RESUMO
Therapeutic drug monitoring (TDM) for anticancer drug imatinib has been suggested as the best way to improve the treatment response and minimize the risk of adverse reactions in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST) patients. TDM of oncology treatments with standard analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) is, however, complex and demanding. This paper proposes a new method for quantitation of imatinib in human plasma, based on surface enhanced raman spectroscopy (SERS) and multivariate calibration using partial least-squares regression (PLSR). The best PLSR model was obtained with three latent variables in the range from 123 to 5000 ng/mL of imatinib, providing a standard error of prediction (SEP) of 510 ng/mL. The method was validated in accordance with international guidelines, through the estimate of figures of merit, such as precision, accuracy, systematic error, analytical sensitivity, limits of detection, and quantitation. Moreover, the feasibility and clinical utility of this approach have also been verified using real plasma samples taken from deidentified patients. The results were in good agreement with a clinically validated LC-MS/MS method. The new SERS method presented in this preliminary work showed simplicity, short analysis time, good sensitivity, and could be considered a promising platform for TDM of imatinib treatment in a point-of-care setting.
Assuntos
Antineoplásicos/sangue , Mesilato de Imatinib/sangue , Análise Espectral Raman/métodos , Calibragem , Monitoramento de Medicamentos/métodos , Humanos , Análise dos Mínimos Quadrados , Limite de Detecção , Análise Multivariada , Reprodutibilidade dos TestesRESUMO
In the history of medicine, nature has represented the main source of medical products. Indeed, the therapeutic use of plants certainly goes back to the Sumerian and Hippocrates and nowadays nature still represents the major source for new drugs discovery. Moreover, in the cancer treatment, drugs are either natural compounds or have been developed from naturally occurring parent compounds firstly isolated from plants and microbes from terrestrial and marine environment. A critical element of an anticancer drug is represented by its severe toxicities and, after administration, the drug concentrations have to remain in an appropriate range to be effective. Anyway, the drug dosage defined during the clinical studies could be inappropriate for an individual patient due to differences in drug absorption, metabolism and excretion. For this reason, personalized medicine, based on therapeutic drug monitoring (TDM), represents one of most important challenges in cancer therapy. Mass spectrometry sensitivity, specificity and fastness lead to elect this technique as the Golden Standard for pharmacokinetics and drug metabolism studies therefore for TDM. This review focuses on the mass spectrometry-based methods developed for pharmacokinetic quantification in human plasma of anticancer drugs derived from natural sources and already used in clinical practice. Particular emphasis was placed both on the pre-analytical and analytical steps, such as: sample preparation procedures, sample size required by the analysis and the limit of quantification of drugs and metabolites to give some insights on the clinical practice applicability. © 2015 Wiley Periodicals, Inc. Mass Spec Rev. 36:213-251, 2017.
Assuntos
Antineoplásicos/farmacocinética , Produtos Biológicos/farmacocinética , Monitoramento de Medicamentos/métodos , Espectrometria de Massas/métodos , Animais , Antineoplásicos/sangue , Produtos Biológicos/sangue , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Monitoramento de Medicamentos/instrumentação , Desenho de Equipamento , Humanos , Espectrometria de Massas/instrumentação , Neoplasias/tratamento farmacológicoRESUMO
The advent of nanocarriers for drug delivery has given rise to new intriguing scenarios in the cancer field. Nanocarriers indeed partly overcome the limits of traditional cytotoxic drugs principally changing the pharmacokinetic behavior of the parental drug. The peculiar characteristics of these systems strongly minimize the adverse reactions and ensure a more precise release of the compound to the tumor site. Several nanocarriers have been developed for the delivery of cytotoxic drugs such as paclitaxel and doxorubicin in order to improve both the outcome and the patients' quality of life. The aims of this review are to describe in detail the pharmacokinetics of nanocarriers, already marketed or in advanced clinical phases, for paclitaxel and doxorubicin, to highlight the main differences with the parental drugs, and to underline, in a critical manner, benefits and disadvantages related to the use of these new drug delivery systems.
Assuntos
Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos/farmacocinética , Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias/tratamento farmacológico , Paclitaxel/administração & dosagem , Qualidade de VidaRESUMO
Irinotecan is a widely used antineoplastic drug, mostly employed for the treatment of colorectal cancer. This drug is a feasible candidate for therapeutic drug monitoring due to the presence of a wide inter-individual variability in the pharmacokinetic and pharmacodynamic parameters. In order to determine the drug concentration during the administration protocol, we developed a quantitative MALDI-MS method using CHCA as MALDI matrix. Here, we demonstrate that MALDI-TOF can be applied in a routine setting for therapeutic drug monitoring in humans offering quick and accurate results. To reach this aim, we cross validated, according to FDA and EMA guidelines, the MALDI-TOF method in comparison with a standard LC-MS/MS method, applying it for the quantification of 108 patients' plasma samples from a clinical trial. Standard curves for irinotecan were linear (R (2) ≥ 0.9842) over the concentration ranges between 300 and 10,000 ng/mL and showed good back-calculated accuracy and precision. Intra- and inter-day precision and accuracy, determined on three quality control levels were always <12.8 % and between 90.1 and 106.9 %, respectively. The cross-validation procedure showed a good reproducibility between the two methods, the percentage differences within 20 % in more than 70 % of the total amount of clinical samples analysed.
Assuntos
Camptotecina/análogos & derivados , Neoplasias Colorretais/sangue , Monitoramento de Medicamentos/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Algoritmos , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Camptotecina/administração & dosagem , Camptotecina/sangue , Camptotecina/farmacocinética , Neoplasias Colorretais/tratamento farmacológico , Humanos , Irinotecano , Taxa de Depuração Metabólica , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Drug levels in patients' bloodstreams vary among individuals and consequently therapeutic drug monitoring (TDM) is fundamental to controlling the effective therapeutic range. For TDM purposes, different analytical approaches have been used, mainly based on immunoassay, liquid chromatography- ultraviolet, liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. More recently a matrix-assisted laser desorption/ionisation method has been proposed for the determination of irinotecan levels in the plasma of subjects under therapy and this method has been cross- validated by comparison with data achieved by LC-MS/MS. However, to reach an effective point-of-care monitoring of plasma drug concentrations, a TDM platform technology for fast, accurate, low-cost assays is required. In this frame, recently the use of paper spray mass spectrometry, which is becoming a popular and widely employed MS method, has been proposed. In this paper we report the results obtained by the development of a paper spray-based method for quantitative analysis in plasma samples of imatinib, a new generation of anticancer drug. Preliminary experiments showed that poor sensitivity, reproducibility and linear response were obtained by the "classical" paper spray set-up. In order to achieve better results, it was thought of interest to operate in presence of a higher and more homogeneous electrical field. For this aim, a stainless steel needle connected with the high voltage power supply was mounted below the paper triangle. Furthermore, in order to obtain valid quantitative data, we analysed the role of the different equilibria participating to the phenomena occurring in paper spray experiments, depending either on instrumental parameters or on the chemical nature of analyte and solvents. A calibration curve was obtained by spiking plasma samples containing different amounts of imatinib (1) with known amounts of deuterated imatinib (1d3) as internal standard, with molar ratios [1]/[1d3] in the range 0.00-2.00. A quite good linearity was obtained (R2 = 0.975) and some experiments performed on spiked plasma samples with known amounts of 1 confirmed the validity of this method.
Assuntos
Monitoramento de Medicamentos/métodos , Mesilato de Imatinib/sangue , Papel , Testes Imediatos , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Análise Química do Sangue/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The development of surface-assisted laser desorption/ionization (SALDI) methodologies in mass spectrometry allows, in principle, the development of new analytical approaches to qualitative and quantitative measurements on small molecules. Some of these methods have been applied to characterize two antineoplastic drugs: irinotecan (1) and sunitinib (2), and also 6-α-hydroxy-paclitaxel (3), the main metabolite of paclitaxel. Three different SALDI approaches have been tested employing nanostructure- assisted laser desorption/ionization (NALDI), carbon nanohorns (NHs) and carbon nanohorns covered by liquid additives. The results so obtained have been compared to those observed under matrix-assisted laser desorption/ionization (MALDI) conditions. Compounds 1 and 2 show the easy formation of protonated molecular species under all the experimental conditions, but the highest absolute intensity was achieved by NALDI. On the contrary, ionic species of low intensity are present for 3, among which are those that exhibit the highest intensity caused by [M+K](+) ions. After a critical evaluation of the obtained data, the linear response of the [M+H](+) ion intensity of 1 versus different deposited sample amounts was investigated, and the best results (R(2) = 0.9889) were obtained under MALDI conditions. The analysis of plasma samples spiked with 1 showed, again, that the MALDI approach was the best one (R(2) = 0.9766). The failure of NALDI measurements could be rationalized by the presence of ion suppression effects.
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A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of sorafenib (SORA), its N-oxide active metabolite and of regorafenib (REGO) and its two active metabolites regorafenib N-oxide and N-desmethyl regorafenib N-oxide in hepatocellular carcinoma patients' plasma. A proper analytes' separation was obtained with Synergi Fusion RP column (4⯵m, 80â¯Å, 50â¯×â¯2.0â¯mm) using a gradient elution of 10â¯mM ammonium acetate with 0.1% formic acid (mobile phase A) and methanol:isopropanol (90:10, v/v, mobile phase B) containing 0.1% formic acid. The analysis was then performed by electrospray ionization in negative mode coupled with a triple quadrupole mass spectrometry, API 4000QT, monitoring two transitions for each analyte, one for the quantification and the other for confirmation. The method could be easily applied to the clinical practice thanks to the short run (7â¯min), the low amount of patient plasma necessary for the analysis (5⯵L) and the fast sample processing based on protein precipitation. The method was therefore fully validated according to FDA and EMA guidelines. The linearity was assessed (R2≥0.998) over the concentration ranges of 50-8000â¯ng/mL for SORA and REGO, and 30-4000â¯ng/mL for their metabolites, that appropriately cover the therapeutic plasma concentrations. The presented method also showed adequate results in terms of intra- and inter-day accuracy and precision (CVâ¯≤â¯7.2% and accuracy between 89.4% and 108.8%), recovery (≥85.5%), sensitivity, analytes stability under various conditions and the absence of the matrix effect. Once the validation was successfully completed, the method was applied to perform the Cmin quantification of SORA, REGO and their metabolites in 54 plasma samples collected from patients enrolled in a clinical study ongoing at the National Cancer Institute of Aviano.
Assuntos
Cromatografia Líquida/métodos , Compostos de Fenilureia/análise , Piridinas/análise , Sorafenibe/análise , Espectrometria de Massas em Tandem/métodos , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/análise , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Carcinoma Hepatocelular/tratamento farmacológico , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Masculino , Compostos de Fenilureia/farmacocinética , Piridinas/farmacocinética , Reprodutibilidade dos Testes , Sorafenibe/farmacocinéticaRESUMO
Sunitinib is approved for advanced renal cell cancer, imatinib-resistant or -intolerant gastrointestinal stromal tumors and pancreatic neuroendocrine cancers. It is prescribed at a fixed dose but its plasma exposure shows large inter-individual variations. Taking into account the narrow therapeutic window and the positive exposure-efficacy relationship, there is a robust rationale for its therapeutic drug monitoring. In fact, a target plasma concentration of sunitinib plus its active metabolite, N-desethyl sunitinib, ≥50â¯ng/mL was suggested. In order to quantify sunitinib and N-desethyl sunitinib in patients' plasma, we developed and validated a new LC-MS/MS method applicable to clinical routine. In solution, sunitinib and N-desethyl sunitinib undergo to photo-isomerization and many published methods overcome this problem by conducting the entire procedures of samples collection and handling under strictly light-protection. Our method is based on a simple and fast procedure that quantitatively reconverts the E-isomer of both analytes, obtained during sample draw and processing without light-protection, into their Z-forms. Moreover, our method uses a small plasma volume (30⯵L) and the analytes are extracted by a rapid protein precipitation. It was validated according to EMA-FDA guidelines. The calibration curves resulted linear (R2 always >0.993) over the concentration ranges (0.1-500â¯ng/mL for sunitinib, 0.1-250â¯ng/mL for N-desethyl sunitinib) with a good precision (within 7.7 % for sunitinib and 10.8% for N- desethyl sunitinib) and accuracy (range 95.8-102.9% for sunitinib and 92.3-106.2% for N-desethyl sunitinib). This method was applied to a pharmacokinetic study in one patient treated with sunitinib. Moreover, as incurred samples reanalysis is an established part of the bioanalytical process to support clinical studies, its assessment was performed early in order to assure that any reproducibility issues was detected as soon as possible. The percentage difference between the two runs resulted within ±20% in all the re-analysed samples for both sunitinib and N- desethyl sunitinib.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Indóis/análise , Pirróis/análise , Sunitinibe/análise , Espectrometria de Massas em Tandem/métodos , Antineoplásicos/análise , Antineoplásicos/sangue , Antineoplásicos/química , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Indóis/sangue , Indóis/química , Isomerismo , Masculino , Pirróis/sangue , Pirróis/química , Reprodutibilidade dos Testes , Sunitinibe/sangue , Sunitinibe/químicaRESUMO
Fluorescent, imprinted nanosized polymers for the detection of irinotecan have been synthesised using a napthalimide polymerisable derivative (2-allyl-6-[2-(aminoethyl)-amino] napthalimide) as functional monomer. The imprinted polymers contain ethylene glycol dimethacrylate (EGDMA) as a cross-linker and were prepared by high dilution radical polymerisation in dimethylsulphoxide (DMSO). The material was able to rebind irinotecan up to 18 nmol/mg with good specificity. Fluorescence emission at 525 nm (excitation at 448 nm) was quenched by increasing concentrations of irinotecan via a static mechanism and also in analytically useful environments as mixtures of human plasma and organic solvents. This allowed the direct detection of irinotecan (in the 10 nM-30 µM range) in human plasma treated with acetonitrile; the limit of detection (LOD) was 9.4 nM, with within-run variability of 10% and day-to-day variability of 13%.
RESUMO
The standard of care for the first-line treatment of advanced gastrointestinal stromal tumor (GIST) is represented by imatinib, which is given daily at a standard dosage until tumor progression. Resistance to imatinib commonly occurs through the clonal selection of genetic mutations in the tumor DNA, and an increase in imatinib dosage was demonstrated to be efficacious to overcome imatinib resistance. Wild-type GISTs, which do not display KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations, are usually primarily insensitive to imatinib and tend to rapidly relapse in course of treatment. Here we report the case of a 53-year-old male patient with gastric GIST who primarily did not respond to imatinib and that, despite the administration of an increased imatinib dose, led to patient death. By using a deep next-generation sequencing barcode-aware approach, we analyzed a panel of actionable cancer-related genes in the patient cfDNA to investigate somatic changes responsible for imatinib resistance. We identified, in two serial circulating tumor DNA (ctDNA) samples, a sharp increase in the allele frequency of a never described TP53 mutation (c.560-7_560-2delCTCTTAinsT) located in a splice acceptor site and responsible for a protein loss of function. The same TP53 mutation was retrospectively identified in the primary tumor by digital droplet PCR at a subclonal frequency (0.1%). The mutation was detected at a very high allelic frequency (99%) in the metastatic hepatic lesion, suggesting a rapid clonal selection of the mutation during tumor progression. Imatinib plasma concentration at steady state was above the threshold of 760 ng/ml reported in the literature for the minimum efficacious concentration. The de novo TP53 (c.560-7_560-2delCTCTTAinsT) mutation was in silico predicted to be associated with an aberrant RNA splicing and with an aggressive phenotype which might have contributed to a rapid disease spread despite the administration of an increased imatinib dosage. This result underlies the need of a better investigation upon the role of TP53 in the pathogenesis of GISTs and sustains the use of next-generation sequencing (NGS) in cfDNA for the identification of novel genetic markers in wild-type GISTs.
RESUMO
A novel LC-MS/MS method was developed for the quantification of the new cyclin dependent kinase inhibitors (CDKIs) palbociclib and ribociclib and the aromatase inhibitor letrozole used in combinatory regimen. The proposed method is appropriate to be applied in clinical practice due to the simple and fast sample preparation based on protein precipitation, the low amount of patient sample necessary for the analysis (10 µL) and the total run time of 6.5 min. It was fully validated according to FDA and EMA guidelines on bioanalytical method validation. The linearity was assessed (R2 within 0.9992-0.9983) over the concentration ranges of 0.3-250 ng/mL for palbociclib, 10-10000 ng/mL for ribociclib and 0.5-500 ng/mL for letrozole that properly cover the therapeutic plasma concentrations. A specific strategy was implemented to reduce the carryover phenomenon, formerly known for these CDKIs. This method was applied to quantify the Cmin of palbociclib, ribociclib and letrozole in plasma samples from patients enrolled in a clinical study. The same set of study samples was analysed twice in separate runs to assess the reproducibility of the method by means of the incurred samples reanalysis. The results corroborated the reliability of the analyte concentrations obtained with the bioanalytical method, already proved by the validation process. The percentage differences were always within ±10% for all the analytes and the R2 of the correlation graph between the two quantifications was equal to 0.9994.
Assuntos
Aminopiridinas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Letrozol/sangue , Piperazinas/sangue , Purinas/sangue , Piridinas/sangue , Espectrometria de Massas em Tandem/métodos , Estabilidade de Medicamentos , Humanos , Reprodutibilidade dos TestesRESUMO
The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 µL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 µm, 2x50 mm) while the trapping column was a POROS R1/20 (20 µm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.
Assuntos
Teste em Amostras de Sangue Seco , Tumores do Estroma Gastrointestinal/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Cromatografia Líquida , Teste em Amostras de Sangue Seco/métodos , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Humanos , Mesilato de Imatinib/administração & dosagem , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Reprodutibilidade dos Testes , Espectrometria de Massas em TandemRESUMO
Gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC) is a pyrimidine antimetabolite employed against several human malignancies. It undergoes intracellular activation to the pharmacologically active triphosphate form (dFdCTP) and metabolic inactivation to the metabolite 2',2'-difluorodeoxyuridine (dFdU). In order to investigate the human plasma pharmacokinetics of dFdC and dFdU, we developed and validated an HPLC-MS/MS method, adding 2'-deoxycytidine as internal standard and simply precipitating the protein with acetonitrile. The method requires a small sample (125 microl), and it is rapid and selective, allowing good resolution of peaks from the plasma matrix in only 7 min. It is sensitive, precise and accurate, with overall precision, expressed as CV%, always less than 10.0% for both analytes and high recovery: > or = 80%. The limits of detection for dFdC and dFdU were 0.1 and 1.1 ng/ml, but considering the high concentrations in the plasma of patients investigated, we set the limit of quantitation at 20 ng/ml (0.08 microM) for dFdC and 250 ng/ml for dFdU, and validated the assay up to the dFdC concentration of 6.0 microg/ml (22.8 microM). The method was successfully used to study the drug pharmacokinetics in patients with advanced non-small-cell lung cancer in a phase II trial with gemcitabine administered as a fixed dose-rate infusion.
Assuntos
Antimetabólitos Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Desoxicitidina/análogos & derivados , Floxuridina/análogos & derivados , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cromatografia Líquida de Alta Pressão , Cisplatino/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/sangue , Desoxicitidina/farmacocinética , Floxuridina/sangue , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Sensibilidade e Especificidade , GencitabinaRESUMO
Sunitinib malate, an oral multi-targeted tyrosine kinase inhibitor approved for the treatment of metastatic renal cell carcinoma, gastrointestinal stromal tumor, and well-differentiated pancreatic neuroendocrine tumors, has been identified as a potential candidate for therapeutic drug monitoring approach. Nevertheless, the development of an analytical assay suitable for clinical application for the quantification of the plasma concentration of sunitinib and its active metabolite, N-desethyl sunitinib, is limited by its Z/E isomerization when exposed to light. Several LC-MS/MS methods already published require protection from light during all sample handling procedures to avoid the formation of E-isomer, which makes them not suitable for clinical practice. In order to obtain a simple and fast procedure to reconvert the E-isomer, formed during sample collection and treatment without light protection, and, thus, to have only Z-isomer peak to quantify, we studied the Z/E photodegradation with special attention to the condition allowing the reverse reaction in plasma matrix. After 30 min of light exposure, the E-isomer maximum percentage of both the analytes was reached (44% of E-sunitinib and 20% of E-N-desethyl sunitinib; these percentages were calculated with respect to the sum of E + Z). Moreover, the formation of the E-isomer increased up to 20% after lowering the pH of the solution. Since the reverse reaction takes place when the pre-exposed solution is placed in dark, we followed the E to Z-isomer kinetics into the autosampler. The conversion rate was very slow when the autosampler was set at 4 °C (after 4 h the mean percentages of E-isomer were 50% for sunitinib and 22% for N-desethyl sunitinib). The reconversion rate was considerably accelerated with the increasing of the temperature: incubating the analytical solution in a heated water bath for 5 min at 70 °C we obtained the quantitative (99%) reconversion of the E- to the Z-isomer. No effect of concentration was observed, while the presence of acids inhibited the reconversion. Based on these results, a simple and fast procedure was setup to quantitatively reconvert the E-isomer formed during sample collection and processing without light protection into its Z-form thus leading to a single peak to quantify. The application of this additional step allows to develop a LC-MS/MS method suitable to clinical practice, due to its practicality and speed.
Assuntos
Indóis/sangue , Isomerismo , Luz/efeitos adversos , Pirróis/sangue , Sunitinibe/sangue , Cromatografia Líquida , Humanos , Concentração de Íons de Hidrogênio , Espectrometria de Massas em Tandem , Temperatura , Fatores de TempoRESUMO
One of the main targets in current clinical oncology is the development of a cheap device capable of monitoring in real-time the concentration of a drug in the blood of a patient. This would allow fine-tuning the dosage according to the patient's metabolism, a key condition to reduce side effects. By using surface plasmon resonance and fluorescence spectroscopy we here show that short peptides designed in silico by a recently developed algorithm are capable of binding the anticancer drug irinotecan (CPT-11) with micromolar affinity. Importantly, the recognition takes place in the denaturating solution used in standard therapeutic drug monitoring to detach the drug from the proteins that are present in human plasma, and some of the peptides are capable of distinguishing CPT-11 from its metabolite SN-38. These results suggest that the in silico design of small artificial peptides is now a viable route for designing sensing units, opening a wide range of applications in diagnostic and clinical areas.
Assuntos
Antineoplásicos/metabolismo , Camptotecina/análogos & derivados , Monitoramento de Medicamentos/métodos , Peptídeos/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Sequência de Aminoácidos , Antineoplásicos/sangue , Sítios de Ligação , Camptotecina/sangue , Camptotecina/metabolismo , Humanos , Irinotecano , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Paclitaxel belongs to the taxanes family and it is used, alone or in multidrug regimens, for the therapy of several solid tumours, such as breast-, lung-, head and neck-, and ovarian cancer. Standard dosing of chemotherapy does not take into account the many inter-patient differences that make drug exposure highly variable, thus leading to the insurgence of severe toxicity. This is particularly true for paclitaxel considering that a relationship between haematological toxicity and plasma exposure was found. Therefore, in order to treat patients with the correct dose of paclitaxel, improving the overall benefit-risk ratio, Therapeutic Drug Monitoring is necessary. In order to quantify paclitaxel and its main metabolite, 6α-hydroxy-paclitaxel, in patients' plasma, we developed a new, sensitive and specific HPLC-MS/MS method applicable to all paclitaxel dosages used in clinical routine. The developed method used a small volume of plasma sample and is based on quick protein precipitation. The chromatographic separation of the analytes was achieved with a SunFire™ C18 column (3.5 µM, 92 Å, 2,1 x 150 mm); the mobile phases were 0.1% formic acid/bidistilled water and 0.1% formic acid/acetonitrile. The electrospray ionization source worked in positive ion mode and the mass spectrometer operated in selected reaction monitoring mode. Our bioanalytical method was successfully validated according to the FDA-EMA guidelines on bioanalytical method validation. The calibration curves resulted linear (R2 ≥0.9948) over the concentration ranges (1-10000 ng/mL for paclitaxel and 1-1000 ng/mL for 6α-hydroxy-paclitaxel) and were characterized by a good accuracy and precision. The intra- and inter-day precision and accuracy were determined on three quality control concentrations for paclitaxel and 6α-hydroxy-paclitaxel and resulted respectively <9.9% and within 91.1-114.8%. In addition, to further verify the assay reproducibility, we tested this method by re-analysing the incurred samples. This bioanalytical method was employed with success to a genotype-guided phase Ib study of weekly paclitaxel in ovarian cancer patients treated with a wide range of drug's dosages.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão/métodos , Paclitaxel/sangue , Paclitaxel/farmacocinética , Espectrometria de Massas em Tandem/métodos , Taxoides/sangue , Humanos , Limite de Detecção , Paclitaxel/metabolismo , Taxoides/metabolismo , Fatores de TempoRESUMO
Purpose:UGT1A1*28 confers a higher risk of toxicity in patients treated with irinotecan. Patients with *1/*1 and *1/*28 genotypes might tolerate higher than standard doses of irinotecan. We aimed to identify the MTD of irinotecan in patients with metastatic colorectal cancer (mCRC) with *1/*1 and *1/*28 genotypes treated with FOLFIRI plus bevacizumab, and to determine whether bevacizumab alters irinotecan pharmacokinetics.Experimental Design: Previously untreated patients with mCRC (25 *1/*1; 23 *1/*28) were given FOLFIRI plus bevacizumab every 2 weeks. The irinotecan dose was escalated using a 3 + 3 design in each genotype group as follows: 260, 310, and 370 mg/m2 The MTD was the highest dose at which <4/10 patients had a dose-limiting toxicity (DLT). Pharmacokinetics of irinotecan and SN-38 were measured on days 1 to 3 (without bevacizumab) and 15 to 17 (with bevacizumab).Results: For *1/*1 patients, 2 DLTs were observed among 10 patients at 310 mg/m2, while 370 mg/m2 was not tolerated (2 DLTs in 4 patients). For *1/*28 patients, 2 DLTs were observed among 10 patients at 260 mg/m2, while 310 mg/m2 was not tolerated (4 DLTs in 10 patients). Neutropenia and diarrhea were the most common DLTs. Changes in the AUCs of irinotecan and SN-38 associated with bevacizumab treatment were marginal.Conclusions: The MTD of irinotecan in FOLFIRI plus bevacizumab is 310 mg/m2 for UGT1A1 *1/*1 patients and 260 mg/m2 for *1/*28 patients. Bevacizumab does not alter the pharmacokinetics of irinotecan. The antitumor efficacy of these genotype-guided doses should be tested in future studies of patients with mCRC treated with FOLFIRI plus bevacizumab. Clin Cancer Res; 23(4); 918-24. ©2016 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Camptotecina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Glucuronosiltransferase/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab/administração & dosagem , Bevacizumab/efeitos adversos , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Genótipo , Humanos , Irinotecano , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Masculino , Pessoa de Meia-Idade , Metástase NeoplásicaRESUMO
PURPOSE: A73-year-old woman with metastatic colon cancer experienced a complete response to chemotherapy with dose-intensified irinotecan that has been durable for 5 years. We sequenced her tumor and germ line DNA and looked for similar patterns in publicly available genomic data from patients with colorectal cancer. PATIENTS AND METHODS: Tumor DNA was obtained from a biopsy before therapy, and germ line DNA was obtained from blood. Tumor and germline DNA were sequenced using a commercial panel with approximately 250 genes. Whole-genome amplification and exome sequencing were performed for POLE and POLD1. A POLD1 mutation was confirmed by Sanger sequencing. The somatic mutation and clinical annotation data files from the colon (n = 461) and rectal (n = 171) adenocarcinoma data sets were downloaded from The Cancer Genome Atlas data portal and analyzed for patterns of mutations and clinical outcomes in patients with POLE- and/or POLD1-mutated tumors. RESULTS: The pattern of alterations included APC biallelic inactivation and microsatellite instability high (MSI-H) phenotype, with somatic inactivation of MLH1 and hypermutation (estimated mutation rate > 200 per megabase). The extremely high mutation rate led us to investigate additional mechanisms for hypermutation, including loss of function of POLE. POLE was unaltered, but a related gene not typically associated with somatic mutation in colon cancer, POLD1, had a somatic mutation c.2171G>A[p.Gly724Glu]. Additionally, we noted that the high mutation rate was largely composed of dinucleotide deletions. A similar pattern of hypermutation (dinucleotide deletions, POLD1 mutations, MSI-H) was found in tumors from The Cancer Genome Atlas. CONCLUSION: POLD1 mutation with associated MSI-H and hyper-indel-hypermutated cancer genome characterizes a previously unrecognized variant of colon cancer that was found in this patient with an exceptional response to chemotherapy.
RESUMO
Several fluorescent molecularly imprinted nanogels for the detection of the anticancer drug sunitinib were synthesized and characterized. A selection of functional monomers based on different aminoacids and coumarin allowed isolation of polymers with very good rebinding properties and sensitivities. The direct detection of sunitinib in human plasma was successfully demonstrated by fluorescence quenching of the coumarin-based nanogels. The plasma sample simply diluted in DMSO allowed the recovery of various amounts of sunitib, as determined by an averaged calibration curve. The LOD was 400nM, with within-run variability <9%, day to day variability <5%, and good accuracy in the recovery of sunitinib from spiked samples.
Assuntos
Análise Química do Sangue/métodos , Monitoramento de Medicamentos/métodos , Indóis/sangue , Impressão Molecular/métodos , Polietilenoglicóis/química , Polietilenoimina/química , Pirróis/sangue , Espectrometria de Fluorescência/métodos , Antineoplásicos/sangue , Antineoplásicos/química , Indóis/química , Nanogéis , Pirróis/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SunitinibeRESUMO
Irinotecan is currently used in several cancer regimens mainly in colorectal cancer (CRC). This drug has a narrow therapeutic range and treatment can lead to side effects, mainly neutropenia and diarrhea, frequently requiring discontinuing or lowering the drug dose. A wide inter-individual variability in irinotecan pharmacokinetic parameters and pharmacodynamics has been reported and associated to patients' genetic background. In particular, a polymorphism in the UGT1A1 gene (UGT1A1*28) has been linked to an impaired detoxification of SN-38 (irinotecan active metabolite) to SN-38 glucuronide (SN-38G) leading to increased toxicities. Therefore, therapeutic drug monitoring of irinotecan, SN-38 and SN-38G is recommended to personalize therapy. In order to quantify simultaneously irinotecan and its main metabolites in patients' plasma, we developed and validated a new, sensitive and specific HPLC-MS/MS method applicable to all irinotecan dosages used in clinic. This method required a small plasma volume, addition of camptothecin as internal standard and simple protein precipitation. Chromatographic separation was done on a Gemini C18 column (3 µM, 100 mm x 2.0 mm) using 0.1% acetic acid/bidistilled water and 0.1% acetic acid/acetonitrile as mobile phases. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring. The standard curves were linear (R2 ≥0.9962) over the concentration ranges (10-10000 ng/mL for irinotecan, 1-500 ng/mL for SN-38 and SN-38G and 1-5000 ng/mL for APC) and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy, determined on three quality control levels for all the analytes, were always <12.3% and between 89.4% and 113.0%, respectively. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method was successfully applied to a pharmacokinetic study in metastatic CRC patients enrolled in a genotype-guided phase Ib study of irinotecan administered in combination with 5-fluorouracil/leucovorin and bevacizumab.