Prognostic value of protein C concentrations in neutropenic patients at high risk of severe septic complications.

OBJECTIVE: To assess the prognostic value of protein C, endogenous activated protein C, and D-dimer concentrations in patients at high risk of developing severe septic complications secondary to cytostatic chemotherapy. DESIGN: Prospective, comparative, single-center study. SETTING: Specialized ward for treating patients with acute leukemia and associated intensive care unit at a university hospital. SUBJECTS: Twenty-six consecutive patients who developed either severe sepsis (n = 13) or septic shock (n = 13) during chemotherapy-induced neutropenia (leukocytes <1,000/microL). INTERVENTION: None, other than standard care. MEASUREMENTS AND MAIN RESULTS: Baseline blood samples were obtained from 97 adult patients treated with intensive cytostatic chemotherapy. Serial blood sampling was performed in 62 of 97 patients who developed fever (>38.3 degrees C). Thirteen patients progressed to severe sepsis and 13 patients to septic shock. Protein C, endogenous activated protein C, and D-dimer were measured in these 26 patients. At fever onset, protein C concentrations decreased from normal baseline concentrations and were significantly lower in the group of patients who progressed to septic shock compared with those who developed severe sepsis (medians for protein C activity: 23.1% vs. 69.5%; p = .0003). The median elapsed time between detection of fever and the diagnosis of severe sepsis or septic shock was 16 hrs and 12 hrs, respectively. All septic shock patients died, whereas patients who progressed only to severe sepsis survived. CONCLUSIONS: Septic shock in neutropenic patients is associated with increased protein C consumption. The data demonstrate that the coagulation cascade is activated and produces a hypercoagulable state before the onset of clinical symptoms of severe sepsis and septic shock. Low protein C concentrations at the onset of fever and before the onset of clinical symptoms of severe sepsis or septic shock may have prognostic value in predicting an unfavorable outcome. Protein C measurements may help identify patients at risk in an early phase of neutropenic sepsis. It is also attractive to speculate that because low protein C concentrations were seen in these patients, protein C replacement may be beneficial in sepsis.

Fused bicyclic Gly-Asp beta-turn mimics with potent affinity for GPIIb-IIIa. Exploration of the arginine isostere.

6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Dibasic benzo[b]thiophene derivatives as a novel class of active site directed thrombin inhibitors: 2. Sidechain optimization and demonstration of in vivo efficacy.

Potent, subnanomolar thrombin inhibitors 4, 5, and 6 are developed through side chain optimization of novel, benzo[b]thiophene-based small organic entities 2 and 3 and through SAR additivity studies of the new structural elements identified. X-ray crystallographic studies of 4b-thrombin complex revealed a hydrophobic and an electrostatic interaction of these new elements with thrombin at the S2 and S3 binding sites. In vitro and in vivo pharmacological studies showed that 4, 5, and 6 are potent anticoagulants in human plasma with demonstrated antithrombotic efficacy in a rat model of thrombosis.

Development of dual-acting agents for thromboxane receptor antagonism and thromboxane synthase inhibition. 3. Synthesis and biological activities of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives and related compounds.

A novel series of oxazolecarboxamide-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acid derivatives was discovered as potent dual-acting agents to block the TXA2 receptor and to inhibit the thromboxane synthase (TRA/TSI). Synthesis, structure-activity relationship (SAR), and in vitro and in vivo pharmacology of this series of compounds are described. Modification of the series revolved around the oxazole moiety to increase the hydrophilicity of the compounds and to correlate the biological activity with lipophilicity of the compounds. The most potent in the series was (E)-7-[4-[4-[[(4-cyclohexylbutyl)amino]carbonyl]-2-oxazolyl] phenyl]-7 -(3-pyridyl)hept-6-enoic acid (14) with Kd = 9.9 +/- 0.4 nM for the thromboxane receptor antagonism and IC50 = 55.0 +/- 17.9 nM for thromboxane synthase inhibition. The compound 14 was a selective TRA/TSI which exhibited desirable characteristics for oral activity, "shunt" effect to elevate PGI2 level, and absence of agonist activity.

Use of conformationally restricted benzamidines as arginine surrogates in the design of platelet GPIIb-IIIa receptor antagonists.

The use of 5,6-bicyclic amidines as arginine surrogates in the design of a novel class of potent platelet glycoprotein IIb-IIIa receptor (GPIIb-IIIa) antagonists is described. The additional conformational restriction offered by the bicyclic nucleus results in 20-400-fold increases in potency compared to the freely flexible, acyclic benzamidine counterpart. The design, synthesis, structure-activity relationships (SAR), and in vitro activity of this novel class of GPIIb-IIIa antagonists are presented.

Non-peptide RGD surrogates which mimic a Gly-Asp beta-turn: potent antagonists of platelet glycoprotein IIb-IIIa.

Cyclic heptapeptide 1, which contains an Arg-Gly-Asp sequence, has good affinity for the platelet receptor GPIIb-IIIa and was chosen for study by 1H NMR techniques. The key RGD sequence of this molecule was found to reside in a conformationally defined type II' Gly-Asp beta-turn, and this information was used in the design of simple non-peptide RGD mimics. Disubstituted isoquinolones, bearing an acidic side chain at position 2 and a basic side chain at position 6, were prepared and were found to have modest affinity for GPIIb-IIIa. Systematic modification of the basic residue contained in these molecules yielded compounds with high affinity for GPIIb-IIIa.

Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2.

The thrombin receptor agonist peptide SFLLRN was less effective than thrombin in eliciting the liberation of arachidonic acid and the generation of thromboxane A2 by human platelets. We found that while SFLLRN evokes an initial transient increase in cystolic free calcium concentration ([Ca2+]i) of similar magnitude as that caused by thrombin, the SFLLRN-induced elevation of [Ca2+]i declines more rapidly to near resting levels than that evoked by thrombin, suggesting that disparate levels of [Ca2+]i may contribute to the attenuated arachidonic acid release. Furthermore, we observed that SFLLRN is less effective than thrombin in mediating the "activating" phosphorylation of cytolic phospholipase A2 (cPLA2). Both thrombin and SFLLRN rapidly and transiently activated kinases that phosphorylate the 21-residue synthetic peptide Thr669 derived from the epidermal growth factor receptor, but the maximal activation of proline-directed kinases by SFLLRN was less pronounced than that by thrombin. MonoQ chromatography and immunoblot analysis of extracts from stimulated platelets revealed that while thrombin induced a prominent activation of the mitogen-activated protein kinases ERK1 and ERK2, SFLLRN completely failed to do so. On the other hand, SFLLRN, like thrombin, stimulated the activity of a proline-directed kinase distinct from ERK1/2, but the activation of this kinase was less pronounced following stimulation of platelets with SFLLRN compared with thrombin. We conclude 1) that the partial activation of cPLA2 and the subsequent attenuated mobilization of arachidonic acid in response to SFLLRN may be the consequence of a less prolonged elevation of [Ca2+]i and insufficient activation of proline-directed kinase(s) by SFLLRN and 2) that the ability of SFLLRN to mediate the activating phosphorylation of cPLA2 in the absence of ERK1/2 stimulation suggest that, at least in human platelets, proline-directed kinases other than ERK1/2 may phosphorylate and activate cPLA2.

Development of dual-acting agents for thromboxane receptor antagonism and thromboxane synthase inhibition--I. Synthesis, structure-activity relationship, and evaluation of substituted omega-phenyl-omega-(3-pyridyl)alkenoic acids.

A series of arylsulfonamido-substituted omega-phenyl-omega-(3-pyridyl)alkenoic acids were synthesized and evaluated in vitro for their ability to act as both a thromboxane A2 receptor antagonist (TRA) and thromboxane synthase inhibitor (TSI). Variations of alkenoic acid chain length, olefin geometry, substituent effect on the benzenesulfonamido group, and conformational flexibility of the substituted arylsulfonamido group were examined. Among the various substituents, iodo-substitution gave the most potent compound. Conformational flexibility between the arylsulfonamido group and the phenyl ring attached to the alkenoic acid side chain significantly enhanced the dual activities. The compound (E)-21c was identified as the most potent TRA/TSI (TRA: Kd = 53 nM; TSI: IC50 = 23 nM) in the series studied. The compounds 9c and 10c have indicated that these series of compounds are orally active and are specific TSIs as exhibited by the so-called 'shunt' effect on prostacyclin synthesis in vitro.

Biochemical and pharmacological activity of arene-fused prostacyclin analogues on human platelets.

Human platelets have been employed as an assay system to evaluate the pharmacological activity of a group of stable, arene-fused prostacyclin analogs. Prostacyclin (PGI2) is a highly active member of the eicosanoid family and is relatively unstable under physiological conditions. Prostacyclin's best characterized activities are those of inhibition of platelet aggregation and relaxation of vascular smooth muscle. These activities are mediated in large part via elevation of intracellular levels of cyclic AMP subsequent to receptor occupation and activation of adenylate cyclase. We previously described the synthesis of a series of arene-fused prostacyclin analogs with stability in aqueous media at physiological pH. Several of these compounds have prostacyclin-like activities, i.e., competitive binding at the platelet prostacyclin receptor, elevation of intraplatelet cyclic AMP levels and inhibition of human platelet aggregation. One compound in particular (11a) demonstrated these activities with potency similar to PGI2, i.e., Kd at platelet receptor of 3.7 nM and IC50 for inhibition of collagen-induced human platelet aggregation in plasma of 2.9 nM.

Correlation of molecular shape with GPIIb-IIIa receptor antagonist activity in RGD peptides.

Arg-Gly-Asp-Ser (RGDS) 1, Arg-Val-Asp-Ser (RVDS) 2, Arg-dVal-Asp-Ser (R[d]VDS) 3, and a cyclic RGD peptide, cyclo S,R [H-Pen-Arg-Gly-Asp-Pen-Gly-OH] 4, were tested for their ability to antagonize GPIIb-IIIa function. The activities were found to fall in the order 4 >> 1 >> 3 > 2. Simulated annealing and molecular dynamics studies were carried out to estimate the most populated conformations of each molecule. The acyclic molecules 1-3 were found to populate a much wider range of conformations than the cyclic molecule 4. The backbones of all four molecules were found to approximate beta-turn structures in the most populated conformations. In 4 the beta-turn intramolecular hydrogen bond between C = O of the i residue (Arg) and NH of the i + 3 residue (Ser) did not appear to be present. The distance between the beta-carbons of the critical Arg and Asp groups was found to be shorter in 4 (average 7.98 A) than in the less active acyclic molecules (averages of 8.65-9.33 A).

In vitro characterization of a novel TXA2/PGH2 receptor ligand (S-145) in platelets and vascular and airway smooth muscle.

The stereoisomers of S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor ligand, were compared to TXA2/PGH2 receptor antagonists, SQ29548 and BM13505 in guinea pig platelets, aortas and trachea. Equilibrium binding assays in platelets yielded Kd values (nanomolar) for (+)-S-145 (0.57 +/- 0.04), (-)-S-145 (9.2 +/- 1.3), SQ29548 (11.1 +/- 0.70) and BM13505 (118 +/- 16). In aortas, the corresponding Kb values (nanomolar) were (0.014 +/- 0.002), (1.90 +/- 0.31), (16.8 +/- 3.3) and (142 +/- 29), respectively, whereas in trachea, the Kd values (nanomolar) were (0.019 +/- 0.004), (1.12 +/- 0.18), (1.94 +/- 0.30) and (18.99 +/- 2.59), respectively. S-145 stereoisomers elicited platelet shape change stereoselectively that was characterized by EC50 values 8 to 16-fold higher than the EC50 values for these ligands to block aggregation induced by TXA2/PGH2 mimetic, U44069. S-145 (+)- and (-)-isomers stereoselectively induced transient aortic contraction at concentrations 214,000- and 16,000-fold higher, respectively, than the corresponding Kb values in this tissue. S-145-induced platelet shape change and aortic contraction were inhibitable by low concentrations of SQ29548. We postulate that S-145 may elicit partial agonist activity in platelets and aorta via lower affinity for the active than inactive state of the TXA2/PGH2 receptor in those tissues. S-145 had no agonist activity in isolated trachea possibly indicating different TXA2/PGH2 recognition sites in aorta and trachea or a smaller preligand ratio of active to inactive TXA2/PGH2 receptors in trachea than in aorta.

Pharmacological assessment of the antithrombotic activity of the peptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), in a canine model of coronary artery thrombosis.

The antithrombotic activity of the tripeptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), was compared to heparin in a model of canine coronary artery thrombosis. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. Drug administration was started 15 min before initiation of intimal injury. Clotting times and ex vivo platelet aggregation were determined on citrated blood samples. Gingival template bleeding times were determined. Clotting times (thrombin time; activated partial thromboplastin time, APTT; prothrombin time, PT) increased in a dose-dependent manner with both anticoagulants. The two anticoagulants selectively inhibited thrombin-induced platelet aggregation. GYKI-14766 and heparin were found to delay thrombosis significantly when compared to vehicle-treated animals; minimum effective antithrombotic doses were 0.25 mg/kg/h and 80 U/kg + 30 U/kg/h, respectively. GYKI-14766 (0.25 mg/kg/h) had no effect on template bleeding time, APTT or PT. Heparin (80 U/kg + 30 U/kg/h), however, was associated with a 2.5- to 3.0-min increase in template bleeding time, a 1.8-fold and 1.7-fold increase in APTT and PT, respectively. Antithrombotic efficacy was achieved at doses of GYKI-14766 that did not affect APTT, PT or template bleeding time, whereas antithrombotic efficacy observed with heparin was associated with significant increases in APTT, PT and template bleeding time. These data demonstrate that the tripeptide thrombin inhibitor, GYKI-14766, could potentially prove to be a safer and more effective antithrombotic agent than heparin.

LY249933: a cardioselective 1,4-dihydropyridine with positive inotropic activity.

Compound LY249933 and its component diastereomers, (RR) and (SR), were studied for their vascular and cardiac effects in vitro and in vivo. In guinea pig cardiac ventricular membranes, LY249933, (RR), and (SR) potently displaced bound [3H]nitrendipine (Kd values = 2-6 nM). In isolated guinea pig right ventricular strips, LY249933 produced a small but significant increase in contraction, whereas (RR) substantially increased (-log EC50 (M) = 4.6 +/- 0.8) and (SR) decreased contraction (-log EC50 (M) = 4.1 +/- 0.8). In isolated canine cephalic vein, contracted with 80 mM KCl, an increase in contraction was produced by (RR), whereas relaxation was produced by LY249933 (-log EC50 (M) = 5.9 +/- 0.9) and (SR) (-log EC50 (M) = 6.0 +/- 0.7). At 20 mM KCl, (RR) increased, (SR) decreased, but LY249933 did not alter contraction. In anesthetized dogs, LY249933 (200 micrograms/kg/min, i.v.) increased dP/dt60, decreased heart rate, but did not change vascular resistance or rate pressure product. At the same dose, (RR) and (SR) both tended to increase dP/dt60 nonsignificantly, whereas (RR) increased and (SR) decreased vascular resistance. Both (RR) and (SR) tended to decrease heart rate nonsignificantly, whereas (RR) did not change and (SR) decreased rate pressure product. Thus, LY249933 produced potentially beneficial cardiovascular changes resulting from the combined actions of its (RR) and (SR) diastereomers that are postulated to be calcium agonist and antagonist, respectively.

Characterization and pharmacological relevance of high affinity binding sites for [3H]LY186126, a cardiotonic phosphodiesterase inhibitor, in canine cardiac membranes.

[3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd = 4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r = 0.94; p less than 0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic reticulum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoximone, selectively displaced bound [3H]LY186126 caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kd values from displacement curves were highly correlated with IC50 values for inhibition of SR-PDE (r = 0.92; p less than 0.001). In addition, Kd values correlated well with published ED50 values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r = 0.94; p less than 0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic reticulum.

Synthesis of a tritium-labeled indolidan analogue and its use as a radioligand for phosphodiesterase-inhibitor cardiotonic binding sites.

We have radiolabeled a structural analogue of indolidan, a potent phosphodiesterase-inhibitor cardiotonic, to permit biochemical studies regarding the interaction of this class of drugs with their pharmacological receptor. [3H]-LY186126 (1,3-dihydro-3,3-dimethyl-1-[3H3]methyl-5-(1,4,5,6-tetrahydro-4-me thyl-6- oxo-3-pyridazinyl)-2H-indol-2-one; [3H]-3) was selected as a synthetic target because of its potency as a cardiotonic and the ability to readily incorporate three tritia via the indolone N-CH3 substituent. Alkylation of a desmethyl precursor with tritium-labeled iodomethane resulted in [3H]-3 with a radiochemical purity of 98% and a specific activity of 79.2 Ci/mmol. This radioligand binds with high affinity to myocardial membrane vesicles. The binding was saturable, and Kd and Bmax values of 4.1 nM and 383 fmol/mg protein were obtained. A series of indolidan congeners displaced [3H]-3 bound to myocardial vesicles, and Ki values for inhibition of binding were highly correlated with canine inotropic ED50 values, suggesting the specific binding of [3H]-3 to cardiac vesicles is pharmacologically relevant.

Specific binding of [3H]LY186126, an analogue of indolidan (LY195115), to cardiac membranes enriched in sarcoplasmic reticulum vesicles.

LY186126 was found to be a potent inhibitor of type IV cyclic AMP phosphodiesterase located in the sarcoplasmic reticulum of canine cardiac muscle. This compound, a close structural analogue of indolidan (LY195115), was prepared in high specific activity, tritiated form to study the positive inotropic receptor(s) for cardiotonic phosphodiesterase inhibitors such as indolidan and milrinone. A high-affinity binding site for [3H]LY186126 was observed (Kd = 4 nM) in purified preparations of canine cardiac sarcoplasmic reticulum vesicles. Binding was proportional to vesicle protein, was inactivated by subjecting membranes to proteolysis or boiling, and was dependent on added Mg2+. Scatchard analysis suggested the presence of a single class of binding sites in the membrane preparation. Indolidan, milrinone, and LY186126 (all at 1 microM) produced essentially complete displacement of bound [3H]LY186126, while nifedipine, propranolol, and prazosin had little or no effect at this concentration. This represents the first reported use of a radioactive analogue to label the inotropic receptor for cardiotonic phosphodiesterase inhibitors. The results suggest that [3H]LY186126 is a useful radioligand for examining the subcellular site(s) responsible for positive inotropic effects of these drugs.

In vitro vascular relaxation by new inotropic agents: relationship to phosphodiesterase inhibition and cyclic nucleotides.

Several novel cardiotonic vasodilators including bipyridines (amrinone and milrinone), imidazolones (enoximone and piroximone), dihydropyridazinones (Cl-914, Cl-930 and LY195115) and an imidazopyridine (isomazole) relaxed rat aortic strips contracted previously with 30 microM serotonin. LY195115 and Cl-930 were the most potent vasorelaxant agonists (ED50 approximately 10(-7) M), whereas piroximone and amrinone were the least potent (ED50 approximately 10(-5) M). In addition to these positive inotropic agents, vascular relaxation was examined further for a series of novel dihydropyridazinones, and relaxant potencies correlated directly with the ability of these agents to inhibit an isozyme of cyclic nucleotide phosphodiesterase (PDE) located in the sarcoplasmic reticulum of cardiac muscle (SR-PDE) (r = 0.87, P less than .01). This excellent correlation suggests that vascular relaxation produced by these agents is related to their ability to inhibit a vascular enzyme similar or identical to SR-PDE. Furthermore, LY195115, milrinone and isomazole (10(-4) M) produced significant increases in both aortic cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Time courses for these changes were consistent with a role for cyclic nucleotides in relaxation; however, differences between the relative increases in cAMP or cGMP produced by these drugs were evident. Removal of the aortic endothelium had no effect upon relaxation produced by milrione and only a modest (approximately 2-fold decrease in potency) effect on relaxation produced by LY195115 and isomazole, indicating that the relaxant effect of these cardiotonics is primarily an endothelium-independent event.(ABSTRACT TRUNCATED AT 250 WORDS)

LY195115: a potent, selective inhibitor of cyclic nucleotide phosphodiesterase located in the sarcoplasmic reticulum.

LY195115 selectively inhibited the peak III isozyme of cardiac cyclic nucleotide phosphodiesterase (PDE) eluted from DEAE-cellulose columns. Inhibition curves were biphasic, suggesting heterogeneity within this preparation. Since peak III PDE is reported to be derived from membranes, effects of LY195115 upon PDE associated with cardiac membranes were examined. LY195115-sensitive PDE measured in the various membrane fractions correlated well with the sarcoplasmic reticulum marker Ca2+-ATPase (r = 0.94; p less than 0.001), but not with Na+,K+-ATPase or azide-sensitive ATPase. Membrane disruption failed to reveal latent LY195115-sensitive PDE in sarcolemmal vesicles known to be primarily right side out. The results suggest that LY195115-sensitive PDE is located within sarcoplasmic reticulum membranes with a distribution similar or identical to that of Ca2+-ATPase. Accordingly, LY195115-sensitive PDE was referred to as SR-PDE. A subfraction of sarcoplasmic reticulum vesicles (free SR vesicles) was sufficiently homogeneous with respect to SR-PDE activity to carry out steady state kinetic studies. Double reciprocal plots of cAMP hydrolysis were linear, yielding Km and Vmax values of 0.46 +/- 0.03 microM and 700 +/- 90 pmol/min/mg of vesicle protein, respectively. LY195115 was a linear competitive inhibitor of SR-PDE with a Ki of 80 +/- 10 nM. -LogIC50 values for inhibition of SR-PDE by a series of structural analogues of LY195115 correlated highly with published -logED50 values for stimulation of cardiac contractility in vivo (r = 0.91, p less than 0.001). Consequently, in vivo effects of LY195115 upon the heart appear to result primarily from competitive inhibition of SR-PDE, or from binding to a site with a topography similar or identical to that of the catalytic site of SR-PDE.