Biomedical subjects
Publications and source records attributed to P D Winocour.
Peptidomimetic inhibitors of thrombin lacking the important Ser195-carbonyl interaction have been prepared. The binding energy lost after the removal of the activated carbonyl was recaptured through a series of modifications of the P1 residues of the bicyclic lactam inhibitors. Selected substituted compounds displayed useful pharmacological profiles both in vitro and in vivo.
Bicyclic piperazinone based thrombin inhibitors of general structure 2 were prepared and evaluated in vitro and in vivo. These inhibitors, having in common an electrophilic basic trans-cyclohexylamine P1 residue, displayed high thrombin affinity, high selectivity against trypsin and good in vivo efficacy in the rat arterial thrombosis model.
We have developed potent and selective thrombin inhibitors with a novel non-peptidic structure. A bicyclic lactam was used as the scaffold on which various P1 and P3 motifs were substituted. Herein, we report the in vitro and in vivo properties of four representatives of this novel class of inhibitors. Their Ki values were less than 10 nM, they inhibited equally both free and clot-bound thrombin, and they displayed high level of specificity for thrombin over other serine proteases (trypsin, factor Xa, activated Protein C, and plasmin). They prolonged the clotting time of human plasma to twice the control value in coagulation assays (TT, APTT, and PT) at a concentration below 3 microM. Their anticoagulant activities using rat plasma were similar to, although slightly weaker, than with human plasma. Furthermore, they inhibited thrombin-induced platelet aggregation (human and rat) at concentrations close to their Ki values for thrombin. These molecules demonstrated similar dose response antithrombotic efficacy in rat arterial and venous thrombosis models when given as i.v. bolus followed by infusion. Antithrombotic efficacy of 85% and greater was observed at a dose of 5-7 microM/kg/hour in each model. Bicyclic lactam inhibitor 3, at a dose which caused a complete inhibition of visible thrombus formation in the venous and arterial models of thrombosis, showed a 1.9-2.1 and a 4.0-4.8-fold shift in APTT and TT, respectively. Unfortunately, the bicyclic lactam inhibitors exhibited low oral bioavailability in rats. Therefore, this novel class of bicyclic lactam thrombin inhibitor has the potential to be promising intravenous antithrombotic agents for the treatment of arterial as well as venous thrombosis and warrants further investigation.
We investigated the effect on in vitro platelet function of hirutonin, a modified hirutonin with an RGD-like motif, a pseudo-RGDS peptide and a linear RGDS peptide. Inhibition of expression of surface fibrinogen on ADP-activated platelets with 40 microM of the peptide was as follows: hirutonin 10+/-3%, modified chimeric peptide 26+/-5%, pseudo-RGDS 66+/-11% and linear RGDS 93+/-13%. Both hirutonin and the chimeric peptide significantly inhibited ADP-induced platelet activation as detected by CD62 expression. Unlike the RGDS and pseudo-RGDS controls, neither the chimeric peptide nor the parent hirutonin inhibited ADP-induced platelet aggregation even at 140 microM. The chimeric hirutonin peptide reduced ATP release from ADP-stimulated platelets by 40+/-4%. This inhibition was stronger than that caused by hirutonin (23+/-13%), but less than the RGDS (90+/-2%) and pseudo RGDS-peptides (59+/-11%). Primary platelet haemostasis was slightly but not significantly affected by the peptide at 40 and 80 microM. However, shear-induced platelet adhesion to vWF and especially subsequent aggregate formation was interrupted after the addition of the chimeric peptide. Similar results were obtained with hirutonin. This inhibition was not as marked as with the RGDS- and pseudo-RGDS peptides. Both the parent hirutonin and the chimeric peptide caused prolongation of the clinical coagulation assays aPTT and TT. In conclusion, the chimeric hirutonin peptide with introduction of the RGD motif retained its anticoagulant effect but had little formal disintegrin activity. Instead, it appeared to have novel anti-platelet effects that may be of therapeutic use.
Utilizing X-ray crystallography and molecular modeling, highly potent and selective peptidomimetic thrombin inhibitors have been designed containing a rigid piperazinedione template. The synthesis and biological activity of these compounds will be described.
Potent and selective thrombin inhibitors have been prepared with a piperazinedione template and L-amino acids. Likewise, incorporation of D-amino acids led to potent inhibitors with a novel mode of binding. Herein, the structure activity relationships and structural aspects of these compounds will be described.
We have developed novel synthetic peptides that display both antithrombin and disintegrin activity. These peptides were derived from hirutonins, a class of potent proteolytically resistant thrombin inhibitors, in which a dipeptidyl sequence, Asp-Phe or Asp-Ser, was introduced after the proteolytically resistant ketomethylene arginyl glycine isostere. These modified hirutonins inhibited the amidolytic activity of alpha-thrombin (Ki approximately 35 nM), prevented fibrinogen clotting (dTT approximately 100 nM) and inhibited human platelet aggregation and 5-hydroxytryptamine secretion induced by alpha-thrombin (IC50 approximately 600 nM). Unlike their parent hirutonins, they inhibited SFLLR-NH2-induced human platelet aggregation (IC50 approximately 45 microM) without inhibition of 5-HT secretion. These peptides also competed for fibrinogen binding to purified GpIIbIIIa integrin (IC50 approximately microM) and prevented attachment of B16-F10 mouse melanoma cells to vitronectin. We conclude that addition of the dipeptidyl sequence, Asp-Phe or Asp-Ser, in hirutonin molecules confers disintegrin activity. However, this activity was not superior to the activity observed with the linear RGDS peptide and was achieved at the expense of direct antithrombin activity. Additional modifications around the RGD-like adhesion sequence may permit identification of the appropriate conformation for optimal binding to thrombin and to specific integrin receptors.
Deep venous thrombosis (DVT) is a common cardiovascular disease, resulting in significant mortality each year in the United States. Direct thrombin inhibitors represent a new class of drugs that could potentially be better than conventional antithrombotic therapy based on indirect inhibition of coagulation factors with heparin and warfarin. BCH 2763 is a potent, selective bifunctional thrombin inhibitor that blocks both the active catalytic site and the anion binding exosite. The objective of this study is to test the antithrombotic efficacy of BCH 2763 in a canine model of DVT induced through electrolytic injury to the femoral vein. BCH 2763 was administered at three dose levels: 0.125 mg/kg bolus followed by 10 microg/kg/min IV infusion (low-dose; n = 5), 0.25 mg/kg bolus followed by 20 microg/kg/min infusion (mid-dose; n = 5), and 0.75 mg/kg bolus followed by 60 microg/kg/min (high-dose; n = 5). The control group (n = 5) received a 5-ml intravenous bolus of saline followed by a 1 mL/kg/h infusion. The parameters evaluated were changes in activated partial thromboplastin time (aPTT), thrombin time (TT), prothrombin time (PT), time to formation of an occlusive thrombus in the femoral vein, and the amount of venous blood flow delivered over the course of the experiment. There were significant dose-dependent increases in aPTT, TT, and PT in the BCH 2763-treated animals compared with the control group. The time to formation of an occlusive thrombus in the control group averaged 69.6 +/- 9 minutes. Treatment with BCH 2763 prolonged the time to occlusion to 126.4 +/- 13 minutes in the low-dose group, 155.4 +/- 17 minutes in the mid-dose group, and 229 +/- 7 minutes in the high-dose group (80% remained patent for the duration of the study), which were all significantly greater than the controls. Femoral venous blood flow was significantly greater in the mid-dose (51 +/- 8%) and the high-dose (70 +/- 6%) groups compared with the control vessels (22 +/- 3%). In conclusion, the results of this study indicate that BCH 2763 is an effective intravenous antithrombotic agent in the canine electrolytic injury model of venous thrombosis.
The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors is described. We have explored the SAR with modifications to the P1 site. The introduction of arginine mimetics at the P1 site led to potent and selective thrombin inhibitors.
Peptidomimetic inhibitors of general structure 1 have been prepared. Optimization of the binding affinities of these compounds through variation of the P3 hydrophobic residue is described. Selected substituted bicylic lactams displayed interesting pharmacological profiles both in vitro and in vivo.
Current clinical use of heparin as an antithrombotic agent is limited by suboptimal efficacy and safety considerations. Thrombin's central role in thrombosis makes it an attractive target to develop more effective and safer antithrombotic agents. BCH-2763 is a novel, potent (Ki: 0.11 nM), low molecular weight (1.51 kDa), bivalent direct thrombin inhibitor. The antithrombotic efficacy of BCH-2763 in vivo following i.v. bolus plus infusion in rats was compared in arterial and venous thrombosis models with two other bivalent direct thrombin inhibitors, r-hirudin and hirulog, with two catalytic site-directed thrombin inhibitors, inogatran and argatroban, and with heparin. In vivo efficacy was related to inhibition in vitro of fibrin clot formation, thrombin-induced aggregation of rat or human washed platelets and activity of free and plasma clot-bound thrombin. All the direct thrombin inhibitors were effective on both arterial and venous thrombosis at markedly lower fold aPTT increases than heparin. The antithrombotic doses of all inhibitors against venous thrombosis were less than against arterial thrombosis. The rank order of potency based on doses (mg/kg/h) required for full efficacy against arterial thrombosis was BCH-2763 (1.2) > inogatran (1.5) > r-hirudin (1.8) > hirulog (3.3) > argatroban (> 3.0); heparin required a markedly higher dose (5.7). In venous thrombosis the doses required for full efficacy were substantially lower for the bivalent (BCH-2763: 0.12; r-hirudin: 0.12; hirulog: 0.18) than for the catalytic site-directed (inogatran: 0.48; argatroban: 0.90) thrombin inhibitors; the dose required for heparin was 0.19. All the direct thrombin inhibitors caused similar shifts in aPTT at doses required to inhibit arterial thrombosis, but BCH-2763 inhibited venous thrombosis at lower aPTT fold increases. In vivo antithrombotic efficacy of direct thrombin inhibitors correlated with their inhibitory activity in vitro against fibrin clot formation and platelet aggregation. In contrast to heparin, all the direct thrombin inhibitors inhibited plasma clot-bound thrombin, but the relative IC50s did not correlate with their antithrombotic efficacy. In summary, direct thrombin inhibitors are more effective than heparin in inhibiting arterial and venous thrombosis in rats with less aPTT increases. BCH-2763 is effective at lower doses than the other direct thrombin inhibitors and for venous thrombosis at a smaller aPTT increase. BCH-2763 may offer an improved therapeutic index in the treatment of thromboembolic complications over heparin and other direct thrombin inhibitors.
Current therapeutic use of heparin as an adjunct to thrombolytic therapy for myocardial infarction is suboptimal with respect to efficacy and bleeding risk. In a rat carotid arterial thrombolysis model (FeCl3-induced injury) we evaluated the combined effect of tPA (2.0 mg/kg/30 min) with our potent injectable direct thrombin inhibitor, BCH-2763 (Ki 0.11 nM; MW 1.5 kDa), which, unlike heparin, inhibits bound and free thrombin; comparisons were with standard heparin (SH), other direct thrombin inhibitors, r-hirudin (MW 6.5 kDa) and hirulog (MW 2.3 kDa), or tPA alone. Time to lysis (TL), patency time (PT), aPTT (fold increase) and bleeding time (BT) were determined. ED100 (100% of rats reperfused) for BCH-2763, hirulog or r-hirudin was 1, 3 or 2 mg/kg/60 min, respectively; 67% of rats reperfused with SH at the highest dose tested (220 U/kg/60 min) and 43% with tPA alone. At these doses, TL (min) was shorter (p < 0.01) with BCH-2763 (0.5 +/- 0.1), hirulog (3.3 +/- 2.3) or r-hirudin (2.3 +/- 1.0) than SH (66.3 +/- 30.8) or tPA alone (93.4 +/- 21.4). The aPTT fold increase after 15 min infusion was markedly greater (p < 0.001) for SH (32.0 +/- 0.8) than BCH-2763 (3.7 +/- 0.5), hirulog (5.2 +/- 0.3) or r-hirudin (4.5 +/- 0.8) in combination with tPA or tPA alone (1.1 +/- 0.1). In addition, the BT (min) for BCH-2763 (3.0 +/- 0.4) was similar to tPA alone (1.6 +/- 0.3), but prolonged (p < 0.05) for hirulog (7.5 +/- 2.7), r-hirudin (6.6 +/- 0.8) or SH (7.3 +/- 1.8). Comparisons at same aPTT fold increase revealed that in combination with tPA, BCH-2763 required a lower anticoagulant level to shorten the TL and prolong the PT than hirulog, r-hirudin or SH. Thus, in this rat arterial thrombolysis model direct thrombin inhibitors are more effective than SH as antithrombotic adjuncts to tPA. BCH-2763 is effective at a lower gravimetric dose and more modest aPTT fold increase than hirulog or r-hirudin with less alteration in haemostasis, which may confer an improved safety index.
Previous studies have shown that alloxan-induced diabetes in rabbits effects a slower release of plasma proteins from the liver, a slower synthesis of 35S-glycosaminoglycan in the extracellular matrix of the arterial wall, and a concurrent reduction in the fractional catabolic rates of several plasma proteins. In the present study, the catabolism of two hemostatic proteins, prothrombin and antithrombin, are compared in alloxan-induced diabetic rabbits (of 6 months' duration) and age-matched control rabbits. Differentially radiolabeled prothrombin and antithrombin were injected intravenously, and arterial blood was sampled over a 7-day period to measure the clearance from plasma. A three-compartment model was used to determine the fractional catabolic rate and compartmental distribution of the two proteins. As observed for other plasma proteins, the whole-body fractional catabolic rates (jt) for prothrombin and antithrombin were significantly less in diabetic rabbits (prothrombin, 0.33 d-1; antithrombin, 0.27 d-1) than in control rabbits (prothrombin, 0.37 d-1; antithrombin, 0.30 d-1; P < .001 and P < .005, respectively). In absolute terms, the catabolism of antithrombin and prothrombin in diabetic rabbits was 5.1 and 6.2 mg.kg-1.d-1, respectively, equivalent to a molar ratio for antithrombin to prothrombin of 0.94. For the control rabbits, catabolism accounted for 6.3 mg.kg-1.d-1 of antithrombin and 7.3 mg.kg-1.d-1 of prothrombin, equivalent to a molar ratio of 1.01. The fractional distribution of these proteins was not significantly different within the intravascular and extravascular spaces in diabetic and control rabbits. The decreased catabolic rates observed for prothrombin and antithrombin in diabetic rabbits conform with results obtained previously for other plasma proteins, and probably reflect a generally decreased rate of plasma protein production by diabetic rabbit liver compared with control liver.
Proteoglycans and glycosaminoglycans in the aortic intima of diabetic rabbits and age-matched controls were examined at 2 weeks and 3, 6, and 12 months after alloxan (or saline) treatment. Measurements were made by morphometric analysis of ruthenium red-stained large proteoglycan granules (LPG) in electron micrographs and by analysis of 35S-labeled glycosaminoglycans, extracted and purified from the intima-media of aortas of rabbits which had been injected with 35S-sulfate 18 hr before exsanguination. There was a progressive increase in the area of the aortic intima with time which was greater in diabetic than in control rabbits. The concentration of proteoglycan (LPG/microns 2) and the concentration of the 35S-glycosaminoglycans in diabetic intima-media were similar to respective values of control intima-media throughout the 12 months. However, the specific radioactivity of the [35S]glycosaminoglycan pool from intima-media of diabetic rabbits was significantly less than that from controls (P < 0.001) at 6 and 12 months. In addition, the staining intensity of LPG of the diabetic compared to control extracellular matrix was decreased at these times. The profile and electrophoretic mobility of the glycosaminoglycan types were similar in diabetic and control intima-media. We conclude that the onset of diabetes in the rabbit has altered the metabolic turnover but not the concentration, sulfate content or profile of aortic intima-media proteoglycan and glycosaminoglycans.
Diabetic patients are at greater risk for restenosis following angioplasty, but the mechanisms involved remain unclear. Early proliferative changes in vessels following angioplasty contribute to the subsequent intimal thickening associated with restenosis. Materials released from platelets which interact with the injured vessels and thrombin generated on the injured vessels could be contributory factors. Clinical restenosis occurs following injury to atherosclerotic vessels and, therefore, more relevant animal models of this clinical problem are those involving injury to already diseased vessels. In the present study the effect of spontaneous diabetes in BB Wistar rats was examined on early smooth muscle cell proliferation, as detected by an antibody to PCNA, in the media and intima after re-injury with a balloon catheter to the aorta similarly injured 3 weeks earlier. In studies in which aortae were re-injured 2 weeks after the first injury 51Cr-labeled platelet accumulation was determined and the vessels were examined morphologically for evidence of fibrin, platelet aggregate, or thrombus formation. The number of PCNA-positive cells (P < 0.01) and the percentage of total cells (P < 0.01) in the aortic media of diabetic rats were greater 1 day after re-injury compared to the 2-hr value; after 3 or 7 days this increase had returned to the 2-hr value. No increase occurred in the control rats. The mean percentage of PCNA-positive cells in the aortic intima in diabetic rats was greater 1 (P < 0.025), 3 (P < 0.05), or 7 (P < 0.01) days after re-injury, compared with the 2-hr value. In contrast, a nonsignificant increase occurred later in control rats. Morphological examination of the aortic surface in control and diabetic rats 30 min after re-injury showed only a monolayer of platelets with no evidence of fibrin or thrombus formation. 51Cr-Labeled platelet accumulation and turnover on the re-injured aortic surface were similar in the control and diabetic rats. Therefore, spontaneous diabetes in rats is associated with small increases in the early proliferative response of smooth muscle cells in the aorta to balloon catheter-induced re-injury, which did not result from increased platelet accumulation or thrombosis on the vessel surface. This altered response of vessels to re-injury due to the diabetic state in rats could be a contributory factor to the greater risk for restenosis seen in diabetic patients following clinical angioplasty.
The metabolism of plasminogen glycoforms I and II was measured in alloxan-induced diabetic and in age-matched control rabbits. Radiolabeled plasminogen I and II were degraded significantly more slowly in diabetic compared with control rabbits; plasminogen II [half-time (T1/2), 1.31 days] was degraded faster than plasminogen I (T1/2), 1.86 days) in diabetic rabbits and in control rabbits (T1/2, 1.18 and 1.58 days, respectively). From the catabolic rates and relative quantities in plasma, we calculated that approximately four molecules of plasminogen II were degraded for one molecule of plasminogen I in the diabetic and control rabbits. To verify this later observation, plasminogen I and II production by diabetic rabbit livers was compared with that by the control livers in vitro. During perfusion with [3H]leucine, 3H-labeled protein was released more slowly from diabetic than from control livers, but no quantitative difference in total plasminogen yield between diabetic and control livers was found. Nevertheless, plasminogen II was produced 0.7 +/- 0.4 and 4.3 +/- 0.3 times faster than plasminogen I by diabetic and control livers, respectively. Plasminogen metabolism in the diabetic rabbit did not differ qualitatively from that in the control rabbit except that catabolism was slowed.
There is considerable information to indicate that when there is vessel injury, platelets can play a major role in the development of atherosclerosis and its thromboembolic complications in the non-diabetic population. Diabetes is a disease associated with an increased risk of vascular complications, but the nature of this association is unclear. Platelets from diabetic humans and animals are hypersensitive to agonists in vitro, but it is unclear if this hypersensitivity persists in vivo. Platelets survival and turnover in the circulation have been used as indicators of platelet behaviour in vivo, but platelet survival measurements do not necessarily correlate with platelet sensitivity in vitro and these measurements may be too insensitive to reflect platelet turnover on injured vessels or in thrombi, unless there is extensive vascular disease. Studies in rats and rabbits indicate that diabetes causes increased platelet interaction with experimentally-injured vessels and accumulation in thrombi. It is unclear, however, whether this altered behaviour of platelets at the injured vessel surface reflects changes in the platelets or in the vessel wall and whether it contributes to the increased risk for vascular complications in diabetic patients.
Diabetes is associated with increased risk for atherosclerosis and its thromboembolic complications. Theories about mechanisms of atherosclerosis in diabetes are similar to those in the nondiabetic population. Platelets contribute to atherosclerosis through effects on vessels by materials released from the platelets, which interact with injured or altered vessels. In diabetes, platelets could contribute to enhanced atherosclerosis through hypersensitivity to agonists at sites of vessel injury and increased release of materials from adherent platelets. Diabetic platelets are hypersensitive to agonists in vitro, and alterations in a number of mechanisms involved in platelet activation occur in these platelets, which could contribute to the hypersensitivity. These alterations include increased presence of glycoprotein receptors for agonists and adhesive proteins on the platelet surface, increased fibrinogen binding, decreased membrane fluidity, enhanced arachidonate pathway activation with increased thromboxane A2 formation, and increased phosphoinositide turnover leading to increased inositol trisphosphate production, Ca2+ mobilization, and protein phosphorylation. There is some evidence for increased platelet activity in vivo in diabetes, but it is unclear whether this reflects platelet hypersensitivity or increased platelet turnover on already diseased vessels. Studies in diabetic animals indicate greater interaction of platelets with injured vessels and incorporation into experimentally induced thrombi, but it is unclear if this reflects changes in platelets or other factors. These changes could be contributing to the enhanced atherosclerosis and its clinical complications in diabetic patients.