Inhibition of platelet adhesion and thrombus formation on a collagen-coated surface by novel carbamoylpiperidine antiplatelet agents.

The inhibitory effect of two novel antiplatelet agents, 1,10-bis[3-(N,N-diethylcarbamoyl)piperidino]decane dihydrobromide (G-110) and 1,6-bis[3-(N,N-diethylcarbamoyl)piperidino]hexane dihydrobromide (G-112), on platelet adhesion and subsequent aggregation on collagen-coated surface was evaluated under controlled flow. Glass coverslips coated with bovine fibrillar collagen type I were exposed to heparinized human whole blood that had been preincubated with either aqueous solutions of one of the two carbamoylpiperidine congeners or corresponding amounts (1.0-4.0 microliters/ml blood) of distilled water, at a wall shear rate of 1000 s-1, in a parallel-plate perfusion chamber. Epifluorescence video microscopy with a microphotometric measurement technique was used to visualize and quantify deposition of fluorescently-labeled platelets from flowing whole blood onto the collagen-coated surface. At concentrations of 100 and 200 microM, G-110 inhibited platelet accumulation by 30 +/- 9% (+/- S.E.) and 63 +/- 3% (+/- S.E.), respectively; while G-112 reduced platelet deposition by 19 +/- 3% (+/- S.E.) and 31 +/- 2% (+/- S.E.) at concentrations of 200 and 400 microM, respectively. Digital image processing techniques were used to analyze the dynamics of thrombus growth on the collagen-coated surfaces. It was found that the compounds reduced the rate of thrombus growth by impeding both surface coverage and the number of platelets per thrombus in a concentration-dependent manner. This study, together with others on related compounds GT-12, BPAT-117 and BPAT-143, corroborates the nature of pivotal features in the molecular structure of carbamoylpiperidine and nipecotoylpiperazine derivatives which enhance desirable antithrombotic effects, e.g. intramolecular distance between two tertiary amines (ring nitrogens) and levels of hydrophobicity.

Novel perspectives for structuring synthetic inhibitors of human blood platelet aggregation and potential antithrombotic agents.

An overview is presented on using molecular probes in tracing platelet function response patterns to specific structural features and physicochemical characteristics. Our carbamoylpiperidine nipecotoylpiperazine and related congeners are yielding new insights for as yet uncharted mechanisms of thrombocyte interaction dynamics, and the findings indicate promise for the development of novel potential antithrombotic agents.

Ethanol inhibition of thrombus formation on collagen-coated glass.

Epi-fluorescent video microscopy was used to evaluate the effect of ethanol on platelet mural thrombus formation. Whole blood, treated with ethanol, was perfused over collagen coated glass in a parallel-plate flow chamber at a shear rate of 1,000/s. Digital image processing and photodiode measurements were used to analyze the dynamics of thrombus growth on this surface. Ethanol concentrations as low as 0.02% v/v were found to inhibit 45 +/- 23% (+/- S.D.) of normal platelet accumulation on the slide while 0.2% v/v ethanol effected an 82 +/- 15% inhibition of mural thrombus formation. While platelet adhesion to the collagen surface appeared unaffected by ethanol concentrations up to 0.1% v/v, 0.2% v/v ethanol had an effect on adhesion as well as aggregation. These results imply that low ethanol concentrations inhibit the formation of mural thrombi in a model of a damaged blood vessel at physiological shear rates. This inhibition would not be detected in systems which measure bulk aggregation, e. g. in aggregometric determinations.

Inhibition of mural thrombus formation by novel nipecotoylpiperazine antiplatelet agents.

The effectiveness of two closely related nipecotoylpiperazine derivatives, BPAT-143 and BPAT-117, as antiplatelet agents was measured by their ability to inhibit the accumulation of human blood platelets on collagen-coated (type 1) glass in a parallel plate flow chamber. Whole human blood, with fluorescently labeled platelets, was perfused through the flow chamber, and epi-fluorescent video microscopy was used to visualize the dynamics of individual platelet adhesion and thrombus formation on the collagen-coated surface. Digital image processing was used to analyze the dynamics of thrombus growth on the surfaces. The collagen-coated surface serves as a model for the damaged blood vessel wall, as collagen is a primary component of the matrix beneath endothelial cells. At a concentration of 50 microM, BPAT-117 (the considerably more hydrophobic molecule) inhibited platelet accumulation by striking 90 +/- 2% (+/- S.E.), while it took 2- to 4-fold higher concentrations of BPAT-143 to register meaningful to comparable effects (52 +/- 6% and 80 +/- 4%, respectively). This further corroborates the substantial impact of hydrophobic features within the matrix of appropriately structured molecules on their ability to alter platelet function.

Inhibition of thrombus formation in vivo by novel antiplatelet agent.

The antithrombotic and antihemostatic effects of the novel antiplatelet compound, alpha, alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene dihydrobromide (GT-12), were investigated in a baboon model of platelet-dependent thrombosis under high flow conditions. In this model, segments of collagen-coated tubing and Dacron vascular graft were placed as thrombus-inducing extension devices in exteriorized femoral arteriovenous shunts. The deposition of 111In-labeled platelets was measured for each thrombogenic segment throughout 1 hour by using gamma camera imaging. In addition, the 125I-fibrin retained in the forming thrombus was measured. Intravenous infusion of GT-12 (100 mumol/kg, 63.3 mg/kg) over a 15-minute period before the insertion of the test segments prolonged the bleeding time from a baseline value of 4.4 +/- 0.4 min to 7.6 +/- 1.0 min (p = 0.036) and inhibited platelet aggregation ex vivo induced by adenosine diphosphate (ED50 4.7 +/- 0.9 to 10.3 +/- 2.2 microM; p less than 0.02) and collagen (ED50 2.0 +/- 0.4 to 8.0 +/- 2.4 micrograms/ml; p less than 0.05). Deposition of platelets and fibrin was decreased in concert by 30% (p less than 0.05) for vascular grafts and possibly collagen segments at the end of the 60-minute observation period. We conclude that GT-12 is antithrombotic for Dacron graft-induced thrombus formation in vivo.

The effect of carbamoylpiperidine and nipecotoylpiperazine analogs on ADP-stimulated human thrombocyte aggregation and platelet factor 3 availability.

There is a striking congruence between the inhibitory effects of three synthetic entities on ADP-induced (i) human blood platelet aggregation and (ii) platelet factor 3 availability as evidenced by prolonged 'Stypven time'. The pronounced parallel between each compound's potency in inhibiting aggregation (e.g. IA 48.9 +/- 1.3, S.E., %; n = 16) and in impeding platelet factor 3 availability (e.g. IPF-3av 42.3 +/- 2.5, S.E., %; n = 12), determined concurrently in platelet-rich plasma of four different donors, further substantiates that the antiplatelet activity of our carbamoylpiperidine and nipecotoylpiperazine congeners is exerted through their interaction with anionic phospholipids.

Some novel inhibitors of platelet aggregation: acute toxicity in mice and its relationship to in vitro efficacy and toxicity. II. Nipecotoylaminoalkane and nipecotoylpiperazine congeners.

Four closely related nipecotoyl congeners are employed as molecular probes to evaluate the effects of systematic molecular changes upon lethal potency of the compounds. The in vivo toxicities, effected by changes in molecular structure, are compared to their in vitro concentrations inhibiting ADP-induced aggregation and epinephrine-induced primary aggregation of human blood platelets and their toxicities to mouse fibroblasts (L-929 cells) in culture. To assist in the selection of compounds which offer the greatest promise as therapeutic agents for further evaluation and to guide future development of optimal molecular structures, a ratio of acute ip LD50 (mumol/kg) [Tm] to concentration inhibiting 50% ADP-induced platelet aggregation (mumol/liter) [A] is calculated for each compound. These ratios range from 2.41 to 24.92 for the four compounds included in this study.

Relationships between chemical structure and inhibition of epinephrine-induced human blood platelet aggregation.

The effect of structural features in a series of carbamoylpiperidine and nipecotoylpiperazine congeners on epinephrine-induced aggregation of human blood platelets is examined. Epinephrine-induced primary aggregation is effectively inhibited by the nipecotoylpiperazine derivatives (culminating at an IPA50 of 11.9 microM). While among nipecotoylpiperazine as well as carbamoylpiperidine congeners there are potent inhibitors of ADP-stimulated platelet function (cresting at an IA50 of 12.4 and 11.4 microM, respectively), the carbamoylpiperidine analogs are much less active (e.g., IPA50 of 298.1), or practically inactive, in impeding epinephrine-induced primary aggregation (PA).

Relationships between chemical structure and inhibition of ADP-stimulated human thrombocyte release of serotonin and platelet factor 4.

The inhibitory potencies of carbamoylpiperidinoalkane and N-alkylnipecotoylpiperazine derivatives on ADP-stimulated human blood platelet aggregation, serotonin (5-HT) release and platelet factor 4 (PF-4) release were evaluated. The procedure was designed to allow concurrent determination of all three sets of values. Most compounds were more than twice as potent in blocking PF-4 (X = 91 +/- 1 (S.E., n = 7)%) compared to their inhibition of 5-HT (X = 38 +/- 1(S.E., n = 6)%) release; the one compound which failed to meet these criteria was still decidedly more powerful in impeding PF-4 than 5-HT release. Since the compounds' platelet aggregation-inhibitory values were within the same range as their 5-HT release-blocking potencies, but had a strikingly greater impact in arresting PF-4 release, it is suggested that the platelet plasma membrane and the lining enveloping the dense bodies may share certain commonalities, while the sheathing encasing the alpha-granules may differ from both in a tangible manner.

Effects of a novel antiplatelet agent in mural thrombogenesis on collagen-coated glass.

A parallel plate flow chamber and an epifluorescence video microscopy system were used to investigate the inhibitory effect of a novel antiplatelet agent (GT-12), a carbamoylpiperidine congener, on surface platelet aggregation and on the kinetics of thrombus growth induced by collagen-coated glass under controlled flow. Both macroscopic and microscopic measurements revealed that increasing concentrations of the drug correspondingly decreased the reaction rate between platelets at the surface, thereby reducing thrombus rate of growth at the surface. Because of decreased platelet/platelet adhesion, there was some embolization of the larger thrombi near the inlet of the reactive surface. In the presence of GT-12, average thrombus size and number of platelets per thrombus were both strikingly lowered. In addition, the net rate of growth of individual thrombi decreased to zero after short exposure times (about 60 seconds), in sharp contrast to controls. In contrast to chlorpromazine, GT-12 was effective in inhibiting platelet aggregation and thrombus rate of growth at relatively low concentrations (less than 100 mumol/L) in whole blood. The drug's effectiveness relative to controls in impeding platelet/platelet interactions was found to increase with decreasing incubation time and increasing perfusion time.

Some novel inhibitors of platelet aggregation: acute toxicity in mice and its relationship to in vitro activity and toxicity.

Five carbamoylpiperidine congeners and one nipecotoylpiperazine derivative, which inhibit ADP-induced aggregation of human blood platelets in vitro, were evaluated in vivo for acute toxicity to male and female ICR mice. Although the in vitro platelet aggregation inhibiting potency of these compounds covered about a 1150-fold range, the acute ip LD50s (microM/kg) in mice showed only a fourfold range of values. An increase in platelet aggregation inhibiting potency (in vitro) was not paralleled by an increase in acute toxicity in vivo for these compounds; in fact, the most potent aggregation inhibitor (microM/liter) was the least toxic (microM/kg) to mice. A comparison of acute LD50s (microM/kg) to concentrations which produce 50% inhibition of mouse fibroblast cell growth in culture (microM/liter) did not yield a consistent value, nor was the rank order of toxicity the same from these two tests. In hematoxylin and eosin stained slides of major organs from treated mice, no histopathologic lesions were observed which were attributable to administration of these compounds.

Serotonin efflux-inducing effects on human blood platelets by carbamoylpiperidine and nipecotoylpiperazine aggregation inhibitors.

The serotonin efflux-inducing potency of carbamoylpiperidine and nipecotoylpiperazine aggregation inhibitors was evaluated in human blood platelets. Findings were interpreted in terms of structure-activity relationships, and related to actions exerted by the compounds on other human blood platelet functions. Termination of the active efflux period in the presence and absence of acetylsalicylic acid allowed assessment of the influence of aspirin on the quenching process.

Effects of novel aggregation inhibitors on serotonin uptake by human blood platelets.

Novel aggregation inhibitors blocked serotonin uptake by human blood platelets in concentrations ranging from 0.7 +/- 0.1 microM to 237.5 +/- 35.7 microM; a modified procedure, validated by kinetic analysis, was employed in which pH drift was minimized to 0.03 during the active assay period. Structural features in carbamoylpiperidine and nipecotoylpiperazine derivatives which actually constitute molecular probes, and show remarkable specificity for aggregation-inhibitory target sites, disclosed striking differences between the latter and serotonin receptors or other loci affecting serotonin uptake.

Interrelationships between the chemical structure of synthetic entities, their platelet aggregation inhibitory potency and their cellular toxicity, based on in vitro experiments.

Striking relationships were observed, in vitro, between the molecular constitution of synthetic entities, their aggregation-inhibitory potency (as determined in ADP-induced human blood platelet aggregation), and their cellular toxicity (as assessed by their inhibition of cultured mouse fibroblast L-cell growth). Effects exerted on platelets tended to reflect interactions between the molecules' aggregation-inhibitory specific functions and the platelets' corresponding target sites, while fibroblasts were generally more susceptible to the molecular constitution's hydrophobic character. The hyperbolic relationships between concentrations effecting 50% inhibition and slopes of concentration-response curves reflect net activity from both specific and nonspecific receptor site interactions, with the latter being dominant, and indicated that the assays approximated equilibrium systems. Plotting logarithm values of concentrations effecting 50% inhibition against logarithms of reciprocal slopes for concentration response curves yielded multiple regression coefficients of R = 0.97 (platelet aggregation-inhibitory potency) and R = 0.98 (fibroblast growth-inhibitory potency).

The effect of alpha, alpha'-bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene on human blood platelet structural physiology.

alpha, alpha'-Bis[3-(N,N-diethylcarbamoyl)piperidino]-p-xylene enhances human blood platelet membrane integrity by exerting a stabilizing action at the level of the dense tubular system in surface membrane complexes known to sequester platelet calcium.

Blood platelet function, medicinal agents, and other chemical entities.

An overview is presented on blood platelet function, including the interaction of platelets with the vessel wall and with medicinal agents and related chemical entities. Current interpretations are discussed in terms of the most recent biological, chemical, and physicochemical concepts.

Relationships between the chemical constitution of aggregation inhibitors and human blood platelet response profile.

Gradually altered synthetic entities were employed as molecular probes, and arachidonic acid, ADP, human alpha-thrombin and the Ca2+ ionophore A23187 as aggregation-inducing agents, in a comprehensive study on the response profile of human blood platelets with an emphasis on the effects of exogenous and increased intracellular Ca2+. Corroborating further previous conclusions, some representative carbamoylpiperidine derivatives, at concentrations effecting substantial inhibition of ADP-induced aggregation, failed to retain that effect when 5.0 mM Ca2+ was introduced into the otherwise identical test medium; reference compounds chlorpromazine and propranolol registered corresponding inhibitory patterns. At increased concentrations the compounds' inhibitory potency was regenerated even in the presence of 5 mM Ca2+. In fact, in sufficiently high concentrations, the compounds were even capable of inhibiting aggregation elicited by 15 microM of the ionophore A23187; so did chlorpromazine and propranolol. Another set of congeners revealed the striking sensitivity of ionophore A23187-induced human blood platelet aggregation to the surface active potencies of inhibitor molecules. The loss in inhibitory potency was directly related to the lesser hydrophobic character of the molecule.

The influence of novel platelet aggregation inhibitors on human blood platelet clustering and retention effected by some polymer materials, in vitro.

Human blood platelet clustering and retention in vitro induced by medical grade Cuprophane and Silastic and by non-medical grade polyethylene and Mylar, are discussed. The influence of synthetic compounds on polymer-induced platelet clustering and retention has been evaluated, interpreted in terms of physicochemical parameters, and correlated with ADP- and human alpha-trombin-stimulated aggregation; the Pearson product-moment correlation coefficient for a series of bis (alkylnipecotoylamino) alkanes was computed at r = 0.98 between (i) mylar-induced cluster formation and (ii) ADP-effected aggregation, and at r = 0.94 in corresponding correlations with thrombin as an inducing agent. All experiments were carried out in vitro. Due to the paucity of polymer specimens meeting the stipulated specifications, several evaluations had to be limited to triplicate and duplicate determinations.

Surface activity and human blood platelet aggregation-inhibitory potency.

Surface and interfacial activity is correlated with molecular constitution and inhibitory potency of mono- and bis(carbamoylpiperidino)alkanes and aralkanes, and of some corresponding quaternary pyridinium congeners, in ADP-induced human blood platelet aggregation. The measurements of surface and interfacial tension were carried out at concentrations and pH-values approximating those employed in the hemodynamic study. The effect of changes in chemical structure, ranging from relatively minor variations in a specific functional group to the alteration of major components in molecular constitution, was examined and interpreted in terms of contemporary theoretical chemistry.