Evaluation of anti-activated protein C antibody development in patients with severe sepsis from four clinical studies with drotrecogin alpha (activated).

BACKGROUND: Drotrecogin alpha (activated) (DAA) is a recombinant human activated protein C (APC), which is an antithrombotic protein. OBJECTIVES: To evaluate the development of anti-APC antibodies in severe sepsis patients in DAA clinical studies. PATIENTS AND METHODS: Serum and plasma samples were collected in placebo-controlled studies (PROWESS, ADDRESS) and studies where all patients were DAA-treated (ENHANCE, XPRESS). An enzyme-linked immunosorbent assay detecting anti-APC IgA/IgG/IgM antibodies was used. IgG isolated from plasma of positive samples was tested for neutralizing activity against DAA-induced prolongation of activated partial thromboplastin time. RESULTS: The proportions of patients with negative baseline but positive postbaseline anti-APC antibodies were 1.5% (27/1855) and 1.6% (24/1493) in the DAA and placebo cohorts, respectively (P = 0.72 for the difference). Of the 27 DAA and 24 placebo patients with positive anti-APC antibodies, all but one (DAA) were alive at day 28, and all but seven (four DAA and three placebo) were alive at hospital discharge, including eight (five DAA and three placebo) patients who tested positive for anti-APC neutralizing antibodies. Two of the 51 patients who tested positive for the development of anti-APC antibodies experienced a thrombotic event (one DAA, one placebo). In ADDRESS, no anti-APC antibody was detected in the six DAA-treated patients who had received a previous course of DAA therapy. CONCLUSIONS: The proportion of patients with anti-APC antibodies was low and was similar between DAA-treated and placebo-treated patients. No relationship between anti-APC antibody development and adverse reactions was observed. There was no evidence that the anti-APC antibodies detected represented a specific immune response to DAA therapy.

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.

Fused bicyclic Gly-Asp beta-turn mimics with specific affinity for GPIIb-IIIa.

Disubstituted isoquinolones 2 and 3 have affinity for GPIIb-IIIa and represent leads for further structural evaluation. Structure-activity studies centered on the bicyclic beta-turn mimic contained in these molecules indicated that this moiety could accommodate a variety of modifications. Specifically, monocyclic, 6, 5-bicyclic, and 6,7-bicyclic structures provide compounds with affinity for GPIIb-IIIa. Within the 6,6-series, isoquinoline, tetralin, tetralone, and benzopyran nuclei yield potent antagonists that are specific for GPIIb-IIIa. Attachment of the arginine isostere (benzamidine) to the supporting nucleus can be accomplished with an ether or amide linkage, although the latter enhances activity. Several compounds in this series provided measurable blood levels after oral dosing. Conversion of the acid moiety in these molecules to an ester generally provided compounds which gave greater systemic exposure after oral administration. Absolute bioavailabilities in the rat for the ethyl ester prodrug derivatives of the tetralin, tetralone, and benzopyran analogues of 3 were 28%, 23%, and 24%, respectively.

Quantitative detection of platelet GPIIb-IIIa receptor antagonist activity using a flow cytometric method.

Platelet-membrane surface receptors are important targets for pharmacologic intervention in cardiovascular disease. Among these, glycoprotein (GP) IIb-IIIa is dominant and integrally involved in platelet aggregation and thrombus formation. When activated, GPIIb-IIIa binds soluble fibrinogen (Fb) in a key, early step of this process. New drugs are under development that block Fb binding to GPIIb-IIIa and inhibit platelet aggregation. A thorough understanding of the relationship between circulating drug levels and the extent of GPIIb-IIIa receptor occupancy in humans is crucial for safe and efficacious use of these agents. Described here is the development of a new technique for measurement of GPIIb-IIIa receptor occupancy. In this assay, activated human platelets are incubated with biotinylated fibrinogen (Fb-biotin) followed by antibiotin-FITC.The extent of Fb binding is determined using flow cytometric analysis. Our results indicate that Fb-biotin binds rapidly to activated platelets and its detection is dependent on incubation temperature. Platelets that were pre-incubated with the GPIIb-IIIa antagonist echistatin were inhibited from binding Fb-biotin in a concentration-dependent manner. The fluorescence of processed samples was stable for two weeks in the cold. The assay described here is simple, cost effective, and can be adapted for use in clinical evaluations of these new drugs.

Human group II 14 kDa phospholipase A2 activates human platelets.

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

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.

p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.

The Ca2+-sensitive 85-kDa cytosolic phospholipase A2 (cPLA2) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA2, thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA2 is not required for its receptor-mediated activation.

Phenothiazines as lipid peroxidation inhibitors and cytoprotective agents.

A series of phenothiazines was synthesized and evaluated as in vitro inhibitors of iron-dependent lipid peroxidation. The MIC (minimum tested concentration that gave greater than or equal to 50% inhibition) for 2-(10H-phenothiazin-2-yloxy)-N,N-dimethylethanolamine methanesulfonate (6) was 0.26 microM. Whereas methyl substitution at N-10 diminished activity nearly 100-fold, other structural modifications such as varying the amine group, the distance separating the amine substituent from the phenothiazine nucleus, and the linking group had little effect. Compound 6 was more effective than probucol, a known antioxidant, in blocking Cu2+ catalyzed oxidation of low-density lipoprotein (LDL) as measured by competitive scavenger receptor mediated degradation of 125I-labeled acetyl-LDL by mouse peritoneal macrophage cells in vitro. At a concentration of 5 microM, compound 6 also protected primary cultures of rat hippocampal neurons exposed to hydrogen peroxide (50 microM) when assessed 18 h later by fluorescein diacetate and propidium iodide uptake.