Penetration of adenosine antagonists into mouse brain as determined by ex vivo binding

Abstract

The penetration of the adenosine antagonists 8-cyclopentyl-1,3-dimethylxanthine (CPT), 8-cyclopentyl-1,3-dipropylxanthine (CPX), 8-(p-sulfophenyltheophylline (8-PST), and 8-[4-[[[[(2-amino-ethyl)amino]carbonyl]methyl]oxy]phenyl]- 1,3-dipropylxanthine (XAC) into mouse brain was determined using ex vivo binding and locomotor studies. CPT and CPX (25 and 0.25 mg/kg, respectively) both penetrated into brain in substantial amounts: 49 and 17% of theoretical levels assuming free penetration throughout the body, 10 min after i.p. injection, respectively. Brain levels of CPT decreased rapidly, declining to undetectable levels by 30 min post-injection, whereas levels of CPX declined much more slowly. As expected, no detectable brain levels of 8-PST were found following i.p. injection of 50 mg/kg. XAC (20 mg/kg) penetrated into brain poorly: 1.6% after 10 min and 3.2% 20 min post-injection. The ability of CPT to stimulate locomotor activity paralleled the brain levels, i.e. it was similar to theophylline at short times and the effect rapidly diminished. These studies demonstrate the usefulness of ex vivo binding in determining CNS penetration of adenosine receptor ligands.

Fig. 1

Adenosine antagonists structurally related to theophylline.

Fig. 2

Competition curve for CPT inhibition of [³H]PIA binding to homogenate of mouse brain. Homogenate was treated with ADA (6 U/mL), and then incubated with 2nM [³H]PIA in 50 mM Tris-HCl buffer (pH 7.4) for 60 min at 37°. Data points represent averages of duplicate values and the line is a non-linear regression analysis of the data. In the absence of CPT, specific binding amounted to about 5500 cpm (100% of control).

Fig. 3

Time course for penetration of xanthines in the mouse brain. Mice were injected i.p. with: (■] CPT, 25 mg/ kg; (●) CPX, 0.25 mg/kg; and (○) 8-PST, 50 mk/kg. After the indicated time, their forebrains were removed, homogenized in 8 vol. of Tris-HCl, pH 7.4, and, after treatment with ADA (6 U/mL) used directlfy for [³H]PIA binding. Concentrations of xanthines in brain were determined by comparing [³H]PIA binding to that from a standard curve. One hundred percent brain penetration is defined as the amount of compound reaching the brain in the absence of any permeability barrier assuming that the injected dose distributes uniformly throughout the mouse. Each determination is the mean ± SD from at least three different animals.

Fig. 4

Dose-dependency for CPT penetration into brain relative to each injected dose. The indicated doses of CPT were injected i.p., and ex vivo binding was performed 10 min after injection. The error bars represent average ± range of duplicate animals.

Fig. 5

Time course of locomotor activity. Mice were treated with 10 mg/kg CFT (○) or 10 mg/kg theophylline (□) compared to vehicle control (△). The total distance travelled (cm/30min) in an Omnitech Activity analyser (dimensions of plexiglass cage: 40 × 40 cm, 30 cm high) was calculated using ILAM software. Animals were placed in the cage immediately after injection, and monitoring for three intervals of 10 min each was initiated after 10 min. Error bars shown represent the standard error of the mean of six to eight animals.