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Small molecule aptamer-based assay meets FDA criteria for drug monitoring

05/01/2021

 

A newly published paper in The Analyst by Dr. Federico Polo, Assistant Professor at Ca’ Foscari University of Venice and former group leader at the National Cancer Institute (Centro di Riferimento Oncologico IRCCS) in Aviano, Italy, demonstrates the use of a small molecule aptamer from Aptamer Group, to develop biosensor platform for the detection and monitoring of the chemotherapeutic, imatinib, in human plasma.

Polo and his team used the small molecule aptamer to produce an assay that could be developed to deliver point-of-care analysis of imatinib concentrations for therapeutic drug monitoring in oncology. The developed biosensor is an SPR-based assay that meets the strict regulatory criteria for ligand binding assays suitable for clinical samples. This is the first time that a biosensing approach has met the required accuracy and precision for the analytical validation of therapeutic concentrations of imatinib.

“Measuring the interactions between small molecules and aptamers or other affinity molecules by means of SPR is considered difficult, due to the small local change of the optical properties on the sensor surface that occurs when small molecule target interacts with the higher molecular weight capturing receptor, such as an aptamer. Yet, the ability to monitor patient plasma concentrations at point of care rather than through high cost, laborious processes currently required offers significant patient and clinician advantages.”

Lead author Dr. Federico Polo

Measuring the interactions between small molecules and aptamers or other affinity molecules by means of SPR is considered difficult, due to the small local change of the optical properties on the sensor surface that occurs when small molecule target interacts with the higher molecular weight capturing receptor, such as an aptamer. Yet, the ability to monitor patient plasma concentrations at point of care rather than through high cost, laborious processes currently required offers significant patient and clinician advantages.

 

Aptamer use drug concentrations

Selecting small molecule aptamers

Aptamer Group used their proprietary small molecule aptamer selection platform to identify an aptamer specific for imatinib. The aptamer was selected to:

  • be target-specific
  • not interact with the human plasma matrix
  • not interact with complimentary drugs used alongside imatinib in standard therapeutic regimes

The selected imatinib aptamer exhibited a KD = 131 ± 11 nM, which is in the typical range for small molecules, and showed no interaction with 12 different co-medications when assayed on the biosensor platform.

The excellent selectivity and performance of this small molecule aptamer allowed Polo’s team to develop the diagnostic assay for this chemotherapeutic in line with strict FDA regulatory guidelines to address the needs of oncologists and clinicians.

Aptamer coated biosensor surface

Validation of the aptamer-assay to regulatory guidelines

The aptamer-based biosensor assay was fitted to a 4-parameter logistic model in accordance with the FDA regulatory guidelines for ligand-binding assays. Within this the linear fitting range of back calculated concentrations was in the range 400-6000 ng/mL allowing the entire therapeutic range of imatnib to be assessed, with an accuracy between 97.1% and 101.8% and precision ranged from 1.3% and 5.2 % as a percentage of the coefficient of variance.

The developed assay was tested using clinical samples taken from 12 patients administered with imatinib at the National Cancer Institute in Aviano, Italy. Dr. Toffoli, co-author and Director of the Experimental and Clinical Pharmacology Unit at the National Cancer Institute in Aviano, and his team analysed these samples in parallel via standard HPLC and mass spectrometry methods for comparison with the small molecule aptamer assay. Samples were processed following the same procedure for standards and quality control. A Pearson coefficient of 0.98 and mean difference of 5.3% between the analytical platforms show excellent correlation and accuracy of the aptamer-based assay compared to standard HPLC-MS values. Using the small molecule aptamer for imatinib detection and quantification the biosensor assay showed excellent linearity, recovery, selectivity, low matrix effects, inter- and intra-day precision and accuracy values.

“We addressed three important and specific aspects in this study:

  • a rapid detection of the analyte by minimizing the sample preparation procedure as much as possible.
  • a validation of the method following regulatory guidelines, an aspect too often underestimated in the literature and academic environment.
  • reduce or avoid the use of chemicals/solvents.

Following our promising findings, we intend to further develop this analytical method, with the possibility of developing new analytical tools for personalized TDM and PoC applications that could reduce the sample volume to 5-10 µL, essentially a drop of blood.”

Lead author Dr. Federico Polo

Using this biosensor approach, employing small molecule aptamers could potentially lead to the development of reliable analytical tools for the therapeutic drug monitoring of many small molecule drugs, offering significant benefits to patients and healthcare systems for rapid point-of-care monitoring and the prevention of adverse effects in treatment.

 

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small molecule detectionsmall molecule monitoring

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