Blood-brain barrier transport of synthetic adenosine A1 receptor agonists in vitro: structure transport relationships.

Transport of 11 structurally related adenosine A(1) receptor agonists was determined in an in vitro BBB model of brain-capillary-endothelial-cells and astrocytes. Inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI) was used to quantify the contribution of the es nucleoside transporter to the overall transport. The N(6)-substituted adenosine analogues N(6)-cyclobutyladenosine (CBA), N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA) showed concentration-dependent clearance and their transport could be inhibited by NBTI. The V(max) was 1.5+/-0.2 pmol min(-1) and the Km values were 2.2+/-0.2, 1.8+/-0.3 and 15+/-4 microM for CBA, CPA and CHA, respectively. Further chemical modification such as substitution in the C8-position or modification at the ribose-moiety resulted in loss of affinity for the es nucleoside transporter. Transport by passive diffusion was slow with clearances ranging from 0.21+/-0.01 microl min(-1) for 8-(methylamino)-CPA (MCPA) to 1.8+/-0.18 microl min(-1) for 5'-deoxy-CPA (5'dCPA). Regression analysis showed no relationship between transport clearance by passive diffusion and the GTP-shift, a non-linear relationship between the transport clearance by passive diffusion and the dynamic polar surface area (Cl=0.469e(-0.071DPSA); R2=0.88) and a linear relationship between transport clearance and prediction of BBB transport on basis of the Abraham equation (logCl=1.53logBB-1.56; R2=0.83). It is concluded that the transport of synthetic A(1) adenosine derivatives across the blood-brain barrier is generally quite slow. In addition, transport by the es nucleoside transporter may contribute to the transport of certain structurally distinct analogues.

Functional role of adenosine receptor subtypes in the regulation of blood-brain barrier permeability: possible implications for the design of synthetic adenosine derivatives.

The objective of this investigation was to determine the functional role of adenosine receptor subtypes in the regulation of blood-brain barrier (BBB) permeability. The presence of the equilibrative es and ei nucleoside transporters at the BBB was also determined. Studies were conducted in an experimental in vitro BBB model comprising bovine brain capillary endothelial cells (BCECs) and rat astrocytes (RAs). The presence of the receptors and transporters was investigated by a combination of RT-PCR and radioligand binding assays. Changes in paracellular permeability were investigated on basis of changes in trans-endothelial-electrical-resistance (TEER) and transport of paracellular markers. In BCECs the presence of A(2A) and A(3) receptors and the es nucleoside transporter was demonstrated. The A(1) receptor was absent, while the presence of the A(2B) receptor and the ei nucleoside transporter remained uncertain. In RAs the presence of all four receptor subtypes and the es and ei nucleoside transporters was demonstrated. Upon application of selective agonists no significant changes in TEER or the transport of the paracellular markers were observed. The functional role of adenosine receptor subtypes in regulating the paracellular permeability of the BBB is probably small. It is unlikely therefore that the BBB transport of synthetic adenosine analogues is modified by permeability changes. The es nucleoside transporter might play a role in the BBB transport of synthetic adenosine analogues.