The Role of Diagnostics

Transcript

1 "Il ruolo della cromatografia liquida e del rilevatore di massa nel TDM (therapeutic drug monitoring) dei farmaci antiretrovirali" Antonio D Avolio Laboratory of Clinical Pharmacology and Pharmacogenetics University of Turin Spettrometria di Massa ed HPLC nel 2012: prospettive ed innovazioni Martedì 28 Febbraio 2012 The Role of Diagnostics Resistance Testing TDM Pharmacogenomics The right drug The right amount of the right drug The right amount of the right drug to the right person From Prof. David Back

2 Definizioni Il Therapeutic Drug Monitoring, meglio noto con l acronimo di TDM, rappresenta un esempio di ricaduta clinica pratica dell attività di laboratorio farmacologica. Come noto consiste nella determinazione delle concentrazioni plasmatiche di un farmaco e nell eventuale variazione posologica sulla base di tali risultanze. Il TDM, per l appunto monitoraggio delle concentrazioni terapeutiche del farmaco, è uno strumento operativo disponibile da tempo per l ottimizzazione dei dosaggi di farmaci come la digitale, gli anticoagulanti, gli anticonvulsivanti, immunosoppressivi, taluni antibiotici, antifungini e antiretrovirali. Definizioni Il concetto fondamentale è rappresentato dalla presenza di un efficacia ed una tossicità entrambe strettamente concentrazione-dipendenti, tali da individuare un range ottimale di concentrazioni entro il quale mantenere i livelli plasmatici del farmaco stesso con gli opportuni aggiustamenti posologici. Nel caso del warfarin il range terapeutico ottimale è rappresentato non tanto dalle concentrazioni di farmaco ma dal suo marker di attività farmacodinamica, ovvero il valore di tempo di protrombina [PD]. 2

3 TDM (Italian Guidelines - October 2011) 3

4 THE QUESTIONS IS How much is used the TDM of antinfective drugs? In Italy it is not widely used! TDM of anti-infective agents Several factors limit the routine use of TDM, these factors include: the lack of large prospective studies showing that TDM improves clinical and microbiological outcomes. the absence of therapeutic range and/or defined toxic concentrations (with few exceptions). intra-patient variability. the lack of widespread availability of clinical laboratories that perform measurements under quality (Certification and use of external quality controls). lack of experts to assist with the interpretation of concentration data and applying these data to revise patients' dosing regimens. high costs for shipment to qualified centers. the assay methods are reliable in chromatography 4

5 TDM of anti-infective agents Several factors limit the routine use of TDM, these factors include: the lack of large prospective studies showing that TDM improves clinical and microbiological outcomes. the absence of therapeutic range and/or defined toxic concentrations (with few exceptions). intra-patient variability. the lack of widespread availability of clinical laboratories that perform measurements under quality (Certification and use of external quality controls). lack of experts to assist with the interpretation of concentration data and applying these data to revise patients' dosing regimens. high costs for shipment to qualified centers. the assay methods are reliable in chromatography FAQ and Answers (7) Which is the best method to quantify drugs? Generally, there is not a best method to quantify a drug It depends on drug, concentration range, and the plasma volume and instrument! Many methods were developed and published, and all could be used to quantify the indicated drug 5

6 FAQ and Answers (8) Which is the best instrument to quantify drugs? HPLC UV or Fluorimetric UPLC UV or Fluorimetric HPLC MS or MS/MS UPLC MS or MS/MS GC MS ELISA (Immunoenzymatic) Others (Capillary Electrophoresis, ) It depends How to quantify drugs (ARVs)? (1) Choose of the instruments (on the basis of result reliability): 1. HPLC/UPLC UV or Fluorimetric (Gold Standard) 2. HPLC/UPLC MS or MS/MS 3. Capillary Electrophoresis 4. GC MS 5. ELISA (Immunoenzymatic) [metabolite?] (RSD or CV <20%) 6

7 How to quantify drugs (ARVs)? (2) Choice of the instruments (on the basis of test cost [including instrument charge]): 1. ELISA (Immunoenzymatic) 2. HPLC/UPLC UV or Fluorimetric (Gold Standard) 3. Capillary Electrophoresis 4. HPLC/UPLC MS or MS/MS 5. GC MS How to quantify drugs (ARVs)? (3) Choice of the instruments (on the basis of test cost [excluding instrument charge]): 1. HPLC/UPLC UV or Fluorimetric (Gold Standard) 2. HPLC/UPLC MS or MS/MS 3. GC MS 4. ELISA (Immunoenzymatic) 5. Capillary Electrophoresis Reagent and consumables expense: from 3 to 10 Euros/sample (excluding labour cost) 7

8 How to quantify drugs (ARVs)? (4) Choice of the instruments (on the basis of use simplicity): 1. ELISA (Immunoenzymatic) 2. HPLC/UPLC UV or Fluorimetric (Gold Standard) 3. Capillary Electrophoresis 4. HPLC/UPLC MS or MS/MS 5. GC MS How to quantify drugs (ARVs)? (3) Choice of the instruments (on the basis of sensitivity): 1. HPLC/UPLC MS or MS/MS 2. GC MS 3. HPLC/UPLC UV or Fluorimetric (Gold Standard) 4. Capillary Electrophoresis 5. ELISA (Immunoenzymatic) 8

9 HPLC/Mass Spectrometry (LC-MS or MS/MS) Moreover, with mass spectrometry you can quantify two or more coeluted peaks. Universal Specific Sensitive FAQ and Answers (7) Message for laboratorians Which is the best instrument to quantify drugs? HPLC/UPLC UV or Fluorimetric (Gold Standard) At same concentrations the UV detector is more precise and accurate when compared to the MS detector, using a good chromatographic run. When should be used the Mass detector? When the drug to be assayed has very low concentrations, using a good chromatographic run. 9

10 HPLC/Mass Spectrometry (LC/MS) Be careful! When two analytes (drugs or plasmatic noise) coeluate, the matrix effect is frequently observed and it may cause an underestmation of drug concentration The Message: Don t rely upon a short run with many drugs. A good peak separation is more important than time of analysis. TDM of anti-infective agents Several factors limit the routine use of TDM, these factors include: the lack of large prospective studies showing that TDM improves clinical and microbiological outcomes. the absence of therapeutic range and/or defined toxic concentrations (with few exceptions). intra-patient variability. the lack of widespread availability of clinical laboratories that perform measurements under quality (Certification and use of external quality controls). lack of experts to assist with the interpretation of concentration data and applying these data to revise patients' dosing regimens. high costs for shipment to qualified centers. the assay methods are reliable in chromatography 10

11 FAQ and Answers Which is the best shipping condition for the sample? To close laboratory: The primary tube has to come as quickly as possible to the laboratory, serum samples and frozen immediately. To external Laboratory: The best condition (Gold Standard) for shipping, for all antiretrovirals TDM, is frozen plasma samples, with dry ice. News about sending samples Which is the best shipping condition for the sample? New way with Dry Plasma Spots (DPS) and Dry Blood Spots (DBS) with delivery at room temperature and low-cost. Not for all drugs, but ONLY for thermostable drugs. DPS DBS 11

12 News about sending samples DPS or DBS? Dry Blood Spots ADVANTAGES It does not require centrifugation or waiting for the sedimentation. DISADVANTAGES Often requires correction by the hematocrit All clinical studies (guidelines) were performed on PLASMA The assay depends upon the thickness of the deposited material News about sending samples DPS or DBS? Dry Plasma Spots ADVANTAGES It does not require correction by the hematocrit The result is not influenced by the thickness of the deposited material It stabilizes better the drugs (for a best dehydration) DISADVANTAGES Require the centrifugation or sedimentation waiting. 12

13 News about sending samples Dry Sample Spots Device? D Avolio et al (JPBA) We have developed (and now it is on sale) a new transport system (at room temperature) for the determination of drugs from Dry Plasma Spots We are studying the stability, even in the long term, at room temperature, at 0/4 C and at -20 C for many drugs. With the Dry Sample Spots Device (DSSD) we have observed for several molecules no significant degradation at room temperature for 2-4 weeks. Dehydrated biological samples with DSSD are then possible to send to laboratories with a simple postal letter. The shipping is possible without dry ice, reducing costs and making more accessible the TDM and/or the determination of drugs in clinical trials too. Dry Sample Spots Device 13

14 Dry Sample Spots Device DPS (TDM) 14

15 Dry Sample Spots Device DBS (TDM e PG) Dry Sample Spots Device List of drugs actually assayed in our laboratory in Turin through the system Dry Sample Spots Device (DSSD): Antiretrovirals (Nevirapine, Efavirenz, Etravirine, Raltegravir, Maraviroc, Atazanavir, Nelfinavir, Darunavir, Amprenavir, Indinavir, Lopinavir, Ritonavir, Saquinavir, Tipranavir) Antivirals (Ribavirin) Antifungals (Voriconazole, Posaconazole, Itraconazole) Antibiotics (Daptomycin, Linezolid,... Other being validated) 15

16 Dry Plasma Spot - [Evolution] Points to be highlighted: DSSD used for environmental analysis? Sampling accuracy Calibrated pipettes Manual ability Samples have to be well distributed on DSSD... or other setting? Instruments H-UPLC-UV/PDA/FL (not optimal?) H-UPLC-MS or MS/MS (low volume!) News about sending samples (final considerations) The current gold standard for ARVs TDM is fresh plasma. Alternatively, plasma should be frozen and sent in dry ice to the Reference Laboratories. The DBS and DPS are new opportunities for low-cost shipping. Not all drugs can be shipped via DPS and / or DBS, but only the most thermo-stable. DPS better than DBS (Clinical studies on Plasma; DPS No need correction by hematocrit) [if we consider the same instrumentation and method of analysis] Several companies provide goods that can be used (DSSD Biomicron Laboratories; FTA Whatman; others ) Choose experienced laboratories in the field, with right instruments (mass detectors). 16

17 TDM of anti-infective agents Several factors limit the routine use of TDM, these factors include: the lack of large prospective studies showing that TDM improves clinical and microbiological outcomes. the absence of therapeutic range and/or defined toxic concentrations (with few exceptions). intra-patient variability. the lack of widespread availability of clinical laboratories that perform measurements under quality (Certification and use of external quality controls). lack of experts to assist with the interpretation of concentration data and applying these data to revise patients' dosing regimens. high costs for shipment to qualified centers. the assay methods are reliable in chromatography Continuous monitoring (External Control Quality) To verify the extraction and instrumental methods To verify calibrators and quality controls 17

18 KKGT From 2003, we monitor our analysis by KKGT International Interlaboratory Quality Control Program for TDM in HIV infection and for Antifungal Drugs ISO or GLP Quality Certification To answer to market and quality requirements In our setting, also for an optimal internal organization 18

19 RINA From 2008, we are UNI EN ISO 9001:2008 certified by RINA (Italian Certification Company) IQNet and by IQNet (International Certification Company) for the following fields of activities: design, development and application of determination methods for anti-infective drugs and now for pharmacogenetic analyses. The certifications and annual monitoring with international quality control are aimed to improve and level up the performance of each laboratory and to give the most reliable results to clinicians 19

20 Conclusioni e Considerazioni (1) La cromatografia (H-UPLC) è una tecnica complessa da utilizzare nei laboratori analisi clinici. Il rivelatore di massa complica la gestione strumentale, ma facilita la messa a punto e l ottenimento di un risultato (che sarà in ogni caso migliore di quello ottenuto con l ELISA) Per avere risultati affidabili e precisi ci vogliono strumenti adeguati e metodiche validate in maniera seria e scrupolosa. Tutti sono in grado di garantire questi risultati? (NO al momento!) Conclusioni e Considerazioni (2) Vi è il bisogno di identificare centri specializzati in ogni regione (qualificati e certificati). Con personale esperto ed addestrato alle problematiche tecniche. Da considerare anche nell ottica del contenimento delle spese, accentrando ed ottimizzando le risorse. Nasce la necessità di inviare i campioni a questi centri. I DPS e DBS sono buone opportunità/mezzi a basso costo. Necessitano di piccoli volumi ed rivelatori di massa sono gli strumenti che meglio si possono adattare. Non per tutte le molecole da analizzare Già nel presente, ma saranno il futuro! 20

21 Acknowledgement Università di Torino Prof. Giovanni Di Perri Prof. Stefano Bonora Prof. Francesco Giuseppe De Rosa Andrea Calcagno Maria Cristina Tettoni Laura Trentini Liverpool University Prof. David Back Prof. Andrew Owen Marco Siccardi The Laboratory Team Mauro Sciandra Lorena Baietto Marco Simiele Jessica Cusato Alessandra Ariaudo and the students Spallanzani Hospital (Rome) Massimo Tempestilli Leo Pucillo 21