Publications by authors named "Carol Gleason"

30 Publications

  • Page 1 of 1

A bridging immunogenicity assay for anti-cabiralizumab antibodies: overcoming the low assay cut point and drug tolerance challenges.

Bioanalysis 2021 Mar 4;13(5):395-407. Epub 2021 Mar 4.

Nonclinical Disposition & Bioanalysis, Bristol Myers Squibb, Princeton, NJ 08543, USA.

To support the clinical studies of cabiralizumab, an immunogenicity assay for detecting anti-cabiralizumab antibodies is required. Strategies were developed to overcome two major bioanalytical challenges: poor drug tolerance of the anti-drug antibodies assay and very low cut point observed in the screening and confirmatory assays. By using acid dissociation (400 mM glycine solution at pH 2.0), drug tolerance of 200 μg/ml drug was achieved for both the screening and confirmatory assays. Effects of biological matrix (disease state vs normal serum) and assay conditions (capture/detector reagent concentration, minimum required dilution, acid pretreatment) on assay cut points were systematically evaluated. A bridging immunogenicity assay for detecting anti-cabiralizumab antibodies in human serum has been successfully developed, validated and applied to clinical studies.
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http://dx.doi.org/10.4155/bio-2020-0300DOI Listing
March 2021

Land O'Lakes Workshop on Microsampling: Enabling Broader Adoption.

AAPS J 2020 10 23;22(6):135. Epub 2020 Oct 23.

US FDA, CDER, Office of Translational Sciences, Office of Clinical Pharmacology, Silver Spring, Maryland, USA.

The microsampling workshop generated recommendations pertaining to blood sampling site (venous blood versus capillary blood), when to conduct a bridging study, statistical approaches to establish correlation/concordance and deciding on sample size, opportunities and challenges with patient-centric sampling, and how microsampling technology can enrich clinical drug development. Overall, the goal was to provide clarity and recommendations and enable the broader adoption of microsampling supporting patients' needs, convenience, and the transformation from clinic-centric to patient-centric drug development. The need and adoption of away-from-clinic sampling techniques has become critical to maintain patient safety during the current COVID-19 pandemic.
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http://dx.doi.org/10.1208/s12248-020-00524-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583552PMC
October 2020

Recommendations for singlet-based approach in ligand binding assays: an IQ Consortium perspective.

Bioanalysis 2020 Jun 19;12(12):823-834. Epub 2020 Jun 19.

Bristol-Myers Squibb Company, Princeton, NJ 08543, USA.

Historically, ligand-binding assays for pharmacokinetic samples employed duplicate rather than singlet-based analysis. Herein, the Translational and absorption, distribution, metabolism and excretion (ADME) Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) presents a study aiming to determine the value of duplicate versus singlet-based testing. Based on analysis of data collected from eight organizations for 20 drug candidates representing seven molecular types and four analytical platforms, statistical comparisons of validation and in-study quality controls and study unknown samples demonstrated good agreement across duplicate sets. Simulation models were also used to assess the impact of sample duplicate characteristics on bioequivalence outcomes. Results show that testing in singlet is acceptable for assays with %CV ≤15% between duplicates. Singlet-based approach is proposed as the default for ligand-binding assays while a duplicate-based approach is needed where imprecision and/or inaccuracy impede the validation of the assay.
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http://dx.doi.org/10.4155/bio-2020-0094DOI Listing
June 2020

Evaluation of correlation between bioanalytical methods.

Bioanalysis 2020 Mar 14;12(6):419-426. Epub 2020 Apr 14.

Eli Lilly & Company, Indianapolis, IN 46285, USA.

Bioanalytical methods evolve throughout clinical development timelines, resulting in the need for establishing equivalency or correlation between different methods to enable comparison of data across different studies. This is accomplished by the conduct of cross validations and correlative studies to compare and describe the relationship. The incurred sample reanalysis acceptance criterion seems to be adopted universally for cross validations and correlative studies; however, this does not identify any trends or biases between the two methods (datasets) being compared. Presented here are graphing approaches suitable for comparing two methods and describing equivalence or correlation. This article aims to generate awareness on graphing techniques that can be adopted during cross validations and correlative studies.
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http://dx.doi.org/10.4155/bio-2020-0019DOI Listing
March 2020

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

Bioanalysis 2019 Dec 10;11(24):2207-2244. Epub 2019 Dec 10.

US FDA, Silver Spring, MD, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of , issues 22 and 23 (2019), respectively.
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http://dx.doi.org/10.4155/bio-2019-0271DOI Listing
December 2019

Bead-extraction and heat-dissociation (BEHD): A novel way to overcome drug and matrix interference in immunogenicity testing.

J Immunol Methods 2018 11 9;462:34-41. Epub 2018 Aug 9.

Bioanalytical Science, Bristol-Myers Squibb, Princeton, NJ 08540, United States.

Biological therapeutics are foreign antigens and can potentially induce immune response resulting in the formation of anti-drug antibodies (ADA), which in turn may lead to a wide range of side effects. Neutralizing Ab (NAb) is a subset of ADA that can bind to the pharmacological activity regions of therapeutic to inhibit or complete neutralize its clinical efficacy. A cell-based functional NAb assay is preferred to characterize its neutralization activity. However, cell-based NAb assays are often vulnerable to drug interference, as well as interference from numerous serum factors, including but not limited to growth factors and disease-related cytokines. Bead Extraction with Acid Dissociation (BEAD) has been successfully applied to remove circulating drug and/or other interfering factors from human serum samples, thereby enriching for ADA/NAb. However, the harsh acid used in the extraction procedure can cause irreversible denaturing of NAb and lead to underestimated NAb measurement. Herein we describe a new approach when acid-dissociation is not optimal for a PEGylated domain antibody (Ab). We further demonstrate that heating at 62 °C can not only dissociate drug/ADA/NAb immune complex but also selectively and irreversibly denature domain Ab drug due to much lower thermal stability of the domain Ab, when compared to that of full antibodies. The irreversible denaturing of the drug favors the formation of an immune complex between ADA/NAb and the added biotinylated drug thus increasing the recovery of ADA/NAb from samples. We call this new procedure Bead Extraction with Heat Dissociation (BEHD), which can potentially be applied to other NAb assays that have poor compatibility with acid dissociation.
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http://dx.doi.org/10.1016/j.jim.2018.08.003DOI Listing
November 2018

Development and validation of a functional cell-based neutralizing antibody assay for ipilimumab.

Bioanalysis 2018 Aug 27;10(16):1273-1287. Epub 2018 Jun 27.

Analytical & Bioanalytical Operations, Bristol-Myers Squibb, Princeton, NJ 08543, USA.

Ipilimumab is the first US FDA-approved immune checkpoint-blocking antibody drug to harness the patient's own immune cells. One of the postmarketing requirements is to develop a cell-based neutralizing antibody assay. Here, we share some of the most challenging aspects encountered during the assay development: new cell line construction; an unexpected inhibition of T-cell activation by low concentrations of ipilimumab; and two issues caused by sample pretreatment with acid dissociation to overcome drug interference: instability of neutralizing antibody positive control at low pH, and incompatibility of commonly used acid dissociation buffers in the cell assay. After troubleshooting and optimization, we successfully validated the assay and used the assay to test clinical samples to date.
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http://dx.doi.org/10.4155/bio-2018-0109DOI Listing
August 2018

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples.

J Immunol Methods 2017 09 16;448:91-104. Epub 2017 Jun 16.

Analytical and Bioanalytical Operations, Bristol-Myers Squibb, Princeton, NJ 08543, United States.

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation.
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http://dx.doi.org/10.1016/j.jim.2017.06.002DOI Listing
September 2017

Accelerating Regulated Bioanalysis for Biotherapeutics: Case Examples Using a Microfluidic Ligand Binding Assay Platform.

AAPS J 2017 01 27;19(1):82-91. Epub 2016 Oct 27.

Immunochemistry and Biomarkers, Analytical and Bioanalytical Operations, Bristol-Myers Squibb, Route 206 and Province Line Road, Princeton, New Jersey, 08543, USA.

The Gyrolab™ xP is a microfluidic platform for conducting ligand binding assays (LBAs) and is recognized for its utility in discovery bioanalysis. However, few reports have focused on the technology for regulated bioanalysis. This technology has the advantage of low reagent consumption, low sample volume, and automated ligand binding methods. To improve bioanalysis testing timelines and increase the speed at which biotherapeutics are delivered to patients, we evaluated the technology for its potential to deliver high-quality data at reduced testing timelines for regulated bioanalysis. Six LBA methods were validated to support bioanalysis for GLP toxicokinetic or clinical pharmacokinetic studies. Validation, sample analysis, and method transfer are described. In total, approximately 4000 samples have been tested for regulated bioanalysis to support 6 GLP toxicology studies and approximately 1000 samples to support 2 clinical studies. Gyrolab™ xP had high run pass rates (≥83%) and high incurred sample reanalysis (ISR) pass rates (>94%). The maximum total error observed across all QC levels for a given assay was <30% for all six LBAs. High instrument response precision (CV ≤5%) was observed across compact discs (CDs), and methods were validated to use a single standard curve across multiple CDs within a Gyrolab™ xP run. Reduced bioanalysis timelines were achieved compared to standard manual plate-based methods, and methods were successfully transferred across testing labs, paving the way for this platform for use in late-stage clinical development.
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http://dx.doi.org/10.1208/s12248-016-0006-zDOI Listing
January 2017

Development and Validation of Electrochemiluminescence Assays to Measure Free and Total sSLAMF7 in Human Serum in the Absence and Presence of Elotuzumab.

AAPS J 2016 07 26;18(4):989-99. Epub 2016 Apr 26.

Bioanalytical Science-Biologics, Bristol-Myers Squibb, L14-03, Route 206 & Province Line Rd, Princeton, New Jersey, 08543, USA.

Elotuzumab is a first in class humanized IgG1 monoclonal antibody for the treatment of multiple myeloma (MM). Elotuzumab targets the glycoprotein signaling lymphocyte activation molecule family 7 (SLAMF7, also described as CS1 or CRACC) which is expressed on the surface of myeloma cells and a subset of immune cells, including natural killer cells. A soluble version of SLAMF7 (sSLAMF7) has also been reported in MM patients but has not been evaluated as a potential biomarker following therapeutic intervention. In order to measure serum levels of sSLAMF7, two immunoassays were developed to monitor changes in circulating sSLAMF7 before and after elotuzumab treatment. Free (drug-unbound) and total (drug-bound and unbound) electrochemiluminescence (ECL) ELISA assays were developed and validated following a fit for purpose (FFP) methodology. Both assays met analytical acceptance criteria for precision, drug interference, dilution linearity, spike recovery, parallelism, and stability. Both exhibited the range and sensitivity necessary to measure clinical samples with an LLOQ of 51.2 pg/mL and ULOQs of 160 (free) and 800 ng/mL (total). Previously described assays were unable to detect sSLAMF7 in healthy individuals. However, due to the increased sensitivity of these new assays, low but measurable sSLAMF7 levels were detected in all normal healthy sera evaluated and were significantly elevated in MM patients. Cohort statistics revealed a significant increase of circulating sSLAMF7 in MM patients versus normal controls and both significant decreases in free and increases in total levels of protein post-elotuzumab treatment.
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http://dx.doi.org/10.1208/s12248-016-9912-3DOI Listing
July 2016

Evaluation of cardiac function in unrestrained dogs and monkeys using left ventricular dP/dt.

J Pharmacol Toxicol Methods 2016 Jul-Aug;80:51-8. Epub 2016 Apr 8.

Bristol Myers Squibb, 3553 Lawrenceville Rd, Princeton, NJ 08540, United States. Electronic address:

Introduction: Preclinical assessment for alterations in cardiac ventricular function for drug candidates has not been a focus of ICH S7b guidelines for cardiovascular safety studies, but there is growing interest given that the cardiovascular risk is associated with positive and negative inotropes.

Methods: From 2003 through 2013, 163 telemetry studies with left-ventricular function analyses were conducted in dogs and monkeys at Bristol Myers Squibb (BMS) in support for drug development programs. The ability of the telemetry system to detect changes in cardiac contractility was verified with positive control agents pimobendan and atenolol. Control data from a subset of studies were analyzed to determine dP/dt reference range values, and minimum detectable mean differences (control vs. treated) for statistical significance.

Results: Median minimum detectable differences for dogs ranged from 14 to 21% for positive dP/dt and 11 to 21% for negative dP/dt. For monkeys, median minimum detectable differences were 25 and 14% for positive and negative dP/dt, respectively. For BMS programs, 15 drug candidates were identified that produced primary effects on contractility. Changes in contractility that were associated with, and potentially secondary to, drug-related effects on heart rate or systemic blood pressure were observed with an additional 29 drug candidates.

Discussion: Changes in contractility have been observed in large animals during drug development studies at BMS over the past 10years. Model sensitivity has been demonstrated and a dP/dt beat-to-beat cloud analysis tool has been developed to help distinguish primary effects from those potentially secondary to systemic hemodynamic changes.
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http://dx.doi.org/10.1016/j.vascn.2016.03.006DOI Listing
May 2017

Impact of menstruation on select hematology and clinical chemistry variables in cynomolgus macaques.

Vet Clin Pathol 2016 Jun 8;45(2):232-43. Epub 2016 Apr 8.

Global Biometric Sciences, Nonclinical Biostatistics, Research & Development, Bristol-Myers Squibb Company, Princeton, NJ, USA.

Background: In preclinical studies with cynomolgus macaques, it is common to have one or more females presenting with menses. Published literature indicates that the blood lost during menses causes decreases in red blood cell mass variables (RBC, HGB, and HCT), which would be a confounding factor in the interpretation of drug-related effects on clinical pathology data, but no scientific data have been published to support this claim.

Objectives: This investigation was conducted to determine if the amount of blood lost during menses in cynomolgus macaques has an effect on routine hematology and serum chemistry variables.

Methods: Ten female cynomolgus macaques (Macaca fascicularis), 5 to 6.5 years old, were observed daily during approximately 3 months (97 days) for the presence of menses. Hematology and serum chemistry variables were evaluated twice weekly.

Results: The results indicated that menstruation affects the erythrogram including RBC, HGB, HCT, MCHC, MCV, reticulocyte count, RDW, the leukogram including neutrophil, lymphocyte, and monocyte counts, and chemistry variables, including GGT activity, and the concentrations of total proteins, albumin, globulins, and calcium. The magnitude of the effect of menstruation on susceptible variables is dependent on the duration of the menstrual phase. Macaques with menstrual phases lasting ≥ 7 days are more likely to develop changes in variables related to chronic blood loss.

Conclusions: In preclinical toxicology studies with cynomolgus macaques, interpretation of changes in several commonly evaluated hematology and serum chemistry variables requires adequate clinical observation and documentation concerning presence and duration of menses. There is a concern that macaques with long menstrual cycles can develop iron deficiency anemia due to chronic menstrual blood loss.
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http://dx.doi.org/10.1111/vcp.12350DOI Listing
June 2016

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 1 - small molecules by LCMS).

Bioanalysis 2015 17;7(22):2913-25. Epub 2015 Nov 17.

AstraZeneca, Cambridge, UK.

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event - a full immersion bioanalytical week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations that emerged from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to advance scientific excellence, improve quality and deliver better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 1 covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (hybrid LBA/LCMS and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis using LBA, biomarkers and immunogenicity) will also be published in volume 7 of Bioanalysis, issues 23 and 24, respectively.
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http://dx.doi.org/10.4155/bio.15.204DOI Listing
September 2016

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development.

AAPS J 2016 Jan 16;18(1):1-14. Epub 2015 Sep 16.

Ampersand Biosciences, LLC, 3 Main St., Saranac Lake, New York, 12983, USA.

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.
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http://dx.doi.org/10.1208/s12248-015-9820-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706274PMC
January 2016

Recommendations for the development and validation of confirmatory anti-drug antibody assays.

Bioanalysis 2015 ;7(13):1619-31

Covance, Inc., 3635 Concorde Parkway, Suite 100 Chantilly, VA 20151, USA.

Identification and characterization of anti-drug antibodies is a critical component of biopharmaceutical drug development. The tiered approach for immunogenicity testing consists of screening, confirmatory, and characterization assays. Herein, we provide recommendations for confirmatory assays by expanding upon published guidance and present common practices across the industry. The authors recommend scientific approaches for development and validation of confirmatory assays using competition methods in ligand-binding assays, along with statistical formulae for routine use and validation. The paper will assist in understanding the confirmatory assay, and carefully implementing validation criteria a priori, as well as during sample analysis. These approaches represent the authors' current knowledge and practices, with the aim that more uniform practices will be applied across the industry.
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http://dx.doi.org/10.4155/bio.15.96DOI Listing
April 2016

Anti-PEG antibody bioanalysis: a clinical case study with PEG-IFN-λ-1a and PEG-IFN-α2a in naive patients.

Bioanalysis 2015 ;7(9):1093-106

3LabCorp, 115 Silvia Street, West Trenton, NJ 08628, USA.

Background: Extensive use of polyethylene glycol (PEG) in consumer products necessitates the assessment of anti-PEG antibodies (APAb).

Methods: In clinical trials comparing PEG-IFN-λ to PEG-IFN-α, conventional bridge and direct assays were assessed.

Results & Conclusion: The bridge assay detected IgM and IgG APAb reactive with common PEG sizes and derivatives at sufficient sensitivity, 15-500 ng/ml. Of subjects evaluated, 6% of PEG-IFN-λ and 9% of PEG-IFN-α subjects had persistent APAb while 60% of PEG-IFN-λ and 33% of PEG-IFN-α subjects had persistent anti-interferon antibodies (AIAb). Pre-existing APAb and AIAb prevalence was comparable (approximately 10% of subjects). APAb were earlier onset, less frequent, less persistent and lower titer than AIAb. No associated hypersensitivity events were reported.
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http://dx.doi.org/10.4155/bio.15.36DOI Listing
February 2016

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

AAPS J 2015 Jul 1;17(4):976-87. Epub 2015 May 1.

Bioanalytical Science-Biologics, Bristol-Myers Squibb, L4.016B, Route 206 & Province Line Rd, Princeton, New Jersey, 08543, USA,

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.
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http://dx.doi.org/10.1208/s12248-015-9762-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476994PMC
July 2015

Development and validation of an LC-MS/MS assay for the quantitation of a PEGylated anti-CD28 domain antibody in human serum: overcoming interference from antidrug antibodies and soluble target.

Bioanalysis 2014 Sep;6(18):2371-83

Analytical & Bioanalytical Development, Bristol-Myers Squibb, Route 206 & Province Line Road, Princeton, NJ 08543, USA.

Aim: To support drug development of a PEGylated anti-CD28 domain antibody, a sensitive and robust LC-MS/MS assay was developed for the first in-human multiple ascending dose study.

Materials & Methods: The procedure consists of a protein precipitation with acidified acetonitrile, followed by trypsin digestion of the supernatant. A surrogate peptide from the complementarity determining region was quantified with an LC-MS/MS assay using a stable isotope-labeled internal standard with flanking amino acids. An acid dissociation step was found to be essential to achieve full analyte recovery in the presence of antidrug antibodies and soluble target CD28.

Results & Conclusion: The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.
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http://dx.doi.org/10.4155/bio.14.181DOI Listing
September 2014

Addressing matrix effects in ligand-binding assays through the use of new reagents and technology.

Bioanalysis 2014 Apr;6(8):1059-67

Bristol-Myers Squibb, Route 206 & Province Line Rd, Princeton, NJ 08543, USA.

Background: Ligand-binding assays (LBAs) used in the quantification of biotherapeutics for pharmacokinetic determinations rely on interactions between reagents (antibodies or target molecule) and the biotherapeutic. Most LBAs do not employ an analyte extraction procedure and are susceptible to matrix interference. Here, we present a case study on the development of a LBA for the quantification of a PEGylated domain antibody where matrix interference was observed. The assay used to support the single ascending dose study was a plate-based electrochemiluminescent assay with a lower limit of quantification of 80 ng/mL. To meet sensitivity requirements of future studies, new reagents and the Gyrolab™ Workstation were evaluated.

Results: Assay sensitivity improved nearly threefold in the final method utilizing new antibody reagents, a buffer containing blockers to human anti-animal antibodies, and the Gyrolab Workstation.

Conclusion: Experimental data indicate that all factors changed played a role in overcoming matrix effects.
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http://dx.doi.org/10.4155/bio.14.75DOI Listing
April 2014

Fit-for-purpose bioanalytical cross-validation for LC-MS/MS assays in clinical studies.

Bioanalysis 2013 Jan;5(1):83-90

Research & Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA.

The paradigm shift of globalized research and conducting clinical studies at different geographic locations worldwide to access broader patient populations has resulted in increased need of correlating bioanalytical results generated in multiple laboratories, often across national borders. Cross-validations of bioanalytical methods are often implemented to assure the equivalency of the bioanalytical results is demonstrated. Regulatory agencies, such as the US FDA and European Medicines Agency, have included the requirement of cross-validations in their respective bioanalytical validation guidance and guidelines. While those documents provide high-level expectations, the detailed implementation is at the discretion of each individual organization. At Bristol-Myers Squibb, we practice a fit-for-purpose approach for conducting cross-validations for small-molecule bioanalytical methods using LC-MS/MS. A step-by-step proposal on the overall strategy, procedures and technical details for conducting a successful cross-validation is presented herein. A case study utilizing the proposed cross-validation approach to rule out method variability as the potential cause for high variance observed in PK studies is also presented.
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http://dx.doi.org/10.4155/bio.12.291DOI Listing
January 2013

Addressing drug effects on cut point determination for an anti-drug antibody assay.

J Immunol Methods 2012 Oct 28;384(1-2):152-6. Epub 2012 Jun 28.

Analytical and Bioanalytical Sciences, Bristol-Myers Squibb Company-BMS, Route 206 & Province Line Road, Princeton, NJ 08543, USA.

The effect of trough levels of a monoclonal antibody drug (drugB) on screening cut point (CP) determination for an anti-drug antibody (ADA) assay was scrutinized and the conclusions substantiated by data from a phase 3 cancer clinical study. The ADA assay utilized an acid dissociation step and either 0 or 100 μg/ml drugB was added to the samples prior to obtaining the signals used for CP calculations. Serum samples from three different drug-naive populations were tested (healthy individuals, cancer patients enrolled in the drugB clinical trial and cancer patients whose serum samples were available commercially). For the same disease state samples, both the screening CP and confirmation CP were different when calculated during validation or from study sample analysis. It is reasonable to assume that variability was due to the patient heterogeneity, as they could have been at distinct stages of disease progression, and/or taking different medications, amongst other differences. The patients enrolled in the clinical trial were stratified as per protocol and hence represented a more homogeneous population. Drug effects on CP may be population dependent and also assay dependent.
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http://dx.doi.org/10.1016/j.jim.2012.06.014DOI Listing
October 2012

In vivo flow cytometric Pig-a and micronucleus assays: highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated-dose designs.

Environ Mol Mutagen 2012 Jul 22;53(6):420-8. Epub 2012 Jun 22.

Litron Laboratories, Rochester, NY 14623, USA.

Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague-Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3-day and 28-day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig-a mutation with a dual labeling method (SYTO 13 in combination with anti-CD59-PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3-day study (43-fold increase) and on Day 42 of the 28-day study (171-fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28-day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate that Pig-a and micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that the Pig-a endpoint is suitable for both repeated-dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on-going toxicology studies, and use as a short-term assay that provides efficient screening and mechanistic information in vivo.
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http://dx.doi.org/10.1002/em.21709DOI Listing
July 2012

International Pig-a gene mutation assay trial (stage III): results with N-methyl-N-nitrosourea.

Environ Mol Mutagen 2011 Dec;52(9):699-710

GlaxoSmithKline, Ware, Hertfordshire, United Kingdom.

N-methyl-N-nitrosourea (MNU) was evaluated in the in vivo Pig-a mutation assay as part of an International Collaborative Trial to investigate laboratory reproducibility, 28-day study integration, and comparative analysis with micronucleus (MN), comet, and clinical pathology endpoints. Male Sprague Dawley rats were treated for 28 days with doses of 0, 2.5, 5, and 10 mg MNU/kg/day in two independent laboratories, GlaxoSmithKline (GSK) and Bristol Myers Squibb (BMS). Additional studies investigated the low-dose region (<2.5 mg/kg/day). Reticulocytes were evaluated for Pig-a phenotypic mutation, CD59-negative reticulocytes/erythrocytes (RETs(CD592-)/ RBCs(CD592-)) on Days 1, 4, 15, 29, 43, and 57, and for micronucleated reticulocytes (MN-RETs) on Days 4 and 29. Comet analysis was conducted for liver and whole blood, and hematology and clinical chemistry was investigated. Dose-dependent increases in the frequency of RETs(CD592-) and RBCs(CD592-) were observed by Day 15 or 29, respectively. Dose-dependent increases were observed in %MN-RET on Days 4 and 29, and in mean %tail intensity in liver and in blood. Hematology/clinical chemistry data demonstrated bone marrow toxicity. Data comparison between GSK and BMS indicated a high degree of concordance with the Pig-a mutation assay results, consistent with previous observations with MNU and N-ethyl-N-nitrosourea. These data confirm that complementary genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that can provide information on gene mutation, chromosome damage, and DNA strand breaks in a single repeat dose rodent study. Collectively, this would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints.
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http://dx.doi.org/10.1002/em.20691DOI Listing
December 2011

International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories.

Environ Mol Mutagen 2011 Dec 11;52(9):690-8. Epub 2011 Sep 11.

Litron Laboratories, Rochester, New York, USA.

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.
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http://dx.doi.org/10.1002/em.20672DOI Listing
December 2011

Effect of body position on limb lead electrocardiographic findings in sedated cynomolgus macaques (Macaca fascicularis).

J Am Assoc Lab Anim Sci 2010 May;49(3):352-6

Veterinary Science, Bristol-Myers Squibb Research and Development, Syracuse, New York, USA.

Electrocardiograms (ECGs) often are collected from sedated cynomolgus macaques (Macaca fascicularis) in drug safety studies to support investigational new drug applications. ECGs are evaluated either manually or electronically, and the quality of the ECG tracing can affect the quality of that evaluation. The body position of the subject sometimes is manipulated to eliminate noise or clarify ECG complex morphology, and typically multiple technicians collect ECG data over time. Both factors-body position and multiple technicians-could affect ECG quality. This study was designed to determine whether body position or multiple technicians affects heart rate, mean electrical axis, or ECG parameters (RR interval, P wave duration, PR interval, QRS duration, QT interval (uncorrected and rate-corrected by using the Bazett [QTcb] and Fridericia [QTcf] formulas), P wave amplitude, R wave amplitude, T wave height, T wave height negative, and ST segment elevation). The results reveal minimal (coefficient of variation [CV] less than 10%) within-animal variation between body positions (ventral, dorsal, right or left lateral), with the exception of P wave amplitude (17.5%), R wave amplitude (23.7%), and ST segment elevation (43%). Minimal variation in ECG parameters (no more than 7%) was detected between technicians, across animals, and across body positions. These findings suggest that neither a change in body position to increase the quality of an ECG tracing nor the use of multiple technicians significantly affect the evaluation of quantitative ECG parameters, especially QTcb (0.1% CV) and QTcf (1.3% CV).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877309PMC
May 2010

Echocardiographic reference ranges for sedated healthy cynomolgus monkeys (Macaca fascicularis).

J Am Assoc Lab Anim Sci 2008 Jan;47(1):22-5

Veterinary School, University of Pennsylvania, Philadelphia, USA.

Echocardiographic imaging has become the primary tool to evaluate cardiac structure and function in human and veterinary medicine. The cynomolgus monkey (Macaca fascicularis) is a nonhuman primate species frequently used in biomedical research, particularly for the study of human cardiovascular disease and toxicology, yet echocardiographic reference ranges are not available for this species. Using standard 2-dimensional and M-mode imaging, we performed echocardiographic evaluation of 118 female and 119 male cynomolgus monkeys under sedation with either ketamine hydrochloride (10 mg/ kg IM) alone or with a combination of tiletamine hydrochloride and zolazepam (4.0 mg/kg IM) and atropine sulfate (0.015 mg/kg IM). Reference ranges were developed (tolerance interval methodology) separately for each gender for heart rate, left ventricular (LV) size (interventricular septum in diastole, LV internal diameter in diastole and systole, LV free wall in diastole), left atrial diameter, and aortic diameter. LV functional parameters (fractional shortening, aortic peak flow velocity, LV ejection time, and LV preejection period) and mitral valve E point to septal separation were also measured. After normalization for body weight (1.7 to 6.3 kg), the data were analyzed for gender- and sedation-associated differences. Using a large number of healthy subjects (118 of each gender), we have developed gender-specific echocardiographic reference ranges for cynomolgus monkeys.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652620PMC
January 2008

Evaluation of immunogenicity of the T cell costimulation modulator abatacept in patients treated for rheumatoid arthritis.

J Rheumatol 2007 Dec 15;34(12):2365-73. Epub 2007 Nov 15.

Immunotoxicology, Drug Safety Evaluation, Bristol-Myers Squibb, Syracuse, New York 13057, USA.

Objective: The immunogenicity of abatacept, a selective costimulation modulator, administered intravenously, was assessed across Phase II and III trials in patients with rheumatoid arthritis (RA).

Methods: Two direct-format enzyme-linked immunosorbent assays evaluated antibody responses [whole abatacept molecule (CTLA-4 and Ig portion) and CTLA-4 portion only (Assay A)] in the Phase II trials. During the Phase III trials and 2-year open-label periods, a similar, but more sensitive, Assay B was employed. Serum samples collected prestudy, during treatment, and 56 and/or 85 days following the last dose were evaluated. Seropositive samples with anti-CTLA-4 reactivity and sufficiently low drug levels were further characterized for neutralizing activity (cell-based bioassay).

Results: A total of 2237 patients with both pre- and post-baseline serum samples were eligible for assessment. Of these, 62 (2.8%) patients demonstrated an anti-abatacept or anti-CTLA-4 response, determined using either Assay A or B. Using the more sensitive Assay B, 60 of 1990 patients (3.0%) demonstrated an antibody response to the whole abatacept molecule (n = 41, 2.1%) or the CTLA-4 portion (n = 19, 1.0%). Of the 1764 RA patients evaluated in the Phase III studies, 203 discontinued therapy and had sera collected 56 and/or 85 days after discontinuation. Patients who discontinued had a higher incidence of immunogenicity versus patients who did not discontinue (7.4% vs 2.6%, respectively). Of 20 patients positive for anti-CTLA-4 reactivity, 13 were eligible for assessment with the neutralization bioassay. Of these, 8 patients exhibited neutralizing activity. Seroconversion occurred with no adverse safety outcomes or effect on pharmacokinetic parameters. No consistent pattern was observed between antibody response and loss of efficacy (American College of Rheumatology 20 and Health Assessment Questionnaire responses).

Conclusion: Abatacept was associated with a low incidence of immunogenicity in patients with RA and lacked any adverse sequelae.
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December 2007

Analysis of the nonclinical telemetered ECG: impact of logging rate and RR bin width in the dog and cynomolgus monkey.

J Pharmacol Toxicol Methods 2007 Jul-Aug;56(1):34-42. Epub 2006 Dec 20.

Bristol-Myers Squibb Pharmaceutical Research Institute, United States.

Introduction: Species-dependent ECG differences may affect QT interval analysis. Among these are the range of QT and RR values, heart rate variability, and respiratory sinus arrhythmia (SA). Importantly, the effects of data logging rates and RR bin width (BW) on QT analysis have not been characterized in dogs and nonhuman primates.

Methods: Digital ECGs were collected for 18-21 h in telemetered dogs (n=7) and cynomolgus monkeys (n=7) employing epicardial ECG leads and analyzed by computerized algorithms. ECG intervals were determined employing beat-to-beat (1 ms BW) and 10 s logging rates (for 1, 10, 20, and 50 ms BW). Diurnal heart rate variability was defined as the beat-to-beat RR ratio (VR) where SA was defined as >+/-10% change in VR.

Results: Canine beat-to-beat QT-RR data were curvilinear and plateaued, with multiple RR values associated to any given QT for RR>or=900 ms. Cynomolgus QT-RR data collected as beat-to-beat or at a 10 s logging rate were linear for all RR intervals. During the day, SA comprised 62.9+/-2.4% and 11.4+/-6.1% of total beats in the dog and cynomolgus, respectively, with no diurnal/nocturnal differences in either species. QT interval changes varied with BW such that 5 ms resolution was maintained for BW
Discussion: Differences in the QT interval, primarily due to SA, were responsible for skewed canine beat-to-beat QT-RR relationships. A 10 s logging rate eliminated this confounding factor, enabling the derivation of accurate QT rate-corrections. Thus, the uniform application of a 10 s logging rate in conjunction with a 10 ms RR BW ensured consistent and accurate QT analysis in both species. In conclusion, optimal BWs were defined which maintained beat-to-beat accuracy, while maximizing the number of QT-RR observations incorporated into QT-RR rate-correction models, a critical factor for robust probabilistic QT rate-corrections.
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http://dx.doi.org/10.1016/j.vascn.2006.12.002DOI Listing
August 2007

Development and validation of a preclinical food effect model.

J Pharm Sci 2007 Feb;96(2):459-72

Pharmaceutical Candidate Optimization, Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492-1951, USA.

A preclinical canine model capable of predicting a compound's potential for a human food effect was developed. The beagle dog was chosen as the in vivo model. A validation set of compounds with known propensities for human food effect was studied. Several diets were considered including high-fat dog food and various quantities of the human FDA meal. The effect of pentagastrin pretreatment was also investigated. The high-fat dog food did not predict human food effect and was discontinued from further evaluation. The amount of FDA meal in the dog was important in the overall prediction of the magnitude of human food effect. Fed/fasted Cmax and AUC ratios using a 50-g aliquot of the FDA meal in the dog were in the closest qualitative agreement to human data. Pentagastrin pretreatment did not affect the AUC in the fed state, but increased the fasted AUC for weakly basic compounds. Pentagastrin pretreatment and a 50-g aliquot of the FDA meal in the dog predicted the human food effect for a validation set of compounds. This model, which is intended for compound screening, will be helpful for determining food effect as a liability when compounds progress from discovery to clinical development.
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http://dx.doi.org/10.1002/jps.20767DOI Listing
February 2007

Novel probabilistic method for precisely correcting the QT interval for heart rate in telemetered dogs and cynomolgus monkeys.

J Pharmacol Toxicol Methods 2007 Mar-Apr;55(2):159-75. Epub 2006 May 27.

Pharmaceutical Research Institute, Bristol-Myers Squibb Co., Syracuse, NY 13221, USA.

Introduction: QT intervals are not regulated on a beat-to-beat cadence, but are strongly influenced by the preceding heart rate history (hysteresis). ECG sampling, when performed over sufficiently long periods, results in the detection of ranges of different QT values for each discrete RR interval. Given the potential impact of QT hysteresis in QT interval rate-correction procedures, we hypothesized that, physiologically, the QT interval exists as a probabilistic variable where the exact value corresponding to any RR interval is precisely estimated from the associated QT population.

Methods: Digital ECGs were collected for 18-21 h in telemetered dogs (n=7) and cynomolgus monkeys (n=7) employing epicardial ECG leads for accurate T(end) detection, and analyzed by computerized algorithms. Descriptive statistics were calculated for raw QT values in 10 ms RR increments. Individual rate-corrected QT (QTc) formulae were derived from the slopes of log-transformed QT-RR data where each QT point was the mean of >250 beats/RR increment. The aptness of this QTc model was assessed by residual analysis.

Results: Beat-to-beat ECG analysis demonstrated that for all discrete cycle lengths, the associated raw QT intervals were normally distributed populations, spanning approximately 30-40 and 45-100 ms in the dog and cynomolgus monkey, respectively. In both species, QTc was stable (< or =5 ms variation) over all physiological RR intervals.

Discussion: The probabilistic treatment of raw QT interval populations natively associated to any RR interval provides hysteresis-free raw QT estimates which can be accurately modeled, allowing the derivation of a precise QTc value. Previous unawareness of the probabilistic nature of the QT interval explains the historical failure of numerous QT rate-correction formulae to correctly solve this scientific issue. Importantly, QT distribution analysis has the potential to provide, for the first time, a universal and sensitive method for QT heart rate-correction, providing a robust method for nonclinical and clinical cardiac safety investigations of repolarization delay.
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http://dx.doi.org/10.1016/j.vascn.2006.05.007DOI Listing
April 2007