EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 537

By means of the integration plot method, early-phase uptake of drugs by several
organs can be accessed, throwing light on the roles of carrier-mediated transport in
drug distribution and accumulation. 18 , 19 In this method, mice are anesthetized and a
radiolabeled test compound is injected with or without 0.5 M unlabeled test compound
in saline. In addition, [ 14 C]mannitol is often coinjected as a paracellular route marker.
At appropriate times, blood is withdrawn from the right jugular vein and plasma
is separated by centrifugation. After 1, 2, 3 or 5 minutes, mice are sacrificed, and
the target organs are excised immediately. The organ are rinsed with ice-cold saline,
blotted with dry filter paper, weighed, and solubilized. The solubilized organs are
mixed with hydrogen peroxide and the solution is neutralized with 5 M HCl. The
solubilized organs and plasma are mixed with scintillation liquid, and the associated
radioactivity is measured with a liquid scintillation counter, followed by calculations
of the tissue uptake clearance.
By using the integration plot method, we studied the distribution of an inhibitor of
xanthine oxidase, Y-700, in the liver. 20 Following the intravenous administration of
[ 14 C]Y-700 to rats, the liver and kidneys were removed and the amounts of [ 14 C]Y-
700 taken up in these organs were measured. The values of tissue uptake clearance
were 1.04 and 0.316 mL/minute per kilogram in the liver and kidney, respectively,
suggesting efficient uptake of Y-700 by the liver in vivo. 21
In comparative studies, alterations of pharmacokinetic parameters can be useful
in evaluating drug behavior and the pharmacological roles of transporters. Here we
focus on the measurement of drug disposition and tissue distribution. Animals are
starved overnight, anesthetized, and bolus-injected with radiolabeled test compound
via the jugular vein. [ 3 H]Inulin is injected simultaneously with the radiolabeled test
compounds to evaluate the glomerular filtration rate (GFR). Serial blood samples
are collected from the intraorbital venous plexus using heparinized capillary tubes at
designated time intervals in individual mice during the experiment. Urine samples are
collected by washing the bladder with saline (0.5 mL) at designated times through a
catheter. Pharmacokinetic parameters, the area under the plasma concentration-time
curve (AUC), the elimination rate constant ( k ), the steady-state distribution volume
( V dss ), and the total body clearance (CL tot ) can be estimated by means of model-
independent moment analysis or compartment analysis using Win-Nonlin (SCI) or
other computer-fitting techniques. GFR is estimated by inulin or creatinine clearance.
The renal clearance (CL r ) and renal secretory clearance (CL s ) are calculated by using
the equations CL r = X/AUC and CL s = CL r − GFR, where X is the urinary excretion

amount of the test compound.
To evaluate the tissue distribution of drugs, the tissue-to-plasma concentration ra-
tio ( K p ) is often adopted. Mice are decapitated at the appropriate time after a single
intravenous injection of radiolabeled test compound. Tissues are quickly excised,
rinsed well with ice-cold saline, blotted to dryness, and weighed. Plasma and urine
samples are mixed with scintillation fluid. Tissue samples are dissolved, mixed with
scintillation fluid, and neutralized with 1 N HCl, then the associated radioactivity
is measured. If nonradiolabeled test compounds are used, quantification can be per-
formed with high-performance liquid chromatography (HPLC) or other analytical
techniques.