Safety and pharmacokinetics of the antisense oligonucleotide (ASO) LY2181308 as a single-agent or in combination with idarubicin and cytarabine in patients with refractory or relapsed acute myeloid leukemia (AML).

Survivin is expressed in tumor cells, including acute myeloid leukemia (AML), regulates mitosis, and prevents tumor cell death. The antisense oligonucleotide sodium LY2181308 (LY2181308) inhibits survivin expression and may cause cell cycle arrest and restore apoptosis in AML. In this study, the safety, pharmacokinetics, and pharmacodynamics/efficacy of LY2181308 was examined in AML patients, first in a cohort with monotherapy (n = 8) and then post-amendment in a cohort with the combination of cytarabine and idarubicin treatment (n = 16). LY2181308 was administered with a loading dosage of three consecutive daily infusions of 750 mg followed by weekly intravenous (IV) maintenance doses of 750 mg. Cytarabine 1.5 g/m(2) was administered as a 4-hour IV infusion on Days 3, 4, and 5 of Cycle 1, and idarubicin 12 mg/m(2) was administered as a 30-minute IV infusion on Days 3, 4, and 5 of Cycle 1. Cytarabine and idarubicin were administered on Days 1, 2, and 3 of each subsequent 28-day cycle. Reduction of survivin was evaluated in peripheral blasts and bone marrow. Single-agent LY2181308 was well tolerated and survivin was reduced only in patients with a high survivin expression. In combination with chemotherapy, 4/16 patients had complete responses, 1/16 patients had incomplete responses, and 4/16 patients had cytoreduction. Nine patients died on study: 6 (monotherapy), 3 (combination). LY2181308 alone is well tolerated in patients with AML. In combination with cytarabine and idarubicin, LY2181308 does not appear to cause additional toxicity, and has shown some clinical benefit needing confirmation in future clinical trials.

Cytochrome p-450 polymorphisms and response to clopidogrel.

BACKGROUND: Clopidogrel requires transformation into an active metabolite by cytochrome P-450 (CYP) enzymes for its antiplatelet effect. The genes encoding CYP enzymes are polymorphic, with common alleles conferring reduced function. METHODS: We tested the association between functional genetic variants in CYP genes, plasma concentrations of active drug metabolite, and platelet inhibition in response to clopidogrel in 162 healthy subjects. We then examined the association between these genetic variants and cardiovascular outcomes in a separate cohort of 1477 subjects with acute coronary syndromes who were treated with clopidogrel in the Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel-Thrombolysis in Myocardial Infarction (TRITON-TIMI) 38. RESULTS: In healthy subjects who were treated with clopidogrel, carriers of at least one CYP2C19 reduced-function allele (approximately 30% of the study population) had a relative reduction of 32.4% in plasma exposure to the active metabolite of clopidogrel, as compared with noncarriers (P<0.001). Carriers also had an absolute reduction in maximal platelet aggregation in response to clopidogrel that was 9 percentage points less than that seen in noncarriers (P<0.001). Among clopidogrel-treated subjects in TRITON-TIMI 38, carriers had a relative increase of 53% in the composite primary efficacy outcome of the risk of death from cardiovascular causes, myocardial infarction, or stroke, as compared with noncarriers (12.1% vs. 8.0%; hazard ratio for carriers, 1.53; 95% confidence interval [CI], 1.07 to 2.19; P=0.01) and an increase by a factor of 3 in the risk of stent thrombosis (2.6% vs. 0.8%; hazard ratio, 3.09; 95% CI, 1.19 to 8.00; P=0.02). CONCLUSIONS: Among persons treated with clopidogrel, carriers of a reduced-function CYP2C19 allele had significantly lower levels of the active metabolite of clopidogrel, diminished platelet inhibition, and a higher rate of major adverse cardiovascular events, including stent thrombosis, than did noncarriers.

Association of long-term administration of the survivin mRNA-targeted antisense oligonucleotide LY2181308 with reversible kidney injury in a patient with metastatic melanoma.

A 57-year-old man with metastatic melanoma was treated with the survivin inhibitor and antisense oligonucleotide LY2181308 as part of a First-in-Human Dose trial. After 18 months of treatment, he developed kidney injury and the treatment was discontinued. At 9 months and before the development of kidney injury, LY2181308 concentrations were 8- to 10-fold higher relative to median predicted values, but within the targeted exposure considered to be safe. However, at 17 months, 28 days after stopping LY2181308 therapy, LY2181308 concentration exceeded the predicted range by 38-fold. His decreased kidney function was slow to improve after stopping treatment. A kidney biopsy showed signs of acute tubular injury with regeneration. Complete recovery of kidney function occurred 6 months after treatment was stopped. The relationship between high exposures and slow LY2181308 clearance with the gradual improvement in kidney function after stopping the antisense treatment suggests that the oligonucleotide was related to the kidney injury. Based on this case report, kidney function should be monitored frequently in patients receiving long-term treatment with antisense oligonucleotides that specifically target survivin, particularly when they receive concomitant angiotensin-converting enzyme inhibitors or nonsteroidal anti-inflammatory drugs.

Cytochrome P450 genetic polymorphisms and the response to prasugrel: relationship to pharmacokinetic, pharmacodynamic, and clinical outcomes.

BACKGROUND: Both clopidogrel and prasugrel require biotransformation to active metabolites by cytochrome P450 (CYP) enzymes. Among persons treated with clopidogrel, carriers of reduced-function CYP2C19 alleles have significantly lower levels of active metabolite, diminished platelet inhibition, and higher rates of adverse cardiovascular events. The effect of CYP polymorphisms on the clinical outcomes in patients treated with prasugrel remains unknown. METHODS AND RESULTS: The associations between functional variants in CYP genes, plasma concentrations of active drug metabolite, and platelet inhibition in response to prasugrel were tested in 238 healthy subjects. We then examined the association of these genetic variants with cardiovascular outcomes in a cohort of 1466 patients with acute coronary syndromes allocated to treatment with prasugrel in the Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition With Prasugrel-Thrombolysis in Myocardial Infarction 38 trial. Among the healthy subjects, no significant attenuation of the pharmacokinetic or the pharmacodynamic response to prasugrel was observed in carriers versus noncarriers of at least 1 reduced-function allele for any of the CYP genes tested (CYP2C19, CYP2C9, CYP2B6, CYP3A5, and CYP1A2). Consistent with these findings, in subjects with acute coronary syndromes treated with prasugrel, no significant associations were found between any of the tested CYP genotypes and risk of cardiovascular death, myocardial infarction, or stroke. CONCLUSIONS: Common functional CYP genetic variants do not affect active drug metabolite levels, inhibition of platelet aggregation, or clinical cardiovascular event rates in persons treated with prasugrel. These pharmacogenetic findings are in contrast to observations with clopidogrel, which may explain, in part, the different pharmacological and clinical responses to the 2 medications.

Genetic variation of CYP2C19 affects both pharmacokinetic and pharmacodynamic responses to clopidogrel but not prasugrel in aspirin-treated patients with coronary artery disease.

AIMS: The metabolic pathways leading to the formation of prasugrel and clopidogrel active metabolites differ. We hypothesized that decreased CYP2C19 activity affects the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. METHODS AND RESULTS: Ninety-eight patients with coronary artery disease (CAD) taking either clopidogrel 600 mg loading dose (LD)/75 mg maintenance dose (MD) or prasugrel 60 mg LD/10 mg MD were genotyped for variation in six CYP genes. Based on CYP genotype, patients were segregated into two groups: normal function (extensive) metabolizers (EM) and reduced function metabolizers (RM). Plasma active metabolite concentrations were measured at 30 min, 1, 2, 4, and 6 h post-LD and during the MD period on Day 2, Day 14, and Day 29 at 30 min, 1, 2, and 4 h. Vasodilator-stimulated phosphoprotein (VASP) and VerifyNow P2Y12 were measured predose, 2, and 24 +/- 4 h post-LD and predose during the MD period on Day 14 +/- 3 and Day 29 +/- 3. For clopidogrel, active metabolite exposure was significantly lower (P = 0.0015) and VASP platelet reactivity index (PRI, %) and VerifyNow P2Y(12) reaction unit (PRU) values were significantly higher (P < 0.05) in the CYP2C19 RM compared with the EM group. For prasugrel, there was no statistically significant difference in active metabolite exposure or pharmacodynamic response between CYP2C19 EM and RM. Variation in the other five genes demonstrated no statistically significant differences in pharmacokinetic or pharmacodynamic responses. CONCLUSION: Variation in the gene encoding CYP2C19 in patients with stable CAD contributes to reduced exposure to clopidogrel's active metabolite and a corresponding reduction in P2Y(12) inhibition, but has no significant influence on the response to prasugrel.

Assessment of P2Y(12) inhibition with the point-of-care device VerifyNow P2Y12 in patients treated with prasugrel or clopidogrel coadministered with aspirin.

BACKGROUND: Variability in response to thienopyridines has led to the development of point-of-care devices to assess adenosine diphosphate (ADP)-induced platelet aggregation. These tests need to be evaluated in comparison to reference measurements of P2Y(12) function during different thienopyridine treatments. METHODS: After a run-in on 75 mg aspirin, 110 subjects were randomized to double-blind treatment with clopidogrel 600 mg loading dose (LD)/75 mg maintenance dose (MD) or prasugrel 60 mg LD/10 mg MD. Antiplatelet effects were evaluated by VerifyNow P2Y12 (VN-P2Y12) device (Accumetrics, San Diego, CA), vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay, and light transmission aggregometry (LTA). Prasugrel's and clopidogrel's active metabolite concentration were also determined. RESULTS: Dose- and time-dependent inhibition of P2Y(12) was evident with VN-P2Y12. There was strong correlation with VN-P2Y12 and VASP or LTA for all treatments through a wide range of P2Y(12) function. At high levels of P2Y(12) inhibition, platelet function measured by VN-P2Y12 was maximally inhibited and could not reflect further changes seen with VASP or LTA methods. Correlation was also observed between exposure to clopidogrel's active metabolite and VN-P2Y12 during MD and LD, whereas it was observed only with prasugrel MD. CONCLUSION: The VN-P2Y12 correlated strongly with inhibition of P2Y(12) function, as measured with either VASP or LTA. VN-P2Y12 also correlated to exposure to the active metabolite of prasugrel and clopidogrel up to levels associated with assumed saturation of the P2Y(12) receptor.

Inhibition of platelet aggregation with prasugrel and clopidogrel: an integrated analysis in 846 subjects.

This integrated analysis compared speed of onset, level of platelet inhibition, and response variability to prasugrel and clopidogrel in healthy subjects and in patients with stable coronary artery disease with data pooled from 24 clinical pharmacology studies. Data from subjects (N = 846) were categorized into the following treatment groups: prasugrel 60 mg loading dose (LD)/10 mg maintenance dose (MD), clopidogrel 300 mg LD/75 mg MD, or clopidogrel 600 mg LD/75 mg MDs. Maximum platelet aggregation (MPA) and inhibition of platelet aggregation (IPA) to 5 and 20 muM ADP were assessed by turbidimetric aggregometry. A linear mixed-effect model compared the MPA and IPA between treatments over time points evaluated in the integrated database, and covariates affecting platelet inhibition were identified. Prasugrel 60 mg LD resulted in faster onset, greater magnitude, and more consistent levels of inhibition of platelet function compared to either clopidogrel 300 mg or 600 mg LDs. Greater and more consistent levels of platelet inhibition were observed with the prasugrel 10 mg MD compared to the clopidogrel 75 mg MD. This integrated analysis confirms the findings of earlier individual studies, that prasugrel achieves faster onset of greater extent and more consistent platelet inhibition compared to the approved and higher loading doses of clopidogrel. Gender, race, body weight, and age were identified as statistically significant covariates impacting platelet inhibition.

A comparison of the antiplatelet effects of prasugrel and high-dose clopidogrel as assessed by VASP-phosphorylation and light transmission aggregometry.

Platelet inhibition as measured by vasodilator-stimulated phosphoprotein (VASP) and light transmission aggregometry (LTA) have shown concordance following dosing of clopidogrel. No reports have directly compared the VASP assay and LTA at the levels of P2Y(12) blockade after loading doses (LDs) of prasugrel or high dose clopidogrel (600 and 900 mg). The aim was to compare the VASP assay and LTA during the loading dose phase of a comparative study of prasugrel and clopidogrel. Prasugrel 60 mg LD/10 mg maintenance dose (MD) and clopidogrel 300 mg/75 mg and 600 mg/75 mg LD/MD regimens were compared in a 3-way crossover study in 41 healthy, aspirin-free subjects. Each LD was followed by seven daily MDs and a 14-day washout period. P2Y(12) receptor blockade was estimated using the VASP assay, expressed as platelet reactivity index (VASP-PRI). Platelet aggregation was assessed by light transmission aggregometry (20 and 5 microM ADP). Twenty-four hours after prasgurel 60 mg or clopidogrel 300 mg and 600 mg, respectively, VASP-PRI decreased from approximately 80% to 8.9%, 54.7%, and 39.0%, and maximal platelet aggregation (MPA) decreased from approximately 79% to 10.8%, 42.7%, and 31.2%, with an overall VASP:MPA correlation of 0.88 (p < 0.01). VASP assay responses after the clopidogrel LDs showed a wider range of values (300 mg: 0-93%; 600 mg: 0-80%) than prasugrel (0-13%); MPA responses followed a similar trend. Pearson's correlation suggested a strong agreement between VASP and LTA (20 microM ADP) for MPA (r = 0.86, p < 0.0001). VASP and LTA demonstrated concordance across the response range of P2Y(12) receptor blockade following thienopyridine LDs.

Greater reduction of platelet activation markers and platelet-monocyte aggregates by prasugrel compared to clopidogrel in stable coronary artery disease.

Prasugrel, a novel P2Y(12) ADP-receptor antagonist, has been reported to achieve greater inhibition of platelet aggregation compared to clopidogrel as assessed by light transmission aggregometry. It was the objective of this study to investigate the effect of prasugrel on alternative markers of platelet activation in comparison to a high loading dose and the approved maintenance dose of clopidogrel. One hundred ten aspirin-treated patients with stable coronary artery disease were randomized to a loading dose (LD, day 1)/ maintenance dose (MD, days 2-29) of prasugrel 60 mg/10 mg or clopidogrel 600 mg/75 mg. Platelet activation markers were analyzed by whole blood flow cytometry pre-dose and at 2 and 24 hours after LD and pre-dose at 14 and 29 days. After stimulation with 20 muM ADP, 2 hours after LD, significantly lower expression of activated GPIIb/IIIa (4.3 vs. 21.8 [mean fluorescent intensity (MFI)], p < 0.001) and P-selectin (2.0 vs. 11.7 MFI, p < 0.001) along with decreased formation of platelet-monocyte aggregates (16.4% vs. 29.6% positive cells, p < 0.001) was observed with prasugrel versus clopidogrel. All these effects were maintained through 24 hours and during the MD period. In conclusion, prasugrel 60 mg LD and 10 mg MD inhibit several markers of platelet activation and the formation of platelet-monocyte aggregates more effectively than a 600 mg LD and 75 mg MD of clopidogrel. Attenuated platelet aggregation and reduced expression of platelet pro-coagulant and pro-inflammatory markers with prasugrel suggest the potential to reduce cardiovascular events both in the acute setting and in long-term treatment.

The use of the VerifyNow P2Y12 point-of-care device to monitor platelet function across a range of P2Y12 inhibition levels following prasugrel and clopidogrel administration.

Variability in response to antiplatelet agents has prompted the development of point-of-care (POC) technology. In this study, we compared the VerifyNow P2Y12 (VN-P2Y12) POC device with light transmission aggregometry (LTA) in subjects switched directly from clopidogrel to prasugrel. Healthy subjects on aspirin were administered a clopidogrel 600 mg loading dose (LD) followed by a 75 mg/d maintenance dose (MD) for 10 days. Subjects were then switched to a prasugrel 60 mg LD and then 10 mg/d MD for 10 days (n = 16), or to a prasugrel 10 mg/d MD for 11 days (n = 19). Platelet function was measured by LTA and VN-P2Y12 at baseline and after dosing. Clopidogrel 600 mg LD/75 mg MD treatment led to a reduction in P2Y(12) reaction units (PRU) from baseline. A switch from clopidogrel MD to prasugrel 60 mg LD/10 mg MD produced an immediate decrease in PRU, while a switch to prasugrel 10 mg MD resulted in a more gradual decline. Consistent with the reduction in PRU, device-reported percent inhibition increased during both clopidogrel and prasugrel regimens. Inhibition of platelet aggregation as measured by LTA showed a very similar pattern to that found with VN-P2Y12 measurement, irrespective of treatment regimens. The dynamic range of VN-P2Y12 appeared to be narrower than that of LTA. With two different thienopyridines, the VN-P2Y12 device, within a somewhat more limited range, reflected the overall magnitude of change in aggregation response determined by LTA. The determination of the clinical utility of such POC devices will require their use in clinical outcome studies.

Effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel.

Prasugrel and clopidogrel, thienopyridine prodrugs, are each metabolized to an active metabolite that inhibits the platelet P2Y(12) ADP receptor. In this open-label, 4-period crossover study, the effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel were assessed in healthy subjects given single doses of prasugrel 60 mg and clopidogrel 300 mg with and without concurrent lansoprazole 30 mg qd. C(max) and AUC(0-tlast) of prasugrel's active metabolite, R-138727, and clopidogrel's inactive carboxylic acid metabolite, SR26334, were assessed. Inhibition of platelet aggregation (IPA) was measured by turbidimetric aggregometry 4 to 24 hours after each treatment. Lansoprazole (1) decreased R-138727 AUC(0-tlast) and C(max) by 13% and 29%, respectively, but did not affect IPA after the prasugrel dose, and (2) did not affect SR62334 exposure but tended to lower IPA after a clopidogrel dose. A retrospective tertile analysis showed in subjects with high IPA after a clopidogrel dose alone that lansoprazole decreased IPA, whereas IPA was unaffected in these same subjects after a prasugrel dose. The overall data suggest that a prasugrel dose adjustment is not likely warranted in an individual taking prasugrel with a proton pump inhibitor such as lansoprazole.

Effect of atorvastatin on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel in healthy subjects.

STUDY OBJECTIVE: To investigate the potential effect of atorvastatin 80 mg/day on the pharmacokinetics and pharmacodynamics of the thienopyridines prasugrel and clopidogrel. DESIGN: Open-label, randomized, crossover, two-arm, parallel-group study. SETTING: Single clinical research center in the United Kingdom. PARTICIPANTS: Sixty-nine healthy men aged 18-60 years. Intervention. Subjects received either a loading dose of prasugrel 60 mg followed by a maintenance dose of 10 mg/day or a loading dose of clopidogrel 300 mg followed by 75 mg/day. The drug was given as monotherapy for 10 days, and after a 6-day run-in period with atorvastatin 80 mg/day, the same dosage of atorvastatin was continued with the respective thienopyridine for 10 days. A 14-day washout period separated the treatment regimens. MEASUREMENTS AND MAIN RESULTS: Blood samples were collected before and at various time points after dosing on days 1 and 11 for determination of plasma concentrations of metabolites and for measurement of platelet aggregation induced by adenosine 5'-diphosphate 20 microM and vasodilator-stimulated phosphoprotein (VASP). Coadministration of atorvastatin did not alter exposure to active metabolites of prasugrel or clopidogrel after the loading dose and thus did not alter inhibition of platelet aggregation (IPA). During maintenance dosing, atorvastatin administration resulted in 17% and 28% increases in the area under the plasma concentration-time curve (AUC) values of prasugrel's and clopidogrel's active metabolites, respectively. These small changes in AUC did not result in a significant change in IPA response to prasugrel but did result in a significant increase in IPA during clopidogrel maintenance dosing at some, but not all, of the time points on day 11. Coadministration of atorvastatin with either prasugrel or clopidogrel had no effect on VASP phosphorylation relative to the thienopyridine alone after the loading dose. CONCLUSION: Coadministration of atorvastatin 80 mg/day with prasugrel or clopidogrel did not negatively affect the antiplatelet response to either drug after a loading dose or during maintenance dosing. The lack of a clinically meaningful effect of high-dose atorvastatin on the pharmacodynamic response to prasugrel after the loading or maintenance dose indicates that no dosage adjustment should be necessary in patients receiving these drugs concomitantly.

Switching directly to prasugrel from clopidogrel results in greater inhibition of platelet aggregation in aspirin-treated subjects.

Prasugrel, a novel P2Y(12) antagonist, achieves faster onset and greater inhibition of platelet aggregation than clopidogrel 300 and 600 mg loading doses (LD). We studied the safety, time course, and level of platelet inhibition when switching directly from clopidogrel 75 mg maintenance dose (MD) to a prasugrel 60 mg LD/10 mg MD or 10 mg MD regimen. Healthy subjects (n = 39) on aspirin (81 mg/d) received a clopidogrel 600 mg LD followed by 10 days of clopidogrel MD (75 mg/d). Subjects were then randomized without a washout period to prasugrel 60 mg LD (n = 16) followed by 10 days of prasugrel MD (10 mg/d) or to prasugrel MD (10 mg/d, n = 19) for 11 days. Maximal platelet aggregation (MPA) to 20 microM ADP was measured by turbidimetric aggregometry. In subjects on clopidogrel 75 mg MD, mean MPA decreased from 39 to 12% by 30 minutes, and to 5% by 1 hour after a prasugrel 60 mg LD (p < 0.001 for both) and from 37 to 28% (p < 0.001) by 1 hour after a prasugrel 10 mg MD. During prasugrel MD, a new pharmacodynamic steady state MPA of approximately 24% (p < 0.01 vs. clopidogrel MD) occurred within four to five days of switching from clopidogrel. Changing from clopidogrel to prasugrel did not increase bleeding episodes or other adverse events. Switching directly from clopidogrel MD to either prasugrel LD or MD was well tolerated and resulted in significantly greater levels of platelet inhibition than a clopidogrel 75 mg MD.

A comparison of prasugrel and clopidogrel loading doses on platelet function: magnitude of platelet inhibition is related to active metabolite formation.

BACKGROUND: The aim of this study was to compare rate of onset, magnitude, and consistency of platelet inhibition after administration of prasugrel or clopidogrel and to relate platelet inhibition to systemic exposure to each active metabolite. Thienopyridines are prodrugs, metabolized in vivo to active metabolites that inhibit the platelet P2Y12 adenosine diphosphate (ADP) receptor. METHODS: This was an open-label, 2-way, crossover study that randomized healthy subjects (n = 68) to an oral loading dose (LD) of prasugrel 60 mg or clopidogrel 300 mg. Platelet aggregation response to 5 and 20 micromol/L of ADP was measured by turbidometric aggregometry. Plasma concentrations of the active metabolites of prasugrel and clopidogrel were quantified by liquid chromatography with tandem mass spectrometry detection methods. RESULTS: Inhibition of platelet aggregation (IPA) after prasugrel was significantly higher (P < .01) than that after clopidogrel from 15 minutes through 24 hours (5 micromol/L ADP) and from 30 minutes through 24 hours (20 micromol/L ADP). For 20 micromol/L ADP, the median time to reach > or = 20% IPA was 30 minutes for prasugrel and 1.5 hours for clopidogrel (P < .001). The maximum IPA was 84.1% +/- 9.5% with prasugrel versus 48.9% +/- 27.0% with clopidogrel for 5 micromol/L ADP and 78.8% +/- 9.2% versus 35.0% +/- 24.5%, respectively, for 20 micromol/L ADP (P < .001). Response to prasugrel was more consistent compared to clopidogrel (P < .01). The lower IPA response to clopidogrel was associated with lower plasma concentrations of its active metabolite (P < .001). CONCLUSIONS: Prasugrel 60 mg LD results in more rapid, potent, and consistent inhibition of platelet function than clopidogrel 300 mg LD. Lower IPA responses to clopidogrel were associated with lower concentrations of its active metabolite.

Comparison of speed of onset of platelet inhibition after loading doses of clopidogrel versus prasugrel in healthy volunteers and correlation with responder status.

Failure to achieve an adequate level of platelet inhibition during percutaneous coronary intervention is associated with an increased risk for periprocedural myocardial injury. This study was conducted to compare the initial rate of platelet inhibition after a loading dose (LD) of prasugrel or clopidogrel and determine the association between the initial rate of inhibition and pharmacodynamic responder status. Data were pooled from 3 studies in which healthy subjects received LDs of prasugrel (60 mg; n = 76) or clopidogrel (300 mg; n = 87). Maximum platelet aggregation (MPA; 20 mumol/L adenosine diphosphate) was measured by turbidimetric aggregometry (0.25 to 24 hours after dosing). A mechanistic model was used to estimate the initial rate of decrease in MPA per hour (fast onset: MPA decrease >20%/hour). Subjects were defined as pharmacodynamic poor responders if the absolute decrease in MPA from baseline was <15% at either 4 to 5 or 24 hours after dosing. The median initial rate of decrease in MPA was greater after prasugrel (203%/hour) than with clopidogrel (23%/hour) (p <0.001). Overall, 76 subjects (100%) receiving prasugrel had fast onset of platelet inhibition compared with 47 subjects (54%) receiving clopidogrel. The initial rate of decrease in MPA was highly correlated with responder status (p <0.001). After prasugrel, subjects had a lower median MPA compared with clopidogrel (p <0.001; from >0.25 to 24 hours after dosing), and intersubject variability in MPA response was less after prasugrel compared with clopidogrel (p <0.001; from >1 to 24 hours after dosing). In conclusion, platelet inhibition after a 60-mg LD of prasugrel was more rapid in onset, less variable, and greater in magnitude than with a 300-mg LD of clopidogrel. After a thienopyridine LD, the initial rate of platelet inhibition was predictive of pharmacodynamic responder status.

Greater inhibition of platelet aggregation and reduced response variability with prasugrel versus clopidogrel: an integrated analysis.

Multiple studies report response variability to a 300-mg clopidogrel loading dose (LD). Pooled platelet aggregometry data compared responses (change in maximal platelet aggregation [DeltaMPA] or inhibition of platelet aggregation [IPA]) to clopidogrel 300-mg (n = 131) or prasugrel 60-mg (n = 109) LDs. Poor responder rates were determined using empiric criteria (IPA < 10% and DeltaMPA < 10% for 20 microM and 5 microM adenosine diphosphate [ADP]) and Bayesian model-based criteria (IPA < 20% and DeltaMPA < 15% for 20 microM ADP; IPA < 25% and DeltaMPA < 20% for 5 microM ADP). Prasugrel achieved greater DeltaMPA and IPA from 2 to 24 hours post-LD (P < .001). For 20 microM ADP, poor responder rates for clopidogrel ranged from 17% to 43%; no prasugrel poor responders were observed. Regardless of the criterion, prasugrel 60 mg achieved greater IPA and fewer poor responders than the clopidogrel 300-mg LD.

Differential reactivity of anti-primate and anti-human secondary antibodies against human and monkey immunoglobulins: implications for determining the sensitivity of immunogenicity assays.

Development of immunogenicity assays for assessment of human antibodies to therapeutic proteins requires a quantitative determination of assay sensitivity. In the absence of true human positive controls, this is usually accomplished by utilizing affinity-purified antibodies from non-human primates or monoclonal antibodies. In the former case, it is generally considered that non-human primate antibodies will be recognized equally to human antibodies by secondary anti-human immunoglobulin reagents used in immunogenicity assays. We present results here demonstrating that this is not the case. In reality, anti-human immunoglobulin secondary antibodies do not recognize primate immunoglobulins as well as human immunoglobulins. As a result, the use of affinity purified primate antibodies to determine the sensitivity of an immunogenicity assay will likely result in the true sensitivity of the assay being underestimated.

Effect of double antigen bridging immunoassay format on antigen coating concentration dependence and implications for designing immunogenicity assays for monoclonal antibodies.

The double antigen bridging immunoassay has been used extensively for detection of immunogenicity responses to therapeutic monoclonal antibodies. We have analyzed parameters affecting performance of this type of immunoassay including microtiter plate antigen coating concentration, enzyme-labeled antigen conjugate dilution and assay format (one-step versus two-step). We present results demonstrating that the format of the assay has a significant impact on the optimal parameters to maximize assay performance. A one-step assay format achieves maximal sensitivity across a broad range of coating concentrations and at a lower concentration of conjugate than that in a two-step format. In contrast, a two-step format requires very low coating concentrations and higher conjugate concentrations to achieve maximal sensitivity and suffers from significantly reduced sensitivity at higher coating concentrations. Together, these findings indicate that a one-step assay format can greatly reduce the effect of coating concentration variation on assay performance.

Development, validation, and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines in human serum.

Quantification of biomarkers can provide important information about the safety and efficacy of candidate drugs. Unfortunately, limited sample volume and excess costs often limit analysis of multiple biomarkers. We developed, optimized, validated, and implemented a multiplex immunoassay for simultaneous measurement of multiple circulating cytokines: IL-1beta, TNFalpha, IL-6, IL-8, and IL-10. Multiplex immuoassays were performed using the Luminex LabMAP instrument. Capture antibodies for each cytokine were covalently bound to distinct microsphere subsets distinguished by differing dye ratios. The concentration of each individual cytokine determined by measuring orange fluorescence produced by a complex of a biotinylated cytokine-specific antibody and streptavidin-phycoerythrin. The lower limit of quantification for all assays was 20 pg/mL with the exception of IL-8 which was 100 pg/mL. The inter-assay precision was less than 25%CV for all analytes at all control levels both pre-study and in-study. The percent recovery ranged from 83 to 108% pre-study and 90 to 125% in-study. In a linearity assessment, a 15,000 pg/mL multi-analyte control could be diluted 1:50 and maintain expected accuracy. We measured the cytokine concentrations in more than 2000 serum samples from patients with sepsis. Multiplex results for IL-6 were compared to a conventional commercially available ELISA kit. The degree of agreement between the two methods as measured by the concordance correlation coefficient was 84.5%. Multiplex results were 2.36-fold higher than ELISA values on the average. After adjusting for this mean difference, the 95% empirical limits of agreement for the ratio of individual sample values were 0.33, 2.65. This multiplex immunoassay provided simultaneous measurement of circulating cytokines using 80% less patient specimen compared to traditional approaches and at a significantly decreased cost. Efficient use of this platform requires process improvements to fully maximize the positive impact of multiplex assays in clinical drug development.

Plasminogen and tissue-type plasminogen activator deficiency as risk factors for thromboembolic disease.

OBJECTIVE: To review the published evidence for an association between a deficiency of plasminogen or tissue-type plasminogen activator (tPA) and the risk of thrombosis. DATA SOURCES: Review of the medical literature, with an emphasis on the last 10 years. DATA EXTRACTION AND SYNTHESIS: After an initial assessment of the literature, including review of clinical study design and laboratory methods, a draft manuscript summarizing the findings was prepared and circulated to participants in the College of American Pathologists Conference on Diagnostic Issues in Thrombophilia. The key findings and each recommendation were presented for discussion at the conference. Recommendations were accepted if a consensus of the 27 experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form. CONCLUSIONS: The consensus of the conference was that routine laboratory assessment of plasminogen and tPA concentration in patients with thrombophilia is not warranted at this time. Analysis of plasminogen and tPA gene alterations in patients with thrombophilia is also not warranted at this time. Determination of plasminogen concentration should be performed in patients suspected of having ligneous conjunctivitis.