This study re-examined the hepatic extraction for diazepam, the only drug for which isolated perfused rat liver (IPRL) studies have been reported not to be consistent with the well stirred model of organ elimination when only entering and exiting liver concentration measurements are available. First, the time dependency of diazepam equilibrium fraction unbound measurements from 4 to 24 hours was tested, reporting the continuing increases with time. The results showed that the time dependency of equilibrium protein-binding measurements for very highly bound drugs may be an issue that is not readily overcome. When examining C _out/C _in (F _obs) measurements for diazepam when no protein is added to the incubation media, IPRL outcomes were consistent with previous reports showing marked underpredictability of in vivo clearance from in vitro measures of elimination in the absence of protein for very highly bound drugs, which is markedly diminished in the presence of albumin. F _obs for diazepam at additional low concentrations of protein that would allow discrimination of the models of hepatic elimination produced results that were not consistent with the dispersion and parallel-tube models. Therefore, although the outcomes of this study were similar to those reported by Rowland and co-workers, when no protein is added to the perfusion media, these IPRL results for diazepam cannot be reasonably interpreted as proving that hepatic organ elimination is model-independent or as supporting the dispersion and parallel-tube models of organ elimination. SIGNIFICANCE STATEMENT: The only drug experiments for which isolated perfusion rat liver studies do not support hepatic clearance being best described by the well stirred model have been carried out with diazepam at zero protein concentration. This study repeated those studies, confirming the previous results at zero protein concentration, but the addition of low protein-binding conditions capable of differentiating the various models of hepatic elimination are more consistent with the well stirred model of hepatic elimination. These experimental studies do not support the preference for alternate models of hepatic elimination or the proposal that hepatic organ clearance is model-independent.

This study re-examined the hepatic extraction for diazepam, the only drug for which isolated perfused rat liver (IPRL) studies have been reported not to be consistent with the well stirred model of organ elimination when only entering and exiting liver concentration measurements are available. First, the time dependency of diazepam equilibrium fraction unbound measurements from 4 to 24 hours was tested, reporting the continuing increases with time. The results showed that the time dependency of equilibrium protein-binding measurements for very highly bound drugs may be an issue that is not readily overcome. When examining C _out/C _in (F _obs) measurements for diazepam when no protein is added to the incubation media, IPRL outcomes were consistent with previous reports showing marked underpredictability of in vivo clearance from in vitro measures of elimination in the absence of protein for very highly bound drugs, which is markedly diminished in the presence of albumin. F _obs for diazepam at additional low concentrations of protein that would allow discrimination of the models of hepatic elimination produced results that were not consistent with the dispersion and parallel-tube models. Therefore, although the outcomes of this study were similar to those reported by Rowland and co-workers, when no protein is added to the perfusion media, these IPRL results for diazepam cannot be reasonably interpreted as proving that hepatic organ elimination is model-independent or as supporting the dispersion and parallel-tube models of organ elimination. SIGNIFICANCE STATEMENT: The only drug experiments for which isolated perfusion rat liver studies do not support hepatic clearance being best described by the well stirred model have been carried out with diazepam at zero protein concentration. This study repeated those studies, confirming the previous results at zero protein concentration, but the addition of low protein-binding conditions capable of differentiating the various models of hepatic elimination are more consistent with the well stirred model of hepatic elimination. These experimental studies do not support the preference for alternate models of hepatic elimination or the proposal that hepatic organ clearance is model-independent.

In reviewing previously published isolated perfused rat liver studies, we find no experimental data for high-clearance metabolized drugs that reasonably or unambiguously support preference for the dispersion and parallel-tube models versus the well-stirred model of organ elimination when only entering and exiting drug concentrations are available. It is likely that the investigators cited here may have been influenced by: 1) the unphysiologic aspects of the well-stirred model, which may have led them to undervalue the studies that directly test the various hepatic disposition models for high-clearance drugs (for which model differences are the greatest); 2) experimental assumptions made in the last century, which are no longer valid today, related to the predictability of in vivo outcomes from in vitro measures of drug elimination and the influence of albumin in hepatic drug uptake; and 3) a lack of critical review of previously reported experimental studies, resulting in inappropriate interpretation of the available experimental data. The number of papers investigating the theoretical aspects of the dispersion, parallel-tube, and well-stirred models of hepatic elimination greatly outnumber the papers that actually examine the experimental evidence available to substantiate these models. When all experimental studies that measure organ elimination using entering and exiting drug concentrations at steady state are critically reviewed, the simple but unphysiologic well-stirred model is the only model that can describe all trustworthy published available data. SIGNIFICANCE STATEMENT: Although the dispersion model of hepatic elimination more adequately reflects physiologic reality, there are no convincing experimental data that unambiguously favor this model. The well-stirred model can describe all well-designed perfusion studies with high-clearance drugs and nondrug substrates, but the field has not recognized this because of hesitation to accept a nonphysiologic model and flawed attempts to utilize in vitro-in vivo extrapolation approaches.

In reviewing previously published isolated perfused rat liver studies, we find no experimental data for high-clearance metabolized drugs that reasonably or unambiguously support preference for the dispersion and parallel-tube models versus the well-stirred model of organ elimination when only entering and exiting drug concentrations are available. It is likely that the investigators cited here may have been influenced by: 1) the unphysiologic aspects of the well-stirred model, which may have led them to undervalue the studies that directly test the various hepatic disposition models for high-clearance drugs (for which model differences are the greatest); 2) experimental assumptions made in the last century, which are no longer valid today, related to the predictability of in vivo outcomes from in vitro measures of drug elimination and the influence of albumin in hepatic drug uptake; and 3) a lack of critical review of previously reported experimental studies, resulting in inappropriate interpretation of the available experimental data. The number of papers investigating the theoretical aspects of the dispersion, parallel-tube, and well-stirred models of hepatic elimination greatly outnumber the papers that actually examine the experimental evidence available to substantiate these models. When all experimental studies that measure organ elimination using entering and exiting drug concentrations at steady state are critically reviewed, the simple but unphysiologic well-stirred model is the only model that can describe all trustworthy published available data. SIGNIFICANCE STATEMENT: Although the dispersion model of hepatic elimination more adequately reflects physiologic reality, there are no convincing experimental data that unambiguously favor this model. The well-stirred model can describe all well-designed perfusion studies with high-clearance drugs and nondrug substrates, but the field has not recognized this because of hesitation to accept a nonphysiologic model and flawed attempts to utilize in vitro-in vivo extrapolation approaches.