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New paper: Small molecule drug monitoring for therapeutic drug monitoring

Researchers at the National Cancer Institute (Centro di Referimento Oncologico IRCCS), Aviano, Italy, have published a new paper in Analytical and Bioanalytical Chemistry. Using aptamers from Aptamer Group, the team detected the small molecule drug, irinotecan, on a biosensor platform to FDA regulatory standards.

Following on from the recent paper in The Analyst, showing the development of an aptamer-based biosensing platform for the small molecule drug, imatinib, Dr Toffolli and his lab, demonstrated the ability to analyze irinotecan at clinically relevant concentrations in human plasma, using SPR as the detection technique and a DNA aptamer as the selective receptor.

The paper represents an innovative approach in the field of drug monitoring. This work involved a significant partnership between the unit of experimental and clinical pharmacology of the National Cancer Institute and Aptamer Group, paving the way for future collaborations, underpinning the relationships between private businesses and public institutions. The use of aptamers to optimise drug dosage looks toward the horizon of personalised and tailored medicine, which is a new frontier in oncology.

Lead Author Dr Giuseppe Toffoli

Why use aptamers in small molecule drug monitoring?

Aptamers are single-stranded oligonucleotides (RNA or DNA), capable of recognizing and binding different targets such as proteins, peptides, drugs, and small molecules with high affinity and specificity. Aptamers are able to fold into a large variety of secondary structures with specific structural motifs (hairpin, pseudo-knot, stem-loop/ bulge, and G-quadruplex), to form a three-dimensional structure that can specifically bind to the desired target molecule.

Irinotecan is a chemotherapeutic used in the treatment of colonic and rectal cancer. The dose of irinotecan administered should be modified in line with any observed patient side effects. Monitoring the small molecule drug levels during treatment can ensure the dose is maintained within the therapeutic window, while preventing toxic effects or treatment failure.

Detecting small molecule drugs via SPR is challenging, as the small molecule biding yields a weaker SPR signal than larger protein targets. Consequently, monitoring small molecule drug interactions with high molecular weight affinity ligands, such as antibodies, is difficult to assess.

To overcome this, Toffoli’s team used aptamers in a dissociation style assay: Binding of the small molecule target to the aptamer releases the aptamer from the biosensor surface to give an improved signal, due to the aptamer’s larger size compared to the small molecule target. This is not possible with standard affinity ligands, such as antibodies, yet offers new potential for small molecule drug monitoring.

We have experience in developing aptamers and Optimers™ for small molecule targets.

Meeting FDA criteria with novel small molecule drug detection assays

Accuracy, Precision and Bias

Following the initial optimisation of the biosensor assay in buffer, Toffoli moved to analyse human plasma samples. Samples were diluted 1:10 and mixed with Tween-20 and micro-filtered through a 30kDa centrifugal filter. The biosensor response to various concentrations of irinotecan was fitted to a 4 parameter logistic curve, as per the FDA guidelines, and showed Pearson’s coefficient of determination (R2) higher than 0.995 for each set of measurements, with accuracy between 94.5% and 105.2% and precision from 1.6 to 5.8% in terms of CV%. Bias was within 15% for all calibrators except at the lower limit of quantification, where it reached ± 17%.

Limit of Detection

The small molecule drug assay achieved a LOD of 55 ng mL−1 in plasma, meaning that this method can be used to measure irinotecan in samples from patients up to 20 h after dosing, as well as high concentrations of the drug, which lead to toxicity, allowing clinicians to adjust the patient’s treatment accurately. The aptasensor showed a working dynamic range within the same order of magnitude as other detection approaches, of 100-7500 ng mL-1.

Highly selective aptamer

To assess the selectivity of the aptamer assay for the small molecule drug, irinotecan, other known interfering low molecular weight compounds found in clinical samples were tested. These included co-medications commonly given with irinotecan, such as dexamethasone, atropine, chlorphenamine, 5-fluorouracil, ondansetron, loperamide and capecitabine, and folinic acid, and the main metabolites of irinotecan SN-38, SN-38G, APC, and NPC. The co-medications showed no interaction with the immobilised aptamer while negligible interactions were noted with the irinotecan metabolites.

Aptamer-based biosensor shows comparable performance with HPLC-MS

Current testing methods use reverse-phase HPLC coupled with mass spectrometry. However, these assays are highly laborious, low throughput and high cost. Point-of-care testing and biosensing technologies could deliver a much-needed strategy to extend therapeutic drug monitoring to a wide number of patients for more rapid and cost-effective treatment.

To thoroughly assess the performance and potential of the biosensor Toffoli tested 72 clinical samples from patients administered with irinotecan the Centro di Riferimento Oncologico di Aviano (CRO), Italy. The samples were processed in parallel using HPLC-MS and the irinotecan biosensor platform showing a correlation of a slope of 1.09 and a Pearson coefficient of 0.945.

Advantages of the aptamer-based assay for the small molecule drug compared to chromatographic and electrochemical methods include the lack of requirement for organic solvents, making this biosensor approach more suitable for transfer to a point-of-care test.

Although the direct detection of small molecules with SPR does not typically give a strong enough signal, using a dissociation style assay allowed the successful quantitation or irinotecan with consistent validation data across the clinical concentration range, using a simple process with inexpensive, non-toxic reagents. The incorporation of aptamers into biosensing technologies could extend the practice of small molecule drug monitoring to optimise drug dosage and administration in oncology.

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