Kaoru Ueda

Which concentration of the inhibitor should be used to predict in vivo drug interactions from in vitro data

When the metabolism of a drug is competitively or noncompetitively inhibited by another drug, the degree of in vivo interaction can be evaluated from the [I]u/Ki ratio, where [I]u is the unbound concentration around the enzyme and Ki is the inhibition constant of the inhibitor. In the present study, we evaluated the metabolic inhibition potential of drugs known to be inhibitors or substrates of cytochrome P450 by estimating their [I]u/Ki ratio using literature data. The maximum concentration of the inhibitor in the circulating blood ([I]max), its maximum unbound concentration in the circulating blood ([I]max,u), and its maximum unbound concentration at the inlet to the liver ([I]in,max,u) were used as [I]u, and the results were compared with each other. In order to calculate the [I]u/Ki ratios, the pharmacokinetic parameters of each drug were obtained from the literature, together with their reported Ki values determined in in vitro studies using human liver microsomes. For most of the drugs with a calculated [I]in,max,u/Ki ratio less than 0.25, which applied to about half of the drugs investigated, no in vivo interactions had been reported or “no interaction” was reported in clinical studies. In contrast, the [I]max,u/Ki and [I]max/Ki ratio was calculated to be less than 0.25 for about 90% and 65% of the drugs, respectively, and more than a 1.25-fold increase was reported in the area under the concentration-time curve of the co-administered drug for about 30% of such drugs. These findings indicate that the possibility of underestimation of in vivo interactions (possibility of false-negative prediction) is greater when [I]max,u or [I]max values are used compared with using [I]in,max,u values.

Differences in Substrate Specificity among Glutathione Conjugates (GS-X) Pump Family Members: Comparison between Multidrug Resistance-associated Protein and a Novel Transporter Expressed on a Cisplatin-resistant Cell Line (KCP-4)

The substrate specificity of primary active transporters expressed on two kinds of human epidermoid KB‐3‐1 derived cell lines, C‐A500 and KCP‐4, was examined; the former expresses multi‐drug resistance‐associated protein (MRP1), whereas the latter is resistant to cis‐diammine‐dichloroplatinum (II) (cisplatin). Northern blot analysis indicated that neither P‐glycoprotein, MRP1, MRP2 (canalicular multispecific organic anion transporter; cMOAT) nor MRP3 was over‐expressed on KCP‐4. Membrane vesicles isolated from C‐A500 and KCP‐4, but not from KB‐3‐1, exhibited the ATP‐dependent uptake of glutathione conjugates (GS‐X) such as leukotriene C4 and 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG), indicating the presence of GS‐X pumps on these cells. The uptake of these GS‐X by membrane vesicles from C‐A500 was approximately twice that in the case of KCP‐4. Kinetic analysis indicated that the Km and Vmax values for DNP‐SG uptake were 2.56 and 1.43 μM, and 570 and 160 pmol/min/mg protein for C‐A500 and KCP‐4, respectively. In marked contrast, significant ATP‐dependent uptake of glutathione‐platinum complex was observed only in membrane vesicles from KCP‐4, but not those from KB‐3‐1 and C‐A500. The transport properties of estradiol‐17β‐d‐glucuronide (E217βG) were also different between the two cell lines. This was reflected in the findings that the ATP‐dependent uptake of this conjugated metabolite in membrane vesicles from C‐A500 (Km= 2.33 μM, Vmax= 34 pmol/min/mg protein) was much more extensive than that in the case of KCP‐4 (Km= 5.5 μM, Vmax= 35 pmol/min/mg protein), and that comparable uptake was observed between KCP‐4 and KB‐3‐1. Overall, a clear difference in substrate specificity among GS‐X pump family members expressed on resistant tumor cells was demonstrated.