CLHEP Research Articles

Direct determination of the ratio of unbound fraction in plasma to unbound fraction in microsomal system ( fup/ fumic) for refined prediction of phase I mediated metabolic hepatic clearance

At the drug discovery stage, in vivo metabolic hepatic clearance (CL hep) is commonly predicted using in vitro parent compound disappearance data generated in liver microsomes or hepatocytes. Correction for the unbound fraction of a compound in the in vitro system and in plasma/serum is known to be critical for the accuracy of metabolic clearance predictions. Discrete generation of these required experimental parameters can be laborious. Herein, we describe a straightforward and direct approach to obtain the ratio of unbound fraction in plasma ( fu p) to unbound fraction in the microsomal system ( fu mic) of a small molecule compound using equilibrium dialysis. Experimental conditions were optimized with respect to incubation time, temperature, and plate shaking speed. Results obtained from this system were validated for a set of test compounds by comparison to individually measured fu p and fu mic data using ultracentrifugation. The correlation for fu p/ fu mic between the two methods for a set of 23 data points was very good with R 2 of 0.94, slope of 1.05 and an intercept of 0.007. The impact of microsomal binding on predicted CL hep was illustrated for a tightly bound compound using a series of incubations with increasing concentration of monkey liver microsomal protein. Alteration of this experimental parameter profoundly affected calculated CL hep using the well-stirred model. Significant differences were observed in the prediction when the model was corrected for fu p only; in contrast, the model corrected for plasma protein and microsomal protein binding predicted clearance values independent of the microsomal protein concentration.

Pharmacokinetic studies of Gln117 tissue-type plasminogen activator in rats.

Gln117 t-PA is a mutant type of tissue-type plasminogen activator (mt-PA), which is generated by the removal of a high mannose oligosaccharide resulting from the mutation of amino acid #117, from asparagine (Asn) to glutamine (Gln). The plasma concentration, distribution, metabolism and excretion of Gln117 t-PA were investigated after a single intravenous administration of 125I-Gln117 t-PA or Gln117 t-PA, comparing with wild-type t-PA (WT t-PA). The plasma concentration of Gln117 t-PA decreased more slowly than that of WT t-PA, plasma clearance (CL(p)), and that of Gln117 t-PA in rats was approximately 2.6 times lower than that of WT t-PA. The highest radioactivity was found in the liver at 5 min after intravenous administration of [125I]Gln117 t-PA to rats, but the radioactivity in the liver was lower than that after intravenous administration of [125I]WT t-PA in our previous paper. Within 288 h after intravenous administration of [125I]Gln117 t-PA to rats, 88.5 and 5.5% of administered radioactivity were excreted into urine and feces, respectively. In a gel-filtration chomatographic (GFC) analysis of plasma, Gln117 t-PA formed complexes with plasma proteins, similarly to WT t-PA. The hepatic clearance (CL(hep)) of both t-PAs was evaluated by comparing the plasma concentration after a constant intravenous infusion with that after a constant intraportal venous infusion. The CL(hep) of Gln117 t-PA was 6.5 times lower than that of WT t-PA. These results indicate that the low uptake of Gln117 t-PA to the liver reduces CL(p) compared with WT t-PA.