Debora Pensi (Presenter)
University of Turin
Bio: I am a PhD Student of Molecular Medicine of the University of Turin. I work in the Laboratory of Clinical Pharmacology and Pharmacogenetic of Amedeo di Savoia Hospital (Turin, Italy).
Authorship: D. Pensi, A. De Nicolò, F. Favata, C. Pisciotta, G. Di Perri, A. D'Avolio
Unit of Infectious Diseases, University of Turin, Department of Medical Sciences, Amedeo di Savoia Hospital, Turin, Italy (Laboratory of Clinical Pharmacology and Pharmacogenetic)
| Short Abstract For many drugs, blood or plasma measurements have a poor prediction of their efficacy and toxicity in the site of action. This method is capable of obtain concentration as ng/mL from different kind of tissues: we described the example of tacrolimus. After tissue lysation, done by a MagNA Lyser Instrument (Roche), tacrolimus was extracted with on-line SPE platform and then separated and detected through a UHPLC-MS/MS. This method could be used for evaluating the intra tissue concentrations of different drugs and for understanding the correlation between plasmatic/blood/PBMCs and tissue concentrations, with the same results expression (ng/mL). |
Long Abstract
INTRODUCTION: Intra-tissue drug concentrations are fundamental to evaluate drug efficacy and toxicity in the site of action, as well as for monitoring drug interactions and inter-subject variability in drug response. Blood or plasma drug concentrations are typically used as a surrogate measure for the ones at the site of action. Unfortunately, this is not valid for poorly permeable, actively transported and highly protein-bound drugs (majority of the drugs). For this reason, efficiently quantifying drugs at the real sites of action could be important. However, current methods for drugs intra-tissue quantification express results as “ng/mg of tissue”, making these not comparable with plasma or blood concentrations, as volume ratio [1-4]. Conversely, in this study we aimed to develop a method to correctly normalize intra-tissue analytical results on the real volume of cells (as “ng/mL of cells”): Tacrolimus (TAC) was used as example.
MATERIAL and METHODS: Blank PBMCs for calibration curve were collected using Cell-Preparation-Tubes and counted with a Beckman Coulter Counter. Swine biopsies were washed with PBS and divided in portion of 20 mg. After Internal Standard (6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline) and TAC addition, samples were homogenated into MagNA Lyser Green Beads by a MagNA Lyser Instrument (Roche): 200 ul of supernatant underwent automated DNA extraction through MagNA Pure Compact (Roche). Cell number was quantified by evaluating DNA absorbance using a spectrophotometer (Biophotometer, Eppendorf), comparing it to a calibration curve (PBMCs). Other 50uL of the homogenate were diluted with 450uL H2O:MeOH [30:70] and 140uL H2O:MeOH [50:50] to obtain the same conditions of the previously published UPLC-MS/ MS coupled with on-line SPE method used for TAC quantification in PBMCs[5]. H2O and MeOH (both with 2mM/L ammonium acetate and 1mL/L formic acid) were used as mobile phases. The monitored mass transitions were 821.53 > 768.3, and 313.09 > 246.46 for TAC and IS, respectively. From each biopsy, a part of tissue after collagenase-dispase treatment (Roche) was used to evaluate tissue cells Mean Cellular Volume (MCV) using a Beckman Coulter Counter.
RESULTS: This method, applied to different kinds of tissue (liver and kidney), resulted capable of quantifying TAC, expressing it as ng/mL, and ensures a high sensitivity and high specificity (LLOQ of 0.39 ng/mL). The calibration curve (0.39 - 40 ng/mL) was found to be linear (r2>0.998) and performed on the basis of peak area, forced through the origin of the axes. No interfering peaks were observed at TAC and IS retention times of 4.19 ± 0.07 and 2.26 ± 0.07 min, respectively. The accuracy and intra- and inter-day precision were evaluated for both tissues: high accuracy and high precision were found at all three QC levels. Preliminary data on recovery and matrix effect were obtained. Recovery was evaluated both as extracted sample vs post homogenation spike sample (PHSS) and also as extracted sample vs post extraction spike sample (PESS) at QCL and QCH levels: it resulted stable and reproducible (mean recovery 69%, mean RSD 5.5%). The matrix effect for all tissues at QCL and QCH levels resulted stable and reproducible (mean matrix effect 41.6%, mean RDS 6.5 %). No significant differences in applicability of this method to PBMC and tissue samples can be observed.
CONCLUSION:
Using the last generation UHPLC-MS/MS coupled with SPE on-line technology, we have developed a simple, specific, sensitive, precise, rapid and accurate method. Since this protocol allows to correctly normalize intracellular analytical results, it could be useful in the near future to verify the correlation between plasma/blood/PBMC and tissue concentrations for many drugs, as TAC, to help clinicians to improve therapy and clinical outcome, and/or to evaluate their potential intracellular accumulations on the basis of data obtained from the target cells for research and clinic purposes.
References & Acknowledgements:
(1)Pellegatti and Pagliarusco, 2011
(2)Xue et al., 2012
(3)Mouton et al.,2008
(4)Timmerman et al.,2014
(5) Pensi et al.,2015
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