Discovery of an orally efficaceous 4-phenoxypyrrolidine-based BACE-1 inhibitor.

Based on a lead compound identified from the patent literature, we developed patentably novel BACE-1 inhibitors by introducing a cyclic amine scaffold as embodied by 1a and 1b. Extensive SAR studies assessed a variety of isophthalamide replacements including substituted pyrrolidinones and ultimately led to the identification of 11. Due to its favorable overall profile, 11 has been extensively profiled in various in vivo settings.

Rational design of novel, potent piperazinone and imidazolidinone BACE1 inhibitors.

Guided by structure-based design, we synthesized two novel series of potent inhibitors of BACE1 and generated extensive SAR around both the prime and non-prime side binding pockets. The key feature of both series is a cyclic amine motif specifically crafted to achieve interactions with both the flap and with the S2' pocket.

Sulfur-dioxide exposure increases TRPV1-mediated responses in nodose ganglia cells and augments cough in guinea pigs.

The objective of the present experiments was to study the effects of pulmonary inflammation induced by subacute Sulfur-dioxide (SO(2)) exposure on capsaicin-induced responses in isolated primary vagal sensory neurons and cough. Additionally, we examined the effects of SO(2) exposure on respiratory function and lung histology. All experiments were conducted 24 h after 4 days of subacute SO(2) (1000 ppm, 3 h/day for 4 days) exposure. In in vitro experiments, intracellular Ca(2+) concentrations were measured in single nodose ganglia cells isolated from SO(2) treated and control guinea pigs, using a fluorescence-based methodology. In nodose ganglia cells from SO(2)-exposed animals, intracellular Ca(2+) responses evoked by capsaicin (1 x 10(-7) and 1 x 10(-6) M) were significantly augmented (87% and 59%, respectively) compared to nodose ganglia from control animals. In vivo experiments, cough responses induced by a submaximal dose of aerosolized capsaicin (30 microM) were increased approximately 50% in SO(2) exposed animals compared to control animals. The enhanced cough response produced by SO(2) was inhibited by the corticosteroid, dexamethasone (10 mg/kg, p.o. b.i.d for 4 days and 10 mg/kg, p.o. once on day 5). In separate experiments, guinea pigs exposed to SO(2) displayed a decrease in respiratory frequency and minute ventilation and an increase in enhanced pause (PenH), a surrogate measure for pulmonary obstruction. Associated with the SO(2)-induced increase in cough and changes in respiratory parameters was an increase in BAL neutrophils. BAL neutrophil counts were 5+/-4 and 691+/-141 cells x 10(3)/ml for air and SO(2)-exposed animals, respectively. The neutrophillic inflammation induced by SO(2) was attenuated by dexamethasone treatment. Finally, staining for collagen, smooth muscle and goblet cells showed inflammation, remodeling and goblet cell metaphasia in the SO(2)-exposed animals. Our results demonstrate that SO(2) exposure enhances TRPV1 receptor function at the level of the nodose ganglia. This effect occurs in parallel with an increase sensitivity of the cough response to capsaicin.

Role of CXCR2 in cigarette smoke-induced lung inflammation.

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.

Estrogen diminishes postischemic hydroxyl radical production.

Reperfusion of blood flow to an ischemic myocardium is imperative to survival; ironically, it may also manifest several pathophysiological conditions. The most important of these are reperfusion arrhythmias and tissue injury and/or death. The mechanisms involved in reperfusion arrhythmias remain to be fully elucidated; however, increasing evidence indicates that reperfusion-induced arrhythmias are a free radical-mediated phenomenon. Acute administration of conjugated equine estrogen to dogs attenuates ischemia- and reperfusion-induced arrhythmias. The cardioprotective effect of estrogens in postmenopausal women is well documented, and recent studies suggest that estrogens possess strong antioxidant properties, with equine estrogens most potent. In this study we show that administration of conjugated equine estrogen to fully anesthetized dogs abolishes the burst of .OH radicals typically produced on reperfusion of the myocardium. This indicates that estrogen might attenuate reperfusion-induced ventricular arrhythmias by virtue of its antioxidant properties, suggesting a novel cardioprotective effect of the hormone.

Cardiac and coronary vascular effects of chronically administered estrogen in the dog.

The acute administration of conjugated equine estrogen (CEE) to dogs significantly attenuated the severity and incidence of ventricular arrhythmias during ischemia and reperfusion. We hypothesized that one of the cardioprotective mechanisms of estrogen might be the ability to maintain electrical stability of the heart during ischemia. The current study was conducted to determine the effect of chronic administration of estrogen, simulating hormone replacement therapy, on the ventricular arrhythmias of ischemia and reperfusion. Chronically-treated (100 micrograms/kg/week CEE, or vehicle) male beagles were anesthetized and subjected to regional ischemia (20 min) and reperfusion. Although there was a trend towards a lower incidence of arrhythmias during ischemia in estrogen-treated dogs, values did not achieve significance at P < 0.05. Baseline coronary vascular resistance was significantly higher in estrogen-treated dogs (2.3 vs 1.5 mmHg/ml/min/100 g, P < 0.05) indicating an increase in vasomotor tone. There was also an increase in the time it took hyperemic coronary blood flow to reach a peak value upon reperfusion (71 sec in estrogen-treated dogs vs 12 sec in vehicle-treated dogs, P < 0.05). This slower reflow is consistent with increased coronary vascular resistance upon reflow in estrogen-treated dogs. We conclude that the chronic administration of CEE to male dogs increased coronary vascular tone, and impaired the rate of reperfusion, but did not decrease the incidence of ventricular arrhythmias caused by ischemia.

Acute coronary vascular and myocardial perfusion effects of conjugated equine estrogen in the anesthetized dog.

Women generally exhibit angina rather than myocardial infarction as the first manifestation of heart disease. Postmenopausal use of hormone replacement therapy, specifically estrogens, is associated with reduced incidence of major cardiac events suggesting estrogen may protect the heart during ischemia. We recently showed that acute administration of conjugated equine estrogens prior to ischemia attenuated the ventricular arrhythmias of ischemia as well as those of reperfusion. This study looks at basal effects of estrogen on coronary blood flow and the effects of estrogen on regional blood flow during ischemia to determine if estrogen exerts its antiarrhythmic effects during ischemia by altering blood flow. Under conditions of natural blood flow, estrogen caused cyclic changes in blood flow. When coronary blood flow was controlled and limited, estrogen increased coronary perfusion pressure (118 +/- 8 mmHg vs. 85 +/- 10 mmHg in non-treated dogs, P < 0.05) demonstrating an overall vasoconstrictor effect. Coronary blood flow and regional myocardial perfusion were determined before and during ischemia in anesthetized dogs with and without acutely-administered estrogen. Colored microspheres were injected at steady state prior to ischemia, and during steady state myocardial ischemia. Conjugated equine estrogen (10 micrograms/kg), administered about 6 min before ischemia, had no effect on regional perfusion under steady state conditions, nor in the non-ischemic zone during ischemia. Perfusion in the subepicardial and subendocardial ischemic zones in estrogen-treated dogs was significantly lower than in non-treated dogs [0.14 +/- 0.01 ml/min/g vs. 0.23 +/- 0.02 ml/min/g (P < 0.05) in the epicardial ischemic zone; and, 0.15 +/- 0.02 ml/min/g vs. 0.22 +/- 0.03 ml/min/g (P < 0.05) in the endocardial ischemic zone]. We conclude that the acute, systemic administration of estrogen in the anesthetized dog decreases regional perfusion in the ischemic myocardium and causes significant coronary vaso-constriction when flow is controlled and limited.

Ischemia- and reperfusion-induced ventricular arrhythmias in dogs: effects of estrogen.

The purpose of this investigation was to determine if exogenous estrogen could attenuate the ventricular arrhythmias caused by myocardial ischemia and reperfusion. Conjugated equine estrogen, administered as an intravenous bolus injection (100 micrograms) to anesthetized, instrumented beagles of both genders, significantly attenuated the incidence of ventricular arrhythmias during a 20-min period of ischemia (2 +/- 1 vs. 19 +/- 16% ectopy) and in the first 5 min of reperfusion (15 +/- 9 vs. 69 +/- 20% ectopy). By 15-20 min of ischemia, ventricular salvos and nonsustained ventricular tachycardia were frequently observed in nontreated dogs. One dog in this group fibrillated during ischemia. In contrast, estrogen-treated dogs exhibited only an occasional ventricular premature beat during the same period of ischemia. When compared with baseline values, the percent ectopy during ischemia in estrogen-treated dogs was insignificant. During reperfusion, nontreated dogs displayed severe, life-threatening arrhythmias such as sustained ventricular tachycardia. In two of these dogs ventricular tachycardia deteriorated to ventricular fibrillation. In comparison, estrogen-treated dogs displayed only innocuous ventricular arrhythmias during reperfusion, i.e., ventricular premature beats, ventricular salvos, and ventricular bigeminy. In addition to the effect of estrogen on arrhythmias, there was a gradual increase in coronary blood flow on reperfusion in estrogen-treated dogs. This effect of estrogen was preceded by a significantly higher coronary perfusion pressure during ischemia (31 +/- 2 vs. 18 +/- 4 mmHg, P < 0.05). In conclusion, our findings suggest that antiarrhythmic effects of estrogen treatment might stabilize ventricular rhythmicity during ischemia and reperfusion.