Cell-Penetrating Pepducin Therapy Targeting PAR1 in Subjects With Coronary Artery Disease

Abstract

Objective: Pepducins are membrane-tethered, cell-penetrating lipopeptides that target the cytoplasmic surface of their cognate receptor. Here, we report the first human use of a protease-activated receptor-1-based pepducin, which is intended as an antiplatelet agent to prevent ischemic complications of percutaneous coronary interventions.

Approach and results: PZ-128 was administered by 1 to 2 hours continuous intravenous infusion (0.01-2 mg/kg) to 31 subjects with coronary artery disease or multiple coronary artery disease risk factors. Safety, antiplatelet efficacy, and pharmacokinetics were assessed at baseline and 0.5, 1, 2, 6, 24 hours, and 7 to 10 days postdosing. The inhibitory effects of PZ-128 on platelet aggregation stimulated by the protease-activated receptor-1 agonist SFLLRN (8 μmol/L) at 30 minutes to 6 hours were dose dependent with 20% to 40% inhibition at 0.3 mg/kg, 40% to 60% at 0.5 mg/kg, and ≥ 80% to 100% at 1 to 2 mg/kg. The subgroup receiving aspirin in the 0.5 and 1-mg/kg dose cohorts had 65% to 100% inhibition of final aggregation to SFLLRN at 30 minutes to 2 hours and 95% to 100% inhibition by 6 hours. The inhibitory effects of 0.5 mg/kg PZ-128 were reversible with 50% recovery of aggregation to SFLLRN by 24 hours. There were no significant effects of PZ-128 on aggregation induced by AYPGKF, ADP, or collagen, indicating that the observed effects were specific to protease-activated receptor-1. The plasma half-life was 1.3 to 1.8 hours, and PZ-128 was nondetectable in urine. There were no effects on bleeding, coagulation, clinical chemistry, or ECG parameters.

Conclusions: PZ-128 is a promising antiplatelet agent that provides rapid, specific, dose dependent, and reversible inhibition of platelet protease-activated receptor-1 through a novel intracellular mechanism.

Clinical trial registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01806077.

Keywords: aspirin; collagen; coronary artery disease; lipopeptides; risk factors.

Conflict of interest statement

Disclosures

The other authors report no conflicts.

© 2015 American Heart Association, Inc.

Figures

Figure 1

Disposition of subjects. A total of 32 volunteers with vascular disease or coronary artery disease risk factors were prospectively assigned to 8 sequential dose levels and all subjects completed the study protocol. One subject became ineligible to receive the study drug after the administration of the pretreatment regimen in the predose setting. PD indicates pharmacodynamics; and PK, pharmacokinetics.

Figure 2

Inhibition of platelet aggregation (IPA) in the 0.5 mg/kg PZ-128 dose cohort to 8, 12, and 20 μmol/L protease-activated receptor-1 (PAR1) agonist peptide, SFLLRN. A, The structure of the PZ-128 pepducin (green) aligns closely (backbone root mean square deviation 1.4 Å) with the i3-loop/cytoplasmic α-helical extension of TM6 based on the rhodopsin, (red), and PAR1-vorapaxar (blue) x-ray structures. Ex vivo aggregation was conducted in platelet-rich plasma by standard light transmission aggregometry and maximal and final (at 6–7 minutes) aggregation (mean±SD) normalized to baseline (t=0) for each agonist for (B) all subjects, and the subset receiving (C) aspirin (acetylsalicylic acid [ASA]).

Figure 3

Inhibition of platelet aggregation (IPA) in the 1 mg/kg PZ-128 dose cohort to 8, 12, and 20 μmol/L protease-activated receptor-1 agonist peptide, SFLLRN. Ex vivo aggregation was conducted in platelet-rich plasma by light transmission aggregometry and maximal and final (at 6 minutes) aggregation (mean±SD) normalized to baseline (t=0) for each agonist for (A) all subjects, and the subset receiving (B) aspirin (acetylsalicylic acid [ASA]).

Figure 4

Dose dependence of mean inhibition of platelet aggregation (IPA) in the 0.3 to 2 mg/kg PZ-128 dose cohorts to 8 μmol/L protease-activated receptor-1 agonist peptide, SFLLRN. Ex vivo aggregation was conducted in platelet-rich plasma by light transmission aggregometry and maximal and final (at 6–7 minutes) aggregation (mean±SD) normalized to baseline (t=0) for each agonist for either all subjects or the subset receiving aspirin (acetylsalicylic acid) as indicated. Saline was the IPA response at 30 minutes for subject TMC-27 who received a 1-hour infusion of saline only.

Figure 5

Pharmacokinetics of PZ-128 (μmol/L) in plasma for 0.01 to 2 mg/kg dose cohorts.

Figure 6

Hemostasis parameters: Effect of PZ-128 dose on (A) activated partial thromboplastin time (APTT), (B) international normalized ratio (INR), (C) activated clotting time (ACT), and (D) bleeding time for all subjects. Hemostasis parameter (at all time points after drug infusion was initiated) versus dose for all subjects TMC1-32 was analyzed with PZ-128 dose as a continuous variable and correlations and P values for each slope were not significant (P>0.01) using repeated measures, mixed effects models, based on a Bonferroni post hoc test correction. Changes in bleeding time between baseline and 2 hours for all individuals in D was also analyzed for significance by 2-tailed, paired t test, with the mean bleeding times shown as horizontal lines.