**4.1.2 The importance of free drug measurement**

The free drug is responsible for pharmacokinetic and pharmacodynamic properties of drugs and relation between dose and response is correct when free drug supplies in target tissue get into account. In this regard interstitial fluid and intra cellular fluid drug levels in brain are important data for drug discovery.

The traditional methods of brain homogenization destroy all compartments of brain (including brain tissue binding and plasma protein binding) and drug levels in specific compartments cannot be measured (Reichel, 2009). The plasma free fractions data cannot be used in CNS drug discovery studies, because of the different physiological properties, blood brain interstitial fluid free fractions. Some researchers used cerebrospinal drug levels (CSF sampling) as an estimate of the unbound drug levels in brain which is not so reliable because of lower tightness of cerebra-spinal blood barrier which leads to higher diffusion and overestimation of free drug concentration in brain (Read & Braggio, 2010). The microdialysis is the only *in vivo* method to provide such data directly, which is limited by its practicability.
