Tandem mass spectrometry techniques for the TDM of antifungals : new perspectives and challenges

Transcription

1 Tandem mass spectrometry techniques for the TDM of antifungals : new perspectives and challenges 1 st International Workshop on Clinical Pharmacology of Antifungal Drugs and Fungal Diseases Berlin, April 26, 2013 Dr Laurent Decosterd, PhD Innovation & Development Unit, Laboratory of Clinical Pharmacology, Service of Biomedicine, CHUV Lausanne

2 Therapeutic Drug Monitoring (TDM) Rationale and usefulness of TDM Principle of Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) Multiplex analysis of antifungal drugs Selected examples of applications Monitoring of antifungal drugs combination Identification of altered metabolism of voriconazole Detection of previous treatments Conclusions

3 Therapeutig Drug Monitoring (TDM) : Measurement of plasma concentration under treatment Dosage individualization based on plasma levels, targeting a pre-defined range Better chances of achieving therapeutic efficacy Reduced risks of meeting toxicity Candidate Drugs : Therapeutic Drug Monitoring (TDM) General principles Drugs administrated on the long-term (chronic conditions) Drugs administrated to critically ill patients, with limited remaining therapeutic options (ICU). Significant inter-patient pharmacokinetic variability Low intra-patient pharmacokinetic variability Consistent concentration - efficacy / toxicity relationships

4 Therapeutic Drug Monitoring (TDM) Should be considered in case of lack of early, or easily accessible pharmacodynamic surrogates Unexpected clinical toxicities Less than expected clinical response Drug-drug or drug-food interactions Changes in patho-physiological state, that may impair gastrointestinal, hepatic or renal function Pregnancy and pediatric patients Monitoring short-term adherence

5 Therapeutic Drug Monitoring (TDM) Antifungal drugs meet the overall criteria for the implementation of a routine TDM program Measurements and interpretations!!! Reliable analytical methods must be available

6 Development of UPLC-tandem MS method for the assay of antifungals drugs Results must be available for TDM interpretations within 24-48h hours The analytical method must be: Rapid (and user-friendly) Precise (to limit the number of replicates) Accurate (obviously )

7 Instrument of High or Ultra Performance Liquid Chromatography (HPLC, UPLC ) High pressure pump ( bar) Auto-sampler (samples) Chromatography column (drugs and metabolites separation) Detector: UV, fluorescence electrochemical MS, MS/MS ml/min Solvents (MeCN, MeOH, H 2 O) Data processing (PC)

8 Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) From HPLC column The Ionisation Step To the first quadrupole of the MS instrument

9 Selection of parent ion: (i.e. molecular mass : m/z (fluconazole) Selection of product ion (for instance: caracteristic fragment: m/z 238 daughter mass for fluconazole) N N N OH + N N OH N N F N N F F F We observe (and actually measure) The transition m/z [M+H + ] m/z 238

10 (Triple Stage Quadripole Mass Spectrometer) TSQ Quantum, Thermo Scientific Corporation

11 Strength and Limitations of Tandem Mass Spectrometry Ionisation From HPLC column To the first quadrupole of the MS instrument Strength: ultimate selectivity (thanks to triple quadripole mass detection) Limitations: Ionisation of the analyte may be influenced by the presence of endogenous matrix components co-eluting with the target analyte that may influence its ionisation: = the so-called MATRIX EFFECT (Achilles' heel) This influence must be eliminated or at least normalised.

12 Stable isotope-labeled internal Standards (I.S.) and IS calibration method Ionisation step From HPLC column To the first quadrupole of the MS instrument The use of stable isotope-labeled internal standards is the first choice approach to minimize the influence of matrix effect due to endogenous components co-eluting with the analytes that affects signal intensity, and hence, the accuracy and precision of a quantitative method. Of particular importance when using electrospray ionisation mass spectrometry. The potential influence of can be normalised using the corresponding stable isotope-labeled Internal Standards (deuterium, 13 C).

13 «Multiplex» Analyses of antifungal drugs Fluconazole Voriconazole Voriconazole-N-oxide Posaconazole Itraconazole Hydroxy-itraconazole Caspofungine Anidulafungine Hydroxy-itraconazole Posaconazole Antimicrob Agents Chemother, 54, (2010)

14 «Multiplex» assay: Analysis of drugs from the same therapeutic class in a single chromatographic run Advantages : Unique sample extraction procedure and short analytical run. Saves times by the establishment of simultaneous calibration curves Applicable for blood samples from patients receiving either different single-drug, or combination regimens Results obtained on a daily basis: TDM dosing adjustments performed within shortest time intervals. Detection of other drugs possibly present in sample.

15 An Ultra-Performance Liquid Chromatography - tandem MS method for the simultaneous determination of 6 antifungal agents and their metabolites Fluconazole Posaconazole Fluconazole-d4 Posaconazole-d4 Voriconazole-NO HO-itraconazole Voriconazole-NO-d3 HO-itraconazole-d5 Voriconazole Anidulafungin Voriconazole-d3 Caspofungin Itraconazole Caspofungin analogue Itraconazole-d5 The chromatographic separation of 8 compounds takes place in less than 5 minutes

16 Voriconazole N-oxide : in source metabolite dissociation voriconazole 2.7 µg/ml Chromatogram from an Acute Lymphoblastic Leukemia ( ALL) patient on oral voriconazole 400 mg BID. 0.4 µg/ml Voriconazole trough level was 0.4 µg/ml (below the proposed therapeutic range) Voriconazole N-oxide level was 2.7 µg/ml suggesting an accelerated hepatic metabolism with first pass effect on oral bioavailability Importance of the chromatographic separation to avoid cross-talk!

17 MULTIPLEX ASSAY 1. Monitoring of antifungals drugs combination 2. Detection of previous treatment Fluconazole stopped 50 days before sampling 0.5 µg/ml 0.16 µg/ml 3.0 µg/ml 1.9 µg/ml 2.6 µg/ml 1.0 µg/ml Antimicrob Agents Chemother, 54, (2010)

18 The simultaneous assay of voriconazole and voriconazole-no is an index of the phenotyping activity of CYP2C19, that integrates : genetic background, liver function, and drugs interactions Hyland, R., B. C. Jones, and D. A. Smith Identification of the cytochromep450 enzymes involved in the N-oxidation of voriconazole. Drug Metab. Dispos. 31:

19 MULTIPLEX ASSAY 3. High voriconazole levels with impaired liver metabolism 4. Increased voriconazole metabolism as evidenced by High voriconazole-no levels C:\Xcalibur\...\Fong-b205\fong-b205_018 2/27/2013 5:13:00 PM P 6676 RT: SM: 15B Relative Abundance RT: 2.48 AA: RT: 2.93 AA: RT: 2.93 AA: NL: 3.12E5 TIC F: + c ESI SRM ms [ ] MS ICIS fong-b205_018 Voriconazole-NO NL: 1.54E6 TIC F: + c ESI SRM ms [ ] MS ICIS fong-b205_018 Voriconazole 7.2 µg/ml NL: 2.90E6 TIC F: + c ESI SRM ms [ ] MS ICIS fong-b205_018 C:\Xcalibur\...\Fong-b220\fong-b220c_020 4/15/2013 8:36:52 PM P 6894 RT: SM: 15B Relative Abundance RT: 2.20 AA: µg/ml µg/ml RT: 2.66 AA: RT: 2.65 AA: Voriconazole-NO Voriconazole 2.0 µg/ml NL: 2.56E6 TIC F: + c ESI SRM ms [ ] MS ICIS fong-b220c_020 NL: 2.97E5 TIC F: + c ESI SRM ms [ ] MS ICIS fong-b220c_020 NL: 2.57E6 TIC F: + c ESI SRM ms [ ] MS ICIS fong-b220c_ Voriconazole-d Voriconazole-d Time (min) Time (min)

20 How accurate is the multiplex assay? External Quality Control Program KKGT, Stichting Kwaliteitsbewaking Klinische Geneesmiddelanalyse en Toxicologie: Association for Quality Assessment in TDM and clinical Toxicology, The Hague, The Netherlands. International Interlaboratory External Quality Assurance Program for antifungals agents (organized at present for azoles and flucytosine)

21 International Interlaboratory External Quality Assurance Program for antifungals agents (57 labos): LAUSANNE CHUV Results for 2012

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23 Probability of response to voriconazole (solid line) and of grade 3 neurotoxicity (dashed line) as a function of voriconazole trough plasma concentrations in adult patients with invasive fungal infections Pascual A, Csajka C et al & Marchetti O, Clin Infect Dis 2012 Therapeutic range : 1.5 to 4.5 mg/l

24 «Multiplex» assay: LIMITATIONS Restricted to analytes contained in the calibration samples. Total number of drugs in calibration is not infinite, because of solubility issues of drugs in the matrix-match calibration samples, especially for lipophilic drugs at high concentrations (requiring the addition of nonmatrix components (i.e. solvents) : < 10% of total biological matrix volume of calibrators. Calibration curves established for each analytical series, but used only in case of patients samples. Possibly requires an additional step of validation. i.e. cross-validation calibration: «single analyte» versus «multiplex» analysis. Important in case of co-elution of analytes to exclude the occurence of reciprocal competition at the ionisation step. Easily circumvented, if stable isotopically labelled (D, 13 C,) Internal Standards of drugs are available.

25 LC tandem MS assay of further antifungal drugs Isavuconazole Micafungin Amphotericin B (free and total levels and liposomal fraction) New antifungal agents of current and future classes

26 Tandem mass spectrometry techniques for the TDM of antifungals: challenges Availability to isotopically labelled (D, 13 C) Internal Standards (IS) for echinocandins (and new antifungal agents is the future) is a recurring issues. Efforts for a facilitated access must be prioritized. The assay of amphotericin B (administered as liposomal formulation) is an additional analytical challenge. Total amphotericin B plasma levels (free + liposomal) (ie. after liposomal disruption with suitable methodology (i.e. detergent, sonication)) Free plasma levels of amphotericin B

27 TDM of antifungals: Conclusions The concentration of antifungal drug measured in plasma is at present the best marker of exposure in patient, integrating both genetic and non-genetic (drug interactions, patients pathophysiology, etc) influences. Strong arguments exist for implementing TDM for antifungal drugs, based on patients plasma concentrations. Randomised trials of prospective TDM intervention integrating clinical outcome, emergence of resistance and morbidity/mortality are still lacking. In the emerging field of Personalized Medicine, the development of TDM for patient-tailored dosage adjustment of antifungal therapy should allow to maximize both the therapeutic benefit and the tolerability of drugs. The right dose of the right drug to the right patient.

28 Acknowlegments Laboratory Thomas Mercier Dr Boris Zanolari Beatrice Ternon Sandra Cruchon Nicole Guignard Research Scientists Manel Aouri, PhD student Jaurès Blanc, PhD student Dr Aurélie Fayet Mello Dr Nicolas Widmer Partners within the Lausanne University Hospital Prof Oscar Marchetti Prof Chantal Csajka Prof Thierry Buclin PD Dr Matthias Cavassini Prof Amalio Telenti Prof Chin Bin Eap

29 Financial Supports and Collaborations Centre Coordonné d Oncologie ambulatoire, CCO Lausanne Swiss Tropical Institute Basel