Unbound plasma compound concentration

A similar strategy was employed to identify a DPP-IV inhibitor (6) with good in vivo potency in a mouse model of diabetes [44], Plasma protein binding, as assessed by shift assay (50% serum), was presented for all final compounds. The compound selected as having the best overall profile was active in vivo at 0.1 mg/kg. The activity at 1 h post-dose was consistent with the free drug principle - total plasma concentration 269 nM murine-free fraction 4% unbound plasma concentration 11 nM in vitro potency versus murine DPP-IV 6nM. 

True steady state is usually only achieved for a prolonged period with intravenous infusion. If we assume that we wish for a similar steady value after oral administration, then we need to balance our dosing frequency with the rate of decline of drug concentration and the rule of thumb referred to earlier (dosing interval equal to drug half-life) can be applied. Unbound clearance and free drug are particularly applicable to drugs delivered by the oral route. For a well-absorbed compound the free plasma concentrations directly relate to Cli (intrinsic unbound clearance). 

The lack of information conveyed by total brain concentration is indicated by studies on KA-672 [6], a lipophilic benzopyranone acetylcholinestrase inhibitor. The compound achieved total brain concentrations of 0.39 iM at a dose of 1 mg kg" equivalent to the IC50 determined in vitro (0.36 juM). Doses up to 10 mg kg were without pharmacological effect. Analysis of CSF indicated concentrations of the compound were below 0.01 juM readily explaining the lack of activity. These low concentrations are presumably due to high (unbound) free drug clearance and resultant low concentrations of free drug in the plasma (and CSF). 

The use of microdialysis has enabled unbound drug concentrations to be determined in ECF, providing another measurement of penetration across the blood-brain barrier and one more closely related to activity. A review of data obtained by microdialysis [7], showed that free drug exposure in the brain is equal to or less than free drug concentration in plasma or blood, with ratios ranging from 4% for the most polar compound (atenolol) to unity for lipophilic compounds (e.g. carbamazepine). This largely supports the similar conclusions from the CSF data shown above. This relationship is illustrated in Figure 4.4. 

There was a direct relationship between the effect and the plasma concentration in the rat pharmacodynamic data and it was well described by a simple Emax model. Based on preclinical models for efficacy, a 90% effect was considered as the target for therapeutic effect. Finally the human C90 (human concentration corresponding to 90% effect, C90 man) was estimated by accounting for the different affinities and unbound fractions of each compound for the rat and human receptors as follows ... 

Figure 6 Well-stirred model of hepatic clearance. The exchange of a drug between plasma and hepatocyte and its removal from this cell involves an unbound compound. Intrinsic clearance, CLint, relates the rate of the elimination (by formation of metabolites, CLint>f, and secretion of unchanged compound into bile, CLint5ex) to the unbound drug in the cell, CUr Cbout and CUout are the bound and unbound concentrations of the drug leaving the liver at total concentration Cout.

The plasma levels one gets from PK studies say nothing at all about protein binding. They represent total, that is, bound plus unbound, concentrations. Traditionally, a couple of methods have been used to determine protein binding. The first, equilibrium dialysis, is considered the gold standard method. A sample of the compound in plasma at pH 7.4 and at 37°C is dialyzed through a membrane with a 10 kDa cutoff into buffer for some hours. 

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