both surgical groups but not in the conservatively treated human and animal groups. RYGB leads to a characteristic metabolomic profile, which differs distinctly from that following non-surgical intervention. Analysis of the human and rat data revealed that RYGB induces specific changes in the metabolome independent of weight loss.
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Cirurgia Bariátrica , Derivação Gástrica , Obesidade Mórbida , Humanos , Ratos , Animais , Derivação Gástrica/métodos , Metabolômica/métodos , Dieta , Redução de Peso , Obesidade Mórbida/metabolismoRESUMO
Serum liquid chromatography-tandem mass spectrometry (LC-MS/MS) steroid profiling is used for the diagnosis of adrenocortical carcinoma (ACC). Guidelines recommend endocrine work-up in addition to radiological imaging for follow-up in ACC, but data on this topic are scarce. Patients were included in this retrospective study if pre-therapeutic hormone values, regular tumour evaluation by imaging, steroid measurements by LC-MS/MS, and details on therapies were available. The utility of steroid profiles in detecting recurrence or disease progression was assessed, whereby "endocrine progress" was defined by an elevation of at least 3 of 13 analysed hormones. Cohort A included 47 patients after R0 resection, of whom 15 experienced recurrence and 32 did not. In cohort B, 52 patients with advanced disease (including 7 patients of cohort A with recurrence) could be evaluated on 74 visits when progressive disease was documented. In 20 of 89 cases with documented disease progression, "endocrine progress" was detectable prior to radiological progress. In these cases, recurrence/progression was detected at a median of 32 days earlier by steroid measurement than by imaging, with 11-deoxycortisol and testosterone being the most sensitive markers. Notably, these patients had significantly larger tumour burden. In conclusion, steroid profiling by LC-MS/MS is of value in detecting recurrent/progressive disease in ACC.
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Ruxolitinib (RUX) is approved for the treatment of steroid-refractory acute and chronic graft versus host disease (GvHD). It is predominantly metabolized via cytochrome P450 (CYP) 3A4. As patients with GvHD have an increased risk of invasive fungal infections, RUX is frequently combined with posaconazole (POS), a strong CYP3A4 inhibitor. Knowledge of RUX exposure under concomitant POS treatment is scarce and recommendations on dose modifications are inconsistent. A physiologically based pharmacokinetic (PBPK) model was developed to investigate the drug-drug interaction (DDI) between POS and RUX. The predicted RUX exposure was compared to observed concentrations in patients with GvHD in the clinical routine. PBPK models for RUX and POS were independently set up using PK-Sim® Version 11. Plasma concentration-time profiles were described successfully and all predicted area under the curve (AUC) values were within 2-fold of the observed values. The increase in RUX exposure was predicted with a DDI ratio of 1.21 (Cmax) and 1.59 (AUC). Standard dosing in patients with GvHD led to higher RUX exposure than expected, suggesting further dose reduction if combined with POS. The developed model can serve as a starting point for further simulations of the implemented DDI and can be extended to further perpetrators of CYP-mediated PK-DDIs or disease-specific physiological changes.
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Patients treated with dabrafenib and trametinib for BRAFV600-mutant melanoma often experience dose reductions and treatment discontinuations. Current knowledge about the associations between patient characteristics, adverse events (AE), and exposure is inconclusive. Our study included 27 patients (including 18 patients for micro-sampling). Dabrafenib and trametinib exposure was prospectively analyzed, and the relevant patient characteristics and AE were reported. Their association with the observed concentrations and Bayesian estimates of the pharmacokinetic (PK) parameters of (hydroxy-)dabrafenib and trametinib were investigated. Further, the feasibility of at-home sampling of capillary blood was assessed. A population pharmacokinetic (popPK) model-informed conversion model was developed to derive serum PK parameters from self-sampled capillary blood. Results showed that (hydroxy-)dabrafenib or trametinib exposure was not associated with age, sex, body mass index, or toxicity. Co-medication with P-glycoprotein inducers was associated with significantly lower trough concentrations of trametinib (p = 0.027) but not (hydroxy-)dabrafenib. Self-sampling of capillary blood was feasible for use in routine care. Our conversion model was adequate for estimating serum PK parameters from micro-samples. Findings do not support a general recommendation for monitoring dabrafenib and trametinib but suggest that monitoring can facilitate making decisions about dosage adjustments. To this end, micro-sampling and the newly developed conversion model may be useful for estimating precise PK parameters.
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Introduction: Preoperative diagnostic workup of adrenal tumors is based on imaging and hormone analyses, but charged with uncertainties. Steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 24-h urine has shown potential to discriminate benign and malignant adrenal tumors. Our aim was to develop and validate a specific and accurate LC-MS/MS method for the quantification of deconjugated urinary marker steroids, to evaluate their pre-analytical stability and to apply the method to clinical samples of patients with adrenal tumors. Methods: A method for the quantification of 11 deconjugated steroids (5-pregnenetriol, dehydroepiandrosterone, cortisone, cortisol, α-cortolone, tetrahydro-11-deoxycortisol, etiocholanolone, pregnenolone, pregnanetriol, pregnanediol, and 5-pregnenediol) in human urine was developed and validated based on international guidelines. Steroids were enzymatically deconjugated and extracted by solid phase extraction before LC-MS/MS quantification in positive electrospray ionization mode. Results: Excellent linearity with R2 > 0.99 and intra- and inter-day precisions of < 10.1 % were found. Relative matrix effects were between 96.4 % and 101.6 % and relative recovery was between 98.2 % and 115.0 %. Sufficient pre-freeze stability for all steroids in urine was found at 20-25 °C for seven days and at 4-6 °C for up to 28 days. Samples were stable during long-term storage at -20 °C and -80 °C for 6 months. Conclusions: A sensitive and robust LC-MS/MS method for the quantification of 11 urinary steroids was developed and validated according to international guidelines. Pre-analytical matrix stability was evaluated and the suitability of the method for the analysis of clinical samples and prospective validation studies was shown.
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OBJECTIVE: Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a highly selective and sensitive method for the quantification of kinase inhibitors, yet not widely available in clinical routine for therapeutic drug monitoring (TDM). To provide a more accessible alternative, a high-performance liquid chromatography method with ultraviolet/diode array detection (HPLC-UV/DAD) to quantify cabozantinib, dabrafenib, nilotinib and osimertinib, was developed and validated. Results were compared to LC-MS/MS. METHOD: After liquid-liquid-extraction and reconstitution of the residue in 20 mM potassium dihydrogen phosphate (KH2PO4) (pH4.6), acetonitrile and methanol (50:25:25,v/v/v), chromatographic separation was achieved in 20.0 min using a Luna® C18(2)-HST column (100 × 2 mm, 2.5 µm), protected by a C18 guard column (4 × 2 mm) (column temperature: 30 °C, autosampler: 10 °C). Mobile phase A and B consisted of 20 mM KH2PO4 (pH4.9) and acetonitrile (9:1,v/v) and acetonitrile:20 mM KH2PO4 (pH4.9) (7:3,v/v), respectively. Gradient elution was performed at 200 µL/min. Analytes were quantified at 250, 280 and 330 nm, using sorafenib as internal standard. RESULTS: Calibration curves were linear (35-2,000 ng/mL). Method validation assays met requirements by U.S. Food and Drug Administration and European Medicines Agency. Compared to the more sensitive and specific LC-MS/MS, HPLC-UV/DAD showed a good correlation and a strong positive association (Kendall's tau 0.811¬-0.963, p < 0.05). Bland-Altman-plots revealed 100% (cabozantinib), 98.6% (dabrafenib), 98.6% (nilotinib) and 96.2% (osimertinib) of relative differences inside the limits of agreement. Regulatory agency criteria for sample reanalysis and cross validation were met (±20%-criterion:100% (cabozantinib), 94.3% (dabrafenib), 92% (nilotinib) and 84.6% (osimertinib). CONCLUSION: The developed HPLC-UV/DAD method is "fit-for-TDM" in clinical routine and serves as a genuine alternative to LC-MS/MS.
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Monitoramento de Medicamentos , Espectrometria de Massas em Tandem , Acetonitrilas , Acrilamidas , Anilidas , Compostos de Anilina , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Humanos , Imidazóis , Indóis , Oximas , Piridinas , Pirimidinas , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
CONTEXT: Diagnosis of primary aldosteronism (PA) for many patients depends on positive results for the saline infusion test (SIT). Plasma aldosterone is often measured by immunoassays, which can return inaccurate results. OBJECTIVE: This study aimed to establish whether differences in aldosterone measurements by immunoassay versus mass spectrometry (MS) might impact confirmatory testing for PA. METHODS: This study, involving 240 patients tested using the SIT at 5 tertiary care centers, assessed discordance between immunoassay and MS-based measurements of plasma aldosterone. RESULTS: Plasma aldosterone measured by Liaison and iSYS immunoassays were respectively 86% and 58% higher than determined by MS. With an immunoassay-based SIT cutoff for aldosterone of 170 pmol/L, 78 and 162 patients had, respectivel, negative and positive results. All former patients had MS-based measurements of aldosteroneâ <â 117 pmol/L, below MS-based cutoffs of 162 pmol/L. Among the 162 patients with pathogenic SIT results, MS returned nonpathologic results in 62, including 32 under 117 pmol/L. Repeat measurements by an independent MS method confirmed nonpathogenic results in 53 patients with discordant results. Patients with discordant results showed a higher (Pâ <â 0.0001) prevalence of nonlateralized than lateralized adrenal aldosterone production than patients with concordant results (83% vs 28%). Among patients with nonlateralized aldosterone production, 66% had discordant results. Discordance was more prevalent for the Liaison than iSYS immunoassay (32% vs 16%; Pâ =â 0.0065) and was eliminated by plasma purification to remove interferents. CONCLUSION: These findings raise concerns about the validity of immunoassay-based diagnosis of PA in over 60% of patients with presumed bilateral disease. We provide a simple solution to minimize immunoassay inaccuracy-associated misdiagnosis of PA.
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Aldosterona , Hiperaldosteronismo , Humanos , Hiperaldosteronismo/diagnóstico , Imunoensaio/métodos , Espectrometria de Massas , Renina , Solução SalinaAssuntos
Glândulas Suprarrenais/fisiologia , Prolactina/metabolismo , Sequenciamento Completo do Genoma/estatística & dados numéricos , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/fisiopatologia , Humanos , Prolactina/genética , Prolactina/farmacologia , Sequenciamento Completo do Genoma/métodosRESUMO
Context: Pheochromocytomas and paragangliomas (PPGL) cause catecholamine excess leading to a characteristic clinical phenotype. Intra-individual changes at metabolome level have been described after surgical PPGL removal. The value of metabolomics for the diagnosis of PPGL has not been studied yet. Objective: Evaluation of quantitative metabolomics as a diagnostic tool for PPGL. Design: Targeted metabolomics by liquid chromatography-tandem mass spectrometry of plasma specimens and statistical modeling using ML-based feature selection approaches in a clinically well characterized cohort study. Patients: Prospectively enrolled patients (n=36, 17 female) from the Prospective Monoamine-producing Tumor Study (PMT) with hormonally active PPGL and 36 matched controls in whom PPGL was rigorously excluded. Results: Among 188 measured metabolites, only without considering false discovery rate, 4 exhibited statistically significant differences between patients with PPGL and controls (histidine p=0.004, threonine p=0.008, lyso PC a C28:0 p=0.044, sum of hexoses p=0.018). Weak, but significant correlations for histidine, threonine and lyso PC a C28:0 with total urine catecholamine levels were identified. Only the sum of hexoses (reflecting glucose) showed significant correlations with plasma metanephrines.By using ML-based feature selection approaches, we identified diagnostic signatures which all exhibited low accuracy and sensitivity. The best predictive value (sensitivity 87.5%, accuracy 67.3%) was obtained by using Gradient Boosting Machine Modelling. Conclusions: The diabetogenic effect of catecholamine excess dominates the plasma metabolome in PPGL patients. While curative surgery for PPGL led to normalization of catecholamine-induced alterations of metabolomics in individual patients, plasma metabolomics are not useful for diagnostic purposes, most likely due to inter-individual variability.
Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Células Sanguíneas/metabolismo , Metaboloma , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico , Neoplasias das Glândulas Suprarrenais/sangue , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Análise Química do Sangue/métodos , Estudos de Casos e Controles , Catecolaminas/metabolismo , Cromatografia Líquida , Feminino , Alemanha , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Paraganglioma/sangue , Paraganglioma/metabolismo , Feocromocitoma/sangue , Feocromocitoma/metabolismo , Espectrometria de Massas em TandemRESUMO
PURPOSE: Knowledge on Ruxolitinib exposure in patients with graft versus host disease (GvHD) is scarce. The purpose of this prospective study was to analyze Ruxolitinib concentrations of GvHD patients and to investigate effects of CYP3A4 and CYP2C9 inhibitors and other covariates as well as concentration-dependent effects. METHODS: 262 blood samples of 29 patients with acute or chronic GvHD who were administered Ruxolitinib during clinical routine were analyzed. A population pharmacokinetic model obtained from myelofibrosis patients was adapted to our population and was used to identify relevant pharmacokinetic properties and covariates on drug exposure. Relationships between Ruxolitinib exposure and adverse events were assessed. RESULTS: Median of individual mean trough serum concentrations was 39.9 ng/mL at 10 mg twice daily (IQR 27.1 ng/mL, range 5.6-99.8 ng/mL). Applying a population pharmacokinetic model revealed that concentrations in our cohort were significantly higher compared to myelofibrosis patients receiving the same daily dose (p < 0.001). Increased Ruxolitinib exposure was caused by a significant reduction in Ruxolitinib clearance by approximately 50%. Additional comedication with at least one strong CYP3A4 or CYP2C9 inhibitor led to a further reduction by 15% (p < 0.05). No other covariate affected pharmacokinetics significantly. Mean trough concentrations of patients requiring dose reduction related to adverse events were significantly elevated (p < 0.05). CONCLUSION: Ruxolitinib exposure is increased in GvHD patients in comparison to myelofibrosis patients due to reduced clearance and comedication with CYP3A4 or CYP2C9 inhibitors. Elevated Ruxolitinib trough concentrations might be a surrogate for toxicity.
Assuntos
Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Interações Medicamentosas , Doença Enxerto-Hospedeiro/tratamento farmacológico , Nitrilas/farmacocinética , Padrões de Prática Médica/estatística & dados numéricos , Mielofibrose Primária/tratamento farmacológico , Pirazóis/farmacocinética , Pirimidinas/farmacocinética , Doença Aguda , Adulto , Idoso , Doença Crônica , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP3A/química , Feminino , Seguimentos , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Humanos , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Nitrilas/administração & dosagem , Nitrilas/sangue , Mielofibrose Primária/metabolismo , Mielofibrose Primária/patologia , Prognóstico , Estudos Prospectivos , Pirazóis/administração & dosagem , Pirazóis/sangue , Pirimidinas/administração & dosagem , Pirimidinas/sangue , Distribuição Tecidual , Adulto JovemRESUMO
BACKGROUND: Adrenocortical carcinoma is an orphan but aggressive malignancy with limited treatment options. Cabozantinib (CAB), a tyrosine kinase inhibitor, has emerged as a new potential treatment. However, no data are available on whether and how CAB can be administered to patients undergoing hemodialysis. METHODS: An liquid chromatography with tandem mass spectrometry detection method was developed and validated according to the European Medicines Agency and United States Food and Drug Administration guidelines for bioanalytical method validation. The samples were prepared using protein precipitation and online solid-phase extraction. The method was applied to clinical samples of an adrenocortical carcinoma patient receiving CAB treatment (80 mg daily). During the 10 days of observation, the patient received periodic hemodialysis on 7 days. Pharmacokinetic (PK) simulations were performed using Bayesian forecasting according to an existing population PK model for CAB. RESULTS: Based on the PK simulation, a mean plasma trough concentration of 1375 ng/mL [90% prediction interval (PI), 601-2602 ng/mL] in the steady state at a daily dose of 80 mg was expected for CAB. However, an individual simulation involving the measured plasma levels of the patient resulted in a mean trough concentration of 348 ng/mL (90% PI, 278-430 ng/mL). The model based on individual PK parameters estimated accessible plasma levels of 521, 625, and 834 ng/mL by dose adjustment to 100, 120, and 160 mg, respectively. CONCLUSIONS: After establishing an liquid chromatography with tandem mass spectrometry detection method for therapeutic drug monitoring of CAB, our analyses involving a single patient undergoing hemodialysis indicated that higher than expected doses of CAB were required to achieve reasonable plasma concentrations. Our study demonstrates the usefulness of therapeutic drug monitoring for the evaluation of "new" drugs in patients with renal impairment.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Anilidas/farmacocinética , Piridinas/farmacocinética , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Carcinoma Adrenocortical/tratamento farmacológico , Anilidas/sangue , Teorema de Bayes , Simulação por Computador , Humanos , Piridinas/sangue , Diálise RenalRESUMO
BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.