EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 538

In our study of systemic carnitine defficiency (SCD) by using jvs mice lacking the
organic cation transporter Octn2, 22 we compared the K p values in jvs and wild-type
mice at 4 hours after tetraethylammonium administration. In kidney, jvs mice showed
significantly a higher K p value of TEA (25.0 ±

1.16), and the K p values in brain, lung, liver, and spleen of jvs mice were significantly
lower. We proposed that the increase in kidney may be explained in terms of decreased
renal apical secretory transport activity, and that the decrease of K p values in other
tissues may be explained in terms of decreased tissue-uptake activities. 23

± 4.6) than did wild-type mice (9.89

18.3. ISOLATED TISSUE METHODS

To predict the involvement of transporters in absorption and distribution, studies with
isolated tissues are often performed. To investigate intestinal absorption mechanisms,
the loop method, Ussing-type chamber method, or everted sac method can be adopted.
The loop method is an in situ approach, whereas the Ussing-type chamber and everted
sac methods are performed with isolated intestinal tissues. Sliced organs are often
used, except in the case of intestine.

18.3.1. Loop Method

The loop method is an in situ method for studying the intestinal absorption mecha-
nisms of drugs. Typically, mice are anesthetized, the intestine is exposed by midline
abdominal incision, and the bile duct is ligated. A 5-cm closed loop of intestine is
prepared by ligation at both ends after clearing the gut by passing warmed isotonic
2-morpholinoethanesulfonic (MES) acid buffer (pH 6.4, 290 mOsm/kg) slowly
through it until the effluent is clear, and expelling the remaining solution with air
pumped through a syringe. Each loop is filled with 0.2 mL of isotonic MES buffer. In
the case of intestinal absorption study, the mouse is kept on a warm plate at 37 ◦ C for
a 10-minute recovery period. The test compound is introduced into the loop. After
15 minutes the solution in the loop is collected and the loop is rinsed with isotonic
MES buffer to give a total effluent volume of 5 mL. To estimate the remaining amount
of the test compound, the concentration in the effluent is measured by HPLC or by
other analytical techniques. 24
Using the loop method, we compared the absorption of grepafloxacin in mouse
small intestine of mdr1a/1b double-knockout and wild-type mice. The knockout mice
showed significantly lower fractional absorption in the intestinal loop, suggesting that
mdr1 restricts the uptake of grepafloxacin from mouse small intestine. 24 , 25

18.3.2. Ussing-Type Chamber Method

A Ussing-type chamber consists of two buffer tanks with isolated tissues (intestine,
skin, etc.) mounted between them. 26 Drug movements from the apical to the ba-
solateral side and from the basolateral to the apical side can be measured to study
directional drug transport in tissues. We have used the Ussing-type chamber method
to investigate the intestinal absorption of a -lactam antibiotic, cefadroxil, which is