About one third of patients with epilepsy are pharmacoresistant and suffer from poor quality of life, seizure-related accidents and comorbidities, increased risk of death and disabling psychosocial consequences. Clinical outcomes for patients unresponsive to current pharmacological tools could be improved by characterization and development of biomarkers that could assist physicians in either (i) optimizing response to treatment with available antiepileptic drugs or (ii) identifying early those patients who are pharmacoresistant and could benefit from earlier referral to alternative therapies such as epilepsy surgery or neurostimulation. The first part of the research relates to pharmacokinetic biomarkers aimed at optimizing response to pharmacological treatment. Specifically, plasma concentrations of antiepileptic drugs have been shown to provide valuable indicators of pharmacological response, although their usefulness has not yet been clearly established for some second-generation drugs, partly due to lack of availability of assay methods easily applicable to therapeutic drug monitoring in the routine setting. In the present research, the latter deficiency was addressed by developing and validating two assay techniques for the determination of retigabine, known also as ezogabine, and perampanel in body fluids. For retigabine, an HPLC-UV method suitable for therapeutic drug monitoring was developed which permits sensitive quantitation of the drug in human plasma. Although therapeutic drug monitoring of antiepileptic drugs is usually based on their determination in plasma samples, alternative matrices, such as dried plasma spots, may offer advantages in some settings. For this reason, a HPLC-UV method for the quantitation of perampanel in dried plasma spots was developed and validated. Perampanel concentrations assessed in dried plasma spot samples obtained from patients receiving therapeutic doses were comparable to those measured in plasma samples. The second part of the research described in the present thesis relates to the investigation of microRNA (miRNA) profiles in plasma-derived extracellular vesicles as potential epigenetic biomarkers for early differentiation between patients resistant to antiepileptic drugs and patients responsive to these drugs. Additionally, miRNA expression profiles were also assessed in healthy controls in order to ascertain potential differences related to the disease itself rather than drug responsiveness. By applying a cross-sectional non-interventional open-label study design, miRNAs profiles in exosomes and microvesicles isolated from plasma were compared among 20 patients with drug-resistant focal epilepsy, 20 patients with drug-responsive focal epilepsy and 20 healthy control subjects. Extracellular purity, concentration and size distribution in each sample were ascertained by applying a combination of techniques (Western blot analysis, nanoparticle tracking analysis and transmission electron microscopy). Total RNA was extracted by exosome and microvesicle pellets, and sequenced on NGS platforms. The potential involvement of identified miRNAs in epilepsy-relevant pathways (predicted target genes and predicted pathways) was investigated by using the miRWalk 3.0 database and KEGG database. Statistical analysis of the results did not identify significant differences across groups in extracellular vesicle size or count. Five miRNAs were found to be de-regulated in exosomes isolated from pharmacoresistant patients compared with healthy controls (one was up-regulated and four were down-regulated). Four miRNAs were found to be de-regulated in microvesicles obtained from pharmacoresistant patients compared with healthy controls (two up-regulated miRNAs and two down-regulated). These findings, which require confirmation in a larger sample size, may provide valuable clues for a better understanding of processes involved in epileptogenesis as well as mechanisms implicated in pharmacoresistance.

Biomarcatori farmacologici e molecolari nell'epilessia farmacoresistente

BARUFFI, KATIA
2019-12-19

Abstract

About one third of patients with epilepsy are pharmacoresistant and suffer from poor quality of life, seizure-related accidents and comorbidities, increased risk of death and disabling psychosocial consequences. Clinical outcomes for patients unresponsive to current pharmacological tools could be improved by characterization and development of biomarkers that could assist physicians in either (i) optimizing response to treatment with available antiepileptic drugs or (ii) identifying early those patients who are pharmacoresistant and could benefit from earlier referral to alternative therapies such as epilepsy surgery or neurostimulation. The first part of the research relates to pharmacokinetic biomarkers aimed at optimizing response to pharmacological treatment. Specifically, plasma concentrations of antiepileptic drugs have been shown to provide valuable indicators of pharmacological response, although their usefulness has not yet been clearly established for some second-generation drugs, partly due to lack of availability of assay methods easily applicable to therapeutic drug monitoring in the routine setting. In the present research, the latter deficiency was addressed by developing and validating two assay techniques for the determination of retigabine, known also as ezogabine, and perampanel in body fluids. For retigabine, an HPLC-UV method suitable for therapeutic drug monitoring was developed which permits sensitive quantitation of the drug in human plasma. Although therapeutic drug monitoring of antiepileptic drugs is usually based on their determination in plasma samples, alternative matrices, such as dried plasma spots, may offer advantages in some settings. For this reason, a HPLC-UV method for the quantitation of perampanel in dried plasma spots was developed and validated. Perampanel concentrations assessed in dried plasma spot samples obtained from patients receiving therapeutic doses were comparable to those measured in plasma samples. The second part of the research described in the present thesis relates to the investigation of microRNA (miRNA) profiles in plasma-derived extracellular vesicles as potential epigenetic biomarkers for early differentiation between patients resistant to antiepileptic drugs and patients responsive to these drugs. Additionally, miRNA expression profiles were also assessed in healthy controls in order to ascertain potential differences related to the disease itself rather than drug responsiveness. By applying a cross-sectional non-interventional open-label study design, miRNAs profiles in exosomes and microvesicles isolated from plasma were compared among 20 patients with drug-resistant focal epilepsy, 20 patients with drug-responsive focal epilepsy and 20 healthy control subjects. Extracellular purity, concentration and size distribution in each sample were ascertained by applying a combination of techniques (Western blot analysis, nanoparticle tracking analysis and transmission electron microscopy). Total RNA was extracted by exosome and microvesicle pellets, and sequenced on NGS platforms. The potential involvement of identified miRNAs in epilepsy-relevant pathways (predicted target genes and predicted pathways) was investigated by using the miRWalk 3.0 database and KEGG database. Statistical analysis of the results did not identify significant differences across groups in extracellular vesicle size or count. Five miRNAs were found to be de-regulated in exosomes isolated from pharmacoresistant patients compared with healthy controls (one was up-regulated and four were down-regulated). Four miRNAs were found to be de-regulated in microvesicles obtained from pharmacoresistant patients compared with healthy controls (two up-regulated miRNAs and two down-regulated). These findings, which require confirmation in a larger sample size, may provide valuable clues for a better understanding of processes involved in epileptogenesis as well as mechanisms implicated in pharmacoresistance.
19-dic-2019
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1301288
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