Mary Ann Mascelli

Platelet GP IIIa Pl(A) polymorphisms display different sensitivities to agonists

BACKGROUND Both inherited predisposition and platelet hyperreactivity have been associated with ischemic coronary events, but mechanisms that support genetic differences among platelets from different subjects are generally lacking. Associations between the platelet Pl(A2) polymorphism of GP IIIa and coronary syndromes raise the question as to whether this inherited variation may contribute to platelet hyperreactivity. METHODS AND RESULTS In this study, we characterized functional parameters in platelets from healthy donors with the Pl(A) (HPA-1) polymorphism, a Leu (Pl(A1)) to Pro (Pl(A2)) substitution at position 33 of the GP IIIa subunit of the platelet GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)). We studied 56 normal donors (20 Pl(A1,A1), 20 Pl(A1,A2), and 16 Pl(A2,A2)). Compared with Pl(A1,A1) platelets, Pl(A2)-positive platelets showed a gene dosage effect for significantly greater surface-expressed P-selectin, GP IIb/IIIa-bound fibrinogen, and activated GP IIb/IIIa in response to low-dose ADP. Surface expression of GP IIb/IIIa was similar in resting platelets of all 3 genotypes but was significantly greater on Pl(A2,A2) platelets after ADP stimulation (P=0.003 versus Pl(A1,A1); P=0.03 versus Pl(A1,A2)). Pl(A1,A2) platelets were more sensitive to inhibition of aggregation by pharmacologically relevant concentrations of aspirin and abciximab. CONCLUSIONS Pl(A2)-positive platelets displayed a lower threshold for activation, and platelets heterozygous for Pl(A) alleles showed increased sensitivity to 2 antiplatelet drugs. These in vitro platelet studies may have relevance for in vivo thrombotic conditions.

Pharmacodynamic Profile of Short-term Abciximab Treatment Demonstrates Prolonged Platelet Inhibition With Gradual Recovery From GP IIb/IIIa Receptor Blockade

BACKGROUND The glycoprotein (GP) IIb/IIIa receptor antagonist abciximab is approved for use in high-risk percutaneous coronary interventions. The purpose of the present study was to establish the pharmacodynamic profile and platelet-bound life span of abciximab. METHODS AND RESULTS The pharmacodynamics of abciximab (inhibition of ex vivo platelet aggregation and GP IIb/IIIa receptor blockade) were measured in 41 individuals who were randomized to receive a 0.25-mg/kg bolus and a 12-hour infusion of either 10 microg/min (EPIC regimen) or 0.125 microg x kg(-1) x min(-1) (EPILOG regimen) of the antiplatelet agent. At extended times, the amount and distribution of platelet-bound abciximab were monitored by flow cytometry. The EPIC and EPILOG infusion regimens exhibited equivalent blockade of both GP IIb/IIIa receptors and platelet aggregation throughout the duration of abciximab treatment. Flow cytometry revealed a single, highly fluorescent platelet population during treatment, consistent with complete saturation and homogeneous distribution of abciximab on circulating platelets. For 15 days after treatment, the fluorescence histograms remained unimodal with gradually diminishing fluorescence intensity, indicating decreasing levels of platelet-bound abciximab. At 8 and 15 days, which exceeds the normal circulating life span of platelets, median relative fluorescence intensity corresponded to 29100 (29% GP IIb/IIIa receptor blockade) and 13300 (13% GP IIb/IIIa receptor blockade) abciximab molecules bound per platelet, respectively. CONCLUSIONS These results are consistent with continuous reequilibration of abciximab among circulating platelets and may explain the gradual recovery of platelet function and long-term prevention of ischemic complications by abciximab after coronary intervention.

Abciximab Suppresses the Rise in Levels of Circulating Inflammatory Markers After Percutaneous Coronary Revascularization

Background—Previous investigators have shown that systemic markers of inflammation may be increased in patients with acute ischemic syndromes or after percutaneous coronary revascularization and that persistent elevation in these markers is predictive of excess risk of subsequent adverse cardiac events. By virtue of its cross-reactivity with the glycoprotein IIb/IIIa, av&bgr;3, and &agr;M&bgr;2 receptors, abciximab may reduce inflammatory processes. Methods and Results—Assays for the inflammatory markers C-reactive protein, interleukin-6, and tumor necrosis factor-&agr; were performed on serum samples obtained from 160 patients in a placebo-controlled, randomized trial of abciximab during angioplasty. Eighty patients each had received a placebo or abciximab bolus plus a 12-hour infusion. Serum samples were drawn at baseline (before revascularization), 24 to 48 hours after study drug administration, and 4 weeks after study drug administration. Between baseline and 24 to 48 hours, the increase in C-reactive protein was 32% less in patients receiving abciximab than placebo (P =0.025); the rise in interleukin-6 levels was 76% less in the abciximab group (P <0.001); and the rise in tumor necrosis factor-&agr; levels was 100% less with abciximab therapy (P =0.112). By 4 weeks, most marker levels had returned to baseline, with no significant differences between placebo and abciximab groups. Conclusions—Systemic markers of inflammation increase in the first 24 to 48 hours after angioplasty, but the magnitude of that rise is diminished by periprocedural abciximab. Some of the long-term clinical benefit derived from this agent may be related to an anti-inflammatory effect.

An Anti-IL-12p40 Antibody Down-Regulates Type 1 Cytokines, Chemokines, and IL-12/IL-23 in Psoriasis

Psoriasis is characterized by activation of T cells with a type 1 cytokine profile. IL-12 and IL-23 produced by APCs are essential for inducing Th1 effector cells. Promising clinical results of administration of an Ab specific for the p40 subunit of IL-12 and IL-23 (anti-IL-12p40) have been reported recently. This study evaluated histological changes and mRNA expression of relevant cytokines and chemokines in psoriatic skin lesions following a single administration of anti-IL-12p40, using immunohistochemistry and real-time RT-PCR. Expression levels of type 1 cytokine (IFN-γ) and chemokines (IL-8, IFN-γ-inducible protein-10, and MCP-1) were significantly reduced at 2 wk posttreatment. The rapid decrease of these expression levels preceded clinical response and histologic changes. Interestingly, the level of an anti-inflammatory cytokine, IL-10, was also significantly reduced. Significant reductions in TNF-α levels and infiltrating T cells were observed in high responders (improvement in clinical score, ≥75% at 16 wk), but not in low responders. Of importance, the levels of APC cytokines, IL-12p40 and IL-23p19, were significantly decreased in both responder populations, with larger decreases in high responders. In addition, baseline levels of TNF-α significantly correlated with the clinical improvement at 16 wk, suggesting that these levels may predict therapeutic responsiveness to anti-IL-12p40. Thus, in a human Th1-mediated disease, blockade of APC cytokines by anti-IL-12p40 down-regulates expression of type 1 cytokines and chemokines that are downstream of IL-12/IL-23, and also IL-12/IL-23 themselves, with a pattern indicative of coordinated deactivation of APCs and Th1 cells.

Pharmacokinetics and Safety of Golimumab, a Fully Human Anti‐TNF‐α Monoclonal Antibody, in Subjects With Rheumatoid Arthritis

Golimumab is a fully human antitumor necrosis factor alpha (TNF‐α) monoclonal antibody that is being developed for intravenous and subcutaneous administration. To assess the pharmacokinetics and safety of the intravenous formulation of golimumab, 36 adult subjects with rheumatoid arthritis were randomly assigned to receive a single infusion of placebo or golimumab (0.1, 0.3, 1, 3, 6, or 10 mg/kg). Serum concentrations of golimumab were determined using a validated enzyme‐linked immunosorbent assay method. In addition to the noncompartmental analysis and compartmental modeling, a population pharmacokinetics analysis using NONMEM was also conducted. Both the maximum serum concentration and the area under the serum concentration‐time curve appeared to increase in a dose‐proportional manner. The median half‐life ranged from 7 to 20 days. A 2‐compartment population pharmacokinetic model adequately described the pharmacokinetics of golimumab. The following pharmacokinetic parameters (typical value [% coefficient of variation]) were estimated from the population pharmacokinetic model: clearance (CL: 0.40 [10.1%] L/d), volume of distribution in the central compartment (Vc: 3.07 [6.4%] L), intercompartmental clearance (Q: 0.42 [15.5%] L/d), and volume of distribution in the peripheral compartment (Vp: 3.68 [11.8%] L). Interindividual variability of the pharmacokinetic parameters was quantified for CL (44.3%), Vc (25.5%), Q (44.6%), and Vp (44.6%). Residual variability was estimated to be 15.0%. Body weight was found to be an important covariate on Vc. Golimumab was generally well tolerated. The pharmacokinetics of golimumab appeared to be linear over the dose range evaluated in this study.

Occurrence and clinical significance of pseudothrombocytopenia during abciximab therapy

OBJECTIVES This study determined the incidence of pseudothrombocytopenia during abciximab therapy administered for percutaneous coronary interventions and compared the clinical course of patients with pseudothrombocytopenia with the clinical courses of patients with thrombocytopenia and patients with normal platelet counts. BACKGROUND Although pseudothrombocytopenia has been previously reported during therapy with abciximab, the incidence and significance of this occurrence are unknown. The failure to differentiate pseudothrombocytopenia from thrombocytopenia could lead to unnecessary interruption of abciximab infusions or to platelet transfusions. METHODS The incidences of pseudothrombocytopenia and thrombocytopenia were determined in four large placebo-controlled abciximab trials: c7E3 Fab Antiplatelet Therapy in Unstable Refractory Angina (CAPTURE), Evaluation of 7E3 for the Prevention of Ischemic Complications (EPIC), Evaluation of Percutaneous Transluminal Coronary Angioplasty to Improve Long-term Outcome of c7E3 GpIIb/IIIa Receptor Blockade (EPILOG) and Evaluation of Platelet IIb/IIIa Inhibitor for Stenting (EPISTENT). The clinical features, bleeding complications and major clinical outcomes of patients with pseudothrombocytopenia and those with thrombocytopenia were compared with each other and with those of patients with normal platelet count. RESULTS Pseudothrombocytopenia occurred in 2.1% (95% confidence intervals [CI]: 1.7%, 2.5%) of abciximab-treated patients and in 0.6% of placebo-treated patients (p < 0.001). Thrombocytopenia occurred in 3.7% (95% CI: 3.2%, 4.2%) of abciximab-treated patients and in 1.8% (95% CI: 1.3%, 2.3%) of placebo-treated patients (p < 0.001). Patients with thrombocytopenia had significantly higher rates of major bleeding, major decreases in hemoglobin and increased transfusion requirements of both blood and platelets compared with those without thrombocytopenia. By contrast, pseudothrombocytopenic patients did not differ from patients with normal platelet counts in any of the measures of blood loss or transfusion requirements. Thrombocytopenic patients, but not those with pseudothrombocytopenia, had increased rates of revascularization at 30 days and six months. As previously reported, there was also a higher rate of death and myocardial infarction in the thrombocytopenic patients. CONCLUSIONS Pseudothrombocytopenia is the cause of more than one third (36.3%) of low platelet counts in patients undergoing coronary interventions who are treated with abciximab. This study demonstrates that pseudothrombocytopenia is a benign laboratory condition that does not increase bleeding, stroke, transfusion requirements or the need for repeat revascularization. It is important to recognize pseudothrombocytopenia so that the beneficial effects of abciximab are not lost by premature termination of therapy.

Anti-Platelet Factor 4/Heparin Antibodies An Independent Predictor of 30-Day Myocardial Infarction After Acute Coronary Ischemic Syndromes

Background—We postulated that antibodies to platelet factor 4/heparin complex might contribute to recurrent ischemic events in patients with acute coronary syndrome. Methods and Results—We analyzed serum from patients enrolled in the placebo/unfractionated heparin arm of the GUSTO IV-ACS trial who had high likelihood of prior heparin exposure. We selected 109 patients without thrombocytopenia with the 30-day primary end point (death, myocardial infarction [MI], or revascularization) and 109 age-, gender-, and race-matched controls who did not achieve the primary end point. An ELISA for anti-platelet factor 4/heparin antibodies was performed using 48-hour serum samples. The analyses were done by blinded investigators, and the results were correlated with clinical outcomes. Twenty-three of 218 patients (10.6%) had anti-PF4/heparin antibodies. Patients with anti-PF4/heparin antibodies were more likely to have death or MI (30.4% versus 11.3%, P =0.011) or MI (21.7% versus 6.2%, P =0.008) than patients who were negative for the antibody. After multiple logistic regression analysis, anti-PF4/heparin antibodies remained a predictor of 30-day death or MI (odds ratio, 4.0; 95% CI, 1.4 to 11.3;P =0.0093) and MI (odds ratio, 4.6; 95% CI, 1.4 to 15.0;P =0.0108). The antibody was not associated with the composite end point (death, MI, or revascularization) or with death or revascularization alone. Conclusions—Antibodies to the platelet factor 4/heparin complex are a novel, independent predictor of myocardial infarction at 30 days in patients presenting with acute coronary ischemic syndromes. This finding may explain the previous association between thrombocytopenia and adverse events in patients with acute coronary syndrome and may have important implications for the choice of anticoagulant regimens.

A phase 1, double-blind, placebo-controlled study evaluating single subcutaneous administrations of a human interleukin-12/23 monoclonal antibody in subjects with plaque psoriasis

ABSTRACT Objective: To evaluate safety, pharmacokinetics, pharmacodynamics, and clinical response of single subcutaneous (SC) administrations of a human monoclonal antibody against the p40 subunit of IL‑12/23 (IL‑12/23 mAb) in subjects with moderate-to-severe psoriasis. Methods: Twenty-one subjects were enrolled sequentially into 4 dose cohorts (0.27, 0.675, 1.35, and 2.7 mg/kg) and randomized to IL‑12/23 mAb or placebo in a 4:1 ratio. Laboratory/clinical parameters and pharmacokinetics were evaluated through Week 24; mRNA cytokine expression was measured in psoriatic plaques at Week 1. Results: Mostly mild adverse events and no serious adverse events were reported. The pharmacokinetics (Cmax and AUC) of IL‑12/23 mAb increased in an approximately dose-proportional manner. Of the 17 subjects who received IL‑12/23 mAb, 13 achieved PASI 75 (compared with no placebo subjects). mRNA expression of IL‑8, IL‑18, and IFN‑γ in psoriatic plaques decreased in subjects with sustained Psoriasis Area and Severity Index (PASI) improvement. Limitations: Interpretation of results is limited due to the small sample size in each dose cohort. Conclusion: A single SC administration of IL‑12/23 mAb was well tolerated and showed clinical response in subjects with moderate-to-severe psoriasis.

Molecular, biologic, and pharmacokinetic properties of monoclonal antibodies: impact of these parameters on early clinical development.

Currently, 14 intact, unconjugated, monoclonal antibodies (Mabs) are approved for therapeutic use in the United States, and more than 100 Mabs are presently undergoing clinical development or regulatory review. Mabs are large molecular weight glycoproteins that embody structural, biochemical, and pharmacologic properties distinct from other biologics or chemically synthesized compounds. Early therapeutic Mabs were murine proteins, and clinical testing of these agents revealed serious immune‐mediated toxicities. The side effect profile of murine Mab therapeutic agents restricted the clinical development of these agents to indications with high morbidity and/or mortality (ie, oncology, graft vs host rejection). Advances in genetic engineering and protein expression technologies resulted in the development of Mabs composed either predominately (ie, mouse/human chimeric, “humanized”) or completely (ie, “fully human” Mabs) of the human amino acid sequence. The production of chimeric, humanized, and fully human Mabs significantly reduced the immune‐mediated toxicities and expanded the utility for these agents in numerous therapeutic areas, particularly in chronic disorders requiring either long‐term administration (ie, rheumatoid arthritis) or treatment upon the flare up of disease (Crohns disease, psoriasis). This review provides an overview of the molecular, biochemical, and pharmacokinetic properties and clinical development history of Mabs and details how these factors currently affect the scope and design of early clinical development strategies for these drug candidates. Emphasis is placed on the criteria for selecting appropriate subject populations for phase I testing of Mabs.

Therapeutic targeting of the IL-12/23 pathways: generation and characterization of ustekinumab

Preclinical and clinical studies conducted in the mid-1990s reported strong association and causality between the T-cell helper (TH) 1 inductor cytokine interleukin (IL)-12 and numerous immune-mediated disorders, which spurred the development of therapeutic agents targeting IL-12 function. One of the first to enter the clinic, ustekinumab, is a human monoclonal antibody (mAb) that binds to the p40 subunit of IL-12. Subsequent to the generation of ustekinumab, it was discovered that IL-23 also contains the p40 subunit. Thus, although ustekinumab was designed to target IL-12, it also modulates IL-23, a cytokine important to the development and/or maintenance of TH17 cells. Clinical observations established that IL-12/23p40 is integral to the pathologies of psoriasis, psoriatic arthritis and Crohns disease. The molecular and cellular evaluations conducted in ustekinumab clinical programs have provided numerous insights into the pathologic processes of these disorders, illustrating how a novel molecular entity can contribute to our understanding of disease. The individual contributions of these cytokines to specific pathologies require investigation and clinical evaluation of the role of IL-12– and IL-23–specific inhibitors.