Thomas M. Reilly

Selective αvβ3 integrin blockade potently limits neointimal hyperplasia and lumen stenosis following deep coronary arterial stent injury1

Lumen loss from vascular restenosis remains a leading cause of chronic revascularization failure. Objective : We hypothesized that cell-matrix adhesion, migration, and differentiation events that underlie restenosis are mediated by αvβ3 integrin-ligand interactions. Methods : Using immunohistochemistry and in situ hybridization, we examined the spatial and temporal vessel wall expression of αvβ3 and osteopontin following deep coronary arterial injury. Cell migration and adhesion assays were performed to demonstrate the affinity and specificity of XJ735 for various vessel wall integrins. The effects of XJ 735 (a selective cyclic Arg-Gly-Asp (RGD) peptidomimetic αvβ3 antagonist) on neointimal hyperplasia and lumen stenosis were tested in a porcine coronary injury model. Normolipemic swine underwent oversized stent injury followed by XJ 735 administration (9 animals, 28 lesions; 1 mg/kg bolus+7 days 4 mg/kg/d infusion+21 days 2 mg/kg i.v. bolus 12 hourly) or placebo (10 animals, 30 arterial lesions). Results : Maximal αvβ3 immunoreactivity was observed between 7–14 days following injury in the neointima, media, and adventitia. Maximal osteopontin mRNA signal in the neointima, media, and adventitia was observed at 14, 7 and 28 days respectively. IC50 for XJ 735 αvβ3-mediated inhibition of human and porcine endothelial cell adhesion, and vascular smooth muscle cell migration, ranged from 0.6 to 4.4 μM. In contrast, IC50 for porcine or human αIIb/β3, α4β1, αvβ5, and α5β1 inhibition exceeded 100 μM. Steady state XJ 735 plasma levels exceeded 5 μM. Despite slightly higher injury scores in XJ 735 treated animals, significant reductions in mean neointima area (43% reduction; p = 0.0009), and mean percent lumen stenosis (∼2.9 fold reduction; p = 0.04) were observed in XJ 735 treated animals. XJ 735 treatment did not significantly alter the relative size of the arterial injury and reference sites (geometric remodeling). Comparison of neontima area vs. injury score regression lines revealed significant reductions in slope ( p = 0.0001) and intercept ( p = 0.0001) for XJ 735. Conclusions : Selective αvβ3 blockade is an effective anti-restenosis strategy that potently limits neointimal growth and lumen stenosis following deep arterial injury. The co-ordinate spatial and temporal upregulation of αvβ3 expression following vessel wall injury, and the high affinity and specificity of XJ 735 for αvβ3, confirms the importance of this integrin in adhesive and migratory cell-matrix events underlying coronary restenosis.

CONSTRAINED PEPTIDE LIBRARIES AS A TOOL FOR FINDING MIMOTOPES

The monoclonal antibody (mAb) KAA8 recognizes the peptide angiotensin II (AII). The KAA8 mAb was biopanned using two phage-displayed peptide libraries, one unconstrained, the other constrained by a disulfide bond. After several cycles of biopanning, both libraries showed enrichment for phage that bind KAA8. Phage isolated from the unconstrained library contain a consensus sequence that matches the sequence of AII. A consensus sequence was also identified from the constrained library that does not resemble the AII sequence, and represents a mimotope of AII. We have also demonstrated that monovalent phage display can be used to discriminate between modest and high-affinity binding peptides.

Antiplatelet Efficacy and Specificity of DMP728, a Novel Platelet GPIIb/llla Receptor Antagonist

The present study was undertaken to define the platelet GPIIb/IIIa affinity and specificity of DMP728, the cyclic [(D-2-aminobutyrate-N-methyl-L-arginyl-glycyl-L-aspartyl)-3-aminomethyl- benzoic acid] methane sulfonate. DMP728 demonstrated similar potency (IC50 = 0.046 +/- 0.002 microM) in inhibiting human platelet aggregation induced by various agonists or combination of agonists as assessed either by light transmittance aggregometry or impedance techniques. Similarly, DMP728 inhibited (IC50 = 2.3 +/- 0.8 nM) with equipotency in inhibiting 125I-fibrinogen binding to human gel-purified platelets regardless of the agonist used. In purified human GPIIb/IIIa ELISA, DMP728 demonstrated a competitive high affinity binding (Ki = 0.4 nM). Additionally, a high binding affinity (Kd = 0.1 nM) of 3H-DMP728 was demonstrated in human platelets. Furthermore, a platelet deaggregatory efficacy was shown. DMP728 demonstrated a high degree of specificity for platelet GPIIb/IIIa (alpha 2/beta 3) as compared to other integrins on endothelial cells (vitronectin receptors), platelets GPIb/1X, alpha v/beta 3, and other integrins on leukocytes or nonintegrin-related systems. In conclusion, DMP728 is a novel antiplatelet agent with high affinity and specificity for platelet GPIIb/IIIa.

Novel nonpeptide antiplatelet glycoprotein llb/llla receptor antagonist, DMP754: receptor binding affinity and specificity

ObjectiveTo define the antiplatelet efficacy and specificity of the glycoprotein llb/llla complex (GPIIb/llla) antagonist prodrug DMP754 and its free acid form, XV459. Methods and materialsDMP754 has an IC50 > 1 μmol/l, and, upon its conversion with esterases to its free acid form, demonstrated high potency (IC50 20–45 nmol/l) in inhibiting human platelet aggregation induced by 10 μmol/l adenosine diphosphate, 20 μg/ml collagen, 1 mmol/l epinephrine, 10 μmol/l platelet activating factor or 0.5 lU/ml thrombin. The in-vitro rate of hydrolysis of DMP754 or XV459 is much faster with human or canine liver esterases (t1/2 = 2.4–23 min) than with plasma esterases (t1,2 = 5.5–7.6 h). Platelet Gpllb/llla integrin binding affinity and specificity for XV459 were determined using cell binding/adhesion assays. ResultsThe range of IC50 values of XV459 in inhibiting platelet aggregation in platelet-rich plasma obtained from 12 subjects was 0.035–0.069 μmol/l with a mean IC50 of 0.050 ± 0.003 μmol/l. Additionally, XV459 inhibited platelets obtained from mongrel dogs, baboons, sheep, guinea pigs, and mice with IC50 in the range 0.024–0.06 μmol/l, and IC50 in the range 0.16–5.8 μmol/l in pigs, rabbits, and rats. XV459 inhibited [125l]-fibrinogen binding to activated human platelets with an IC50 of 0.011 ± 0.003 μmol/l. XV459 demonstrated a high degree of selectivity in specifically inhibiting fibrinogen binding to the platelet integrin, GPIIb/llla (IC50 = 0.00025 ± 0.00005 μmol/l) compared with inhibiting other integrins (αvβ3, IC50 > 10 μmol/l; or αvβ5, α5β1, or α5β1, for which the IC50 exceeded 100 μmol/l). ConclusionDMP754 is a potent antiplatelet agent in inhibiting platelet aggregation, and has a high specificity and affinity for human platelet GPIIb/llla receptors.

Hypoxia induces differential expression of the integrin receptors ?v?3 and ?v?5 in cultured human endothelial cells

The integrins αvβ3 and αvβ5 have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits β3 and β5. β3 and β5 messenger RNA (mRNA), protein levels, and αvβ3 function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O2) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of αV and β3, peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, β5 mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in αvβ3 protein levels, measured by blotting with LM609, evident by 24 h. αvβ5 protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an αvβ3 mediated process. These results indicate that hypoxia can increase expression of αvβ3 in HUVECs, and that hypoxic regulation of αvβ3 may be an important regulator of angiogenesis. J. Cell. Biochem. 78:674–680, 2000.

Effect of Single Oral Dose of Aspirin on Human Platelet Functions and Plasma Plasminogen Activator Inhibitor-1

Previous reports documented the inhibitory efficacy of different doses of aspirin on arachidonic acid (AA)-induced platelet aggregation, however, the sensitivity of platelets toward other agonists as well as the effects of aspirin on platelet and plasma plasminogen activator inhibitor-1 (PAI-1) release and levels were not investigated. Hence, the present study was undertaken to investigate the effect and duration of action of a single oral dose (650 mg) of aspirin on human platelet functions (n = 34, normal healthy male and female volunteers) including aggregation, fibrinogen binding and PAI-1 release, and on the plasma level of PAI-1. Aspirin demonstrated a rapid onset of action (at 2 h after ingestion) in specifically inhibiting ex vivo AA-mediated functions including (a) fibrinogen binding to gel-purified platelets, (b) platelet aggregation, and (c) platelet PAI-1 release. A peak reduction of plasma PAI-1 level at 2 h was demonstrated as well. The effect of aspirin on the ex vivo AA-mediated effects (a-c) was shown to last for up to 4 days. However, aspirin treatment resulted in a rebound effect in platelet function (a-c) to other platelet agonists such as adenosine diphosphate or the combination of agonists including adenosine diphosphate, epinephrine, and AA. In conclusion, a single oral dose of aspirin has long-acting effects on AA-induced platelet activation and reduces plasma levels of PAI-1 as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic structural and functional relationships in recombinant plasminogen activator inhibitor-1 (rPAI-1)

The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity.

αvβ3 integrin binding affinity and specificity of SM256 in various species

This study was undertaken to define the alphavbeta3 binding affinity and specificity of the low-molecular-weight nonpeptide integrin antagonist, SM256. SM256 demonstrated high potency (IC50, 0.057+/-0.030 nM) in inhibiting vitronectin binding to purified human alphavbeta3 receptors. Additionally, SM256 inhibited alphavbeta3-mediated human umbilical vein endothelial cell (HUVEC) or 293/beta3 (beta3-transfected cell line) adhesion to fibrinogen with IC50 values of 0.0054+/-0.0058 and 0.0023+/-0.0012 microM, respectively. SM256 demonstrated a relatively high degree of specificity for human alphavbeta3-mediated functions as compared with other human integrins including alphavbeta5 (IC50, 0.92+/-0.69 microM), alphaIIbbeta3 (IC50, 0.72+/-0.07 microM), alpha4/beta1 (IC50, >100 microM) and alpha5/beta1 (IC50, 2.3+/-2.1 microM). SM256 demonstrated different degree of species specificity in blocking alphavbeta3-mediated cellular adhesion with relatively higher affinity to dog (IC50, 0.005+/-0.002 microM), rabbit (IC50, 0.021+/-0.01 microM), mouse (IC50, 0.035+/-0.01 microM), and pig (IC50, 0.41+/-0.24 microM) endothelial or smooth-muscle cell alphavbeta3-mediated adhesion. Additionally, SM256 demonstrated high degree of alphavbeta3 specificity as compared with alphavbeta5, alpha5beta1, or alphaIIbbeta3-mediated binding in these species. SM256 is a potent alphavbeta3, antagonist with high affinity and specificity for alphavbeta3-mediated functions. Additionally, a comparable alphavbeta3 affinity for SM256 was demonstrated with endothelial cells obtained from various species (dog, mouse, rabbit, and pig) as compared with that from human.

Oral antiplatelet efficacy and specificity of a novel nonpeptide platelet GPIIb/IIIa receptor antagonist, DMP 802

This study was undertaken to define the platelet glycoprotein alphaIIb beta3 integrin (GPII/IIIa) affinity, specificity, and oral antiplatelet efficacy of DMP 802, a small-molecule nonpeptide antiplatelet agent. Platelet GPIIb/IIIa integrin binding affinity and specificity for DMP 802 were determined by using binding and adhesion assays with cells from various species, including human. DMP 802 demonstrated a potent antiplatelet efficacy [median inhibitory concentration (IC50), 0.029 +/- 0.0042 microM] in inhibiting human platelet aggregation induced by 10 microM adenosine diphosphate (ADP), as assessed by light-transmittance aggregometry. DMP 802 inhibited 125I-fibrinogen binding to activated (ADP, epinephrine, and arachidonic acid at 100 microM each) gel purified human platelets with an IC50 of 0.012 +/- 0.003 microM. DMP 802 demonstrated tight association with unactivated human, baboon, or canine platelets (t(1/2) of dissociation, 32 +/- 2, 32 +/- 13, and 11 +/- 1 min, respectively). DMP 802 binds with high affinity to both unactivated and activated human platelets (Kd = 0.61 +/- 0.17, 0.57 +/- 0.21 nM, respectively). DMP 802 demonstrated species specificity in inhibiting platelet aggregation with IC50 values ranging from 0.025 to 0.092 microM (human, guinea pig, dog, swine, hamster) and 0.88-1.0 microM (rabbit and rat) in platelets obtained from these various species. DMP 802 demonstrated a high degree of specificity for platelet GPIIb/IIIa (alphaIIb/beta3) as compared with other integrins including alpha(v)beta3 (IC50, >10 microM), alpha(v)beta5 (IC50, >100 microM), alpha4beta1 (IC50, >100 microM), and alpha5beta1 (IC50, >10 microM). Oral antiplatelet efficacy of DMP 802 was examined after single oral (0.05-0.20 mg/kg) and after repeated oral dosing at 0.05 mg/kg daily for 5 days in mongrel dogs. Dose-dependent antiplatelet efficacy with an extended duration of antiplatelet efficacy was demonstrated based on ex vivo inhibition of platelet aggregation induced by 100 microM ADP. DMP 802 has an oral bioavailability of 14.9% in dogs. In conclusion, the alpha sulfonamide isoxazoline analog, DMP 802, is a novel oral antiplatelet agent with high affinity, relatively slow dissociation rate and specificity for human platelet GPIIb/IIIa receptors.

Intravenous antiplatelet efficacy and safety of the platelet GPIIb/IIIa antagonist, DMP 728 in anesthetized dogs.

DMP 728, cyclo (D-2-aminobutyrate-N-Methyl-L-Arginyl-Glycyl-L-Aspartyl- 3-amino-methyl-benzoic acid) methanesulfonate salt, is a novel antiplatelet agent with high affinity and specificity for human and canine platelet GPIIb/IIIa (alpha 2/beta 3) receptors. DMP 728 demonstrated a potent antiplatelet efficacy in inhibiting ADP-induced platelet aggregation in either human or canine PRP with an IC50 of 0.046 and 0.015 microM, respectively. The IC50 of DMP 728 in inhibiting human platelet aggregation in PRP ranged from 0.02-0.05 microM regardless of the agonist used or even their combinations. Additionally, DMP 728 displayed a much greater affinity in inhibiting 125I-fibrinogen binding to stimulated human platelets as compared to the linear peptide RGDS or fibrinogen. The present study was undertaken to examine the i.v. antiplatelet efficacy and safety of DMP 728 in anesthetized dogs. In anesthetized mongrel dogs, DMP 728 (0.001-1.0 mg/kg, i.v. bolus) produced a dose-dependent inhibition of ex vivo platelet aggregation induced by ADP. The onset of inhibition was immediate, and the duration of antiplatelet effects was dose-dependent. A maximal inhibition of platelet aggregation and a reversible prolongation of bleeding time at 0.01 mg/kg were shown. Additionally, the antiplatelet efficacy/safety of DMP 728 was examined after i.v. administration at different infusion rates ranging from 0.008 to 0.833 micrograms/kg/min for 2 hours. A minimal antiplatelet effect was observed at the 0.008 micrograms/kg/min for 2 hours, while a maximal inhibition of platelet aggregation along with a reversible prolongation of bleeding time was achieved at 45-60 min post-infusion of 0.08 micrograms/kg/min x 2 hours. Prolongation of bleeding time was significantly reduced upon the cessation of the infusion while maximal inhibition of platelet aggregation was maintained longer. At all of the above regimens, DMP 728 did not result in any significant effects on platelet counts. Furthermore, DMP 728 did not elicit any other platelet unrelated adverse effects over wide range of doses. These data suggest that DMP 728, a low molecular weight platelet GPIIb/IIIa receptor antagonist, is a potent and systemically active antiplatelet agent with reversible effects on bleeding time.