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

Abstract

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...

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

“…Here, we have identified the key amino acids within the 16mer that are essential for binding. We also show that CP‐316819, a member of the indole‐2‐carboxamide series of phosphorylase inhibitors [8] is capable of modulating the interaction of the G L 16mer peptide with phosphorylase a .…”

Astrocyte Glycogen Sustains Neuronal Activity during Hypoglycemia: Studies with the Glycogen Phosphorylase Inhibitor CP-316,819 ([R-R*,S*]-5-Chloro-N-[2-hydroxy-3-(methoxymethylamino)-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide)

“…CP-316,819 and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) were prepared as described previously (Overkleeft et al, 1998;Yu et al, 2006). CP-316,819 was purified by chromatography on silica gel to greater than 98% purity, as assessed by HPLC analysis.…”

“…Thus, as an initial step, primary astrocyte cultures were used to evaluate the effects of four compounds, each of which blocks glycogenolysis by a different mechanism. These compounds, CP-316,819 Yu et al, 2006), MOR-14 (Arai et al, 1998), DAB (Fosgerau et al, 2000), and methionine sulfoximine (MSO) (Sellinger and Weiler, 1963), each produced a dose-dependent increase in astrocyte glycogen content when added for 24 h to standard culture medium containing 5 mM glucose (Fig. 1A).…”

Thermodynamic Stability Considerations for Isostructural Dehydrates

“…1) is a glycogen phosphorylase inhibitor that is being developed for the treatment of Type 2 diabetes mellitus. 12 Polymorph screening was conducted during early development to identify two crystalline forms (Forms A and C) that were physically stable under ambient conditions. Form A was identified as an anhydrous form and Form C was identified as a nonstoichiometric channel hydrate.…”