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Purpose To characterize the pharmacokinetics (PK) of naloxegol in paediatric subjects (≥ 6 months to Methods Subjects in three age groups (≥12 to Results Naloxegol exhibits comparable PK characteristics in paediatric and adult subjects. Neither age nor body weight was identified as a significant covariate in the prior (adult only data), or current model. Naloxegol NCA- and PPK-derived AUC0–∞ values normalized to the adult 12.5 mg or 25 mg dose in the ≥6 to 0–∞ values for the 25 mg adult equivalent dose for all paediatric age groups were comparable to adults. Conclusion The PK of naloxegol was well characterized in paediatric subjects ≥ 6 months of age utilizing both NCA and PPK analysis and was shown to be comparable to adult subjects.

Background The innate immune system senses viral infection through pattern recognition receptors (PRRs), leading to type I interferon production. The role of type I interferon and PPRs in rhinovirus-induced asthma exacerbations in vivo are uncertain. Objectives We sought to compare bronchial mucosal type I interferon and PRR expression at baseline and after rhinovirus infection in atopic asthmatic patients and control subjects. Methods Immunohistochemistry was used to detect expression of IFN-α, IFN-β, and the PRRs: Toll-like receptor 3, melanoma differentiation–associated gene 5, and retinoic acid–inducible protein I in bronchial biopsy specimens from 10 atopic asthmatic patients and 15 nonasthmatic nonatopic control subjects at baseline and on day 4 and 6 weeks after rhinovirus infection. Results We observed IFN-α/β deficiency in the bronchial epithelium at 3 time points in asthmatic patients in vivo. Lower epithelial IFN-α/β expression was related to greater viral load, worse airway symptoms, airway hyperresponsiveness, and reductions in lung function during rhinovirus infection. We found lower frequencies of bronchial subepithelial monocytes/macrophages expressing IFN-α/β in asthmatic patients during infection. Interferon deficiency at baseline was not accompanied by deficient PRR expression in asthmatic patients. Both epithelial and subepithelial PRR expression were induced during rhinovirus infection. Rhinovirus infection–increased numbers of subepithelial interferon/PRR-expressing inflammatory cells were related to greater viral load, airway hyperresponsiveness, and reductions in lung function. Conclusions Bronchial epithelial IFN-α/β expression and numbers of subepithelial IFN-α/β–expressing monocytes/macrophages during infection were both deficient in asthmatic patients. Lower epithelial IFN-α/β expression was associated with adverse clinical outcomes after rhinovirus infection in vivo. Increases in numbers of subepithelial cells expressing interferon/PRRs during infection were also related to greater viral load/illness severity.

Approximately two-thirds of patients with the lysosomal storage disease mucopolysaccharidosis II have progressive cognitive impairment. Intravenous (i.v.) enzyme replacement therapy does not affect cognitive impairment because recombinant iduronate-2-sulfatase (idursulfase) does not penetrate the blood-brain barrier at therapeutic concentrations. We examined the safety of idursulfase formulated for intrathecal administration (idursulfase-IT) via intrathecal drug delivery device (IDDD). A secondary endpoint was change in concentration of glycosaminoglycans in cerebrospinal fluid. Sixteen cognitively impaired males with mucopolysaccharidosis II who were previously treated with weekly i.v. idursulfase 0.5 mg/kg for ≥6 months were enrolled. Patients were randomized to no treatment or 10-mg, 30-mg, or 1-mg idursulfase-IT monthly for 6 months (four patients per group) while continuing i.v. idursulfase weekly. No serious adverse events related to idursulfase-IT were observed. Surgical revision/removal of the IDDD was required in 6 of 12 patients. Twelve total doses were administrated by lumbar puncture. Mean cerebrospinal fluid glycosaminoglycan concentration was reduced by approximately 90% in the 10-mg and 30-mg groups and approximately 80% in the 1-mg group after 6 months. These preliminary data support further development of investigational idursulfase-IT in MPS II patients with the severe phenotype who have progressed only to a mild-to-moderate level of cognitive impairment.Genet Med advance online publication 02 April 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.36.

Preclinical and clinical studies conducted in the mid-1990s reported strong association and causality between the T-cell helper (T(H)) 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 T(H)17 cells. Clinical observations established that IL-12/23p40 is integral to the pathologies of psoriasis, psoriatic arthritis and Crohn's 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.

Intravenous enzyme replacement therapy with iduronate-2-sulfatase is an approved treatment for Hunter syndrome, however, conventional intravenous delivery cannot treat the neurologic manifestations of the disease due to its limited central nervous system penetration. Intrathecal administration of iduronate-2-sulfatase for delivery to the central nervous system is currently under investigation. The objective of this study was to evaluate the pharmacokinetics of idursulfase in the central nervous system of cynomolgus monkeys (Macaca fasicularis) after intravenous and intrathecal administration. Twenty-seven monkeys, treatment-naive to enzyme replacement therapy, were placed into 4 groups according to body weight: Group 1 was administered 0.5 mg/kg idursulfase intravenously, Groups 2–4 were administered an intrathecal formulation (1-, 10-, and 30-mg doses). Blood samples and cerebrospinal fluid (sampled at the cisterna magna or lumbar level) were collected at the same time points for 72 hours post dosing. Following intravenous administration, a high maximum serum concentration and rapid distribution of iduronate-2-sulfatase out of the central compartment were observed (elimination half-life: 4.3 hours). Iduronate-2-sulfatase exposure in the cerebrospinal fluid was limited, suggesting intravenous administration provided minimal penetration of the blood–brain barrier. Following intrathecal administration, a high maximum observed concentration was immediately noted and elimination half-life ranged between 7.8–10 hours and 5.9–6.7 hours (cisterna magna and lumbar sampling, respectively). Cerebrospinal fluid pharmacokinetic profiles at different doses of iduronate-2-sulfatase were similar and the dose/exposure relationship was proportional. After intrathecal administration, movement of iduronate-2-sulfatase from cerebrospinal fluid to serum was observed (systemic bioavailability was 40–83%). The clear penetration of iduronate-2-sulfatase into the cerebrospinal fluid and the dose response suggest that intrathecal delivery of iduronate-2-sulfatase may be suitable for treating the central nervous system manifestations associated with Hunter syndrome.

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.

The potential therapeutic activity of a human monoclonal antibody to the human interleukin-12 p40 subunit (anti-IL-12p40) has been established both in vitro and in vivo, warranting a first-in-human investigation in psoriasis. This phase I, first-in-human, non-randomized, open-label study evaluated the short-term safety, pharmacokinetics, and clinical response of single, ascending, intravenous (IV) doses of anti-IL-12p40 in subjects with moderate-to-severe psoriasis vulgaris. Eighteen subjects with at least 3% body surface area involvement were enrolled in four dose groups (0.1, 0.3, 1.0, and 5.0 mg per kg). Safety, pharmacokinetics, and clinical response (e.g., Psoriasis Area and Severity Index (PASI)) were monitored at baseline and at specific time points over a 16-wk follow-up period. Anti-IL-12p40 was generally well tolerated. No related serious adverse events or infusion reactions were reported, and most adverse events were mild. IV anti-IL-12p40 yielded linear pharmacokinetics, with a mean terminal half-life of approximately 24 d. Dose-dependent associations with both the rate and extent of clinical response were observed across the four dose groups. Twelve of 18 subjects (67%) achieved at least a 75% improvement in PASI between 8 and 16 wk after study agent administration. Significant and sustained concentration-dependent improvements in psoriatic lesions were observed in most subjects.

We postulated that patients with recent acute coronary syndromes and antibodies to the platelet factor 4/heparin complex would have an increased risk of myocardial infarction (MI), even in the absence of thrombocytopenia. We analyzed sera from patients enrolled in the placebo/unfractionated heparin arm of the GUSTO IV-ACS trial who had a high likelihood of prior heparin exposure. We selected 109 patients without thrombocytopenia with the 30-day primary endpoint (death, MI, or revascularization) and 109 age-, gender-, and race-matched controls who did not achieve the primary endpoint. Twenty-three of 218 patients (10.6%) had anti-PF4/heparin antibodies. Patients with anti-PF4/heparin were more likely to have death or MI (30.4% vs. 11.3%, p = 0.011) or MI (21.7% vs. 6.2%, p = 0.008) than patients who were negative for the antibody. Antibody-positive patients had higher levels of sVCAM-1 (892 +/- 263 microg/L vs. 780 +/- 228 microg/L; p = 0.04) and sICAM-1 (246 +/- 50 microg/L vs. 222 +/- 71 microg/L; p = 0.02) than antibody-negative patients. In a multiple logistic regression model that included inflammatory markers and clinical risk factors, antibodies to PF4/heparin were a strong predictor of 30-day MI (odds ratio, 9.0; 95% confidence intervals, 2.1 to 38.6; p < 0.01), with IL-6 being the only other predictor (odds ratio, 1.1; 95% confidence intervals, 1.0 to 1.2; p = 0.03). Antibodies to the platelet factor 4/heparin complex are a novel, independent predictor of MI at 30 days in patients presenting with acute coronary ischemic syndromes. Antibodies to PF4/heparin are a stronger predictor of MI than clinical characteristics or inflammation markers.

Because of its potential for antigenicity, theoretical concerns related to readministration of abciximab have been raised. We conducted the ReoPro Readministration Registry to assess the efficacy and safety of abciximab readministration. A total of 1,342 patients who underwent percutaneous coronary intervention and who received abciximab for at least a second time were recruited. Safety end points were hypersensitivity reactions, major bleeding, and thrombocytopenia (TCP). Human antichimeric antibody (HACA) titers were determined before and after readministration. Procedural success was 98% and was not influenced by the number of courses of abciximab or the presence of HACA. There were no cases of anaphylaxis. There were 5 minor allergic reactions, none of which required termination of the infusion. Clinically significant bleeding occurred in 31 patients (2.3%), including 1 (0.07%) with intracranial hemorrhage. TCP (

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.

Objectives Our objectives were to compare and contrast abciximab concentration-effect relationships in healthy volunteer participants with those in patients with coronary atherosclerosis undergoing elective coronary angioplasty. We also aimed to establish abciximab plasma concentrations associated with 80% inhibition of platelet aggregation. Methods Abciximab clearance and concentration-effect relationships were determined from two separate clinical studies, one in 30 healthy subjects aged 21 to 66 years and the other in 32 patients aged 44 to 74 years before they underwent elective coronary angioplasty. After abciximab administration, abciximab plasma concentrations, platelet glycoprotein IIb/IIIa (GP IIb/IIIa) receptor occupancy, and degree of inhibition of platelet aggregation in the presence of 5-μmol/L and 20-μmol/L adenosine diphosphate was determined. With an Emax (receptor occupancy) or inhibitory Emax (inhibition of platelet aggregation) model, abciximab concentrations required for 80% receptor occupancy and 80% inhibition of platelet aggregation were determined. Results Abciximab steady-state clearance in healthy participants was 183 ± 72 ml/min (mean ± SD), and single-dose clearance in patients undergoing angioplasty was 405 ± 240 ml/min (mean ± SD). Abciximab concentration required for 80% GP IIb/IIIa receptor occupancy was 35.2 ± 2.4 versus 72.8 ± 6.4 ng/ml in healthy participants versus patients (P < .01). Concentrations required for 80% inhibition of platelet aggregation stimulated by 5-μmol/L adenosine diphosphate were 25.6 ± 1.6 versus 68.9 ± 9.2 ng/ml (P < .01). Similarly, the concentrations required for 80% inhibition of platelet aggregation stimulated by 20-μmol/L adenosine diphosphate were 56.0 ± 3.2 versus 141 ± 16.8 ng/ml (P < .01). Conclusion Approximately 2-fold greater abciximab exposure is required to achieve the same degree of GP IIb/IIIa occupancy and inhibition of platelet aggregation in patients undergoing angioplasty as compared with healthy participants. The difference between groups may be related either to different states of basal platelet activation or to the effect of heparin that patients received as part of the angioplasty procedure. A therapeutic concentration range for patients is 100 to 175 ng/ml, because this is the concentration consistent with >80% inhibition of platelet aggregation when 20-μmol/L adenosine diphosphate is used as the aggregating stimulus. Clinical Pharmacology & Therapeutics (2002) 71, 186–195; doi: 10.1067/mcp.2002.121775

SummaryThe ability of abciximab to prevent fibrinogen binding to activated platelets indicates it may also promote dissolution of platelet-rich thrombi. The present study examined the capacity of abciximab to reverse platelet aggregation in vitro. Experiments were performed on blood from healthy non-medicated donors. Platelet aggregate formation and disaggregation were monitored turbidimetrically. Platelet-bound fibrinogen was measured by flow cytometry. For disaggregation studies, platelets were first stimulated with either ADP or the 11-mer thrombin receptor activating peptide (TRAP), then varying amounts of abciximab were added at periodic intervals after agonist addition. Platelet disaggregation was detected by comparing the extent of light transmittance at 4 min after addition of either abciximab or saline to PRP. ATP release was simultaneously monitored by chemi-luminescence. When added 1 min after low concentrations of ADP, abciximab rapidly (These data indicate that abciximab facilitates the dispersal of newly formed platelet aggregates in vitro, by partially displacing fibrinogen from activated GPIIb/IIIa receptors. In vivo, abciximab may destabilize coronary thrombi by preventing aggregate formation and dispersing mural thrombi.

Objective The purpose of the study was to establish a rebolus regimen for abciximab that restores pharmacologic glycoprotein (GP) IIb/IIIa receptor blockade within a short time frame (up to 48 hours) after completion of an initial treatment. Methods and Results The study was a single-center, nonrandomized, open-label dose escalation trial in healthy volunteers (n = 30). Each subject received a 0.25 mg/kg bolus and a 0.125 μg/kg per minute infusion of abciximab, followed by incremental bolus doses of the agent at 15-minute intervals up to 48 hours (10 per group) after completion of the infusion, (maximal cumulative rebolus dose of 0.25 mg/kg). Pharmacodynamic measurements (GP IIb/IIIa receptor blockade, turbidimetric and whole blood platelet aggregation with use of a rapid platelet function assay [RPFA]) were obtained at periodic intervals during and after administration of the abciximab bolus and infusion. At the time of the first rebolus, pharmacodynamic measurements were attained immediately before administration of each rebolus and 15 minutes after the last rebolus dose. In subjects who received reboluses 12 hours after infusion, a cumulative dose of 0.05 mg/kg restored >80% blockade of GP IIb/IIIa receptors and >80% inhibition of turbidimetric (5 and 20 μmol/L adenosine diphosphate) and RPFA aggregation in 10 of 10 subjects. At 24 hours after treatment, a cumulative abciximab bolus dose of 0.1 mg/kg restored >80% blockade of all 4 pharmacodynamic measurements in 10 of 10 subjects. At 48 hours after treatment, a cumulative bolus dose of 0.15 mg/kg restored >80% blockade of all 4 pharmacodynamic measurements in 10 of 10 subjects. Conclusions A fraction of the bolus of abciximab restored pharmacologic (>80%) GP IIb/IIIa receptor blockade when readministered at various postinfusion time points. These observations suggest that in the setting where acute readministration of abciximab is required less than a full bolus dose of the agent is warranted. (Am Heart J 2002;143:87-94.)

Background — Platelet glycoprotein IIb/IIIa blockade with abciximab (ReoPro) improves the clinical outcomes of percutaneous coronary intervention. This registry was conducted to characterize the effects of repeated administration of abciximab during intervention. Methods and Results — We recruited 500 consecutive patients at 22 centers in the United States who were receiving abciximab for at least a second time during percutaneous coronary intervention. Safety was measured as the incidence of hypersensitivity reactions, major bleeding, and thrombocytopenia. Efficacy was assessed as event-free clinical success. Human antichimeric antibody (HACA) responses were also characterized. There were no cases of hypersensitivity (95% upper confidence bound, 0.3%), major bleeding, or death. Clinical success was 94.4%. Thrombocytopenia occurred in 23 patients (4.6%; 95% CI, 2.8% to 6.4%), including 12 (2.4%; 95% CI, 1.1% to 3.7%) who developed profound thrombocytopenia (9 cells/L). In 2 patients (0.4%), profound thrombocytopenia did not develop until after hospital discharge; in 4 (0.8%), profound thrombocytopenia recurred despite platelet transfusion. Before a first readministration, a positive HACA titer was present in 22 of 454 patients (4.8%); after a first readministration, an additional 82 of 432 (19.0%) became HACA-positive. HACA did not neutralize the in vitro inhibition of platelet aggregation by abciximab or correlate with clinical events. Conclusions — The results, including overall rates of thrombocytopenia, were consistent with randomized clinical trials of first abciximab treatment. However, there was a shift from mild to profound thrombocytopenia, and cases of delayed presentation and of recurrent thrombocytopenia were seen. These findings suggest that indications and guidelines for first-time use apply to retreatment, particularly the systematic monitoring for thrombocytopenia.

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, avβ3, and αMβ2 receptors, abciximab may reduce inflammatory processes. Methods and Results — Assays for the inflammatory markers C-reactive protein, interleukin-6, and tumor necrosis factor-α 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 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.

Summary Objective. Enhanced GPIIb/IIIa binding and inhibition of platelet aggregation of eptifibatide by the reduction of ionized plasma calcium concentrations have been reported. The present study compared the importance of Ca2+ chelation on the in vitro platelet inhibitory profiles of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban. Methods and Results. Turbidimetric platelet aggregation dose response curves of the various GPIIb/IIIa antagonists were performed using platelet rich plasma (PRP) anticoagulated with either trisodium citrate, or the non-chelating anticoagulant, PPACK. The concentrations of antagonist that resulted in 50% inhibition of TRAP-induced (10 M) platelet aggregation (IC50) were measured in the presence of either citrate or PPACK. In addition, the influence of Ca2+ chelation on the binding properties (relative affinity, on- and off-rates) of abciximab for the GPIIb/IIIa receptor on platelets was measured. For all three agonists, the IC50 concentrations were lower for platelets treated with citrate than PPACK, but the degree of difference varied among the agents. The mean TRAP IC50 values for citrate and PPACK were 88.2 ± 12.2 nM and 126.1 ± 28.4 nM for abciximab (1.4 fold enhancement; p = 0.0007), 75.9 ± 13.3 nM and 142.6 ± 32.6 nM for tirofiban (1.9-fold enhancement; p = 0.001), and 260.2 ± 62.5 nM and 810.3 ± 182.5 nM for eptifibatide (3.1-fold enhancement; p = 0.001). A similar shift in effective inhibitor concentrations for abciximab was observed with ADP (10 M). The relative affinities (EC50), on- and off-rates of abciximab for the platelet GPIIb/IIIa receptor in the presence of trisodium citrate and PPACK were equivalent. Conclusions. These data confirm previous observations that Ca2+ chelation afforded by citrate decreases the effective inhibitor concentrations of GPIIb/IIIa antagonists, as assessed by turbidimetric platelet aggregation. However, the extent of decrease was less for abciximab and tirofiban, compared to eptifibatide.

Background —We evaluated platelet activation and aggregation in patients with acute myocardial infarction (AMI) treated with thrombolytic therapy alone or with reduced-dose thrombolysis and concomitant abciximab. Methods and Results —The study was performed in 20 control subjects and 51 patients with AMI before and after reperfusion with either alteplase or reteplase or reduced doses of these agents with concomitant abciximab. Platelet activation was assayed by platelet surface expression of P-selectin. Turbidometric platelet aggregation in response to ADP was measured in patients before thrombolytic therapy and 90 minutes and 24 hours after the beginning of thrombolytic therapy. P-selectin expression was greater at baseline in patients than normal control subjects (30.4% versus 9.8%, P P =0.37). However, at 24 hours, basal P-selectin expression declined in patients ( P =0.0025 versus baseline), whereas ADP-stimulated P-selectin expression was lower in patients than in control subjects (48% versus 69%, P =0.0004). When combined with reduced doses of either alteplase or reteplase, abciximab achieved 91% and 83% inhibition of 5 and 20 μmol/L ADP–induced platelet aggregation, which decreased to 46% and 40%, respectively, at 24 hours. No appreciable difference in the platelet inhibition profile of abciximab was observed between the 2 thrombolytics. Conclusions —Platelet activation and aggregation are heightened in the setting of thrombolysis for AMI. Despite this enhanced level of platelet activation, abciximab, combined with a reduced-dose thrombolytic, inhibited platelet aggregation similarly to the level reported in elective settings.

Background This study evaluated the effect of heparin on the platelet reactivity and the pharmacodynamic profile of abciximab. Methods and Results Ex vivo studies were performed on patients undergoing elective percutaneous coronary intervention (n = 26) who were at moderate to high risk of ischemic complications. Patients received a 12,000-U bolus of heparin followed by a 0.25-mg/kg bolus of abciximab. Before abciximab treatment, platelet aggregation responses to a variety of stimuli were assessed immediately before and 10 minutes after the heparin bolus. Heparin increased platelet aggregation to 2 and 5 μmol/L adenosine diphosphate (ADP) and 5 μg/ml collagen by 36%, 25%, and 46%, respectively ( P ≤ .001), but did not influence platelet reactivity to thrombin receptor-activating peptide or 20 μmol/L ADP and had no appreciable effect on platelet surface glycoprotein (GP) IIb/IIIa receptor numbers. To assess the impact of heparin on the pharmacodynamic profile of abciximab, GP IIb/IIIa receptor blockade and platelet aggregation inhibition estimates obtained after abciximab administration were calculated relative to the basal levels observed both before and after the heparin bolus. At 2 and 24 hours after the abciximab bolus, GP IIb/IIIa receptor blockade measurements normalized to either the preheparin or postheparin baseline determinations were equivalent. For all ADP concentrations tested, the 2-hour post-abciximab bolus platelet aggregation inhibition estimates based on the preheparin and postheparin baseline values were comparable. However, for 2 and 5 μmol/L ADP, the 24-hour post-abciximab platelet aggregation inhibition measurements based on preheparin baseline values were significantly lower than postheparin baseline determinations (both P ≤ .003). In vitro studies revealed that therapeutic heparin doses induced a concentration-dependent reduction in the extent of platelet inhibition produced by amounts of abciximab that elicit partial inhibition of platelet aggregation. However, at abciximab concentrations that achieved platelet aggregation blockade of >80%, the levels of inhibition of platelet aggregation in the presence and absence of heparin were equivalent. Conclusions The cumulative ex vivo and in vitro data indicate that for certain stimuli, heparin alters the platelet inhibitory profile of abciximab at concentrations of the agent that yield partial suppression of platelet function.

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.

Ticlopidine and abciximab are two antiplatelet agents that are frequently administered during percutaneous coronary interventions. Although they have different mechanisms of action and pharmacological profiles, the two agents are often concomitantly used in complicated stent placements. The purpose of the study was to evaluate the effect of ticlopidine therapy on the capacity of abciximab to inhibit platelet aggregation, in vitro. Blood samples from 13 ticlopidine-treated stent placement patients and 8 patients undergoing PTCA who did not receive ticlopidine were obtained prior to, 12–36 hours and 7–10 days after initiating ticlopidine treatment. For each patient, the minimal ADP and the thrombin receptor activating peptide (TRAP) concentrations that elicited maximal platelet aggregation responses at baseline were used to measure the extent of platelet aggregation and the abciximab concentration that gave a 50% decrease in aggregation (IC50) for both agonists at the three time points. The ticlopidine group baseline and 12–36 hour mean ADP aggregation responses were equivalent, but decreased by 34% (P = 0.009) at 7–10 days. The control group ADP and TRAP, as well as the ticlopidine group TRAP aggregation responses, were equivalent at all time points. The ticlopidine group baseline and 12–36 hour abciximab IC50 values for ADP were comparable (1.58 ± 1.1 ng/mL vs. 1.23 ± 0.5 ng/mL; P = 0.266), but decreased to 1.00 ± 0.6 ng/mL (36%; P = 0.004) at 7–10 days. In contrast, the abciximab IC50 for TRAP increased from 1.48 ± 1.0 ng/mL to 1.85 ± 1.1 ng/mL (25%; P = 0.033) at 12–36 hours, but returned to baseline at 7–10 days (1.40 ± 0.8; P = 0.975). The control group IC50 abciximab values for ADP and TRAP were comparable throughout the monitoring period. The results demonstrate that ticlopidine elicits subtle potentiation of the platelet-inhibitory capacity of abciximab to the agonist ADP, but not TRAP, at 1 week after initiation of treatment.

SummaryIn addition to inhibition of platelet aggregation, GPIIb-IIIa antagonists may reduce thrombotic events via other mechanisms. In a novel whole blood flow cytometric system, we investigated the effects of GPIIb-IIIa antagonists, in the presence or absence of thrombin inhibitors, on platelet surface-bound factor V/Va and platelet surface phospholipids. Diluted venous blood was incubated with either buffer or a GPIIb-IIIa antagonist (abciximab, tirofiban, or eptifibatide). Some samples were pre-incubated with clinically relevant concentrations of unfractionated heparin (UFH), a low molecular weight heparin, a direct thrombin inhibitor, or buffer only. Platelets were then activated and labeled with mAb V237 (factor V/Va-specific) or annexin V (binds phosphatidylserine), fixed, and analyzed by flow cytometry. In the absence of thrombin inhibitors, GPIIb-IIIa antagonists (especially abciximab) significantly reduced agonist-induced platelet procoagulant activity, as determined by reduced binding of V237 and annexin V. At high pharmacologic concentrations, unfractionated heparin and enoxaparin, but not hirudin, further reduced factor V/Va binding to the surface of activated platelets in the presence of GPIIb-IIIa antagonists. Agonist-induced platelet procoagulant activity was reduced in a patient with Glanzmann’s thrombasthenia. We conclude that GPIIb-IIIa antagonists reduce platelet procoagulant activity in whole blood and heparin and enoxaparin augment this reduction. Fibrinogen binding to GPIIb-IIIa is important in the generation of platelet procoagulant activity.

Abciximab is the first of a new class of therapeutic agents that specifically block the function of the platelet cell adhesion receptor, glycoprotein (GP) IIb/IIIa and the subsequent platelet aggregation that contributes to thrombosis. Abciximab displays high avidity for the GPIIb/IIIa receptor, which results in a prolonged platelet-bound half-life, and a sustained duration of platelet inhibition resulting from the redistribution and equilibration of abciximab molecules among circulating platelets at gradually diminishing levels of GPIIb/IIIa receptor occupancy. In contrast to its extended platelet bound life-span, unbound abciximab is cleared rapidly (half-life

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 μg/min (EPIC regimen) or 0.125 μg · kg −1 · 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 29 100 (29% GP IIb/IIIa receptor blockade) and 13 300 (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.

Background The glycoprotein (GP) IIb/IIIa receptor antagonist abciximab (c7E3 Fab, ReoPro) is approved for use in high-risk percutaneous transluminal coronary angioplasty (PTCA). At present, no “point of care” exists for measuring pharmacological GP IIb/IIIa blockade. To address this need, the Chrono-log Whole Blood Aggregometer, which measures platelet aggregation by electrical impedance, was adapted to test platelet function at the bedside. Methods and Results GP IIb/IIIa receptor blockade, impedance (5 μg/mL collagen), and turbidimetric aggregation (5 and 20 μmol/L ADP) measurements were obtained on 14 PTCA patients who received the standard bolus plus a 12-hour infusion of abciximab. During abciximab administration, mean GP IIb/IIIa receptor blockade was >91%, and both impedance and turbidimetric aggregation were inhibited by ≥90%. At 12 hours after abciximab treatment, the mean inhibition of turbidimetric platelet aggregation to 5 and 20 μmol/L ADP was 65±20% and 49±14%, respectively, and inhibition of impedance aggregation was 69±12%. GP IIb/IIIa receptor blockade was 67±8%. At 36 hours after abciximab treatment (n=8), the mean inhibition of turbidimetric platelet aggregation to 5 and 20 μmol/L ADP was 44±21% and 30±14%, respectively, whereas impedance aggregation was inhibited by 60±14%. GP IIb/IIIa receptor blockade was 57±7%. Conclusions During and at 12 hours after abciximab therapy, impedance and turbidimetric platelet aggregation to 5 μmol/L ADP were comparable and closely correlated with GP IIb/IIIa receptor blockade. However, at 36 hours after abciximab treatment, impedance platelet aggregation more closely paralleled GP IIb/IIIa receptor blockade and indicated a slower recovery of platelet function than turbidimetric aggregometry.

Immune-mediated inflammatory diseases encompass a broad and diverse spectrum of serious chronic disorders, many of which have significant need for safe and effective pharmacotherapies. The classes of conventional drugs used to treat immune-mediated inflammatory diseases include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, sulfasalazine, 5-aminosalicylates, methotrexate, azathioprine, and 6-mercaptopurine; however, in many instances, these agents have exhibited limited efficacy or are associated with significant serious on-target and off-target side effects. The initial rationale and promise of complex biologics, such as monoclonal antibodies (mAbs), as pharmacotherapy was focused on oncology and organ transplantation (Ehrlich 1891; Gura 2002); however, recent decades have witnessed the successful development of a number of complex biologics as both anti-allergic and anti-inflammatory therapies. Five of the top-selling mAbs are for the treatment of chronic inflammatory conditions, and this area of research and development is rapidly expanding. Complex biologics are a subclass of protein therapeutics. They are large molecular weight glycoproteins designed and produced through recombinant DNA technology and require production in eukaryotic cells using bioreactor technology. These modalities have provided many targeted and efficacious therapeutic options for patients and are providing significant insights into the underlying complex pathological processes of these disorders, which, in turn, are identifying new targets for treatment of these diseases. A significant translational insight derived from the clinical development programs of complex biologic pharmacotherapy is the dysregulation of common proinflammatory mediators, such as tumor necrosis factor alpha (TNFα), across diverse rheumatologic, dermatologic, and gastroenterologic pathologies. In addition, the observation of patient subsets within a disease that are refractory to a particular therapy indicates that dysregulation of different mediators can drive the underlying pathological processes within a disease.

A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab′)2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab′)2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab′)2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab′)2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.

Since the original publication of the article "Development of the IL-12/23 antagonist ustekinumab in psoriasis: Past, present and future perspectives" in March 2011 (see Appendix),(1) there have been several new publications and developments of note. A number of new reports from the ustekinumab psoriasis clinical development program have been published. The analysis of efficacy and safety in the PHOENIX 1 long-term extension demonstrated that continuous stable maintenance dosing of ustekinumab was generally well tolerated and sustained durable efficacy through up to three years of treatment.(2) Pooled safety data from the phase 2 and phase 3 global trials showed that the safety profile of long-term continuous ustekinumab treatment through up to three years(3,4) and four years(5) of follow-up was favorable and comparable to what has been reported previously in the shorter-term ustekinumab psoriasis studies.(6-8) This represents the greatest exposure and longest follow-up of psoriasis patients treated with a biologic published to date. Additional phase 3 trials in Asian populations demonstrated similar high levels of efficacy and favorable safety profiles in Japanese,(9,10) Korean,(11,12) and Taiwanese(11,12) patients as those observed in trials conducted in mostly White populations in North America and Europe.(6-8) These data support the positive benefit:risk profile and consistency of response to ustekinumab over years of usage, and in multiple ethnic groups. Results from up to five years of treatment with ustekinumab in the long-term extensions of the phase 3 trials, and the efficacy, safety, and effect on quality of life in Chinese patients will be available in 2012. In addition to clinical trials of ustekinumab for the treatment of psoriasis, 24-week data from one phase 3 study of ustekinumab for the treatment of psoriatic arthritis has recently been presented(13) and another study is ongoing. A Phase 2b trial in Crohn's disease has also been presented,(14) and three phase 3 studies in Crohn's disease are currently in progress.

Monoclonal antibody (mAb) therapy was first established upon the approval of a mouse antibody for treatment of human acute organ rejection. However, the high incidence of immune response against the mouse mAb restricted therapeutic utility. Development of chimeric, "humanized" and human mAbs broadened therapeutic application to immune-mediated diseases requiring long-term treatment. Indeed, mAb therapeutics targeting soluble cytokines are highly effective in numerous immune-mediated disorders. A recent example is ustekinumab, a first-in-class therapeutic human immunoglobulin G1 kappa mAb that binds to the interleukins (IL)-12 and IL-23, cytokines that modulate lymphocyte function, including T-helper (Th) 1 and Th17 cell subsets. Ustekinumab was generated via recombinant human IL-12 immunization of human immunoglobulin (hu-Ig) transgenic mice. Ustekinumab binds to the p40 subunit common to IL-12 and IL-23 and prevents their interaction with the IL-12 receptor β1 subunit of the IL-12 and IL-23 receptor complexes. Ustekinumab is approved for treatment of moderate-to-severe plaque psoriasis and has demonstrated efficacy in Crohn disease and psoriatic arthritis. The clinical characterization of ustekinumab continues to clarify our understanding of human immune pathologies and may offer a novel therapeutic option for certain immune-mediated diseases.

The concept of monoclonal antibodies (mAbs) as pharmacotherapeutics was validated in the mid-1980s with the successful clinical development of the fully murine mAb muromonab-CD3 for prevention of acute organ rejection. However, clinical applications of fully murine mAbs are restricted, owing to the high incidence of serious immune-mediated side effects, particularly upon repeated exposure. The rate and severity of immune-mediated toxicities of these agents were significantly attenuated by the development of mouse/human chimeric, fully human, and humanized mAbs. These refinements in molecular structure allowed repeated, long-term administration, where therapeutically warranted, which, in turn, broadened the scope of indications for this class of therapy. Presently, numerous mAbs are approved or are undergoing clinical evaluation for treatment of oncologic and chronic inflammatory diseases. The current experimental development landscape spans respiratory, metabolic, and central nervous system disorders as well as infectious disease. A consequence of the expanding numbers of mAb indications is concomitant administration of these agents with established small molecule pharmacotherapies, which necessitates a comprehensive understanding of mAb–small molecule drug interactions as well as mAb-mAb interactions. Current knowledge indicates that mAbs do not elicit a direct effect on the metabolic/clearance pathways for small molecular therapeutics. However, the immunomodulatory properties of mAbs can indirectly alter clearance of certain small molecule entities through the attenuation of noncatabolic enzymatic pathways.

Ustekinumab, a fully human immunoglobulin (Ig) G1K monoclonal antibody directed against the p40 subunit of interleukin (IL)-12/23, has demonstrated efficacy in patients with moderate-to-severe psoriasis.To evaluate the effect of IL-12/23 inhibition on immunocompetency by antigen-recall response in a preclinical multiple-dose toxicology study and three single-dose, phase 1 studies.Cynomolgus monkeys (Mauritius; n = 32) treated with subcutaneous (s.c.) placebo or ustekinumab 22.5 or 45 mg/kg twice weekly for 26 weeks were assessed for antibody responsiveness to keyhole limpet hemocyanin (KLH). Patients with psoriasis or multiple sclerosis who received a single-dose of placebo (n = 8) or ustekinumab (n = 46) 0.09-4.5 mg/kg intravenous (i.v.) or 0.27-2.7 mg/kg s.c. were assessed by pneumococcal and tetanus antigen challenge. Primary T-cell response was not assessed in humans.Anti-KLH antibody responses in ustekinumab-treated cynomolgus monkeys were comparable to those observed in placebo-treated animals. A normal antibody response (or = two-fold increase from baseline) to pneumococcal antigen was seen in 34/46 (73.9%) ustekinumab-treated versus 4/8 (50%) placebo-treated patients. A normal antigen-recall response (or = four-fold increase from baseline) was seen in 12/20 (60%) ustekinumab- and 4/5 (80%) placebo-treated patients following tetanus toxoid exposure. Percentages of circulating immune cells were not affected by ustekinumab treatment.Results in nonhuman primates and human patients suggest that ustekinumab treatment does not significantly impair recall humoral immune system functions.

The proximate cause of autoantibodies characteristic of systemic autoimmune diseases has been controversial. One hypothesis is that autoantibodies are the result of polyclonal nonspecific B cell activation. Alternatively, autoantibodies could be the result of antigen-driven B cell activation, as observed in secondary immune responses. We have approached this question by studying monoclonal anti-DNA autoantibodies derived from unmanipulated spleen cells of the autoimmune MRL/lpr mouse strain. This analysis shows that anti-DNAs, like rheumatoid factors (19), are the result of specific antigen-driven stimulation. In addition, correlation of sequences with fine specificity shows that: (a) somatic mutations can cause specificity for dsDNA and that such mutations are selected for; (b) arginine residues play an important role in determining specificity; and (c) anti-idiotypes that recognize the majority of anti-DNA are probably not specific for any one family of V regions.

To evaluate safety, pharmacokinetics, pharmacodynamics, and clinical response of single subcutaneous (s.c.) 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.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.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-gamma in psoriatic plaques decreased in subjects with sustained Psoriasis Area and Severity Index (PASI) improvement.Interpretation of results is limited due to the small sample size in each dose cohort.A single s.c. administration of IL-12/23 mAb was well tolerated and showed clinical response in subjects with moderate-to-severe psoriasis.

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 (Crohn's 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.

Golimumab is a fully human antitumor necrosis factor alpha (TNF-alpha) 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 concentrationtime 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 (V(c): 3.07 [6.4%] L), intercompartmental clearance (Q: 0.42 [15.5%] L/d), and volume of distribution in the peripheral compartment (V(p): 3.68 [11.8%] L). Interindividual variability of the pharmacokinetic parameters was quantified for CL (44.3%), V(c) (25.5%), Q (44.6%), and V(p) (44.6%). Residual variability was estimated to be 15.0%. Body weight was found to be an important covariate on V(c). Golimumab was generally well tolerated. The pharmacokinetics of golimumab appeared to be linear over the dose range evaluated in this study.

A phase I–II trial (NCT00920647) evaluating the effects of investigational recombinant iduronate-2-sulfatase formulated for intrathecal administration (idursulfase-IT) in cognitively impaired children with Hunter syndrome (MPS II), and its extension (NCT01506141), has been ongoing since 2010. In addition to safety assessments, changes in glycosaminoglycan (GAG) concentrations in cerebrospinal fluid (CSF) and change from baseline in standardized neurocognitive assessments are being assessed. Sixteen cognitively impaired children with MPS II were originally enrolled. Four patients per dose group received either no treatment, 10 mg, 30 mg, or 1 mg idursulfase-IT monthly for 6 months while continuing intravenous idursulfase 0.5 mg/kg weekly. Patients (currently n = 14) continued into an ongoing extension study, receiving either 10 or 30 mg monthly idursulfase-IT doses. To date, the range of patient exposure time to idursulfase-IT is approximately 2 to 4.5 years. Mean CSF GAG concentration was reduced by about 90% in the 10and 30-mg groups and by about 80% in the 1-mg group at month 6. Long-term follow-up indicates that these low CSF GAG values are maintained, despite occasional skipped doses or, in 1 patient, the development of neutralizing CSF antibodies. Only 5/16 patients were originally cognitively testable over time using the Differential Abilities Scale (2nd edition). Currently, there are 4 patients with cognitive data obtained by this assessment. Although the cognitive data shows large variability, all 4 patients, including 1 patient who was switched from 1 to 10 mg, appear to have experienced stabilization or decreased rate of decline. These data support further development of idursulfase-IT for the stabilization or decreased decline of cognitive function in moderately affected MPS II patients with the severe phenotype. These studies are funded by Shire.

To assess the safety, tolerability and pharmacokinetics of an interleukin (IL)-12/23 monoclonal antibody (mAb) in subjects with a relapsing form of multiple sclerosis (MS).A phase I, double-blind, placebo-controlled, sequential dose escalation study was conducted in 20 subjects with MS. Subjects were randomized (4:1) to receive a single subcutaneous injection of either IL-12/23 mAb (0.3, 0.75, 1.5, and 3.0 mg/kg) or placebo. Clinical and laboratory evaluations were performed through 16 weeks following administration.IL-12/23 mAb was well tolerated in this study. Adverse events were generally mild or moderate, with no apparent dose-related trends. One subject with a family history of breast cancer was diagnosed during the study with breast cancer 21 days after IL-12/23 mAb administration. There were no significant changes in laboratory indicators of systemic or neurotoxicity. There was a large degree of variability in T2 lesion volume and total number of gadolinium-positive lesions, both unaffected by dose escalation. Three relapses of MS occurred in two placebo-treated subjects. Over the range of single doses studied, the median Tmax ranged from 9.0 to 16.5 days, and the median T1/2 ranged from 20.2 to 30.9 days.Single subcutaneous administrations of IL-12/23 mAb in this first study of relapsing MS were generally well tolerated. Safety of the agent will need to be tested in a study of longer duration and involving a larger cohort of subjects.

To assess safety, clinical response, and pharmacokinetics of subcutaneous (SC) and intramuscular (IM) doses of an experimental formulation of infliximab [including experimental SC doses following administration of commercially-formulated intravenous (IV) infliximab] in patients with rheumatoid arthritis (RA) refractory to methotrexate.In this randomized, open-label, 3-stage design, 43 subjects were enrolled in 7 dose groups. In Stage I, 15 subjects received single SC doses of 0.5, 1.5, or 3.0 mg/kg. In Stage II, 21 subjects received one of 3 regimens: 100 mg SC every 2 weeks (3 injections); 3 mg/kg commercially-formulated IV infliximab every 2 weeks (2 infusions) followed by 100 mg SC every 2 weeks (3 injections); or 100 mg IM every 2 weeks (3 injections). In Stage III, 7 subjects received 100 mg SC every 4 weeks (3 injections).No treatment-related serious adverse events were observed, and there were no serious injection site reactions. A low-titer infliximab antibody response was detected in 27% of subjects receiving single SC doses, 5% receiving multiple SC doses, and 43% receiving IM doses. SC administration yielded roughly dose-proportional increases in Cmax and AUC. American College of Rheumatology 20% response (ACR20) was achieved 2 weeks after the last injection by 86.7% of subjects receiving single SC doses, 85.7% receiving SC doses every 2 weeks, 85.7% receiving both IV and SC doses, 57.1% receiving multiple IM doses, and 80.0% receiving SC doses every 4 weeks.SC and IM treatment with this experimental infliximab formulation was well tolerated and was associated with a favorable ACR response.

We postulated that antibodies to platelet factor 4/heparin complex lead to a heightened inflammatory state, contributing to an increased risk of recurrent thrombotic events.We analyzed serum from a subset of patients in the placebo/unfractionated heparin arm of the GUSTO IV-ACS trial who had prior heparin exposure. We selected 109 patients with the 30 day primary endpoint (death, MI or revascularization) and an equal number of controls, excluding patients with thrombocytopenia. Anti-platelet factor 4/heparin antibodies and inflammatory markers (sVCAM-1, sICAM-1, sE-selectin, sP-selectin, us-CRP, IL-6) were measured on serum samples.Patients with anti-PF4/heparin antibodies were more likely to have death or MI (30.4% vs. 11.3%, p = 0.01), or MI (21.7% vs. 6.2%, p = 0.01) than patients who were antibody negative. In a multiple logistic regression model that included inflammatory markers and clinical risk factors, antibody to PF4/heparin was a strong predictor of 30 day MI (odds ratio: 9.0; 95% confidence intervals 2.1-38.6; p0.01), with IL-6 being the only other predictor (odds ratio: 1.1; 95% confidence intervals 1.0-1.2; p = 0.03). Antibody positive patients had higher levels of sVCAM-1 (892 +/- 263 microg/l versus 780 +/- 228 microg/l; p = 0.04) and sICAM-1(246 +/- 50 microg/l versus 222 +/- 71microg/l; p = 0.02) than antibody-negative patients.Antibodies to the platelet factor 4/heparin complex were associated with elevated levels of endothelial but not platelet activation markers, or markers of a systemic inflammatory state. Anti-PF4/heparin antibodies were associated with a 9-fold increased risk of recurrent MI at 30 days. We measured soluble cell adhesion molecules, CRP and IL-6 from 218 non-thrombocytopenic patients, 23 of whom had antibodies to PF4/heparin. Antibody positive patients had higher levels of sVCAM-1 (892 +/- 263 microg/l versus 780 +/- 228 microg/l; p = 0.04) and sICAM-1(246 +/- 50 microg/l versus 222 +/- 71 microg/l; p = 0.02). In a multiple logistic regression model, antibody to PF4/heparin was a predictor of 30 day MI (odds ratio: 9.0; 95% CI: 2.1-38.6; p0.01). The presence of antibodies to PF4/heparin, even in the absence of thrombocytopenia, is a stronger predictor of 30 day MI than clinical variables or inflammatory markers.

An integrated structural pharmacokinetic/pharmacodynamic (PK/PD) model was developed for the glycoprotein IIb/IIIa antagonist abciximab administered to patients undergoing percutaneous transluminal coronary angioplasty. PK/PD data, in the form of plasma abciximab concentrations and ex vivo platelet aggregation in the presence of 20 microM adenosine diphosphate, were obtained from two previously conducted clinical studies. Study 1 consisted of patients who were given abciximab as a single intravenous injection of 0.25 mg/kg (n = 32). Patients in study 2 received an identical bolus dose, followed by a 36-h infusion at 0.125 microg/kg/min (n = 15). The PK component of the final model included drug-receptor binding, nonspecific distribution, and linear systemic clearance, whereas the PD module assumed that ex vivo dynamics were controlled by free plasma drug concentration. Mean PK/PD data from both studies were fitted simultaneously using nonlinear regression. PK profiles from both studies show rapidly decreasing plasma abciximab concentrations at early time points, but with extended terminal disposition phases. The maximum effect (Emax = 83.6%) was achieved rapidly and gradually returned to baseline values, although inhibition could be measured long after abciximab concentrations dropped below the detection limit. The final model well described the resulting PK/PD profiles and allowed for parameter estimation with relatively small coefficients of variation. Simulations were conducted to assess predicted receptor-occupancy and effects of selected parameters on PK/PD profiles. Models such as the one developed in this study demonstrate how drug binding to pharmacological targets may influence the PK of certain drugs and also provide a suitable paradigm for defining the PK/PD relationships of similar compounds.

Introduction: Platelet aggregates form by fibrinogen binding to the membrane receptor glycoprotein IIb/IIIa (GPIIb/IIIa). While GPIIb/IIIa inhibitors block fibrinogen-platelet binding, stimulation of other functionally important platelet receptors may still occur. Blocking the GPIIb/IIIa receptor prevents platelet aggregation but not activation and the subsequent effect on other platelet pathways is largely unknown. Materials and methods: As activated platelets release reactive oxygen species that may influence thrombosis or vascular function, the effect of GPIIb/IIIa inhibitors on the platelet release of nitric oxide (NO) and superoxide was determined using an electrochemical detector and luminescence, respectively. Location of relevant platelet proteins and the interaction between platelets and leukocytes in the presence or absence of GPIIb/IIIa inhibition was determined. Results: Although incubation with GPIIb/IIIa inhibitors completely abolished platelet aggregation, stimulation dependent NO release was significantly enhanced. Superoxide is known to alter the bioavailability of NO, and its contribution to the GPIIb/IIIa dependent increase in NO release was determined. In the presence of GPIIb/IIIa inhibitors, platelet superoxide release was significantly decreased. Preincubation with GPIIb/IIIa inhibitors also modified aggregation induced membrane translocation of the platelet proteins, endothelial NO synthase (eNOS) and NADPH oxidase (p67phox and p47phox), known to contribute to the generation of NO and superoxide, respectively. In the presence of leukocytes, abciximab incubation led to enhanced NO release and attenuated superoxide generation. Conclusion: These observations suggest that the pharmacological effects of GPIIb/IIIa antagonists on platelet function, apart from inhibition of aggregation, may contribute to their efficacy.

Background The Pl A2 polymorphism of the glycoprotein IIb/IIIa (fibrinogen) receptor has been associated with increased restenosis and stent thrombosis. We postulated that this allele could alter the antiplatelet effect of abciximab in patients undergoing percutaneous coronary intervention. Methods Optical platelet aggregation assays, Ultegra (Accumetrics, San Diego) rapid platelet function assays, and radiometric abciximab binding assays were performed in 66 Pl A1/A1 and 21 Pl A1/A2 patients undergoing percutaneous coronary interventions. The affinity of abciximab for the Pl A1 and Pl A2 receptors was determined with use of transfected cells. Results Compared with Pl A1/A1 homozygotes, Pl A1/A2 platelets were less completely inhibited after abciximab bolus ( P =.002) and at 24 hours ( P =.02) as assessed by the rapid platelet function assays. Optical aggregation assays confirmed that Pl A1/A2 platelets were less completely inhibited after abciximab bolus ( P =.05). The radiometric abciximab binding assay demonstrated that the Pl A1/A2 platelets had fewer baseline fibrinogen receptors than did the Pl A1/A1 platelets ( P =.04) and more free fibrinogen receptors at 24 hours ( P =.008). Cells transfected to express homozygous Pl A1 or Pl A2 demonstrated a nonsignificant trend ( P =.12) for reduced abciximab affinity for Pl A2 . Conclusions Pl A1/A2 platelets are less completely inhibited with abciximab, contributing to the observed interindividual variability in platelet function inhibition. Because the extent of platelet inhibition is an independent predictor for the risk of major adverse coronary events after percutaneous coronary intervention, the relative resistance of Pl A2 -positive platelets may contribute to a less favorable outcome in these patients. (Am Heart J 2002;143:76-82.)

Background We examined the ex vivo platelet aggregation profiles of patients who underwent percutaneous coronary intervention and received either abciximab, ticlopidine, or both agents. Study Design and Methods The trial was a prospective, nonrandomized, single-center, open-label study of 42 patients undergoing percutaneous coronary intervention who received the following regimens: group 1, abciximab (0.25 mg/kg bolus and 12-hour, 0.125 μg/kg per minute infusion); group 2, ticlopidine (250 mg twice daily for 14 consecutive days, initiated 12 to 18 hours before intervention); group 3, abciximab plus ticlopidine initiated 12 to 18 hours before procedure; and group 4, abciximab plus ticlopidine initiated 72 to 96 hours before procedure. Platelet aggregation measurements to adenosine diphosphate (ADP) and a thrombin receptor activating peptide (TRAP, 8 μmol/L) were obtained before ticlopidine treatment, after initiation of ticlopidine, and immediately before abciximab treatment and intervention, then at several time periods after onset of abciximab treatment. Platelet surface abciximab levels were monitored by flow cytometry. Results Neither ticlopidine regimen resulted in appreciable platelet inhibition before intervention and before administration of abciximab. In the ticlopidine-only arm, suppression of platelet aggregation to the weakest stimuli (5 μmol/L ADP; 23% ± 7.5%) was detected within 24 hours after intervention, with maximal inhibition to both 5 and 20 μmol/L ADP observed 7 days after intervention (48% ± 7.9% and 18% ± 8.7%, respectively). In contrast, ticlopidine marginally suppressed TRAP-mediated platelet activation at times when maximal effects on ADP-mediated platelet aggregation were evident. Neither ticlopidine regimen appreciably enhanced platelet inhibition during or shortly after cessation of abciximab treatment. For all 3 abciximab treatment arms, profound inhibition of ADP-induced (>80%) and TRAP-induced (>65%) platelet aggregation was observed 2 hours after treatment. In the abciximab-only arm, platelet aggregation responses gradually recovered, with the rate of response directly proportional to the strength of stimuli. However, in the ticlopidine plus abciximab arms, recovery of platelet aggregation at later times (7 and 14 days) reached a plateau and reflected the extent of inhibition observed in ticlopidine-treated patients. No difference in the clearance of surface-bound abciximab from circulating platelets was observed between the abciximab and abciximab plus ticlopidine arms. Conclusions Concomitant abciximab plus ticlopidine treatment yields a platelet inhibition profile that is a composite of the effects of the 2 agents. In the early stages of treatment, inhibition of ex vivo platelet aggregation was mediated primarily by abciximab; effects were more moderate and were predominately mediated by ticlopidine. (Am Heart J 2000;140:492-501.)

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 α IIb β 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.