Written by Punit Marathe, Bristol-Myers Squibb
The “free drug hypothesis” assumes that the free concentration of a drug at the receptor biophase is responsible for eliciting its pharmacological effect. With some exceptions (e.g. coagulation factors, soluble immuno-modulatory receptors), most drug targets reside outside the vasculature, i.e. in defined target tissues. However, in a clinical setting, direct measurement of drug concentration at the active site is seldom possible due to the inaccessibility of the target tissues. For this reason, the free drug concentration in plasma is used in lieu of free drug concentration at the active site. In a preclinical setting, there exists a greater flexibility in sampling various tissues (e.g. liver, tumor, brain). Over the years, greater emphasis has been placed on collecting target tissues from the preclinical efficacy models to determine total drug concentrations and establishing PK-PD relationships. However, the tissue homogenate concentration does not take into account the fact that tissues are made of distinct compartments (interstitial fluid, cells, subcellular organelles) and a given drug is not likely to distribute homogeneously throughout. Overall the concentration of drug in the tissue homogenate is not informative with respect to drug concentration at the site of action in the tissue. For example, if the site of action is in the extracellular compartment and the drug distribution is primarily restricted to the extracellular compartment, homogenizing the tissue leads to dilution of the drug by mixing the intracellular and extracellular fluids and an underestimate of drug concentration at the site of action. Conversely, if the drug is subject to intracellular accumulation, total tissue concentration leads to an overestimation of its concentration at the site of action in the extracellular compartment. For drugs that distribute solely by passive diffusion, at distribution equilibrium the unbound concentration in tissue will equal the unbound concentration in plasma. Over the years, we have begun to understand the role of transporters not just in drug elimination but more importantly in governing drug tissue distribution. It is clear that both influx and efflux transporters play an important role in the rate and extent of drug distribution. For drugs that are substrates for influx and efflux transporters, the simple relationship between plasma and tissue concentrations will not be valid. The unbound drug concentrations in tissues are expected to be higher, versus unbound drug concentrations in plasma, when active uptake transporters are involved in tissue distribution. On the other hand, the unbound drug concentrations in tissues will be lower than the unbound plasma concentrations when active efflux transporters are involved. When both influx and efflux transporters govern tissue distribution, it is difficult to predict the unbound tissue concentrations in relation to unbound plasma concentrations. Clearly, drug distribution in tissues is a complex phenomenon and is foundational to one’s understanding of any PKPD relationship. Consequently, attempts have been made to measure tissue unbound drug concentrations using tissue homogenates, slices and microdialysis. A high Kp value indicates a relatively high total tissue concentration, but it does not indicate a high unbound tissue concentration or high tissue drug penetration efficiency. Hence the total brain-to-plasma ratio should not be used as a parameter to select compounds for CNS action in drug discovery. The same concept may be extended to other target organs such as the liver, muscle and pancreas. Incorporating tissue free fractions may prove useful for predicting efficacious exposure levels during development of new chemical entities. The tissue homogenate method and tissue slice uptake method are useful to estimate unbound tissue concentrations in a drug discovery setting. Sometimes, prediction of clinical efficacy, toxicity, and drug-drug interactions may be improved by accounting for the intracellular unbound drug concentration in vitro and in vivo. Intracellular unbound drug concentrations determine affinity to targets in the cell interior. Unbound intracellular drug concentrations in cultured cells can be estimated based on parallel measurements of cellular drug binding and steady-state intracellular drug concentrations. Once the relationship of tissue unbound concentrations to in vivo pharmacological activity is established, it can be incorporated prospectively in subsequent preclinical efficacy studies. Physiologically-based models may be leveraged to extend the relationship from preclinical species to human by incorporating species differences in target affinity and transporter mediated uptake or efflux. Integration of predicted human plasma PK and tissue unbound concentrations should facilitate the projection of human efficacious doses in a preclinical setting.
Reference: Mariappan TT, Mandlekar S, Marathe P. Insight into Tissue Unbound Concentration: Utility in Drug Discovery and Development. Curr Drug Metab. 14(3):324-340,2013.