Method of screening a metabolite of a parent candidate compound for susceptibility to biliary excretion

A method of screening a metabolite of a parent xenobiotic compound for susceptibility to biliary excretion, the method comprising the steps of:

• (a) establishing a first set and second set of two cultures of hepatocytes, each culture comprising at least one bile canaliculus, a first culture within each set having intact bile canaliculi and a second culture within each set having disrupted bile canaliculi;
• (b) exposing a parent xenobiotic compound to the first culture and to the second culture of each set for a time (T) sufficient to allow uptake of the parent xenobiotic compound;
• (c) inducing metabolic enzyme activity in the hepatocytes of the first set of cultures;
• (d) washing and lysing the first and second cultures of each set;
• (e) measuring an amount of parent xenobiotic compound present in a lysate obtained from each culture in step (d);
• (f) measuring an amount of the metabolite of the parent xenobiotic compound present in a lysate obtained from each culture in step (d);
• (g) calculating a mass of the parent xenobiotic compound in the bile canaliculi as the difference in the amount of the parent xenobiotic compound present in the lysates from the first culture having intact bile canaliculi and the second culture having disrupted bile canaliculi as measured in step (e) to determine susceptibility to biliary excretion of the parent xenobiotic compound;
• (h) calculating a mass of the metabolite of the parent xenobiotic compound in the bile canaliculi as the difference in the amount of the metabolite of the parent xenobiotic compound present in the lysates from the first culture having intact bile canaliculi and the second culture having disrupted bile canaliculi as measured in step (f);
• and
• (i) calculating a biliary clearance value as the ratio of the mass of the metabolite of the parent xenobiotic compound in the bile canaliculi and the area under the curve (AUC) in culture medium, wherein the AUC represents the integral of compound concentration in the medium from time 0 to time T, to thereby screen the metabolite of the parent xenobiotic for susceptibility to biliary excretion.