Method validation and application of protein biomarkers: basic similarities and differences from biotherapeutics

Protein drug development and biomarkers share common bioanalytical technologies that are applied for different purposes. A fit-for-purpose approach should be used for biomarker assays at various stages of novel biomarker development and their application to drug development. Biomarker quantifications can be absolute or relative, depending upon the characteristics of the standard curve, which include the reference standard, substituted matrix and parallelism. Appropriate method-validation experiments should be carried out on sample collection, relative accuracy and precision, range finding, parallelism, selectivity, specificity and stability in order to meet the need for exploratory or advanced application that is specified for a study. The interaction of a biotherapeutic with the target ligand or inter-related biomarkers should be taken into consideration for method platform choice and validation. Direct adoption of commercial diagnostic kits can produce confounding data. Therefore, kit comparison, modification and appropriate validation experiments are often carried out to meet the specific purpose for drug development. Multiplex assays and physicochemical methods can complement the single-analyte ligand-binding assay for protein drugs and biomarkers.     

DxS Ltd

DxS is a personalized medicine company that meets the needs of the pharmaceutical industry for biomarkers and companion diagnostics to support the development and sales of cancer and other therapies. The company provides both biomarker products, which are used predominately during clinical trials, and companion diagnostics, which aid doctors in selecting therapies for patients. Working in partnership with drug companies, DxS offer validated biomarker assays to support drug development and then regulatory approval, by identifying likely responders to drug therapies. DxS have launched the world's first cancer mutation companion diagnostic to support Amgen's Vectibix colorectal cancer therapy. DxS kits detect mutations in oncogenes associated with cancer drug response. TheraScreen is the range of CE-marked diagnostic products for detecting mutations in the EGFR and K-RAS genes. Validated biomarker kits are available for research use for EGFR, RAS, RAF, BCR-ABL and other genes that show a correlation between patient mutation status and drug response.     

Drug use, risk and urban order: examining supervised injection sites (SISs) as ‘governmentality’

This paper problematises the emergence and functioning of the recent phenomenon of ‘supervised injection sites’ (SISs) as a case study of post-welfarist governmentality. We propose that SISs arose as an unprecedented intervention in the late 20th century to deal with the increasing challenge of ‘urban drug scenes’ towards public order interests ‘entrepreneurial city’. Under predominant discourses of ‘public health’ and ‘harm reduction’, SISs became possible within a wide variety of political interests as a technology for purifying public spaces of ‘disorderly’ drug users to present the ‘new city’ as an attractive consumption space. Thus, SISs can be meaningfully understood as one element of socio-spatial ‘exclusion’ of marginalised populations from urban cores to ghettoised, peripheral spaces, even as they more benignly seek to better meet the unique needs of drug user populations. Further, the inner workings of SISs illustrate these facilities as powerful surveillance and discipline sites, defining the drug user as an agent of omnipresent risk being responsibilized in the care of the self and body, but also multiple aspects of behaviour and lifestyle reaching beyond drug use; thus construing the drug user as a ‘normalised’ citizen/consumer. We suggest that pressures to answer to powerful interests promoting ‘order’ are concretised as practices of ‘risk management’ ‘on the shop floor’, raising serious questions about the extent to which the ability to meet user needs is compromised in the interest of social control, surveillance, ‘management’, ‘education’, and ‘rehabilitation’, particularly in the current socio-political context (characterised as it is by a persistence, and indeed concomitant hardening, of repressive measures ‘on the street’).     

Clinical study designs and patient selection methods based on genomic biomarkers: Points-to-consider documents

Recently, genomic biomarkers have been widely used clinically for prediction of the efficacy and safety of pharmacotherapy and diagnosis and prognosis of pathological conditions. Therefore, genomic biomarkers are anticipated to accelerate not only precision medicine for pharmacotherapy but also development of molecularly targeted drugs. Because the design of clinical studies involving biomarkers may differ from conventional clinical study designs, a concept paper focused on clinical studies and patient selection methods based on genomic biomarkers is desired to prompt innovative drug development. Thus, this concept paper aimed to compile and present current scientific information from the related guidelines regarding application of genomic biomarkers to clinical trials and studies for drug development. We hope that this concept paper will prompt the development of guidelines for biomarker application to drug development by industry, regulatory authorities, the medical profession, and academia.     

Comparison of the Mesoscale Discovery and Luminex multiplex platforms for measurement of urinary biomarkers in a cisplatin rat kidney injury model

IntroductionIn the past years several new urinary nephrotoxicity biomarkers have been qualified for use in preclinical studies by the FDA and EMA. Subsequently, kits have been developed to measure these urinary biomarkers on multiplex platforms such as the electro-chemiluminescent based immunoassay from MesoScale Discovery (MSD) and the bead-based immunoassay using Luminex xMAP technology (LMX). The aim of the present study was to compare the two multiplex platforms with respect to the capability of their qualified urinary biomarker panels to measure an increase of these biomarkers relative to histopathological changes in an animal model of nephrotoxicity.MethodsFor comparison of the two platforms we used urine samples from a study with the well-characterized nephrotoxin cisplatin (Cp) in male Wistar rats. The following five biomarkers were measured on both platforms: glutathione S-transferase α (αGST), clusterin (CLU), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL, a.k.a. lipocalin-2) and osteopontin (OPN). The measurements were compared with respect to both the fold increase observed for each biomarker and the absolute concentrations measured in relation to traditional endpoints for nephrotoxicity in clinical pathology and histopathology.ResultsThe platform comparison revealed the expected increases of urinary biomarkers after Cp treatment with similar results at the fold change level enabling consistent detection of kidney injury. The comparison of the absolute concentrations of biomarkers measured in the two platforms showed differences, the extent of which was analyte-dependent.DiscussionBy comparison of two widely used multiplex platforms, MSD and LMX, for the detection of renal toxicity biomarkers in urine, we observed the expected increases of these biomarkers in response to Cp administration. Depending on the marker, significant differences could be found when comparing the absolute concentrations thus suggesting that baseline levels for each platform will have to be set separately.     

Biomarkers in drug discovery and development: from target identification through drug marketing

Biomarkers of disease play an important role in medicine and have begun to assume a greater role in drug discovery and development. The challenge for biomarkers is to allow earlier, more robust drug safety and efficacy measurements. Their role in drug development will continue to grow for the foreseeable future. For biomarkers to assume their rightful role, greater understanding of the mechanism of disease progression and therapeutic intervention is needed. In addition, greater understanding of the requirements for biomarker selection and validation, biomarker assay method validation and application, and clinical endpoint validation and application is needed. Biomarkers need to be taken into account while the therapeutic target is still being identified and the concept is being formulated. Biomarkers need to be incorporated into a continuous cycle that takes what is learned from the discovery and development of one series of biomarkers and translates it into the next series of biomarkers. Optimum biomarker development and application will require a team approach because of the multifaceted nature of biomarker selection, validation, and application, using such techniques as pharmacoepidemiology, pharmacogenetics, pharmacogenomics, and functional proteomics; bioanalytical method development and validation; disease process and therapeutic intervention assessments; and pharmacokinetic/pharmacodynamic modeling and simulation to improve and refine drug development. The potential for biomarkers in medicine and drug development will be limited by the least effective component of the processes. The team approach will minimize the potential for the least effective component to be fatal to the rest of the process. As scientific/regulatory foundations for biomarkers in medicine and drug development begin to be established, successes and applications will need to be effectively communicated with all of the stakeholders, including not only internal and external drug developers and regulators but also the medical community, to ensure that biomarkers are totally integrated into drug discovery and development as well as the practice of medicine.     

Impact of biomarker development on drug safety assessment

Drug safety has always been a key aspect of drug development. Recently, the Vioxx case and several cases of serious adverse events being linked to high-profile products have increased the importance of drug safety, especially in the eyes of drug development companies and global regulatory agencies. Safety biomarkers are increasingly being seen as helping to provide the clarity, predictability, and certainty needed to gain confidence in decision making: early-stage projects can be stopped quicker, late-stage projects become less risky. Public and private organizations are investing heavily in terms of time, money and manpower on safety biomarker development. An illustrative and “door opening” safety biomarker success story is the recent recognition of kidney safety biomarkers for pre-clinical and limited translational contexts by FDA and EMEA. This milestone achieved for kidney biomarkers and the “know how” acquired is being transferred to other organ toxicities, namely liver, heart, vascular system. New technologies and molecular-based approaches, i.e., molecular pathology as a complement to the classical toolbox, allow promising discoveries in the safety biomarker field. This review will focus on the utility and use of safety biomarkers all along drug development, highlighting the present gaps and opportunities identified in organ toxicity monitoring. A last part will be dedicated to safety biomarker development in general, from identification to diagnostic tests, using the kidney safety biomarkers success as an illustrative example.     

Systematic analytical validation of commercial kits for the determination of novel biomarkers for clinical drug development

The use of biomarkers during clinical drug-development programs may expedite pipeline decision making by adding critical information about the pharmacological mechanism and efficacy of a potential therapeutic agent. Currently, advice for laboratorians conducting method development and analytical validation of biomarker methods is provided by published White Paper recommendations from industry thought leaders. The adaptation of commercial test kits to generate biomarker data to support regulated studies offers unique challenges and limitations. In this perspective, we address these issues, including factors to consider when identifying a kit manufacturer and adapting commercial test kits for use in regulated studies. We offer a logical and systematic approach for defining the extent of analytical validation needed for application of commercial kits based upon the intended use of the biomarker data.     

Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement

Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res 22:499–511, 2005), these and other critical issues were addressed. A practical, iterative, “fit-for-purpose” approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development.     

Biomarkers for the Development of New Medications for Cocaine Dependence

There has been significant progress in personalized drug development. In large part, this has taken place in the oncology field and been due to the ability of researchers/clinicians to discover and develop novel drug development tools (DDTs), such as biomarkers. In cancer treatment research, biomarkers have permitted a more accurate pathophysiological characterization of an individual patient, and have enabled practitioners to target mechanistically the right drug, to the right patient, at the right time. Similar to cancer, patients with substance use disorders (SUDs) present clinically with heterogeneous symptomatology and respond variably to therapeutic interventions. If comparable biomarkers could be identified and developed for SUDs, significant diagnostic and therapeutic advances could be made. In this review, we highlight current opportunities and difficulties pertaining to the identification and development of biomarkers for SUDs. We focus on cocaine dependence as an example. Putative diagnostic, pharmacodynamic (PD), and predictive biomarkers for cocaine dependence are discussed across a range of methodological approaches. A possible cocaine-dependent clinical outcome assessment (COA)—another type of defined DDT—is also discussed. At present, biomarkers for cocaine dependence are in their infancy. Much additional research will be needed to identify, validate, and qualify these putative tools prior to their potential use for medications development and/or application to clinical practice. However, with a large unmet medical need and an estimated market size of several hundred million dollars per year, if developed, biomarkers for cocaine dependence will hold tremendous value to both industry and public health.     

Promises of biomarkers in drug development--a reality check

Biomarkers have been a buzz word in drug development for the last 5 years. But where do we stand now? This perspective article will demonstrate to which extent biomarkers have impacted drug development and the use of drugs. In particular, the different types of biomarkers, their identification, validation and use in different phases of drug development from drug discovery, to approval, to clinical application will be discussed as well as the state‐of‐the‐art biomarker technologies and promising future methods. The high interest in biomarkers has generated the need for development of new technologies and refinement of existing ones. Besides discussing their perspectives of applications, the present article also illustrates the future of biomarker development in terms of qualification for regulatory use and co‐development.     

Molecular approaches to the identification of biomarkers of exposure and effect--report of an expert meeting organized by COST Action B15. November 28, 2003

In the past, the term biomarker has been used with several meanings when used in human and environmental toxicology as compared to pharmaceutical development. However, with the advent of molecular approaches and their application in the field of drug development and toxicology, the concept of biomarkers has to be newly defined. In the meeting, the experts found consent in defining the term and described the application of biomarkers in toxicology, drug development and clinical diagnostics. Molecular approaches to biomarker identification and selection lead to a large amount of data. Hence, the statistical analysis is challenging and special statistical problems have to be solved in biomarker characterization, of particular interest are attempts aiming at class discovery and prediction. Reliability and biological relevance are to be demonstrated for biomarkers of exposure and effect which is also true for biomarkers of susceptibility. It is envisaged that the application of biomarkers will expand from current use in pre-clinical toxicology to the risk characterization and risk assessment of chemicals and from early clinical phases of drug development to later phases and even into daily clinical use in diagnostics and disease classification.     

Diagnostic and predictive performance and standardized threshold of traditional biomarkers for drug‐induced liver injury in rats

Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug‐induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver‐operating characteristic (ROC) curve, using a free‐access database where 142 hepatotoxic or non‐hepatotoxic compounds were administrated to male rats (n = 5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in‐house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI. Copyright © 2014 John Wiley & Sons, Ltd.     

Understanding post 9/11 drug control policy and politics in Central Asia

This paper exposes contemporary drug policy challenges in Central Asia by focusing on a single point in the history of drug control, in a single region of the global war against drugs and terrorism, and on one agency whose mission is to help make the world safer from crime, drugs and terrorism. By looking closely at the post 9/11 security-oriented donor priorities, I conclude that, in Central Asia, the rhetoric of the taking a more ‘balanced approach’ to drug policy is bankrupt. When enacted by the national law enforcement agencies in the Central Asian republics, the ‘Drug Free’ aspirational goal is driving the HIV epidemic among IDUs. The face-saving ‘containment’ thesis does not reflect the drug situation in this region but rather the failure to adopt an evidence-based approach. The harm reduction agenda continues to face many challenges including resistance to substitution treatment, the harm from drug treatment, from poorly designed drug prevention programmes and from repressive counter-narcotics policies and practices.     

The relationship of illicit drug use to HIV-infection among commercial sex workers in the city of Santos, São Paulo, Brazil

Objective: To assess the role of illicit drug use as a risk factor of HIV infection among female commercial sex workers (CSWs) in the city of Santos, Brazil. Design: Cross-sectional survey of 697 chain-referred CSWs. Methodology: The study included information on social-demographic characteristics, sexual practices and use of illicit drugs. Blood samples were tested for HIV. Associations between the response variable and drug-related variables, controlling for potential confounders, were assessed through multiple logistic regression procedures. Results: The univariate analyses for all drug-related variables evidenced that ‘use of injecting drugs’ (odds-ratios (OR)=10.9) and ‘use of crack in the previous month’ (OR=9.0), were the two variables most highly associated to the outcome. The multivariate analysis emphasized the role of crack use, the first variable included in the stepwise procedure (adjusted OR=5.3, P=0.0001). Other relevant predictors were ‘educational level’ (P=0.0003), ‘practice of anal intercourse with customers’ (P=0.0031), ‘use of injecting drugs’ (adjusted OR=3.1, P=0.0647), ‘age at first intercourse’ (P=0.0188) and ‘age’ (P=0.0175). Additional multivariate results showed that crack-users tend to use other drugs simultaneously, to agree more frequently to have unprotected sex and to earn a significantly smaller payment per sexual encounter than crack non-users. Conclusions: The analysis showed the vulnerability of CSWs in relation to drug use, lack of education and unprotected sex. These findings highlight the need for preventive programs focused on the general use of drugs in this population as well as efforts to help CSWs to acquire educational and professional skills.     

Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report

No Heading Biomarkers are increasingly used in drug development to aid scientific and clinical decisions regarding the progress of candidate and marketed therapeutics. Biomarkers can improve the understanding of diseases as well as therapeutic and off-target effects of drugs. Early implementation of biomarker strategies thus promises to reduce costs and time-to-market as drugs proceed through increasingly costly and complex clinical development programs. The 2003 American Association of Pharmaceutical Sciences/Clinical Ligand Assay Society Biomarkers Workshop (Salt Lake City, UT, USA, October 24–25, 2003) addressed key issues in biomarker research, with an emphasis on the validation and implementation of biochemical biomarker assays, covering from preclinical discovery of efficacy and toxicity biomarkers through clinical and postmarketing implementation. This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices. For example, it was agreed that because reliable and accurate data provide the basis for sound decision making, biomarker assays must be validated in a manner that enables the creation of such data. The nature of biomarker measurements often precludes direct application of regulatory guidelines established for clinical diagnostics or drug bioanalysis, and future guidance on biomarker assay validation should therefore be adaptable enough that validation criteria do not stifle creative biomarker solutions.     

Advancing cancer drug development through precision medicine and innovative designs

ABSTRACT

Precision medicine has been a hot topic in drug development over the last decade. Biomarkers have been proven useful for understanding the disease progression and treatment response in precision medicine development. Advancement of high-throughput omics technologies has enabled fast identification of molecular biomarkers with low cost. Although biomarkers have brought many promises to drug development, steep challenges arise due to a large amount of data, complexity of technology, and lack of full understanding of biology. In this article, we discuss the technologies and statistical issues that are related to omics biomarker discovery. We also provide an overview of the current development of biomarker-enabled cancer clinical trial designs.     

Revisioning women and drug use: gender sensitivity, embodiment and reducing harm

The purpose of this paper is ‘to revision’ our approach to women's use of drugs – which means to let go of how we have seen in order to construct new perceptions. Women use a variety of substances for a range of reasons, including pleasure. Yet, women who experience problems are left feeling stigmatised, marginalised and demoralised. The paper includes three inter-related discussions. First, two co-existing approaches to drug use, the classical and the postmodern, are explained. Second, after demonstrating how the postmodern approach is more valuable for the development of a gender-sensitive perspective, I will, with special reference to drug use, explain the complexities of two contemporary concepts, gender and embodiment. Here, I attempt to generate a deeper appreciation of these concepts in the postmodern approach. Third, I ask, ‘How can we develop a gender-sensitive, harm reduction approach’? The contention is that while harm reduction philosophies are admirable, these need to be gender-sensitive in order to be effective. A multi-levelled, ‘gender-sensitive’ view of harm reduction is put forward, as harm reduction is examined at the subjective, treatment, relationship, occupational and leisure levels.     

Tartrate-resistant acid phosphatase (TRACP 5b): A biomarker of bone resorption rate in support of drug development: Modification, validation and application of the BoneTRAP kit assay

A commercial kit assay of tartrate-resistant acid phosphatase (TRACP 5b) used for the diagnosis of bone resorption was modified with a ‘Fit-For-Purpose’ approach for drug development of anti-resorptive therapeutics. The modifications included changing the standard matrix from buffer to serum, using a consistent bulk reference material to prepare standards and quality controls (QC), and adding sample controls (SC) prepared from authentic sample pools.Method validation experiments were conducted for: inter- and intra-assay accuracy and precision, establishment of SC, range finding of different population groups, selectivity tests, parallelism and stability. The analytical range was 1.00–10.0 U/L and the total errors of lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) validation samples were 8% and 21%, respectively. Data of range finding experiment showed that serum samples should be collected in tubes instead of bags. Selectivity results showed accurate spike recovery among the majority of test samples from target populations. Samples were demonstrated to be stable for up to four freeze/thaw cycles and for 24 months at −70 ± 10 °C. Our results show that the modified TRACP 5b method is reliable for the quantification of TRACP 5b in human serum samples to support clinical trials of bone resorptive effect reflected by TRACP 5b activities. The method was robust with similar assay performance characteristics shown in three bioanalytical laboratories.