In this study the technique of laser-Doppler flowmetry was evaluated for the measurement of tissue blood flow by comparing laser-Doppler flow (LDF) signal in the renal cortex, gracilis muscle, and cremaster muscle of anesthetized rats to whole-organ blood flow measured with an electromagnetic flowmeter or radioactive microspheres. In vitro, LDF signal was closely correlated (r = 0.99) to changes in erythrocyte velocity generated with a rotating wheel. Although individual LDF readings varied in situ, mean LDF signal calculated from multiple readings on the tissue surface were significantly correlated (r = 0.74-0.95) with tissue blood flows measured at various perfusion pressures. However, significant differences in the slope of the LDF signal vs. blood flow relationship were observed in different tissues and with different methods of measurement in the same tissue. This study indicates that mean laser-Doppler flow signal provides a good estimate of tissue blood flow, provided a sufficient number of points is scanned. However, there appears to be no universal calibration factor for the method.

In the search for a selective adenosine A1 receptor antagonist with greater aqueous solubility than the compounds currently in clinical trials as diuretics, a series of 1,4-substituted 8-cyclohexyl and 8-bicyclo[2.2.2]octylxanthines were investigated. The binding affinities of a variety of cyclohexyl and bicyclo[2.2.2]octylxanthines for the rat and human adenosine A1, A2A, A2B, and A3 receptors are presented. Bicyclo[2.2.2]octylxanthine 16 exhibited good pharmaceutical properties and in vivo activity in a rat diuresis model (ED50=0.3 mg/kg po). Optimization of the bridgehead substituent led to propionic acid 29 (BG9928), which retained high potency (hA1, Ki=7 nM) and selectivity for the adenosine A1 receptor (915-fold versus adenosine A2A receptor; 12-fold versus adenosine A2B receptor) with improved oral efficacy in the rat diuresis model (ED50=0.01 mg/kg) as well as high oral bioavailability in rat, dog, and cynomolgus monkey.

Piperazine derivatives of 2-furanyl[1,2,4]triazolo[1,5-a][1,3,5]triazine have recently been demonstrated to be potent and selective adenosine A(2a) receptor antagonists with oral activity in rodent models of Parkinson's disease. We have replaced the piperazinyl group with a variety of linear, monocyclic, and bicyclic diamines. Of these diamines, (R)-2-(aminomethyl)pyrrolidine is a particularly potent and selective replacement for the piperazinyl group. With this diamine component, we have been able to prepare numerous analogues with low nanomolar affinity toward the A(2a) receptor and good selectivity with respect to the A(1) receptor (>200-fold in some cases). Selected analogues from this series of [1,2,4]triazolo[1,5-a][1,3,5]triazine have now been shown to be orally active in the mouse catalepsy model.

A 1 adenosine receptor (AR) antagonists are effective diuretic agents that may be useful for treating fluid retention disorders including congestive heart failure. However, antagonism of A 1 ARs is potentially a concern when using these agents in patients with ischemic heart disease. To address this concern, the present study was designed to compare the actions of the A 1 AR antagonists CPX (1,3-dipropyl-8-cyclopentylxanthine), BG 9719 (1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine), and BG 9928 (1,3-dipropyl-8-[1-(4-propionate)-bicyclo-[2,2,2]octyl]xanthine) on acute myocardial ischemia/reperfusion injury and ischemic preconditioning (IPC) in an in vivo dog model of infarction. Barbital-anesthetized dogs were subjected to 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion, after which infarct size was assessed by staining with triphenyltetrazolium chloride. IPC was elicited by four 5-min occlusion/5-min reperfusion cycles produced 10 min before the 60-min occlusion. Multiple-cycle IPC produced a robust reduction (ϳ65%) in infarct size; this effect of IPC on infarct size was not abrogated in dogs pretreated with any of the three AR antagonists. Surprisingly, in the absence of IPC, pretreatment with CPX or BG 9928 before occlusion or immediately before reperfusion resulted in significant reductions (ϳ40 -50%) in myocardial infarct size. However, treatment with an equivalent dose of BG 9719 had no similar effect. We conclude that the A 1 AR antagonists BG 9719, BG 9928, and CPX do not exacerbate cardiac injury and do not interfere with IPC induced by multiple ischemia/reperfusion cycles. We discuss the possibility that the cardioprotective actions of CPX and BG 9928 may be related to antagonism of A 2B ARs.

The [1,2,4]triazolo[1,5-a]triazine derivative 3, more commonly known in the field of adenosine research as ZM-241385, has previously been demonstrated to be a potent and selective adenosine A2a receptor antagonist, although with limited oral bioavailability. This [1,2,4]triazolo[1,5-a]triazine core structure has now been improved by incorporating various piperazine derivatives. With some preliminary optimization, the A2a binding affinity of some of the best piperazine derivatives is almost as good as that of compound 3. The selectivity level over the adenosine A1 receptor subtype for some of the more active analogues is also fairly high, > 400-fold in some cases. Many compounds within this piperazine series of [1,2,4]triazolo[1,5-a]triazine have now been shown to have good oral bioavailability in the rat, with some as high as 89% (compound 35). More significantly, some piperazines derivatives of [1,2,4]triazolo[1,5-a]triazine also possessed good oral efficacy in rodent models of Parkinson's disease. For instance, compound 34 was orally active in the rat catalepsy model at 3 mg/kg. In the 6-hydroxydopamine-lesioned rat model, this compound was also quite effective, with a minimum effective dose of 3 mg/kg po.

Adenosine is known to produce cardiovascular effects such as bradycardia and hypotension via activation of myocardial (A₁) and vascular (A₂) receptors and antilipolytic effects through activation of adipocyte (A₁) receptors. We established the cardiovascular and antilipolytic profile of the adenosine A₁ agonist GR79236 (N⁶-[(lS, trans)-2-hydroxycyclopentyl]-adenosine) and compared it with CPA (N⁶-cyclopentyl-adenosine). GR79236 was approximately 3-fold less potent than CPA in inhibiting in vitro lipolysis. In conscious rats, both agents were shown to have antilipolytic and glucose-lowering properties. In rats instrumented with telemetry transmitters, orally administered CPA was one log unit more potent than GR79236 as a hypotensive and bradycardic agent. In summary, GR79236 is an A₁-selective adenosine agonist which reduces heart rate and mean arterial pressure and produces decreased plasma lipids and glucose levels.

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Stimulation of A 1 adenosine receptors in the kidney reduces glomerular filtration rate (GFR) via tubuloglomerular feedback and increases sodium reabsorption. Blocking A 1 adenosine receptors would therefore maintain GFR and cause natriuresis. These attributes would be especially beneficial in treating congestive heart failure (CHF). BG9928 is a xanthine derivative that binds with high affinity to A 1 adenosine receptors from several species including human and acts as a competitive antagonist at these receptors. This compound is being developed for the treatment of CHF. BG9928 receptor binding across a wide array of human and animal receptors was evaluated in vitro. Oral dose-response was assessed in rats, and oral and iv dose-responses were assessed in monkeys. The functional potency of BG9928 was demonstrated in vivo in rats. The binding affinity (K i ) for the human A 1 adenosine receptor is 12.2 nM. BG9928 is orally active, with a dose of 0.3 mg/kg po achieving full natriuretic response in rats and with iv doses as low as 0.03 mg/kg showing activity in nonhuman primates. The renal protective effects of BG9928 were demonstrated by coadministration with the loop diuretic furosemide. In rats, administration of BG9928 effectively attenuated the reductions in GFR that usually occur with the administration of furosemide. Blockade of A 1 receptors by BG9928 protects renal function against the adverse effects of loop diuretics and exerts additive natriuretic effects. In CHF patients, this profile may translate into significant clinical benefit by enhancing natriuresis with diminished concern of inducing renal insufficiency. Drug Dev. Res. 58:486-492, 2003.