A method to estimate binding constants at variable protein concentrations

Römer, Jürg; Bickel, Marcel H.

doi:10.1111/j.2042-7158.1979.tb13411.x

Cited by 42 publications

(26 citation statements)

References 29 publications

(21 reference statements)

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“…In addition, the difference in free fraction will be proportional to the dilution factor when free fraction is very low and relatively insensitive to dilution as free fraction approaches 1. This nonlinear behavior is entirely consistent with the previously described nonlinear relationship between free fraction and nonspecific binding component concentration [31]. Once free fraction calculations were complete, the following three relationships were examined for the two sets of compounds: …”

Section: Data Treatmentmentioning

confidence: 78%

Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery

Kalvass¹,

Maurer²

2002

Biopharm & Drug Disp

386

385

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Relative plasma, brain and cerebrospinal fluid (CSF) exposures and unbound fractions in plasma and brain were examined for 18 proprietary compounds in rats. The relationship between in vivo brain-to-plasma ratio and in vitro plasma-to-brain unbound fraction (fu) was examined. In addition, plasma fu and brain fu were examined for their relationship to in vivo CSF-to-plasma and CSF-to-brain ratios, respectively. Findings were delineated based on the presence or absence of active efflux. Finally, the same comparisons were examined in FVB vs. MDR 1a/1b knockout mice for a selected P-glycoprotein (Pgp) substrate. For the nine compounds without indications of active efflux, predictive correlations were observed between ratios of brain-to-plasma exposure and plasma-to-brain fu (r(2) = 0.98), CSF-to-brain exposure vs. brain fu (r(2) = 0.72), and CSF-to-plasma exposure vs. plasma fu (r(2) = 0.82). For the nine compounds with indications of active efflux, nonspecific binding data tended to over predict the brain-to-plasma and CSF-to-plasma exposure ratios. Interestingly, CSF-to-brain exposure ratio was consistently under predicted by brain fu for this set. Using a select Pgp substrate, it was demonstrated that the brain-to-plasma exposure ratio was identical to that predicted by plasma-to-brain fu ratio in MDR 1a/1b knockout mice. In FVB mice, plasma-to-brain fu over predicted brain-to-plasma exposure ratio to the same degree as the difference in brain-to-plasma exposure ratio between MDR 1a/1b and FVB mice. Consistent results were obtained in rats, suggesting a similar kinetic behavior between species. These data illustrate how an understanding of relative tissue binding (plasma, brain) can allow for a quantitative examination of active processes that determine CNS exposure. The general applicability of this approach offers advantages over species- and mechanism-specific approaches.

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Section: Data Treatmentmentioning

confidence: 78%

Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery

Kalvass¹,

Maurer²

2002

Biopharm & Drug Disp

386

385

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“…If strong concentration dependence were frequently observed then the commonly used description of the process as nonspecific microsomal binding would clearly be inappropriate. The type of model involving defined binding sites (Romer and Bickel, 1979) that has been suggested as appropriate for modeling microsomal binding of drugs (Mclure et al, 2000;Kalvass et al, 2001) seems to be unnecessarily complicated. Binding that is independent of compound concentration is indicative of a phase equilibrium-type process, where clearly defined individual binding sites do not exist, much like organic/aqueous partitioning systems.…”

Section: ϫ133mentioning

confidence: 99%

The Influence of Nonspecific Microsomal Binding on Apparent Intrinsic Clearance, and Its Prediction from Physicochemical Properties

Austin

Barton²,

Cockroft³

et al. 2002

Drug Metab Dispos

341

366

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ABSTRACT:The apparent intrinsic clearance of 13 drugs has been determined using rat liver microsomes at three different concentrations of microsomal protein. The kinetics was studied using the in vitro half-life method. The nonspecific binding of these drugs to the microsomes was also studied under the same conditions, except for cofactor removal, using equilibrium dialysis. The intrinsic clearances are shown to be dependent on the microsomal concentration, but are approximately constant when corrected for the extent of nonspecific binding to the microsomes. The large difference between observed intrinsic clearance and unbound intrinsic clearance that exists for some compounds, particularly lipophilic bases, is highlighted. A simple model has been developed for understanding the binding of compounds to microsomes and is demonstrated to accurately predict the extent of microsomal binding at one concentration of microsomes from measurement at another. The binding of a further 25 drugs to rat liver microsomes at a microsomal concentration of 1 mg/ml was also studied, along with measurements of lipophilicity using octanol-water partition coefficients. It is shown that the extent of microsomal binding is correlated with lipophilicity, but that basic compounds show a different behavior to acidic and neutral compounds. Microsomal binding is shown to be best predicted using a model where logP is used for basic compounds, and logD 7.4 is used for acidic and neutral compounds. This model has been developed further so that the extent of binding to microsomes of any given concentration can be estimated purely from a knowledge of lipophilicity and ionization.In recent years, kinetic measurements using liver microsomes have been increasingly used as a measure of the rate of metabolic drug oxidation in drug discovery. The results from these in vitro assays are then frequently processed, through a variety of methods, to produce predictions of the in vivo metabolic clearance of compounds in humans (Iwatsubu et al., 1997;Obach et al., 1997;Lave et al., 1999). Methods that are often used for scaling such in vitro data to in vivo predictions are the well stirred model and the parallel tube model, and these models contain plasma free fraction terms that account for the effect of plasma binding on the clearance of compounds (Pang and Rowland, 1977). However, it is frequently found that inclusion of the plasma binding correction can lead to underprediction of the in vivo clearance, particularly for nonacidic compounds, and better performance can often be obtained by ignoring the correction altogether (Obach 1997(Obach , 1999Obach et al., 1997). It has been recognized that nonspecific microsomal binding in the in vitro metabolic assays can significantly affect the observed kinetics of metabolism and hamper the accurate prediction of clearance, and there are now several examples where knowledge of the extent of microsomal binding can lead to a better understanding of the relationship between in vitro metabolism and in vivo pharmacokinet...

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“…Theoretically, the in vivo activity should be driven by the amount of liver GPa bound to a GPa inhibitor, which is determined by the intrinsic K d and the free GPa inhibitor concentration at the target site (liver). Accurate measurement of K d under physiological conditions can be complicated and time-consuming (Romer and Bickel, 1979;Wright et al, 1996). In the present study, a robust method was developed to determine the intrinsic K d of 13 GPa inhibitors to purified human liver GPa in the presence of liver tissue homogenate using a previously validated 96-well equilibrium dialysis apparatus (Cory Kalvass and Maurer, 2002;Banker et al, 2003).…”

mentioning

confidence: 99%

Establishment of Correlation between in Vitro Enzyme Binding Potency and in Vivo Pharmacological Activity: Application to Liver Glycogen Phosphorylase a Inhibitors

Yu¹,

Chen²,

Treadway³

et al. 2006

J Pharmacol Exp Ther

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In drug discovery, establishing a correlation between in vitro potency and in vivo activity is critical for the validation of the selected target and for developing confidence in the in vitro screening strategy. The present study developed a competition equilibrium dialysis assay using a 96-well dialysis technique to determine the intrinsic K d for 13 inhibitors of human liver glycogen phosphorylase a (GPa) in the presence of liver homogenate to mimic the physiological environment. The results provided evidence that binding of an inhibitor to GPa was affected by extra cofactors present in the liver homogenate. A good correlation was demonstrated between the in vitro K d determined under liver homogenate environment and free liver concentration of an inhibitor at the minimum efficacious dose in diabetic ob/ob mice. This study revealed important elements (such as endogenous cofactors missing from the in vitro assay and free concentration at the target tissue) that contributed to a better understanding of the linkage between in vitro and in vivo activity. The approach developed here may be applied to many drugs in pharmacology studies in which the correlation between in vitro and in vivo activities for the target tissue (such as solid tumors, brain, and liver) is critical.In the drug discovery process within the pharmaceutical industry, initial lead compounds are usually identified from high-throughput in vitro biological screens, for example, an inhibition assay against a target enzyme. It is often hoped that the in vitro potency (such as IC 50 ) can be used to predict the in vivo pharmacological activity (such as EC 50 ). However, discovery scientists often face a question: why doesn't an in vitro potent inhibitor work in vivo or only work at a much higher in vivo concentration? Establishment of a correlation between in vitro potency and in vivo activity is crucial for validation of the target enzyme and for achieving confidence in an in vitro screening strategy.According to the fundamental free ligand hypothesis, the average free efficacious concentration at the steady-state in vivo should correlate with the intrinsic (unbound) potency determined from an in vitro assay. This hypothesis has been supported by many researchers (Wagner et al., 1965;Wagner, 1976;DeGuchi et al., 1992;Wright et al., 1996). In practice, however, this relationship is often obscured or confounded because of a variety of factors. For example, nonphysiological conditions, involvement of nonspecific binding within in vitro systems, or a combination may yield an inaccurate estimate of the true intrinsic potency. In addition, complex pharmacokinetic/pharmacodynamic relationships arising from indirect effects or target site disequilibrium may result in the inappropriate determination of in vivo potency.Several of the variables stated above confounded the initial establishment of a correlation between in vitro potency and This work was supported by Pfizer Inc. Article, publication date, and citation information can be found at http://jpe...

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A method to estimate binding constants at variable protein concentrations

Cited by 42 publications

References 29 publications

Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery

Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery

The Influence of Nonspecific Microsomal Binding on Apparent Intrinsic Clearance, and Its Prediction from Physicochemical Properties

Establishment of Correlation between in Vitro Enzyme Binding Potency and in Vivo Pharmacological Activity: Application to Liver Glycogen Phosphorylase a Inhibitors

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