Approach to reduce the non-specific binding in microdialysis

• Published in: Journal of neuroscience methods Vol. 209; no. 2; pp. 379 - 387
• Main Authors: Nirogi, Ramakrishna, Kandikere, Vishwottam, Bhyrapuneni, Gopinadh, Benade, Vijay, Saralaya, Ramanatha, Irappanavar, Shantaveer, Muddana, Nageswararao, Ajjala, Devender Reddy
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.08.2012
• Subjects: Adsorption; Animals; Anticonvulsants; Antipsychotic Agents - pharmacology; Brain - cytology; Brain - drug effects; Brain - metabolism; Brain pharmacokinetics; Carbamazepine; Extracellular Space - metabolism; In Vitro Techniques; Male; Microdialysis; Microdialysis - methods; Non-specific binding; Pharmacokinetics; Probe extraction efficiency; Rats; Rats, Sprague-Dawley; BBB; Non-specific binding; CNS; Probe extraction efficiency; CHAPS; FEP; BSA; Microdialysis; Adsorption; CSF; aCSF; ESI; SEM; Brain pharmacokinetics; PEEK
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2012.06.010

► Steps are explained to evaluate non-specific binding of test compounds. ► Test compound passed through the tubing gives an indication of non-specific binding. ► In vitro probe recovery can be used to assess non-specific binding of test compound. ► PEEK tubing showed less non-specific binding compared to FEP tubing. ► First systematic investigation for troubleshooting adsorption in microdialysis. Measurement of unbound test compound concentrations at the biophase is routinely carried out in the drug discovery. Microdialysis is an established sampling technique for in vivo measurement of endogenous and exogenous compounds and it is commonly used for monitoring true concentrations. Endogenous compounds like neurotransmitters and neuropeptides in the brain are routinely evaluated as a proof of pharmacological activity of test compounds. Although, microdialysis offers several advantages over the conventional techniques for its use in brain pharmacokinetics, the absolute determination of extracellular concentrations of test compound depends on the predictable non-specific binding to the tubing and probe membrane. In the present investigation, we have demonstrated steps to predict non-specific binding and described approaches to reduce while working with compounds having different degree of adsorption properties. Non-specific binding to the tubing was measured in vitro for seven structurally diverse compounds and based on the binding characteristics, changes were adapted in study conditions. In vitro probe extraction efficiency was evaluated by gain and loss, which was further used as a second layer of measurement for non-specific binding. For selected compounds, in vivo probe extraction efficiencies were carried out and brain pharmacokinetics was evaluated in the prefrontal cortex of male Sprague-Dawley rats. Thus, the present approach demonstrates a systematic approach for evaluating and reducing the non-specific binding of test compounds to the microdialysis tubing and probe membranes. The stepwise approach described will strengthen the applicability of microdialysis in brain pharmacokinetics.