Effects of a factor Xa inhibitor, DX-9065a, in a novel rabbit model of venous thrombosis.

The objective of this study was to evaluate the effects of DX-9065a, a nonpeptide, direct inhibitor of factor Xa (FXa), in a novel experimental model of venous thrombosis. The experiments were conducted on anesthetized rabbits in which a veno-venous shunt with cotton threads was inserted into the vena cava. DX-9065a was administered intravenously to the rabbits as an initial bolus followed by a maintenance infusion using the following dosing schedules: DX-I: 0.25 mg/kg + 3 micrograms/kg/min.; DX-II: 0.75 mg/kg + 9 micrograms/kg/min.; DX-III: 1.5 mg/kg + 18 micrograms/kg/min.; DX-IV: 3.0 mg/kg + 36 micrograms/kg/min.; DX-V: 6.0 mg/kg + 72 micrograms/kg/min. DX-9065a induced a dose-dependent increase in the time to occlusion and a dose-dependent decrease in thrombus weight. Because of the unique character of the model, we were also able to show a dose-dependent increase in blood flow through the shunt. In addition, there were dose-dependent increases in prothrombin time (PT) and activated coagulation time (ACT) with more variable responses in the activated partial thromboplastin time (APTT). DX-9065a had little effect on thrombin time (TT) or bleeding time at all doses tested. In conclusion, dose-dependent antithrombotic efficacy was documented with DX-9065a in this new model of venous thrombosis. Although the in vivo potency of the compound was not striking, the results support the utility of FXa inhibition in venous thrombosis and demonstrate the utility of this experimental model for evaluating the efficacy of novel anticoagulants.

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor.

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.

Inhibition of E-selectin-, ICAM-1-, and VCAM-1-mediated cell adhesion by benzo[b]thiophene-, benzofuran-, indole-, and naphthalene-2-carboxamides: identification of PD 144795 as an antiinflammatory agent.

It was previously reported that 3-alkoxybenzo[b]thiophene-2-carboxamides exemplified by 1, 5-methoxy-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide, decreased the adherence of neutrophils to activated endothelial cells by inhibiting the upregulation of the adhesion molecules E-selectin and ICAM-1 on the surface of the endothelium. This finding is extended here to a series of 3-thiobenzo[b]thiophene-2-carboxamides and also heterocyclic analogs of 1, including benzofurans, indoles, and napthalenes. The compounds that inhibited the expression of E-selectin and ICAM-1 had the same effect on the expression of VCAM-1. PD 144795, 5-methoxy-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide 1-oxide (44), the sulfoxide analog of 1, was orally active in several models of inflammation. The in vitro and in vivo activity of PD 144795 resided predominately in the S-enantiomer.

Hydroxylamine analogs of 2,6-di-t-butylphenols: dual inhibitors of cyclooxygenase and 5-lipoxygenase or selective 5-lipoxygenase inhibitors.

The preparation of hydroxylamine analogs of 2,6-di-tert-butylphenols (DTBP) and the inhibition of cyclooxygenase (CO) and 5-lipoxygenase (5-LO) by these compounds is discussed.

A novel prodrug of an impermeant inhibitor of 3-deoxy-D-manno-2-octulosonate cytidylyltransferase has antibacterial activity.

Although 8-amino-2,6-anhydro-3,8-dideoxy-D-glycero-D-talo-octonic acid (2) is a potent inhibitor of 3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase), it is unable to reach its cytoplasmic target and is therefore inactive as an antibacterial agent. However, esterification of 2 with 8-(hydroxymethyl)-1-naphthyl methyl disulfide (8) generates a prodrug (12), which gains entry into bacterial cells. Intracellular reduction of the disulfide leads to a rapid, intramolecular, displacement of the acid 2, which then inhibits the growth of Gram-negative bacteria by interfering with the biosynthesis of lipopolysaccharide.

Induction of delayed afterdepolarizations and triggered activity in canine Purkinje fibers by lysophosphoglycerides.

Lysophosphoglycerides accumulate in ischemic myocardium and induce electrophysiologic alterations in normoxic tissue in vitro closely resembling those seen with ischemia in vivo. Delayed afterdepolarizations and triggered activity may be particularly important in the pathogenesis of arrhythmias in the ischemic heart. The present study was performed to determine whether lysophosphatidylcholine (LPC), at concentrations comparable to those present in ischemic myocardium, can induce delayed afterdepolarizations and/or triggered activity in normoxic canine Purkinje fibers. In the present study, as little as 75 microM LPC was found to induce delayed afterdepolarizations and as little as 100 microM LPC was found to induce delayed afterdepolarizations and triggered activity even at low cycle lengths. The amplitude of the induced delayed afterdepolarizations was enhanced by augmentation of the extracellular concentration of calcium (7 mM) or by exogenous epinephrine (10(-9) to 10(-6) M). The amplitude was decreased by verapamil (1 mg/l) or Mn++ (2.5 mM). Epinephrine at a concentration of 10(-6) M also initiated triggered activity in Purkinje fibers exposed to LPC (75 microM), a response blocked by l-propranolol (2 X 10(-7) M and 10(-6) M) but not by the alpha 1-adrenergic blocking agent BE-2254 (10(-6) M). Delayed afterdepolarizations induced by LPC (75 microM) and epinephrine (10(-6) M) persisted even in the presence of acidosis (pH 6.7) and hyperkalemia ([K+]o = 7 mM). Thus, delayed afterdepolarizations and triggered activity induced by LPC may contribute to the induction and/or maintenance of arrhythmias early after the onset of myocardial ischemia. However, because of the reversal of these effects after superfusion with media devoid of LPC, they may occur with ischemia in vivo but not be seen in tissue isolated from ischemic regions and evaluated in vitro.

Thromboxane A2 does not contribute to arrhythmogenesis during evolving canine myocardial infarction.

During the healing phase of evolving myocardial infarction, inflammatory cells invade the affected region and produce metabolites that may influence electrophysiological parameters and the genesis of malignant arrhythmias. We have recently shown an increased synthetic capacity within an evolving infarct for thromboxane A2 (TXA2), a metabolite that has been implicated in arrhythmias associated with early ischemia. The present study used both in vivo and in vitro procedures to define the electrophysiological and arrhythmogenic effects, if any, of thromboxane during evolving myocardial infarction. Thirty-three dogs divided into three groups were studied 3-7 days after transient left anterior descending coronary artery ligation. One group (n = 24) was examined by programmed electrical stimulation in the conscious state and, of the five dogs in this group with sustained ventricular tachycardia (VT), none demonstrated consistent limitation of inducibility by selective inhibition of thromboxane synthetase using three different agents. In the second group, (n = 5) regional conduction velocity was assessed using detailed three-dimensional activation analysis from 232 simultaneous intramyocardial sites, and no change was induced by the thromboxane synthetase inhibitor OKY-1581 in either normal or infarcted myocardial zones during sinus rhythm or with pacing. In the third group (n = 4), isolated ventricular muscle was studied in vitro using both intracellular transmembrane action potential recordings and surface extracellular maps from 48 simultaneous points. Neither intracellular action potential parameters nor extracellularly recorded activation patterns were altered by superfusion with the stable thromboxane analog STA2, the activity of which was verified by bioassay. Thus, despite increased synthetic capacity for thromboxane generation, the presence of TXA2 does not directly influence either electrophysiological indices or arrhythmogenesis.

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells.

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.

Simultaneous computer mapping to facilitate intraoperative localization of accessory pathways in patients with Wolff-Parkinson-White syndrome.

Sixteen patients with the Wolff-Parkinson-White syndrome underwent simultaneous intraoperative computer mapping from multiple sites before surgical division of the accessory pathways. A 16-bipolar electrode band was positioned around the atrioventricular groove. Ventricular epicardial electrograms from single beats were recorded simultaneously during atrial pacing, resulting in maximal preexcitation, and atrial electrograms were recorded during orthodromic supraventricular tachycardia. Four-level transmural plunge needle electrodes were used concomitantly in 3 patients. Electrograms were processed separately using a guarded signal conditioner that isolates, amplifies, filters and analog-to-digitally converts synchronously at 2 kHz with 12-bit accuracy. Digital data were transmitted by fiber optics to a high-density digital recorder and processed with a computer having rapid interactive graphics. Results in the 16 patients revealed 20 distinct Kent bundles. Two patients had only nonsustained supraventricular tachycardia induced intraoperatively and 1 patient manifested intermittent anterograde ventricular preexcitation. Multiple pathways were identified in 4 patients. This simultaneous multiple electrode mapping procedure facilitates intraoperative mapping by requiring only a single beat for analysis of anterograde and retrograde activation times, decreases cardiac manipulation during mapping and obviates the need for cardiopulmonary bypass, and permits analysis of transmural activation patterns. This approach decreases markedly the time required for mapping and permits accurate study of nonsustained arrhythmias as well as rapid identification of multiple accessory pathways.

Intramural reentry as a mechanism of ventricular tachycardia during evolving canine myocardial infarction.

We evaluated the contribution of intramural electrical events in initiation and maintenance of ventricular tachycardia in 15 dogs 3-8 days after either permanent (n = 2) or transient (n = 13) coronary artery occlusion. Seven of the dogs (47%) demonstrated eight distinct monomorphic ventricular tachycardia patterns which were mapped by means of a recently designed computerized system capable of simultaneously detecting, storing, and assessing information from 232 individual cardiac sites. Using both epicardial and intramural electrodes, we found definitive evidence for intramural reentry in seven of the eight monomorphic tachycardias analyzed. Furthermore, five of these animals (71%) demonstrated microreentry, in which small epicardial conduction loops exited intermittently into nonrefractory subendocardium to initiate succeeding beats, while, in the remaining two dogs, ventricular tachycardia was due to macroreentry, during which the broad subendocardial wavefronts depolarizing the ventricle constituted the proximal (fast) reentry limbs. Detailed anatomical analysis of the resultant infarcts demonstrated the thin surviving epicardial tissue rim to be the site of conduction delay necessary for reentry, whereas "preferred pathways" of exit into the subendocardial plane occurred at the infarct borders and were of variable configuration. Successful interruption of these rhythms should accompany interference with the process of exit into nonrefractory subendocardial tissue.

In vivo inhibition of thromboxane synthetase in infarcted canine myocardium.

The in vivo effectiveness of the thromboxane synthetase inhibitor OKY-1581 was tested in normal and infarcted canine myocardium. A rapid in vitro assay was developed which permits an accurate assessment of the status of the tissue thromboxane synthetase at the time of sacrifice. Reperfused infarcts were created by two hours of coronary artery occlusion followed by release of occlusion and three days of recovery. OKY-1581 was infused at 100 micrograms/kg/min for 15 minutes, a dose previously found to cause an 85% inhibition of canine platelet thromboxane synthetase in vivo. The heart was rapidly excised and transmural tissue plugs of infarcted and normal areas were obtained. These were incubated for 5 minutes with prostaglandin endoperoxide (PGH2) in phosphate buffer. Thromboxane production was inhibited from 16 +/- 1 ng TxB2 per tissue plug to 5 +/- 1 in normal myocardium and from 27 +/- 5 to 6 +/- 1 in infarcted areas of myocardium. Control incubations showed no further inhibition with the in vitro addition of 20 micrograms/ml OKY-1581, confirming the completeness of in vivo inhibition. Thus significant inhibition of thromboxane synthetase by intravenous OKY-1581 occurs even in a reperfused zone of infarction.

Increased alpha-adrenergic receptors in ischemic cat myocardium. A potential mediator of electrophysiological derangements.

We have recently demonstrated enhanced alpha-adrenergic responsiveness assessed electrophysiologically in ischemic and reperfused myocardium. This study was performed to determine whether ischemia alters alpha 1-adrenergic receptor number (Bmax) of affinity (KD) based on [3H]prazosin binding. Within 30 min after occlusion, Bmax increased in ischemic regions to 207% of control to 27 +/- 2 fmol/mg protein, with the increase persisting (+ 141% of control) during early reperfusion (2 min), before returning to control base-line values (13 +/- 1.6) after 15 min of reperfusion. KD was not altered at any interval studied. Beta receptor number of ([3H]dihydroalprenolol) and Na+-K+ ATPase activity were comparable in control compared to ischemic myocardium although beta-receptor Bmax and KD in both regions decreased during early reperfusion. Thus, the enhanced alpha-adrenergic responsivity previously recognized with ischemia and reperfusion is correlated with an increase in alpha 1-adrenergic receptors.