Ultrahigh vacuum deposition of organic molecules by electrospray ionization.

An electrospray apparatus for deposition of organic molecules on surfaces in ultrahigh vacuum is presented. The kinetic energy at the impact and mass to charge ratio of deposited ions can be controlled by an electrostatic quadrupole deflector and an in-line quadrupole mass spectrometer. With an ion funnel in the first two vacuum stages a high ion transmission is achieved. Experiments on porphyrin cations and deoxyribonucleic acid deposited on a Au(111) surface demonstrate the capabilities of the instrument.

Torcetrapib-induced blood pressure elevation is independent of CETP inhibition and is accompanied by increased circulating levels of aldosterone.

BACKGROUND AND PURPOSE: Inhibition of cholesteryl ester transfer protein (CETP) with torcetrapib in humans increases plasma high density lipoprotein (HDL) cholesterol levels but is associated with increased blood pressure. In a phase 3 clinical study, evaluating the effects of torcetrapib in atherosclerosis, there was an excess of deaths and adverse cardiovascular events in patients taking torcetrapib. The studies reported herein sought to evaluate off-target effects of torcetrapib. EXPERIMENTAL APPROACH: Cardiovascular effects of the CETP inhibitors torcetrapib and anacetrapib were evaluated in animal models. KEY RESULTS: Torcetrapib evoked an acute increase in blood pressure in all species evaluated whereas no increase was observed with anacetrapib. The pressor effect of torcetrapib was not diminished in the presence of adrenoceptor, angiotensin II or endothelin receptor antagonists. Torcetrapib did not have a contractile effect on vascular smooth muscle suggesting its effects in vivo are via the release of a secondary mediator. Treatment with torcetrapib was associated with an increase in plasma levels of aldosterone and corticosterone and, in vitro, was shown to release aldosterone from adrenocortical cells. Increased adrenal steroid levels were not observed with anacetrapib. Inhibition of adrenal steroid synthesis did not inhibit the pressor response to torcetrapib whereas adrenalectomy prevented the ability of torcetrapib to increase blood pressure in rats. CONCLUSIONS AND IMPLICATIONS: Torcetrapib evoked an acute increase in blood pressure and an acute increase in plasma adrenal steroids. The acute pressor response to torcetrapib was not mediated by adrenal steroids but was dependent on intact adrenal glands.

Lack of beneficial effects of growth hormone treatment in conscious dogs during development of heart failure.

The effects of chronic treatment with growth hormone (porcine GH, 0.56 mg.kg-1.day-1 s.c.) were examined in dogs with heart failure induced by rapid ventricular pacing (240 beats/min) for 4 wk. Fourteen conscious dogs were studied 2-3 wk after surgical instrumentation with catheters in the descending aorta and left atrium, a pressure gauge in the left ventricle (LV), a flow probe around the ascending aorta, pacing leads on the ventricular free wall and left atrium, and ultrasonic crystals on the opposing anterior and posterior endomyocardium of the LV. GH treatment for 4 wk significantly increased both body weight and plasma insulin-like growth factor 1 (IGF-1) compared with vehicle-treated dogs (P < 0.01, +2.0 +/- 0.5 vs. +0.3 +/- 1.1 kg; 1,043 +/- 218 vs. 241 +/- 64 ng/ml, respectively). However, the changes in resting LV systolic (i.e., both isovolumic and ejection phases) and diastolic function (i.e., isovolumic relaxation time constant tau) and the systemic vascular resistance were similar for the GH- and vehicle-treated groups during the development of heart failure. LV contractile reserve, assessed with step infusion of isoproterenol or dobutamine challenge, was markedly attenuated after heart failure, but there were no differences between the GH- and vehicle-treated groups. During the progression of heart failure, the increases in plasma atrial natriuretic peptide correlated (P < 0.01) directly with left atrial pressure and inversely with LV circumferential fiber shortening. However, GH treatment did not substantially modify these relationships. In addition, renal function and myocardial ultrastructure at the advanced stage of heart failure also showed similar changes for the GH- and vehicle-treated groups. We conclude that in conscious dogs during the development of congestive heart failure produced by rapid ventricular pacing, GH at a dose that increases body weight and plasma IGF-1 levels does not affect LV performance or systemic vascular dynamics.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Potent noncovalent thrombin inhibitors that utilize the unique amino acid D-dicyclohexylalanine in the P3 position. Implications on oral bioavailability and antithrombotic efficacy.

In an effort to prepare orally bioavailable analogs of our previously reported thrombin inhibitor 1, we have synthesized a series of compounds that utilize the unique amino acid D-dicyclohexylalanine as a P3 ligand. The resulting compounds are extremely potent and selective thrombin inhibitors, and the N-terminal Boc derivative 8 exhibited excellent oral bioavailability and pharmacokinetics in both rats and dogs. The des-Boc analog 6 was not orally bioavailable in rats. The high level of oral bioavailability observed with 8 appears to be a direct function of its increased lipophilicity versus other close analogs. Although increased lipophilicity may serve to increase the oral absorption of tripeptide thrombin inhibitors, it also appears to have detrimental effects on the antithrombotic properties observed with the compounds. Compound 6 performed extremely well in our in vivo antithrombotic assay, while the much more lipophilic but essentially equipotent analog 8 performed poorly. We have found that in general with this series of thrombin inhibitors as well as with other unreported series, increased lipophilicity and the associated increases in plasma protein binding have detrimental effects on 2X APTT values and subsequent performance in in vivo antithrombotic models.

Intravenous administration of the endothelin-1 antagonist BQ-123 does not ameliorate myocardial ischaemic injury following acute coronary artery occlusion in the dog.

OBJECTIVE: It has been proposed that myocardial ischaemic injury is modulated in part by the release of endothelin-1 from the coronary endothelium either during ischaemia or following reperfusion. Release of sufficient amounts of endothelin-1 would result in coronary vasoconstriction and could potentiate ischaemic damage. An endothelin-1 antagonist, BQ-123, was given intravenously to evaluate the role of endothelin-1 in postischaemic injury and determine whether blockade of the ETA receptor would afford protection from ischaemia/reperfusion injury. METHODS: Myocardial injury was induced in anaesthetised dogs using 90 min of left circumflex coronary artery occlusion followed by 4 h of reperfusion. Animals treated with a continuous intravenous infusion of BQ-123 (0.1 mg.kg-1.min-1), begun 10 min before ischaemia and continued throughout ischaemia and reperfusion, were compared to saline treated animals. RESULTS: After 4 h of reperfusion the myocardial infarct size measured by triphenyltetrazolium chloride staining was not different between the two groups. Infarct size in the control group was 25.7 (SEM 5.4)% of the area at risk while BQ-123 treatment resulted in an infarct size of 29.2(7.1)% of the area at risk (p = 0.70). Plasma endothelin-1 concentration measured at the coronary sinus was only significantly increased following 5 min of reperfusion. CONCLUSIONS: The intravenous administration of a specific ETA receptor antagonist does not protect against ischaemia/reperfusion injury. These results suggest that endothelin-1 receptor antagonists require access to the area at risk during occlusion to protect the myocardium from ischaemic injury.

The role of the neutrophil and formed elements of the blood in an in vitro model of reperfusion injury.

Using The globally ischaemic isolated guinea-pig heart we conducted studies to assess the role of activated neutrophils (PMNs) and the role of the endothelium in reperfusion injury. Reperfusion injury was induced by a 20 min period of global ischaemia followed by a 30 min reperfusion with Krebs' buffer supplemented with f-Met-Leu-Phe (fMLP) and heparinized blood. Ischaemia alone or blood alone resulted in a complete recovery in contractile function measured by developed pressure, fMLP (500 muM) and blood, administered to normoxic hearts did not affect contractile function. The combination of 100 muM fMLP and blood beginning at reperfusion and continuing for 30 min decreased the recovery in contractile function (max. 33 +/- 6% reovery) while buffer and 100 pM fMLP resulted in a complete recovery in function. In hearts infused with buffer and neutropenic blood incubated with 100 muM fMLP a complete recovery in function was observed. Isolated peritoneal neutrophils, 7-70 x 10(5) PMN/ min, incubated with 100 muM fMLP and Krebs' solution decreased contractile function in a concentration-related manner (max. 44 +/- 11% recovery). Platelets, plasma or red blood cells alone incubated with fMLP did not decrease recovery in developed pressure. Platelets and PMN incubated with 100 muM fMLP did not, while red blood cells and PMN did, elicit a reduction in recovery in contractile function (34 +/- 4% recovery). A 20 min period of global ischaemia destroys the functional integrity of the endothelium (response to Ach). Pre-treatment of the heart with sufficient H(2)O(2) to functionally damage the endothelium, followed by infusion of Krebs' solution supplemented with blood and 100 muM fMLP also elicited a reduction in recovery of contractile function (42 +/- 15% recovery). In summary, partially activated neutrophils play a major role in reperfusion injury and there exists a cooperativity between the RBC and PMN in this model.

Pharmacology of RG W-2938: a cardiotonic agent with vasodilator activity.

The cardiovascular effects of RG W-2938, 6-[6-(3,4-dihydro-3-methyl-2(1H)-2-oxoquinazolinyl)]-4,5-dihydro-3 (2H-pyridazinone, a new nonglycoside, noncatecholamine cardiotonic/vasodilator agent were examined in vivo in anesthetized and conscious dogs and in vitro in isolated guinea pig hearts; in the latter, RG W-2938 5 nmol-5 mumol increased contractility in a dose-related fashion. RG W-2938 30-300 micrograms/kg administered intravenously (i.v.) to anesthetized dogs increased contractile force while decreasing arterial pressure and total peripheral resistance (TPR) in a dose-related manner. Heart rate (HR) was only slightly increased, and aortic flow was not appreciably altered. A single oral dose of RG W-2938 0.3 mg/kg administered to conscious chronically instrumented dogs produced a marked and sustained increase in contractility 15-240 min after treatment while only slightly increasing HR. The effects of RG W-2938 30-300 micrograms/kg, i.v. were studied in a mecamylamine-propranolol-induced model of heart failure. RG W-2938 effectively reversed the drug-induced heart failure by increasing myocardial contractility and decreasing arterial pressure while only slightly affecting HR. These studies show that RG W-2938 is an orally effective positive inotropic/vasodilator agent.

Examination of diltiazem for preservation of myocardial function after brief regional ischemia.

Diltiazem (750 micrograms/kg plus 600 micrograms/kg/h X 1 h, i.v.) and vehicle were examined in open-chest anesthetized dogs subjected to 15 min of occlusion of the left circumflex coronary artery (LCCA). Regional segment lengths in myocardium supplied by the LCCA and by the left anterior descending coronary (LAD) were measured with piezoelectric crystals implanted in the subendocardium. Diltiazem decreased heart rate and mean arterial pressure, and increased coronary blood flow, determined with an electromagnetic flowmeter. Vehicle had no significant effects. Occlusion of the LCCA increased end diastolic segment length (EDL), and produced akinesis or paradoxical systolic lengthening: diltiazem -2.5 +/- 2.7% and vehicle 0.0 +/- 1.2% segmental shortening (SS). EDL and SS in the LAD zone showed small increases. After 15 min, the LCCA was reperfused and recovery of SS was followed for 3 h. Significantly greater recovery of SS was observed with diltiazem compared to vehicle throughout reperfusion: at 5 min, diltiazem 105 +/- 22% and vehicle 43 +/- 7% and at 180 min, diltiazem 73 +/- 0% and vehicle 33 +/- 8% of baseline SS. The LCCA and LAD zones both responded to isoproterenol 0.3 microgram/kg given 2.5 h after reperfusion. During the isoproterenol challenge SS for LCCA in the diltiazem group (122 +/- 21%) was not different than that of vehicle (99 +/- 15% of baseline). Calcium entry blockade with diltiazem resulted in improved myocardial function during reperfusion. The stunned myocardium showed significant stimulation of shortening by isoproterenol in both groups.