Four-year follow-up of the Choice of Health Options In prevention of Cardiovascular Events randomized controlled trial.

OBJECTIVE: To determine if the improved risk factor profile at 1 year attributed to the Choice of Health Options In prevention of Cardiovascular Events (CHOICE) program was maintained at 4 years. DESIGN: Single-blind randomized controlled trial with post-hoc 476 months follow-up (76% complete). SETTING: Australian tertiary referral hospital. PATIENTS: Two hundred and eight acute coronary syndrome survivors. INTERVENTIONS: Acute coronary syndrome survivors not accessing cardiac rehabilitation (CR) were randomized to control (n=72) or CHOICE (n=72) comprising the tailored risk factor reduction packaged as a clinic visit and 3 months phone support. A contemporary CR reference group were also recruited (n=64). Blinded risk assessment occurred at baseline, 1 and 4 years. MAIN OUTCOME MEASURES: Total cholesterol, systolic blood pressure, smoking status, physical activity. RESULTS: One year improvements in all the modifiable risk factors achieved in CHOICE were maintained at 4 years. CHOICE and control were well-matched at baseline. At 4 years, there was a trend towards lower total cholesterol in CHOICE compared with controls (mean 4.0±0.1 vs. 4.2±0.1 mmol/l, P=0.05), significantly better systolic blood pressure (mean 132.2±2.1 vs. 136.8±2.0 mmHg, P=0.01), physical activity scores (1200±209 vs. 968±196 metabolic equivalent min/week, P=0.02) and proportion with three or more risk factors above national targets (20 vs. 42%,P=0.02). Participants in CHOICE were at higher baseline risk than CR but at 4 years they had similar risk factor profiles. CONCLUSION: Participants in CHOICE maintained favorable changes in coronary risk profile at 4 years compared with control, indicating that CHOICE is an effective long-term intervention among those not accessing facility-based CR.

Relationship between enteric pathogens and acute gastroenteritis disease severity: a prospective cohort study.

OBJECTIVES: To evaluate the relationship between individual bacterial and viral pathogens and disease severity. METHODS: Children <18 years with three or more episodes of vomiting and/or diarrhoea were enrolled in two Canadian paediatric emergency departments between December 2014 and August 2016. Specimens were analysed employing molecular panels, and outcome data were collected 14 days after enrolment. The primary outcome was severe disease over the entire illness (symptom onset until 14-day follow-up), quantified employing the Modified Vesikari Scale (MVS) score. The score was additionally analysed in two other time periods: index (symptom onset until enrolment) and follow-up (enrolment until 14-day follow-up). RESULTS: Median participant age was 20.7 (IQR: 11.3, 44.2) months; 47.4% (518/1093) and 73.4% (802/1093) of participants had index and total MVS scores ≥11, respectively. The most commonly identified pathogens were rotavirus (289/1093; 26.4%) and norovirus (258/1093; 23.6%). In multivariable analysis, severe disease over the entire illness was associated with rotavirus (OR = 9.60; 95%CI: 5.69, 16.19), Salmonella (OR = 6.61; 95%CI: 1.50, 29.17), adenovirus (OR = 2.53; 95%CI: 1.62, 3.97), and norovirus (OR = 1.43; 95%CI: 1.01, 2.01). Pathogens associated with severe disease at the index visit were: rotavirus only (OR = 6.13; 95%CI: 4.29, 8.75), Salmonella (OR = 4.59; 95%CI: 1.71, 12.29), adenovirus only (OR = 2.06; 95%CI: 1.41, 3.00), rotavirus plus adenovirus (OR = 3.15; 95%CI: 1.35, 7.37), and norovirus (OR = 0.68; 95%CI: 0.49, 0.94). During the follow-up period, rotavirus (OR = 2.21; 95%CI: 1.50, 3.25) and adenovirus (OR = 2.10; 95%CI: 1.39, 3.18) were associated with severe disease. CONCLUSIONS: In children presenting for emergency department care with acute gastroenteritis, pathogens identified were predominantly viruses, and several of which were associated with severe disease. Salmonella was the sole bacterium independently associated with severe disease.

Polymorphonuclear leukocyte phagocytic function increases in plasminogen knockout mice.

BACKGROUND: Mice lacking plasminogen (PG-/-) require alternative pathways of fibrinolysis for survival. This may depend on polymorphonuclear leukocytes (PMN) that can clear soluble and insoluble fibrin(ogen) through PG-independent processes. Our objective was to demonstrate that PMNs from PG-/- mice exhibit increased Mac-1 dependent phagocytic activity, which may explain their increased fibrin(ogen)lytic activity compared with wild type (PG+/+) mice. METHODS: Phagocytic activity of PMNs from PG-/- and PG+/+ mice was compared following exposure to Staphylococcus aureus (S. aureus) particles and the expression of Mac-1 was assessed in parallel by flow cytometric analysis. Resistance to phorbol-12-myristate-13-acetate (PMA)-induced cell death was compared between PMNs from the different genotypes. RESULTS: Stimulation of PG-/- PMNs by opsonized S. aureus diluted in PG-/- plasma significantly increased phagocytosis (15-fold) compared with stimulation of PG+/+ PMNs in PG+/+ plasma. Incubation of PG-/- PMNs with PG+/+ plasma (control) or PG-/- plasma supplemented with human PG inhibited this increased phagocytic activity. Mac-1 cell surface density increased 6.2+/-1.0-fold in PG-/- PMNs versus 2.9+/-0.6-fold in PG+/+ PMNs (P < 0.01) indicating that Mac-1 may be associated with increased phagocytic activity. Supporting this, treatment of PG-/- PMNs with an anti-Mac-1 antibody in PG-/- plasma inhibited phagocytic activity. In addition, physiologic PG blocked Mac-1 accessibility at the surface of PMNs. Addition of PMA resulted in 33% death of PMNs from PG-/- mice versus 68% in PG+/+ controls (P < 0.001). CONCLUSIONS: PMNs from PG-/- mice exhibit a Mac-1 dependent increase in phagocytic activity that is suppressed with human PG, an anti-Mac-1 antibody or the plasma from PG+/+ mice. The propensity for PMNs from PG-/- mice to be activated in response to PMA together with their relative resistance to PMA-toxicity may contribute to increased PMN half-life and enhanced fibrin(ogen) clearance in the setting of PG deficiency.

The CHOICE (Choice of Health Options In prevention of Cardiovascular Events) replication trial: study protocol.

BACKGROUND: Although morbidity and mortality from coronary heart disease (CHD) are high, only a minority of acute coronary syndrome (ACS) survivors accesses an effective secondary prevention program. We aim to determine whether the previously proven CHOICE program can be replicated at multiple sites and whether ongoing reinforcement further improves risk factor modification. METHODS/DESIGN: Participants eligible for but not accessing standard cardiac rehabilitation will be randomly allocated to either a previously tested 3-month CHOICE program or a 30-month CHOICE program (CHOICE-plus). Both groups will participate in individualised risk factor modules of differing duration that involve choice, goal setting and telephone follow-up for three months. CHOICE-plus will also receive additional face-to-face and telephone reinforcement between three and 30 months. At one site we will recruit a randomised control group, receiving conventional care. Primary outcomes are lipid levels, blood pressure, physical activity levels and smoking rates. Secondary outcomes include readmission rates, death, the number of risk factors, other modifiable risk factors, quality of life and process evaluation measures over three years. DISCUSSION: We present the rationale and design of a multi-centre, replication study testing a modular approach for the secondary prevention of CHD following an ACS. TRIAL REGISTRATION: [Clinical Trial Registration Number, ACTRN12608000182392].

C-reactive protein contributes to the hypercoagulable state in coronary artery disease.

OBJECTIVES: Elevated plasma C-reactive protein (CRP) levels predict coronary events, but it is unclear whether CRP plays a role in thrombosis associated with these events. We investigated tissue factor (TF) induction by CRP on peripheral blood mononuclear cells (PBMC) from patients with coronary disease. PATIENTS AND METHODS: PBMC from 35 patients with stable angina (SA) in study 1, 10 male patients with SA, 10 with unstable angina (UA) and 10 matched controls in study 2, and 25 patients with inflammatory disorders (ID) and 24 normal controls in study 3 were stimulated with CRP, interferon-gamma (IFN) or lipopolysaccharide (LPS), or their combination. PBMC from additional normal donors were also stimulated with CRP in adherent and non-adherent conditions, and TF activity, antigen and mRNA expression detected. RESULTS: CRP (5-25 microg mL(-1)) dose dependently induced more TF on PBMC from SA patients than 42 contemporary controls (P = 0.001, study 1). Compared with controls, patients with SA or UA had higher basal, and much higher CRP- or CRP/LPS-induced monocyte TF activity although serum CRP levels were similar (study 2). IFN induced monocyte TF activity in patients with angina, but not in controls. Basal or CRP-induced TF levels did not differ between controls and ID, even though ID patients had much higher serum CRP levels (study 3). CRP-induced monocyte TF activity correlated with serum CRP levels in controls (P = 0.005) and ID (P = 0.007) in study 3, but not in patients with angina (P =0.84) in study 2. CRP induced more TF activity, protein and mRNA under adherent than non-adherent conditions implying that it may mainly target macrophages in lymphocyte-rich lesions. CONCLUSIONS: Our results indicate that monocytes from patients with angina are preactivated and express TF but CRP is unlikely to be a major priming factor in vivo. IFN and CRP further increase TF levels that may contribute to the hypercoagulable state in coronary disease.

Modular prevention of heart disease following acute coronary syndrome (ACS) [ISRCTN42984084].

BACKGROUND: Coronary heart disease (CHD) is a major cause of morbidity and mortality in Australia and it is recommended that all persons with unstable angina (UA) or myocardial infarction (MI) participate in secondary prevention as offered in cardiac rehabilitation (CR) programs. However, the majority of patients do not access standard CR and have higher baseline coronary risk and poorer knowledge of CHD than those persons due to commence CR. The objective of this study is to investigate whether a modular guided self-choice approach to secondary prevention improves coronary risk profile and knowledge in patients who do not access standard CR. METHODS/DESIGN: This randomised controlled trial with one year follow-up will be conducted at a tertiary referral hospital. Participants eligible for but not accessing standard CR will be randomly allocated to either a modular or conventional care group. Modular care will involve participation in individualised modules that involve choice, goal-setting and coaching. Conventional care will involve ongoing heart disease management as directed by the participant's doctors. Both modular and conventional groups will be compared with a contemporary reference group of patients attending CR. Outcomes include measured modifiable risk factors, relative heart disease risk and knowledge of risk factors. DISCUSSION: We present the rationale and design of a randomised controlled trial testing a modular approach for the secondary prevention of coronary heart disease following acute coronary syndrome.

Interferon-gamma and lipopolysaccharide potentiate monocyte tissue factor induction by C-reactive protein: relationship with age, sex, and hormone replacement treatment.

BACKGROUND: Elevated plasma levels of C-reactive protein (CRP) in population studies and in patients with unstable coronary syndromes are predictive of future adverse events, including cardiac death and myocardial infarction, implicating inflammation in pathogenesis. Although CRP is considered a marker of inflammation, it induces monocyte tissue factor (TF) and may play a prothrombotic role in atherosclerosis and its complications. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMCs) from 79 healthy men and women aged 26 to 83 years and 21 healthy postmenopausal women taking hormone replacement therapy (HRT) were stimulated with CRP, lipopolysaccharide (LPS), interferon-gamma (IFN), or their combination. Levels of CRP in the normal range (1 to 5 microg/mL) increased basal monocyte TF 4- to 6-fold and 40-fold at higher concentrations (25 microg/mL). Coincubation of LPS with CRP produced a greater-than-additive response. IFN did not induce TF but synergized with CRP to approximately double activity. There was a striking positive correlation between age and monocyte TF induction, with a dramatic rise on monocytes from postmenopausal women that was not apparent on cells from women taking HRT. CONCLUSIONS: Synergy between CRP and inflammatory mediators may play a direct prothrombotic role in the pathogenesis of coronary atherosclerosis and its acute complications by increasing monocyte/macrophage TF. This may contribute to age and sex differences in coronary events and to the protective effects of HRT.

The design of full agonists for the cortical muscarinic receptor.

In the present study we describe a novel series of oxadiazole based tertiary amines which include the most efficacious and potent muscarinic ligands known. These compounds possess physicochemical characteristics which enable rapid equilibration into the CNS and are able to fully activate cortical muscarinic receptors. Data obtained from this series have allowed us to propose a pharmacophoric model which distinguishes high and low affinity state binding. This in turn has led us to suggest that agonists and antagonists may bind at two independent sites on the receptor protein and to speculate on the steps putatively involved in agonist-induced receptor activation.

Intermittent transdermal nitrates do not improve ischemia in patients taking beta-blockers or calcium antagonists: potential role of rebound ischemia during the nitrate-free period.

OBJECTIVES: This study was conducted to determine whether rebound ischemia occurs during nitrate-free periods with intermittent cutaneous nitroglycerin therapy in patients with angina pectoris who are receiving background antianginal therapy. BACKGROUND: Rebound angina has been suggested to be a complication of the nitrate-free period with long-term cutaneous nitroglycerin therapy given intermittently to prevent tolerance. METHODS: Fifty-two patients with stable effort angina taking either a beta-adrenergic blocking agent (n = 25) or diltiazem (n = 22) or their combination (n = 5) completed a randomized, double-blind, placebo-controlled crossover study of cutaneous nitroglycerin patches (50 mg). Active or placebo patches were worn for 1 week, applied at 8 AM and removed at 10 PM to provide a 10-h daily nitrate-free (or placebo-free) period. During the last 48 h of each study phase, a Holter monitor was used to detect ischemia. RESULTS: Only 31 patients experienced ischemia during either phase of the study (23 during the patch-off period). A total of 463 ischemic episodes were recorded: 246 during placebo and 217 during nitroglycerin (p = 0.8, for per patient comparison). The majority (88%) of ischemic episodes were silent. Mean (+/- SEM) duration of ischemia during the total 48-h period was similar during active and placebo phases (35.5 +/- 15.0 min/24 h for active therapy vs. 29.7 +/- 9.8 for placebo, p = 0.8). This was due to an increase in duration of ischemia with active therapy during the patch-off period (46.9 +/- 17.9 min/24 h for active therapy vs. 22.5 +/- 9.2 for placebo, p = 0.07) and a decrease during the patch-on period (27.5 +/- 14.0 min/24 h for active therapy vs. 34.5 +/- 11.0 min/24 h for placebo, p = 0.16). The pattern of diurnal distribution of ischemic episodes differed between active and placebo phases. During placebo there was a nadir in the incidence of ischemia in the overnight patch-off period, with a significantly lower incidence between midnight and 6 AM (25 episodes) compared with the mean number of episodes during the three other 6-h periods (73 episodes, p < 0.001). During the nitroglycerin patch-off period, there was a loss of this overnight nadir, with the same incidence of ischemia between midnight and 6 AM (53 episodes) as the mean number of episodes for the three other 6-h periods (54 episodes). CONCLUSIONS: The majority of patients taking background antianginal therapy experienced no ischemia during the patch-off period. In the 44% of patients with ischemia during this period, there was a nonsignificant increase in the duration of ischemia with active therapy. Although this result was statistically inconclusive, the change in the distribution of diurnal ischemia offers suggestive evidence that rebound ischemia may be a problem with regard to intermittent cutaneous nitroglycerin.

Effects of diltiazem and long-term beta 1-adrenergic blockade on hemodynamics and blood flow during exercise in patients with stable angina pectoris.

The short-term effects of oral diltiazem on hemodynamics and distribution of cardiac output at rest and during semiupright bicycle exercise were evaluated in eight patients with stable effort angina on long-term beta 1-adrenergic blockade. Cardiac output and iliofemoral blood flow were measured using thermodilution. The patients were exercised to the same work load on a bicycle before and 2 h after oral diltiazem (60 mg in two patients and 120 mg in six). At maximal exercise, diltiazem reduced heart rate from 94 +/- 5 to 88 +/- 6 beats/min (p less than 0.01), mean arterial pressure from 139 +/- 5 to 127 +/- 4 mm Hg (p less than 0.01) and systemic vascular resistance from 9.7 +/- 0.7 to 8.4 +/- 0.4 x 10(2) dynes.s.cm-5 (p less than 0.05) compared with control. During exercise, cardiac output, iliofemoral blood flow, mean pulmonary wedge pressure and mean right atrial pressure were not altered, but stroke volume increased from 119 +/- 11 to 131 +/- 10 ml (p less than 0.05). Maximal ST segment depression during exercise was decreased and angina was less. Diltiazem does not alter the distribution of the cardiac output during exercise but improves ischemia.

Prognostic significance of a predischarge exercise test in risk stratification after unstable angina pectoris.

The prognostic significance of exercise testing was compared with clinical and electrocardiographic (ECG) variables in a prospective study of 107 patients with unstable angina discharged from the hospital on medical therapy. During a follow-up period of 12.8 +/- 1.4 months, 10 patients (9%) had a nonfatal myocardial infarction (n = 8) or died (n = 2) and 22 (20%) were readmitted with recurrent unstable angina. The relation between 20 clinical, ECG and exercise test variables and the risk of adverse outcome (death, nonfatal myocardial infarction or recurrent unstable angina) was analyzed using both univariate and multivariate (logistic regression) analysis. Univariate predictors of adverse outcome included diabetes mellitus, evolutionary T wave changes, T wave changes on the preexercise ECG and low maximal rate-pressure product during exercise. Independent predictors of adverse outcome in multivariate analysis included diabetes mellitus, evolutionary T wave changes after admission, rest pain during hospitalization, ST depression during exercise and low maximal rate-pressure product. A predictive model constructed using the regression equation and all independent predictors stratified patients into high and low risk groups (41% and 5% risk of adverse outcome, respectively). The result of a predischarge exercise test adds independent prognostic information to clinical and ECG data in medically treated patients with unstable angina and could be used in combination with clinical and ECG data to identify patients at risk of adverse events.

Electrocardiographic measurement of infarct size after thrombolytic therapy.

OBJECTIVES: We examined the utility of the 32-point QRS score from the 12-lead electrocardiogram (ECG) for measurement of the ischemic risk region and infarct size in patients receiving thrombolytic therapy. BACKGROUND: The QRS score offers a means of evaluating the therapeutic benefit of thrombolytic therapy by comparing final infarct size with the initial extent of ischemic myocardium. METHODS: The study included 38 patients (34 men, 4 women; mean [+/-SD] age 54 +/- 10 years) with a first infarction (18 anterior, 20 inferior). The maximal potential QRS score (QRS0) was assigned to all leads with >/= 100-microV ST elevation on the initial ECG. The QRS scores were calculated at 7 and 30 days after infarction. Left ventricular ejection fraction was measured by radionuclide ventriculography at 1 month. Twenty-eight patients had thallium (Tl)-201 and technetium (Tc)-99m pyrophosphate tomographic measurement of the ischemic region and infarct size. RESULTS: The QRS0 was 10.3 +/- 3.1 (mean +/- SD) for anterior and 10.4 +/- 3.5 for inferior infarcts. The QRS scores were similar at 7 and 30 days for both anterior (5.6 +/- 3.4 vs. 5.5 +/- 3.4) and inferior infarcts (3.7 +/- 2.6 vs. 2.9 +/- 2.2). The day 7 QRS score and ejection fraction at 1 month were inversely correlated (r = -0.74, p < 0.01). The Tl-201 perfusion defect was 34 +/- 11% of the left ventricle for anterior and 32 +/- 7% for inferior infarcts. Subsequent Tc-99m pyrophosphate infarct size was 15 +/- 9% of the left ventricle for anterior and 17 +/- 9% for inferior infarcts. The QRS0 was correlated with the extent of the Tl-201 perfusion defect (r = 0.79, p < 0.001), and the day 7 QRS score was correlated with Tc-99m pyrophosphate infarct size (r = 0.79, p < 0.005). CONCLUSIONS: The 32-point QRS score can provide useful immediate measurements of the ischemic risk region and subsequent infarct size.

Spontaneous remyelination following prolonged inhibition of alpha4 integrin in chronic EAE.

Inhibition of alpha(4)beta(1) integrin blocks immune cell influx into the CNS providing benefit to patients with multiple sclerosis and in animal model systems. We have used this mechanism to examine whether the presence of inflammatory cells suppresses spontaneous myelin repair in experimental autoimmune encephalomyelitis. We observed (1) 87% of plaques showed remyelination after 40 days of treatment; (2) myelin repair occurred in half of the total lesion area; (3) half of the animals regained motor function. There was no significant repair or gain of motor function in vehicle-treated animals. Therefore, prolonged inhibition of CNS inflammation, in the absence of targeted myelin repair, facilitates mechanisms of spontaneous remyelination.

Verapamil kinetics in normal subjects and patients with coronary artery spasm.

Verapamil kinetics after intravenous and single and long-term oral dosing were studied in 12 patients with coronary artery spasm and four normal subjects. The decline in plasma concentration after intravenous doses was described by triexponential decay equation, with a terminal half-life (t1/2) of 5 hr. After a single oral dose the bioavailability was only 24%, probably because of the first-pass metabolism. During long-term oral doses of 80 mg every 6 hr, mean peak and trough concentrations were 255 +/- 90 and 105 +/- 38 ng/ml, and mean time at which peak concentration occurred was 1.2 +/- 0.5 hr. Norverapamil, the major active metabolite of verapamil, cumulated during oral dosing and may account for a small proportion of the overall pharmacologic effect. Mean elimination t1/2 during long-term oral dosing was longer than after a single dose (9.6 and 5.7 hr, P less than 0.05). Also, during long-term dosing the area under the curve was more than double that of a single dose, and the apparent oral clearance fell from 4.2 to 1.8 l/min (P less than 0.01). These changes may partly be explained by reduction in presystemic metabolism during long-term therapy. Kinetic predictions based on single doses will not give reliable estimates for long-term oral dosage. Less frequent dose schedule may be possible for prolonged therapy.

Characterisation of a chimeric hD3/D2 dopamine receptor expressed in CHO cells.

The D2 dopamine receptor is known to be functionally coupled when expressed in CHO cells, whereas the effector systems for the D3 dopamine receptor remain unclear. A chimeric, human D3/D2 receptor (hD3/D2) was constructed containing the third intracellular loop region of the D2 receptor. CHO cells stably expressing the D2, D3, or hD3/D2 receptors were created and the pharmacology of the receptors was examined. The chimeric hD3/D2 receptor retained D3-like affinities for dopaminergic ligands. However, in contrast to the D2 receptor neither the D3 receptor nor the hD3/D2 receptor could functionally couple to the adenylate cyclase or arachidonic acid release mechanisms.

Stable expression of human D3 dopamine receptors in GH4C1 pituitary cells.

Human D3 dopamine receptor DNA was stably transfected into GH4C1 pituitary cells. Displacement of iodosulpiride binding in hD3 transfected cells (Kd = 0.3 nM, Bmax = 89 fmol/mg protein) by dopaminergic ligands was indistinguishable from that of hD3 receptors in CHO cells. Only two clonal cell lines exhibited weak GppNHp-dependent shifts in [3H]N-0437 binding, and these were used for functional assays. Neither arachidonic acid metabolism, cAMP levels, inositol phosphate turnover, intracellular calcium, or potassium currents were consistently affected by dopamine (1-10 microM). The paucity of responses indicates that human D3 receptors do not couple efficiently to these second messengers in GH4C1 cells.

2-Methyl-1,3-dioxaazaspiro[4.5]decanes as novel muscarinic cholinergic agonists.

Many nonquaternary ammonium muscarinic agonists have been developed over the last few years, but most of the existing compounds (e.g., arecoline, RS-86, AF-30) behave as weak partial agonists at cholinergic receptors in tissues of limited receptor reserve. The current paper describes the synthesis and biochemical assessment of analogues of AF-30 designed to have sufficient conformational freedom to allow greater receptor flexibility and hence activation. The new compounds and important standards were tested in a new biochemical assay designed to measure both receptor affinity and intrinsic activity of each compound and for their ability to stimulate phosphatidylinositol turnover in rat cerebral cortex. Two azaspirodecanes (5a and 5b) were shown to have far greater predicted efficacy than AF-30.

Tetrahydropyridyloxadiazoles: semirigid muscarinic ligands.

Recent studies have described novel azabicycle-based muscarinic agonists which readily penetrate into the central nervous system and are capable of displaying high efficacy at cortical sites. The current paper describes the synthesis and biochemical assessment of semirigid muscarinic ligands which were used to map the requirements of the cortical muscarinic receptor and to study the degree of conformational flexibility required to cause receptor activation. Analogues 6 and 9 provide high-efficacy muscarinic agonists at cortical sites; however, C-alkylation on the tetrahydropyridine ring resulted in more rigid analogues and showed lower predicted efficacy. Molecular mechanics calculations indicated a preference for the E rotameric form. This conformation was also observed in the X-ray crystal structure of ethenyloxadiazole 12. The new compounds were tested in a biochemical assay designed to measure receptor affinity and to predict cortical efficacy.

Synthesis and characterization of all four isomers of the muscarinic agonist 2'-methylspiro[1-azabicyclo[2.2.2]octane-3,4'-[1,3]dioxolane].

The four separated isomers of the muscarinic agonist 1, previously known as AF30, have been synthesized by a route that has allowed the absolute stereochemistry of each isomer to be assigned. With the chirality of (-)-camphanic acid known, X-ray analysis of the more crystalline intermediate diastereomeric camphanate 5A allowed the absolute stereochemistry at the quinuclidine chiral center to be determined. Each diastereomer was separately transformed into the spirodioxolane with concomitant introduction of the second chiral center. Chromatographic separation followed by a second crystal structure determination revealed the absolute stereochemistry of all four isomers of 1. Detailed biological evaluation of each isomer indicated that while the 3(R),2'(S) isomer was the most active in binding studies, it was the 3(R),2'(R) isomer that displayed the largest functional selectivity between ganglion (M-1 site) and heart (M-2 site). With the same internal chiral standard, the absolute configuration of the more active enantiomer of 3 was shown to be S, confirming earlier literature reports.