Enzyme-Based Electrochemical Biosensor for Therapeutic Drug Monitoring of Anticancer Drug Irinotecan.

Therapeutic drug monitoring (TDM) is the clinical practice of measuring pharmaceutical drug concentrations in patients' biofluids at designated intervals, thus allowing a close and timely control of their dosage. To date, TDM in oncology can only be performed by trained personnel in centralized laboratories and core facilities employing conventional analytical techniques (e.g., MS). CPT-11 is an antineoplastic drug that inhibits topoisomerase type I, causing cell death, and is widely used in the treatment of colorectal cancer. CPT-11 was also found to directly inhibit acetylcholine esterase (AChE), an enzyme involved in neuromuscular junction. In this work, we describe an enzymatic biosensor, based on AChE and choline oxidase (ChOx), which can quantify CPT-11. ACh (acetylcholine) substrate is converted to choline, which is subsequently metabolized by ChOx to give betaine aldehyde and hydrogen peroxide. The latter one is then oxidized at a suitably polarized platinum electrode, providing a current transient proportional to the amount of ACh. Such an enzymatic process is hampered by CPT-11. The biosensor showed a ∼60% maximal inhibition toward AChE activity in the clinically relevant concentration range 10-10 000 ng/mL of CPT-11 in both simple (phosphate buffer) and complex (fetal bovine serum) matrixes, while its metabolites showed negligible effects. These findings could open new routes toward a real-time TDM in oncology, thus improving the therapeutic treatments and lowering the related costs.

Simultaneous telemetric monitoring of brain glucose and lactate and motion in freely moving rats.

A new telemetry system for simultaneous detection of extracellular brain glucose and lactate and motion is presented. The device consists of dual-channel, single-supply miniature potentiostat-I/V converter, a microcontroller unit, a signal transmitter, and a miniaturized microvibration sensor. Although based on simple and inexpensive components, the biotelemetry device has been used for accurate transduction of the anodic oxidation currents generated on the surface of implanted glucose and lactate biosensors and animal microvibrations. The device was characterized and validated in vitro before in vivo experiments. The biosensors were implanted in the striatum of freely moving animals and the biotelemetric device was fixed to the animal's head. Physiological and pharmacological stimulations were given in order to induce striatal neural activation and to modify the motor behavior in awake, untethered animals.

Development and characterization of an implantable biosensor for telemetric monitoring of ethanol in the brain of freely moving rats.

Ethanol is one of the most widespread psychotropic agents in western society. While its psychoactive effects are mainly associated with GABAergic and glutamatergic systems, the positive reinforcing properties of ethanol are related to activation of mesolimbic dopaminergic pathways resulting in a release of dopamine in the nucleus accumbens. Given these neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF) is of great importance. In this study, we describe the development and characterization of an implantable biosensor for the amperometric detection of brain ethanol in real time. Ten different designs were characterized in vitro in terms of Michaelis-Menten kinetics (V(MAX) and K(M)), sensitivity (linear region slope, limit of detection (LOD), and limit of quantification (LOQ)), and electroactive interference blocking. The same parameters were monitored in selected designs up to 28 days after fabrication in order to quantify their stability. Finally, the best performing biosensor design was selected for implantation in the nucleus accumbens and coupled with a previously developed telemetric device for the real-time monitoring of ethanol in freely moving, untethered rats. Ethanol was then administered systemically to animals, either alone or in combination with ranitidine (an alcohol dehydrogenase inhibitor) while the biosensor signal was continuously recorded. The implanted biosensor, integrated in the low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF and represents a new generation of analytical tools for studying ethanol toxicokinetics and the effect of drugs on brain ethanol levels.

Practical fluorimetric assay for the detection of anticancer drug SN-38 in human plasma.

The implementation of therapeutic drug monitoring in the routine clinical practice in oncology is mainly limited by the lack of therapeutic indexes for the majority of the anticancer drugs, and by the absence of suitable analytical tools, which can accurately quantify in real time the concentration of the administered drugs and their relevant metabolites in biological fluids. In this work, a simple and efficient fluorimetric determination of SN-38, the active metabolite of the anticancer drug irinotecan, was developed and applied to human plasma samples. The intrinsic fluorescence of SN-38 allowed its quantification in the range 10-500 ng mL-1 with a LOQ of 5.0 ng mL-1 and a LOD of 1.5 ng mL-1. Low interferences due to main metabolites of irinotecan and comedications, commonly associated with administration of irinotecan, were observed. A validation study, according to FDA and EMA guidelines for bioanalytical method validation, was carried out and, finally, blind samples were analyzed in parallel with a HPLC-MS method obtaining an excellent agreement between the two techniques.

Biosensing Technologies for Therapeutic Drug Monitoring.

BACKGROUND AND RATIONALE: Therapeutic drug monitoring (TDM) is the clinical practice of measuring pharmaceutical drug concentrations in patients' biofluids at designated intervals to allow a close and timely control of their dosage. This practice allows for rapid medical intervention in case of toxicity-related issues and/or adjustment of dosage to better fit the therapeutic demand. Currently, TDM is performed in centralized laboratories employing instruments, such as immunoassay analyzers and mass spectrometers that can be run only by trained personnel. However, the time required for the preparation, samples analysis, and data processing, together with the related financial cost, severely affects the application of TDM in medical practices. Therefore, a new generation of analytical tools is necessary to respond to the timely need of drug administration or reduction aiming at effectively treating oncologic patients. AIM OF THE REVIEW: State-of-the-Art Technologies for TDM: Technological advances in the field of nanosciences and biosensors offer the unique opportunity to address such issues. The interest for the so-called nanobiosensors is considerably increasing, particularly in drug discovery and clinical chemistry, even though there are only few examples reporting their use for TDM. The techniques employing nanobiosensors are mainly based on electrochemical, optical, and mass detection systems. CONCLUSIONS: In this review, we described the most promising methodologies that, in our opinion, will bring TDM towards the next stage of clinical practice in the future.

Effects of the neurotoxin MPTP and pargyline protection on extracellular energy metabolites and dopamine levels in the striatum of freely moving rats.

The neurotoxin MPTP is known to induce dopamine release and depletion of ATP in the striatum of rats. Therefore, we studied the changes induced by MPTP and pargyline protection both on striatal dopamine release and on extracellular energy metabolites in freely moving rats, using dual asymmetric-flow microdialysis. A dual microdialysis probe was inserted in the right striatum of rats. MPTP (25mg/kg, 15mg/kg, 10mg/kg) was intraperitoneally administered for three consecutive days. MAO-B inhibitor pargyline (15mg/kg) was systemically administered before neurotoxin administration. The first MPTP dose induced an increase in dialysate dopamine and a decrease of DOPAC levels in striatal dialysate. After the first neurotoxin administration, increases in striatal glucose, lactate, pyruvate, lactate/pyruvate (L/P) and lactate/glucose (L/G) ratios were observed. Subsequent MPTP administrations showed a progressive reduction of dopamine, glucose and pyruvate levels with a concomitant further increase in lactate levels and L/P and L/G ratios. At day 1, pargyline pre-treatment attenuated the MPTP-induced changes in all studied analytes. Starting from day 2, pargyline prevented the depletion of dopamine, glucose and pyruvate while reduced the increase of lactate, L/P ratio and L/G ratio. These in vivo results suggest a pargyline neuroprotection role against the MPTP-induced energetic impairment consequent to mitochondrial damage. This neuroprotective effect was confirmed by TH immunostaining of the substantia nigra.