Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies

2011 ◽  
Vol 402 (3) ◽  
pp. 1229-1239 ◽  
Author(s):  
Shujie J. Wang ◽  
Steven T. Wu ◽  
Jochem Gokemeijer ◽  
Aberra Fura ◽  
Murli Krishna ◽  
...  
Keyword(s):  
Scaffold Protein ◽  
Plasma Samples ◽  
Post Dose ◽  
Monkey Plasma

Differential Inhibition of FLT3-ITD, FLT3-WT, and KIT By Quizartinib As Assessed By Modified PIA Assay

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 951-951
Author(s):  
Ronald Nepomuceno ◽  
Allison M Rooks ◽  
Michael Gardner ◽  
Robert Armstrong
Keyword(s):  
Western Blot ◽  
Inhibitory Activity ◽  
Ex Vivo ◽  
Single Subject ◽  
Plasma Samples ◽  
Active Metabolites ◽  
Post Dose ◽  
Drug Levels ◽  
Percent Inhibition ◽  
Nm Range

Abstract The plasma inhibitory activity (PIA) assay has been used to determine if sufficient exposure of FLT3 kinase inhibitors have been achieved to inhibit their target following clinical administration. Traditionally, clinical plasma samples have been used undiluted to determine inhibitory activity. In the case of quizartinib (AC220), undiluted plasma often results in full inhibition of ex vivo FLT3 activity, regardless of dose administered. To more closely examine the inhibitory properties of circulating quizartinib in a clinical trial, we modified the western blot PIA assay to utilize MSD-based phospho (p)FLT3 and pKIT ELISAs. This modification allows for the comparison of inhibitory activity between samples, resolution of activity against multiple targets from a single subject sample, as well as demonstrates the differences between administered quizartinib doses. A total of 76 relapsed/refractory FLT3-ITD AML subjects were enrolled in a Phase 2b study. Of those, 56 subjects (30 at 30 mg/day; 26 at 60 mg/day) had sufficient pre-dose and either day 8 or 15 plasma samples available for evaluation. Samples were tested undiluted and serially 4-fold diluted into normal human plasma. FLT3 inhibitory activity was determined in Ba/F3 cells exogenously expressing human FLT3 with an internal tandem duplication (ITD) mutation (ITD cells), and in THP-1 cells, where wild-type (WT) FLT3 phosphorylation was FLT3 ligand induced. Inhibitory activity on c-KIT in H256 cells with SCF-induced phosphorylation was also determined. Percent inhibition was calculated by comparing post-dose samples to equivalently diluted pre-dose samples. As in previous western blot-based PIA assays, undiluted plasma samples contained sufficient quizartinib and its active metabolites to inhibit FLT3 phosphorylation in ITD cells by 98.0 ± 3.3% and 100.4 ± 4.0% at 30 and 60 mg doses, respectively, suggesting exposures that completely inhibit cellular FLT3-ITD activity. Diluting the plasma samples allowed for determination of the IT50 (the plasma dilution titer resulting in 50% inhibition of kinase activity) to assess the excess active drug in each sample. For subjects receiving 30 mg, the median IT50 was 21.0 (range 7.3-45.9) (inverse dilution). For subjects receiving 60 mg, the median IT50 was 44.8 (range 19.1-135.5), indicating the expected 2-fold difference in inhibitory activity. The percent inhibition levels were significantly different between 30 and 60 mg doses at all dilutions in the ITD cells. For FLT3-WT in THP-1 cells, the mean inhibitions of ligand-induced phosphorylation in the undiluted samples were 77.6 ± 9.6% and 90.1 ± 5.6% with the two doses, indicating that FLT3-WT is not as well inhibited by quizartinib. The median IT50s were 4.3 (range 3.0-8.0) and 6.6 (range 3.7-13.6) for the 30 and 60 mg doses, consistent with less excess drug relative to FLT3-WT inhibition. KIT inhibitory activity was weaker at these doses of quizartinib. Undiluted plasmas had a mean inhibition of 43.3 ± 16.1% and 63.9 ± 14.1% at 30 and 60 mg, so IT50s could only be calculated for one subject at 30 mg and 10 subjects at 60 mg. KIT inhibition levels were still significantly different between the two doses in the undiluted and 1:4 diluted plasma samples, but were largely eliminated by the 1:16 dilution. Measured plasma levels of quizartinib and its metabolite AC886 were used to calculate the plasma IC50 values for each cell line. For ITD cells, the median IC50 was 20.5 nM (range 6.9-64.6 nM). In the THP-1 cells, it was 129 nM (range 52.1-552 nM). For KIT inhibition, the median IC50 was 442 nM (range 167-1577 nM). Of note, the median quizartinib + AC886 levels were 370 nM (range 205-802 nM) for 30 mg subjects, and 917 nM (range 291-4234 nM) for 60 mg subjects, indicating on average, the 30 mg subjects were 18-fold above the the IC50 for FLT3-ITD inhibition, and the 60 mg subjects were 45-fold above this IC50, and drug levels were 3-fold and 7-fold above the IC50 for FLT3-WT in the two doses. For KIT inhibition, most 30 mg subjects had drug levels below the KIT IC50, while 60 mg subjects had drug levels only 2-fold above it. These results suggest that quizartinib, when dosed at 30 mg or 60 mg achieved peripheral exposures sufficient for complete inhibition of FLT3-ITD as assessed by the PIA assay. At these same doses the degree of inhibition of FLT3-WT, and especially KIT is reduced. Implications of this data for patient response and myelosuppression will be available for presentation. Disclosures Nepomuceno: Ambit Biosciences Corporation: Employment. Rooks:Ambit Biosciences Corporation: Employment. Gardner:Ambit Biosciences Corporation: Employment. Armstrong:Ambit Biosciences Corporation: Employment, Equity Ownership.

scholarly journals Effect of Dihydroartemisinin-Piperaquine on the Pharmacokinetics of Praziquantel for Treatment of Schistosoma mansoni Infection

2021 ◽  
Vol 14 (5) ◽  
pp. 400
Author(s):  
Omary Mashiku Minzi ◽  
Rajabu Hussein Mnkugwe ◽  
Eliford Ngaimisi ◽  
Safari Kinung’hi ◽  
Anna Hansson ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Schistosoma Mansoni ◽  
Geometric Mean ◽  
Plasma Samples ◽  
Geometric Mean Ratio ◽  
Post Dose ◽  
Follow Up Study ◽  
Intestinal Schistosomiasis

Praziquantel (PZQ) and dihydroartemisinin-piperaquine (DHP) combination recently showed superior effectiveness than PZQ alone to treat intestinal schistosomiasis. In this follow-up study, we investigated the effect of DHP co-administration on the pharmacokinetics of PZQ and its enantiomers among 64 Schistosoma mansoni infected children treated with PZQ alone (n = 32) or PZQ + DHP combination (n = 32). Plasma samples collected at 0, 1, 2, 4, 6, and 8 h post-dose were quantified using UPLCMS/MS. The geometric mean (GM) of AUCs for total PZQ, R-PZQ and S-PZQ were significantly higher among children who received PZQ + DHP than PZQ alone. The geometric mean ratio (GMR) and (90% CI) of AUC0–∞ for PZQ + DHP to PZQ for total PZQ, R-PZQ, and S-PZQ were 2.18 (1.27, 3.76), 3.98 (2.27, 7.0) and 1.86 (1.06, 3.28), respectively. The GMR and (90% CI) of AUC0–8 for total PZQ, R-PZQ, and S-PZQ were 1.73 (1.12, 2.69), 2.94 (1.75, 4.92), and 1.50 (0.97, 2.31), respectively. The GM of Cmax for total PZQ, R-PZQ and S-PZQ were significantly higher among those who received PZQ + DHP than PZQ alone. The GMR (90% CI) of Cmax of PZQ + DHP to PZQ for total PZQ, R-PZQ, and S-PZQ were 1.75 (1.15, 2.65), 3.08 (1.91, 4.96), and 1.50 (1.0, 2.25%), respectively. The 90% CI of the GMRs for both AUCs and Cmax for total PZQ, R-PZQ, and S-PZQ were outside the acceptable 0.80–1.25 range, indicating that the two treatment arms were not bioequivalent. DHP co-administration significantly increases systemic PZQ exposure, and this may contribute to increased effectiveness of PZQ + DHP combination therapy than PZQ alone to treat schistosomiasis.

scholarly journals Pharmacokinetic analysis of an extended-pulsed fidaxomicin regimen for the treatment of Clostridioides (Clostridium) difficile infection in patients aged 60 years and older in the EXTEND randomized controlled trial

2020 ◽  
Vol 75 (4) ◽  
pp. 1014-1018
Author(s):  
Benoit Guery ◽  
Areti Georgopali ◽  
Andreas Karas ◽  
Gbenga Kazeem ◽  
Ingrid Michon ◽  
...  
Keyword(s):  
Controlled Trial ◽  
Plasma Concentrations ◽  
Systemic Exposure ◽  
Pharmacokinetic Profile ◽  
Pharmacokinetic Analysis ◽  
Plasma Samples ◽  
Post Dose ◽  
Recommended Treatment ◽  
Clostridioides Difficile ◽  
Stool Samples

Abstract Background Fidaxomicin is a recommended treatment for Clostridioides difficile infection (CDI) and reduces CDI recurrence incidence versus vancomycin. An extended-pulsed fidaxomicin (EPFX) regimen further reduces recurrence frequency. However, the pharmacokinetic profile of fidaxomicin in an EPFX regimen is unknown. Objectives To evaluate plasma and stool concentrations of fidaxomicin and its metabolite, OP-1118, after EPFX administration for CDI. Methods In the Phase 3b/4 EXTEND trial, patients aged ≥60 years with toxin-confirmed CDI were randomized to receive EPFX (oral fidaxomicin twice daily, Days 1–5; once daily on alternate days, Days 7–25). Fidaxomicin and OP-1118 concentrations were determined using post-dose plasma samples obtained on Days 5 ± 1, 12 ± 1 and 25/26, and post-dose stool samples obtained on Days 5 ± 1, 12 ± 1 and 26 ± 1. Results Plasma samples from 14 patients were included in the pharmacokinetic analysis; 12 of these patients provided stool samples. Median (range) plasma concentrations of fidaxomicin on Day 5 ± 1 and Day 25/26 were 0.0252 (0.0038–0.1220) mg/L and 0.0069 (0–0.0887) mg/L, respectively, and those of OP-1118 were 0.0648 (0.0142–0.3250) mg/L and 0.0206 (0–0.3720) mg/L, respectively. Median (range) stool concentrations of fidaxomicin and OP-1118 on Day 26 ± 1 were 272.5 (0–524) mg/kg and 280.5 (0–1120) mg/kg, respectively. Conclusions EPFX treatment maintained fidaxomicin stool concentrations above the C. difficile MIC90 until Day 26 ± 1. Systemic exposure to fidaxomicin and OP-1118 was low throughout and there was no evidence of accumulation in plasma or stool during treatment.

Comparison of Lipoprotein Separation and Lipid Analysis Methodologies for Human and Cynomolgus Monkey Plasma Samples

2011 ◽  
Vol 5 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Seongah Han ◽  
Amy M. Flattery ◽  
David McLaren ◽  
Richard Raubertas ◽  
Sang Ho Lee ◽  
...  
Keyword(s):  
Cynomolgus Monkey ◽  
Lipid Analysis ◽  
Plasma Samples ◽  
Monkey Plasma

Peroxisome proliferation: Organelles or activity as an endpoint?

1993 ◽  
Vol 51 ◽  
pp. 396-397
Author(s):  
Catherine A. Taylor ◽  
Bruce M. Jarnot
Keyword(s):  
Acid Oxidation ◽  
Peroxisome Proliferation ◽  
Post Dose ◽  
Oxidation Activity ◽  
Wasting Syndrome ◽  
Perfluorodecanoic Acid ◽  
Hypolipidemic Agent ◽  
Commercially Important ◽  
Acyl Coa Oxidase ◽  
Mitochondrial Disruption

Peroxisome induction can be expressed as an increase in peroxisome area (proliferation) or as an increase in peroxisomal fatty acid oxidation (activity). This study compares proliferation and activity as endpoints for hepatic peroxisome induction by perfluorodecanoic acid (PFDA). Fluorocarboxylic acids such as PFDA represent a class of compounds possessing commercially important surfactant properties. A single 50 mg/Kg ip. dose of PFDA produces a characteristic “wasting syndrome” in male F-344 rats. Symptoms include hypophagia, weight loss, hepatomegaly, and delayed lethality. Hepatic studies reveal changes similar to those seen with the hypolipidemic agent clofibrate. These include mitochondrial disruption, endoplasmic reticulum and peroxisome proliferation, and increased peroxisomal acyl-CoA oxidase activity.Male Fisher-344 rats received a single ip. dose of 2, 20, or 50mg/Kg PFDA dissolved in 1:1 propylene glycol/water and were sacrificed 8 days post-dose. All control rats received an equal volume of vehicle ip. Animals were provided food and water ad libitum, except pair-fed controls which received the same restrictive food intake consumed by their weight-paired dosed partners (50mg/Kg PFDA group) to simulate the hypophagia associated with PFDA.

Stability of antioxidant vitamins in whole human blood during overnight storage at 4°C and frozen storage up to 6 months

2018 ◽  
Vol 88 (3-4) ◽  
pp. 151-157 ◽  
Author(s):  
Scott W. Leonard ◽  
Gerd Bobe ◽  
Maret G. Traber
Keyword(s):  
Ascorbic Acid ◽  
Whole Blood ◽  
Healthy Subjects ◽  
Frozen Storage ◽  
Blood Collection ◽  
Plasma Samples ◽  
Optimal Conditions ◽  
Plasma Ascorbic Acid ◽  
Blood Samples ◽  
Sample Handling

Abstract. To determine optimal conditions for blood collection during clinical trials, where sample handling logistics might preclude prompt separation of erythrocytes from plasma, healthy subjects (n=8, 6 M/2F) were recruited and non-fasting blood samples were collected into tubes containing different anticoagulants (ethylenediaminetetra-acetic acid (EDTA), Li-heparin or Na-heparin). We hypothesized that heparin, but not EDTA, would effectively protect plasma tocopherols, ascorbic acid, and vitamin E catabolites (α- and γ-CEHC) from oxidative damage. To test this hypothesis, one set of tubes was processed immediately and plasma samples were stored at −80°C, while the other set was stored at 4°C and processed the following morning (~30 hours) and analyzed, or the samples were analyzed after 6 months of storage. Plasma ascorbic acid, as measured using HPLC with electrochemical detection (LC-ECD) decreased by 75% with overnight storage using EDTA as an anticoagulant, but was unchanged when heparin was used. Neither time prior to processing, nor anticoagulant, had any significant effects upon plasma α- or γ-tocopherols or α- or γ-CEHC concentrations. α- and γ-tocopherol concentrations remained unchanged after 6 months of storage at −80°C, when measured using either LC-ECD or LC/mass spectrometry. Thus, refrigeration of whole blood at 4°C overnight does not change plasma α- or γ-tocopherol concentrations or their catabolites. Ascorbic acid is unstable in whole blood when EDTA is used as an anticoagulant, but when whole blood is collected with heparin, it can be stored overnight and subsequently processed.

Factor XII Determinations in the Presence and Absence of Phospholipid Antibodies

1998 ◽  
Vol 79 (01) ◽  
pp. 87-90 ◽  
Author(s):  
D. W. Jones ◽  
M. Winter ◽  
M. J. Gallimore
Keyword(s):  
Lower Limit ◽  
Lupus Anticoagulant ◽  
Factor Xii ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Presence And Absence ◽  
Lower Limit Of Normal

SummaryFactor XII (FXII) levels were determined in plasma samples from 29 normal donors, 10 patients with inherited FXII deficiency (all lupus anticoagulant [LA] negative) and 67 LA positive patients, using clotting (FXIIct), chromogenic substrate (FXIIcs) and immunochemical (FXIIag) assays. Excellent correlations were obtained in the three FXII assays with the LA negative samples and between the FXIIcs and FXIIag assays in the LA positive samples. Correlations between both the FXIIcs and FXIIag with FXIIct in the LA positive patients were poor. Of 67 LA positive samples studied, 25 (37.3%) showed lower values in the FXIIct assay; 13 (19.4%) of these patients were pseudo FXII deficient with values of FXII below the lower limit of normal.These results indicate that a diagnosis of FXII deficiency can be made inappropriately in the presence of phospholipid antibodies and that such a diagnosis should not be made by FXIIct assay alone.

Fibrinogen-Fibrin-Related Antigen Pattern in Human Blood

1974 ◽  
Vol 32 (02/03) ◽  
pp. 266-276
Author(s):  
Carl D. Jacobsen ◽  
John C. Hoak ◽  
Kenneth K. WU ◽  
Glenna L. Fry
Keyword(s):  
Human Blood ◽  
Plasma Samples

SummaryIn serum from patients with DIC at least 3 different FR-antigenic components could be found. It was difficult to demonstrate these components in the corresponding plasma samples. It is possible that a portion of these antigens formed as a result of in vitro clotting despite the presence of proteolytic inhibitors. These results suggest that the interpretation of “increased split products in serum” may be more complex than current concepts indicate.

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