“CLINICAL BIOMARKERS”

AMIT VERMA
Department of Pharmaceutical Sciences
Dr. Hari Singh Gour Vishwavidyalaya
(A Central University)

Sagar (M.P.) – 470003
DEPARTMENT OF PHARMACEUTICAL SCIENCES DR. HARI SINGH GOUR CENTRAL UNIVERSITY SAGAR (M.P.) (Approved by AICTE, UGC New Delhi)

BIOMARKER AS A DIAGNOSTIC TOOL

Introduction
The challenges faced by the pharmaceutical industry in terms of maintaining delivery of new medicinal products in an environment of escalating research and development (R&D) costs have been well documented. One of the key initiatives of the US Food and Drug Administration (FDA) in response to this pipeline problem, the Critical Path Initiative identified six priority challenges for targeted research to increase efficiency, predictability and productivity in the development of new medicinal products. At the top of the list was the development of biomarkers to address the issues of predictive medicine, allowing the prediction of a potential product‘s performance as early as possible with the greatest degree of certainty. This recognition by regulatory agencies of the importance of biomarkers for the future success of drug discovery and development is reflected both by their prevalence within the literature – a simple search for the term ‗biomarker‘ in Medline giving in excess of 350,000 hits – and in the recent approvals of clinically validated molecular diagnostics.

A brief history of Biomarkers
The idea of using biomarkers to detect disease and improve treatment goes back to the very beginnings of medical treatment. The practice of uroscopy examining a patient‘s urine for signs of disease dates back to the 14th century or earlier, when practitioners would regularly inspect the color and sediment of their patient‘s urine and make a diagnosis based on what they observed(Connor, 2001). Today, your doctor may use spirometry to measure your lung function, take your blood pressure as a measure of your cardiovascular health, or test your blood glucose for diabetes. And

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not only are biomarkers useful at the ‗bedside‘; biomarkers have already proved their worth at the ‗bench‘ as well.

Philadelphia chromosome
In 1960, researchers discovered that some patients with chronic myelogenous leukemia (CML), a form of adult leukemia in which there is a proliferation of myeloid cells in the bone marrow, have a specific genetic change associated with their cancer, a shortened version of chromosome 22. This abnormality, known as the Philadelphia chromosome, is caused by a translocation between chromosomes 9 and 22. The consequence of this genetic swap is the creation of the BCR-ABL ‗oncogene‘; this cancer-causing gene produces a protein with elevated tyrosine kinase activity that induces the onset of leukemia (Capdeville et al., 2002). Researchers were able to use the Philadelphia chromosome as a biomarker to indicate which patients would benefit from drug candidates (tyrosine kinase inhibitors) specifically targeting the rogue protein. The end product was the drug imatinib (Gleevec), which decreases the proliferation of Philadelphia chromosome+ cells and slows the progression of the disease. As a postscript to this story, researchers further found that specific mutations in the BCR–ABL gene were biomarkers that predicted resistance to imatinib, leading to the development of newer tyrosine-kinase inhibitors dasatinib and nilotinib.

HIV viral load
In the late 1980‘s, scientists discovered that HIV viral load could be used as a marker of disease progression, and subsequently, as a measure of antiretroviral treatment efficacy. Viral load was used to show that patients receiving combination therapy had a higher reduction in viral load than those on monotherapy, and was

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therefore more effective in slowing the progression of the disease. Eventually, the viral load biomarker was used in the development and assessment of Highly Active Antiretroviral Therapy (HAART) treatment regimens involving a combination of several drugs used by many people living with HIV today.

HER-2 gene and receptor
Probably the most famous biomarker in recent drug development history is the HER-2 gene and receptor, discovered in the mid 1980‘s. Between 20–30% of breast cancer patients show an overexpression of the HER-2 receptor on their cancer cells. Although this biomarker indicates a higher risk of adverse outcomes, it also gave clinicians a new target for novel therapies. The antibody trastuzumab (Herceptin) was developed to target HER-2 receptors in these ‗overexpressing‘ patients, and successfully reduces the proliferation of cancer cells in many of these women.

What Are Biomarkers and What Use Are They?
The word biomarker is frequently used, often very generically, to cover a wide range of measurements that are applied in a large number of ways. This inherently wide scope of the term biomarker has been a source of confusion in this area. Varying formal definitions for biomarker can be found: these usually involve the concepts of an objective measurement of a characteristic as an indicator of a biological process, whether a normal process, a disease process or a response to treatment. Often, the measurement is implicitly or explicitly thought of as being biochemical in nature, but biomarkers are not limited to this definition; for example, biomarkers based on imaging technologies are proving very powerful in a diverse range of applications, from tumour progression to joint narrowing.
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Biomarkers are proposed to measure the delivery of drugs to their intended targets, and to understand and predict pathophysiology, and how it is altered by therapy, through monitoring variables known to have clinical relevance. Biomarkers are especially valuable, as they can help to prioritise drug discovery resources by enabling early proof-of-concept studies for novel therapeutic targets. Biomarkers enable the characterization of patient populations and quantitation of the extent to which new drugs reach intended targets, alter proposed pathophysiological mechanisms and achieve clinical outcomes. A biomarker is a biological characteristic, which can be molecular, anatomic, physiologic, or biochemical. These characteristics can be measured and evaluated objectively. They act as indicators of a normal or a pathogenic biological process. They allow assessing the pharmacological response to a therapeutic intervention. A biomarker shows a specific physical trait or a measurable biologically produced change in the body that is linked to a disease or a particular health condition. A biomarker may be used to assess or detect: A specific disease as early as possible – diagnostic biomarker (HCV RNA after infection) The risk of developing a disease – susceptibility/risk biomarker (BRCA1-breast cancer) The evolution of a disease (indolent vs. aggressive) – prognostic biomarker (HER-2-breast cancer) – but it can be predictive too The response and the toxicity to a given treatment – predictive biomarker (EGFRNSCLC/ gefitinib, DPD-gastrointestinal cancer/fluoropyrimidines). A useful first distinction can be made between two broad classes: surrogate and predictive biomarkers. Surrogate biomarkers are measured characteristics that can be used as substitutes for other

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characteristics, but that are easier to measure due to cost, time or ease of observation. This is not a new concept; for example, raised cholesterol has been used as a surrogate for heart disease for many years.

Property Explanation
Dynamism Measurements can be static or dynamic

Examples and impact
A static biomarker cannot correlate with a dynamic phenotype if the dynamic phenotype cannot predict itself on repeat measurement. Genotype is a good example of a static (subject level) biomarker which is often proposed to impact on plasma pharmacokinetics a dynamic (subject level)

Level

Average single molecule detection

or

Univariate versus multivariate markers

Continuous/ categorical

Measurements can be made at the level of a thin slice of tissue, single lesion or subject level Classical assay development methods were established on analytes present at the level of billions of molecules. Recent technologies, especially using nucleic acids, take us down orders of magnitude to levels where stochastic variation must be considered. Composite measurements should have clear rules to derive a final easy to interpret multivariate index. Measurements can be intrinsically continuous or discrete.

phenotype. Many candidate predictive tumour markers are measured on single sections of tumour tissue and the results automatically extrapolated as a patient level attribute. The Jak2 mutation assay from Ipsogen is suggested to be used at an input of 25 ng of DNA. Therefore the assay may be useful for detecting the presence of high levels of mutant sequence but will not be useful for monitoring residual disease burden once mutant granulocytes drop to a level where the input recommendations preclude the presence of mutant sequence.

The rules governing the combination of target, non-target and new lesions for determining RECIST measurements are a good example of deriving a single patient level index for decision making. Decisions are invariably discrete and therefore care must be taken to provide a clear message with output from continuous markers.

Table: 1 -Properties of biomarkers and phenotype measurement Predictive biomarkers, on the other hand, are measured
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characteristics that can be used to predict the likely outcome for a patient in terms of either disease progression (prognostic biomarkers) or response to treatment (predictive biomarkers), which can then guide clinical treatment. Predictive biomarkers can work in identifying both patients who would respond positively to a drug (efficacy biomarkers) and those who should not be administered the drug as they would be at increased risk of an adverse event (safety biomarkers). This forms the basis of personalised medicine: selecting the right drug for the right patient. Clearly, while these various classes of biomarkers have similarities, and a particular biomarker may fulfil multiple roles, there are also differences in the ways in which they need to be analysed and interpreted and in how they can be integrated into the drug discovery and development process. Signatures in complex biological mixtures that can be unambiguously correlated to biological events in order to validate novel drug targets and predict drug response. Biomarkers can stratify patient populations or quantify drug benefit in primary prevention or disease-modification studies in poorly served areas such as neurodegeneration and cancer. Clinically useful biomarkers are required to inform regulatory and therapeutic decision making regarding candidate drugs and their indications in order to help bring new medicines to the right patients faster than they are today. A biomarker, or biological marker, is in general a substance used as an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. It is used in many scientific fields (Loukopoulos et al. 2003). A biomarker is a measurement used as an indicator of biological actions. Biomarkers are prevalent in most branches of medicine. Measurement of specific biological features allows practitioners to

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determine diagnoses and prognoses and predict treatment outcomes by providing objective measurements to target. (Gotto, 1998). Type
Diagnostic biomarkers

Characteristics
Discriminate normal from a specific disease or disease state independent of a specific therapy

Sources of Biomarkers
Expression analysis, Disease mechanism studies, Epidemiology studies. Expression analysis, Animal models, Epidemiology studies.

Key Validation Requirements
Correlation with a specific disease or disease state

Prognostic biomarkers

Predict the likely course of disease progression independent of a specific therapy

Correlation with a clinical outcome independent of a specific therapy

Stratificatio n biomarkers

Identify patients likely to respond to a specific drug or suffer from its sideeffects prior to administration of the drug

Pre-clinical studies, Clinical trials, Epidemiology studies.

Correlation with a clinical response (benefit/harm)upo n administration of the drug in controlled clinical trials Correlation with the concentration or activity of a specific drug in animal and human studies Correlation with the concentration or activity of a specific drug in clinical trials with placebo control Correlation with the Concentration or activity of a specific drug in clinical trials

PD/PK Biomarkers

Correlate response to a specific drug with concentrations of the drug or its metabolites

Drugs or metabolites, Animal models.

Efficacy Biomarkers

Monitor the beneficial effects of a specific drug on the intended drug target or medical condition Monitor the adverse effects of a specific drug on any unintended cellular processes, cell, tissues or organs

Molecular targets or downstream molecules, Clinical trials. Histopathology, Clinical chemistry, Toxicology studies, Clinical trials.

Toxicity Biomarkers

Table: 2- Characterization of biomarkers Currently, there are no biomarkers available for psychiatric disorders; therefore, diagnostic tools and treatment decisions are restricted to the evaluation of clinical signs and symptoms that lack

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objectivity. That said, treatments for managing psychiatric symptoms are relatively effective. However, no single treatment works for everyone with a given disorder, and selection of the best treatment in mainstream psychiatry remains a challenge. As in any other disease state, a primary goal in psychiatry is the identification of specific biomarkers that would permit a more precise definition of specific disorders and, in turn, enhance the ability to develop targeted patient treatments. In fact, research has highlighted a need for biomarkers in psychiatry to enhance patient management and ensure treatment success (Holsboer, 2008; Keshavan et al., 2005). A biomarker is a parameter that can be used to measure the progress of disease or the effects of treatment. The parameter can be chemical, physical or biological. In molecular terms biomarker is "the subset of markers that might be discovered using genomics, proteomics technologies or imaging technologies. Biomarkers plays major role in medicinal biology. Biomarker brings the future things in our hand by helping in early diagnosis, disease prevention, drug target identification, drug response etc. Several diseased based biomarkers had been identified for many diseases such as serum LDL for cholesterol, blood pressure, P53 gene and MMPs for cancer etc. Gene based biomarker is found to be an effective and acceptable marker in the present scientific world (Loukopoulos et al., 2003). In medicine, a biomarker can be a substance that is introduced into an organism as a means to examine organ function or other aspects of health. For example, rubidium chloride is used as a radioactive isotope to evaluate perfusion of heart muscle. It can also be a substance whose detection indicates a particular disease state, for example, the presence of an antibody may indicate an infection. More specifically, a biomarker indicates a change in expression or state of a protein that correlates with the risk or progression of a disease, or

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with the susceptibility of the disease to a given treatment. A biomarker can also be used to indicate exposure to various environmental substances in epidemiology and toxicology. In these cases, the biomarker may be the external substance itself (e.g. asbestos particles or NNK from tobacco), or a variant of the external substance processed by the body (a metabolite). In cell biology, a biomarker is a molecule that allows for the detection and isolation of a particular cell type (for example, the protein Oct-4 is used as a biomarker to identify embryonic stem cells). In genetics, a biomarker (identified as genetic marker) is a DNA sequence that causes disease or is associated with susceptibility to disease. This is especially important for therapeutic indications in which assumptions regarding the relevance of animal models to clinical disease are tested only in large late-phase, long-term clinical trials that can require extensive dose ranging. Biomarkers can often be developed using animals in vivo before transferring the methodology to the clinic, although some technologies, such as functional brain imaging, can be compromised by constraints of experimental protocols. . Biomarkers are especially valuable for providing early tests of key programme hypotheses, particularly if changes can be measured in normal volunteer subjects during initial clinical trials. The choice of biomarkers should always factor in the feasibility and ease of clinical use in the specific setting in which the biomarker will be deployed. It is important to anticipate that use by planning, in advance, rigorous validation in appropriate preclinical and clinical study designs. The time required to achieve this can be substantial, and therefore biomarker development should begin simultaneously, and proceed in parallel with, the search for new therapeutics. Biomarkers that monitor specific physiological or pharmacological mechanisms can be used to select between multiple therapeutic targets for a drug by

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identifying those that are most sensitive to the intervention. Biomarkers can also reveal drug targets as well as optimize selection of molecules that interact with these targets for further development. For drugs with a large therapeutic index, these biomarkers can allow fast progression from Phase I to Phase II clinical studies on the basis of quantification of target modulation (exposure–effect relationships), rather than the achievement of maximum tolerated dose. It is now clear that understanding the multivariate nature of disease and drug responses will usually depend on an integrated approach encompassing the use of genetic, messenger RNA expression, clinical, epidemiological, proteomic and related molecular phenotype data collectively termed ‗molecular profiling (Schadt et al., 2003). The use genomic approaches as biomarkers could, however, have an earlier impact on the efficiency and probability of success in the preclinical drug discovery process by identifying profiles characteristic of unwanted toxicity I nearly drug candidate screening. Phenotypic or genotypic biomarkers could assist prognosis5 and underpin population-enrichment strategies that increase the signal in early proof-of-concept clinical trials (Van et al., 2002). Clinical relevance is particularly important for clinical biomarkers of potential toxicities, as spurious findings could unnecessarily restrict dosing regimens if they become a limiting factor in the calculation of the maximum recommended starting dose7 or the rate of dose escalation. Ultimately, however, it is unlikely that secure safety decisions could made on the basis of biomarker data alone (US Department of Health and Human Services 2002).

Biomarker requirements
For chronic diseases, whose treatment may require patients to take medications for years, accurate diagnosis is particularly important, especially when strong side effects are expected from the treatment.
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In these cases, biomarkers are becoming more and more important, because they can confirm a difficult diagnosis or even make it possible in the first place (Pharma Matters White Paper 2008).

Figure: 2-Rationale for using biomarkers to assess risk A number of diseases, such as Alzheimer‘s disease or rheumatoid arthritis, often begin with an early, symptom-free phase. In such symptom-free patients there may be more or less probability of actually developing symptoms. In these cases, biomarkers help to identify high-risk individuals reliably and in a timely manner so that they can either be treated before onset of the disease or as soon as possible thereafter (Craig et al., 2008; Egerer et al., 2009). In order to use a biomarker for diagnostics, the sample material must be as easy to obtain as possible. This may be a blood sample taken by a doctor, a urine or saliva sample, or a drop of blood like those diabetes patients extract from their own fingertips for regular blood-sugar monitoring. For rapid initiation of treatment, the speed
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with which a result is obtained from the biomarker test is critical. A rapid test, which delivers a result after only a few minutes, is optimal. This makes it possible for the physician to discuss with the patient how to proceed and if necessary to start treatment immediately after the test. Naturally, the detection method for a biomarker must be accurate and as easy to carry out as possible. The results from different laboratories may not differ significantly from each other, and the biomarker must naturally have proven its effectiveness for the diagnosis, prognosis, and risk assessment of the affected diseases in independent studies.

Types of biomarkers
Biomarkers validated by genetic and molecular biology methods can be classified into three types:

Type 0
Type 0 biomarkers are markers of the natural history of a disease and correlate longitudinally with known clinical indices, such as symptoms over the full range of disease states. Type 0 markers can be characterized in phase 0 clinical studies, in which a reliable assay is used in a well- defined patient population for a specified period of time. Ideally, linear (positive or negative) relationships established with the ‗gold standard‘ clinical assessor. Such studies are presently underway in osteoarthritis (see Further information, Osteoarthritis imaging initiative) and are planned for Alzheimer‘s disease (see Further information, Alzheimer‘s disease neuroimaging initiative) (Crum et al., 2001).

Type 1
Type I markers capture the effects of an intervention in accordance with the mechanism of action of the drug, even though the

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mechanism might not be known to be associated with clinical outcome. A priori validation of Type I biomarkers is impossible for truly novel targets without an effective positive control treatment. By definition, the more innovative the target, the less validated will be the associated biomarkers (Rolan et al. 1997). Therefore, for novel targets the biomarker will be validated in parallel with the drug candidate, and the risk in decisions made on the basis of that biomarker will improve over time. Alternatively, validating a marker can sometimes be achieved using a closely related treatment. By way of example, a battery of cognition markers validated with the anticholinergic scopolamine were used to confirm the pharmacological effect of a novel agonist directed against the α7 nicotinic acetylcholine receptor, for which there is at present no candidate drug sufficiently well characterized in humans that it could serve as a positive control. Good type I biomarkers will capture the effects of both agonists and antagonists (Huestis et al. 2001).

Type 2
Type II markers are considered SURROGATE END- POINTS because a change in that marker predicts clinical benefit. Unambiguous definitions were proposed to distinguish biomarkers from CLINICAL ENDPOINTS, enabling debate on the validation and application of surrogate. Type II biomarkers (or surrogate end-points) must be relevant both to the mechanism of action of the drug and to the pathophysiology of the disease. Changes in the biomarker should reflect treatment benefit and therefore effective therapy is necessary for this validation. Type II marker to drugs with different mechanisms for the same clinical indication, or to drugs with the same mechanism for different indications, needs careful consideration of those differences. It is

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likely that some bio-markers will be restricted to a particular class of drug defined according to mechanism in a specific indication. The most commonly used surrogate endpoint for regulatory purposes is plasma concentration of drug in ‗bioequivalence‘ studies (Lesko et al., 2001), which proves the limiting case that a biomarker wellcorrelated with both the mechanism of action and the clinical effect needs only a single set of clinical trials for validation; that is to say, the Phase III studies supporting claims of effectiveness for the innovator drug are necessary and sufficient. The quality of evidence supporting the validity of a new biomarker has been described as similar to that used to support the validity of new therapies in terms of the number and size of clinical validation trials and the concordance among those trials. The risk of using bio- markers for decision making is directly related to the level of validation for the purpose to which it is applied. The ideal trial to validate a surrogate endpoint is not necessarily the ideal trial to gain drug approval using the surrogate endpoint. Indeed, it can be argued that to establish a surrogate is to lower the hurdle for competitors coming behind in the same therapeutic area, but has the advantage that the validator is the primary holder of the data that establishes the value of the surrogate with respect to the clinical endpoint (Pearson et al. 2003).

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Table: 3-Biomarker examples

Disease-related Biomarkers and Drug-related Biomarkers
It is necessary to distinguish between disease-related and drugrelated biomarkers. Disease-related biomarkers give an indication of whether there is a threat of disease (risk indicator or predictive biomarkers), if a disease already exists (diagnostic biomarker), or how such a disease may develop in an individual case (prognostic biomarker). In contrast, drug-related biomarkers indicate whether a drug will be effective in a specific patient and how the patient‘s body will process it. In addition to long-known parameters, such as those included and objectively measured in a blood count, there are numerous novel

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biomarkers used in the various medical specialties. Currently, intensive work is taking place on the discovery and development of innovative and more effective biomarkers. These "new" biomarkers have become the basis for preventive medicine, meaning medicine that recognizes diseases or the risk of disease early, and takes specific countermeasures to prevent the development of disease. Biomarkers are also seen as the key to personalized medicine, treatments individually tailored to specific patients for highly efficient intervention in disease processes. Often, such biomarkers indicate changes in metabolic processes. The "classic" biomarker in medicine is a laboratory parameter that the doctor can use to help make decisions in making a diagnosis and selecting a course of treatment. For example, the detection of certain autoantibodies in patient blood is a reliable biomarker for autoimmune disease, and the detection of rheumatoid factors has been an important diagnostic marker for rheumatoid arthritis (RA) for over 50 years (Waaler et al., 2007; Rose et al., 1948).

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Fig: 3-Scope of biomarkers For the diagnosis of this autoimmune disease the antibodies against the body‘s own citrullinated proteins are of particular value. These ACPAs, (ACPA stands for Anti-citrullinated protein/peptide antibody) can be detected in the blood before the first symptoms of RA appear. They are thus valuable and highly predictive biomarkers for the early diagnosis of this autoimmune disease (Bang et al., 2007). In addition, they indicate if the disease threatens to be severe with serious damage to the bones and joints, which is an important tool for the doctor when providing a diagnosis and developing a treatment plan. There are also more and more indications that ACPAs can be very useful in monitoring the success of treatment for RA (Nicaise et al., 2008). This would make possible the accurate use of modern

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treatments with biologicals. Physicians hope to soon be able to individually tailor rheumatoid arthritis treatments for each patient. With the growing number of new biological agents, there is increasing pressure to identify molecular parameters such as ACPAs that will not only guide the therapeutic decision but also help to define the most important targets for which new biological agents should be tested in clinical studies (Haupl et al., 2010). An NIH study group committed to the following definition in 1998: "a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention." In the past, biomarkers were primarily physiological indicators such as blood pressure or heart rate. More recently, biomarker is becoming a synonym for molecular biomarker, such as elevated prostate specific antigen as a molecular biomarker for prostate cancer, or using enzyme assays as liver function tests. There has recently been heightened interest in the relevance of biomarkers in oncology, including the role of KRAS in CRC and other EGFR-associated cancers. In patients whose tumors express the mutated KRAS gene, the KRAS protein, which forms part of the EGFR signaling pathway, is always ‗turned on‘. This overactive EGFR signaling means that signaling continues downstream even when the upstream signaling is blocked by an EGFR inhibitor, such as cetuximab (Erbitux) and results in continued cancer cell growth and proliferation. Testing a tumor for its KRAS status (wild-type vs. mutant) helps to identify those patients who will benefit most from treatment with cetuximab. Biomarkers also cover the use of molecular indicators of environmental exposure in epidemiologic studies such as human papilloma virus or certain markers of tobacco exposure such as 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To date no

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biomarkers have been established for SCCHN (Szodoray et al., 2009; Mathsson et al., 2008)

Biomarkers in Drug Development
Biomarkers predictive of drug toxicity or efficacy have conceivably the greatest impact on successful drug development. However, only a small number of biomarkers can accurately predict clinical safety and efficacy endpoints so as to be considered as surrogate endpoints as defined. On the other hand, biomarkers indicative of the mechanism of action of drug intervention can provide great predictive value in early drug development even if they do not become surrogate endpoints. The major challenge for drug development is how to select candidate biomarkers for a compound and differentiate surrogates from other biomarkers. Clearly, the choice is largely dependent upon the stage of drug development as well as the availability of resources. In addition, the biological materials that can be easily obtained from human subjects consenting to first-time-in-human trials are often limited. Therefore, the emphasis on selecting appropriate candidates should be heavily placed in the early stages of the drug discovery pipeline where both the costs of evaluating many candidate biomarkers and the risk of moving compounds forward that have adverse or toxic effects are reduced. The functions of biomarkers that can be used for pharmacodynamic selection, evaluation, patient toxicology determination, candidate stratification,

identification of new indications for existing drugs and even as early diagnostics or predisposition tests to expand the drug market to presymptomatic individuals can help bring clarity to understanding the

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compounds being developed and therefore significantly enhance researchers capability of decision making, including attrition of drug candidates. The use of biomarkers during the various stages of drug discovery and development are summarized in Table 4 and described here as examples of how transcriptome analysis may be applied in the future to enhance the discovery and development of novel pharmacophores. Biomarker development should follow different pathways depending on the stage of drug development. For early stages of clinical development, biomarkers can identify or confirm molecular targets, help to optimize dose schedules for the anticancer agent and might correlate with clinical benefit. Identifying clinically relevant targets is challenging; in numerous examples, the intended target was found to be irrelevant. As not all molecular targets are legitimate therapeutic targets, however, biomarkers can provide a means of determining which target(s), when inhibited, correlate with tumor control. In the case of some anticancer agents [e.g. cetuximab, gefitinib, erlotinib and inhibitors of vascular endothelial growth factor (VEGF)], it appears that the molecular target is the therapeutic target. In the later stages of clinical development, identified markers could be used to select the patients most likely to respond to the targeted agent. Any biomarker used as a basis for patient selection must demonstrate excellent sensitivity and specificity; otherwise, the risk of not treating patients who might benefit would be unacceptably high. Proper patient selection enables efficient clinical trial design for targeted therapies and ensures that the number of individuals exposed to the risks of anticancer therapy is minimized. The ultimate biomarker would be able to indicate the diseased state and be altered by therapeutic intervention so that clinical outcome could be predicted. However, it is most unlikely that one biomarker could be used for all these purposes; different biomarkers must be

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used at different stages in drug discovery and development. Biomarkers have been classified into three types. The first are markers of the disease state. The second type indicates the effects of a therapeutic intervention based on the mechanism of action for a drug, even though this may not be known to be associated with the desired clinical outcome. This type of biomarker is often used in preclinical screening of drug candidates. The third type of biomarker is used as a surrogate end-point because a change in that marker predicts clinical outcome. Areas like oncology have progressed further than other areas in biomarker discovery and use, and have already proven successful with such examples as HER-2, a biomarker for a subset of more aggressive breast cancers. HER-2, a human epidermal growth factor, not only identifies patients that will benefit from Herceptin but is also the target of the drug. Not all diseases will be amenable to biomarker discovery and use. Biomarkers will be easier to use in diseases that are well defined with more homogenous patient populations. However, the more that is learned about disease mechanisms and genetic differences among populations, the more biomarkers will be derived from this research.

Preclinical stage
Incorporating biomarkers early in the discovery process when a new therapeutic target is being identified is of utmost importance especially if the biomarkers can reflect mechanism-based intervention. To find these discovery biomarkers, researchers can look to their pre-clinical experiments, such as animal and cell culture models. Analysis of expression changes in target versus nontarget tissues in treated animal models may provide possible information to establish biomarkers for a potential therapeutic. High-throughput screening targets may also be used as an initial source of potential discovery biomarkers. It is important to keep in mind that these discovery biomarkers which are used preclinically
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may not be the same biomarkers used after the drug is selected to enter clinical trials. In preclinical studies, biomarkers are used to determine if the drug is hitting the target, after which additional biomarkers must confirm that hitting the target actually alters the pathophysiological mechanism, and that altering this mechanism affects clinical outcome. Biomarkers can also reveal other drug targets as well as optimize the selection of molecules that interact with these targets for further development.

Clinical stage
For one successful drug, there are 60 in discovery, 20-30 in early development, and 5-8 in clinical programmes. One of the biomarker development goals is to assess drug safety and efficacy accurately, thereby reducing attrition of drugs during clinical phases of development and hence reducing the overall cost of drug development. Singulex‘ new, more sensitive technologies developed for protein and metabolite biomarker detection provides a significant benefit for monitoring established safety and toxicity biomarkers, such as troponin I for cardiotoxicity, throughout clinical trials. The current cardiotoxicity tests can only detect large-scale damage, which halts drug development. The benefits of more sensitive assays are not only the detection of mild toxicity, repeated exposure to which could lead to major problems, but also in giving the green light for further development. Being able to detect normal levels of biomarkers, especially for toxicity, but for any deviation from normal provides the total information needed to make accurate decisions on whether to proceed further in drug development. Stage of Drug Development Pre-Discovery Potential Uses of Biomarkers

Study disease mechanisms

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Discovery

Pre-Clinical

Early Clinical Late Clinical Post-Marketing

Define drug targets Explore mechanisms of action for the compound class Establish structure-activity relationship Build pharmacokinetic pharmacodynamic models Highlight mechanisms of drug action Establish safety and efficacy end points Guide compound selection and retention Demonstrate bioavailability and bioequivalence Determine dose response Confirm mechanisms of drug action in humans Define targeted population Allow dose selection and optimization Use for registration Allow product differentiation Stratify patients Monitor therapeutic response Monitor side-effects

Table: 4-Utilities of biomarkers in drug development Once a proposed biomarker has been validated, it can be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (choices of drug treatment or administration regimes). In evaluating potential drug therapies, a biomarker may be used as a surrogate for a natural endpoint such as survival or irreversible morbidity. If a treatment alters the biomarker, which has a direct connection to improved health, the biomarker serves as a surrogate endpoint for evaluating clinical benefit. Some of the main areas in which molecular biomarkers are used in the drug development process are early drug development studies, Safety studies, Proof of concept studies, Molecular profiling (Katz et al., 2001).

Stages
Biomarker Discovery

Major Issues
Molecular profiling – experimental design and data interpretation Imaging biology – throughput and cost Biomarker validation – epidemiology
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Prototype Development

Biomarker Test Development

studies Assay development – platforms, reagents, protocols Analytic validation – sensitivity/specificity and alpha-site testing Production – process standardization and quality assurance Clinical validation – clinical trials and beta site testing Marketing – registration

Table: 5-Biomarkers development issues. Molecular biomarkers are often used in early drug development studies. For instance, they are used in phase I study for establishing doses and dosing regimen for future phase II studies. PD biomarkers are commonly observed to respond (either decrease or increase) proportionally with dose. This data, in conjunction with safety data, help determine doses for phase II studies. In addition, Safety molecular biomarkers have been used for decades both in preclinical and clinical research. Since these tests have become mainstream tests, they have been fully automated for both animal and human testing. Among the most common safety tests are those of liver function(e.g., transaminases, bilirubin, alkaline phosphatase) and kidney function(e.g., serum creatinine, creatinine clearance, cystatin C). Others include markers of skeletal muscle(e.g., myoglobin) or cardiac muscle injury(e.g., CK-MB, troponin I or T), as well as bone biomarkers(e.g., bone-specific alkaline phosphatase) (Lesko et al., 2000).

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Fig: 4-Parallel Paths of Drug Development (left column) and Biomarker Development (right column).

Successful biomarker development

Patient selection can be facilitated through the use of systems that enable selection of patients more likely to benefit from targeted therapy. Herceptest was the first such system developed. It is used to identify patients whose tumours overexpress Her-2/ERB2 and, therefore, who would be most likely to respond to treatment with trastuzumab (Herceptin). Her-2/neu is an example of an efficacy target. Validating the target–biomarker–antibody relationship involved a great deal of effort because the initial diagnostic test was somewhat ineffective. Once the marker was validated, however, only patients whose tumours overexpressed Her2/neu (20–25% of invasive breast cancers) were enrolled in the phase III trial. Consequently, only 470 patients were required; if subjects had been accrued from the general patient population, an estimated 2200 subjects would have been necessary. Significant benefit was

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demonstrated in 1.6 years of follow-up instead of 10 years. The response rate in this subpopulation was 50% compared with 10% in the overall patient population (Schatzkin et al., 2003). The antibodies used for the test system must work on different types of tissue. This needs to be confirmed by testing in multi-tissue arrays to make sure that background staining is not problematic. The final step is standardization of the assay to ensure consistency across laboratories. The keys to successful development of antibodies for use in patient selection are high quality in terms of specificity, functionality and sensitivity and standardization of reagents (no batch-to-batch variation), automated protocols and use of imaging as a means of interpreting the response. Regulatory authorities throughout the world strongly advocate standardization of testing to minimize the number of patients who experience adverse side-effects from treatment. Proper patient selection can also optimize treatment expenditure by selecting the patient population most likely to respond. The clinical development of gefitinib, an orally available epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) is a more complex example of biomarker development. Phase I and II development of gefitinib showed dramatic and unexpected tumor regressions in 10% of patients with advanced non-small-cell lung cancer (NSCLC) but data from early-phase trials did not show a clear correlation between patient outcome and EGFR expression in archived tissue (Bailey et al., 2003). Subsequently, however, data emerged indicating that EGFR mutations and increased gene copy number, as measured by fluorescence in situ hybridization (FISH) are associated with clinical response to gefitinib treatment .One of the challenges in the development of gefitinib was that knowledge of potential biomarkers emerged during the conduct of the pivotal trials. Indeed, increased

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EGFR gene copy number measured by FISH was shown to be a prognostic biomarker for outcome after surgery in patients with NSCLC, and subsequently shown to be predictive of response to gefitinib (Hirsch et al., 2007). Later EGFR mutations also emerged as predictors of response to EGFR TKIs in patients with advanced NSCLC. Evolution of biomarkers during the conduct of large randomized trials might become the rule rather than the exception. Although initial candidate biomarkers are evaluated early in development, knowledge increases exponentially as research and clinical experience become more widespread and increased clinical data with which to correlate the translational work become available. Another example of a biomarker used as a safety target exists (Lynch et al., 2004). Irinotecan (Campto), which is approved for treating metastatic colorectal cancer, was found to cause grade 4 neutropenia in 8% of the general patient population. Subsequent data have shown that uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) affects the drug‘s metabolism and, therefore, its toxicity profile. The UGT1A1*28 polymorphism, characterized by an additional TA repeat in the TATA sequence of the UGT1A1 promoter, was associated with greater toxicity (Ichikawa et al., 2008).

Discovery of molecular biomarker
Molecular biomarkers have been defined as biomarkers that can be discovered using basic and acceptable platforms such as genomics and proteomics. Many genomic and proteomics techniques are available for biomarker discovery and few recently using techniques are given below. Apart from genomics and proteomics platforms biomarker assay techniques. Metabolomics, Lipidomics, and Glycomics are also the most commonly used as techniques in identification of biomarker.

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Fig: 5-Strategies for discovering biomarkers

Genomic Approach
Northern blot Gene expression SAGE DNA Microarray

Proteomic Approach
2D-PAGE LS/MS SELDI-TOF Ab Microarray Tissue Microarray

Metabolomics Approach
The term metabolomics has been recently introduced to address the global analysis of all metabolites in a biological sample. A related term, metabolomics, was introduced to refer specifically to the analysis of metabolic responses to drugs or diseases. Metabolomics

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become a major area of research it is the complex system biological study, used as a to identify the biomarker for various disease. In general most of the disease case some of the metabolic pathway had been activate or deactivated, this parameter can be used as a marker for some diseases. Serotonin production pathway activated in alcoholic drinking person it can be metabolic marker of recent alcohol consumption.

Lipidomics Approach
Lipidomics refers to the analysis of lipids. Since lipids have unique physical properties, they have been traditionally difficult to study. However, improvements in new analytical platforms have made it possible to identify and to quantify most of lipids metabolites from a single sample. Three key platforms used for lipid profiling include mass spectrometry, chromatography, and nuclear magnetic resonance. Mass spectrometry was used to delineate the relative concentration and composition of high-density lipoproteins (HDL) particles from lipid extracts isolated from coronary bypass patients and healthy volunteers. They found that HDL particles from coronary bypass patients contained significantly less sphingomyelin relative to phosphadidylcholine and higher triglycerides relative to cholesterol esters. Lipidomic profiling was also used to study the effect of rosiglitazone, a PPARγ agonist, on lipid metabolism on mice. Rosiglitazone was observed to alter lipid composition in different organs (Egerer et al., 2009).

Imaging biomarkers
Many new biomarkers are being developed that involve imaging technology. Imaging biomarkers have many advantages. They are usually noninvasive, and they produce intuitive, multidimensional results. Yielding both qualitative and quantitative data, they are usually relatively comfortable for patients. When combined with
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other sources of information, they can be very useful to clinicians seeking to make a diagnosis (Smith et al., 2003). Cardiac imaging is an active area of biomarker research. Coronary angiography, an invasive procedure requiring catheterization, has long been the gold standard for diagnosing arterial stenosis, but scientists and doctors hope to develop noninvasive techniques. Many believe that cardiac computed tomography (CT) has great potential in this area, but researchers are still attempting to overcome problems related to ―calcium blooming,‖ a phenomenon in which calcium deposits interfere with image resolution. Other intravascular imaging techniques involving magnetic resonance imaging (MRI); fig-6, optical coherence tomography (OCT), and near infrared spectroscopy are also being investigated (Smith et al., 2000).

Fig: 6-magnetic resonance imaging (MRI) Another new imaging biomarker involves radiolabeled fludeoxyglucose. Positron emission tomography (PET) [fig: 7] can be used to measure where in the body cells take up glucose. By tracking glucose, doctors can find sites of inflammation because macrophages there take up glucose at high levels. Tumors also take up a lot of glucose, so the imaging strategy can be used to monitor them as well. Tracking radiolabeled glucose is a promising technique because
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it directly measures a step known to be crucial to inflammation and tumor growth (Petricoin et al. 2002).

Figure: 7- Positron emission tomography (PET)

Future Challenges of in clinical trials

biomarkers

One of the major challenges to innovation is our ability to discover new biomarkers. Part of the answer lies in gaining a better understanding of the pathophysiology of disease, thereby uncovering potential drug targets and biomarkers in the disease pathway. The fundamental issue we have to deal with, both with target selection

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and developing better biomarkers, is a better understanding of pathophysiology. The clinical need is huge, not least in diseases like chronic obstructive pulmonary disease (COPD), an illness about which we know very little. COPD has very few markers to indicate severity and disease progression. In asthma at least we have biomarkers like sputum eosinophils and allergen challenges, and we‘re able to do short term, proof of concept drug studies. But in COPD, we don‘t have equivalent biomarkers. So there‘s the risk that you do harm because you can‘t measure safety with biomarkers beforehand. But the major risk, he says, is committing to a large phase III study without proof of drug efficacy. Finding biomarkers to improve our assessment of efficacy and safety in small numbers of patients is where all the initiatives are directed at. In 2006, GSK launched the ECLIPSE project, a three-year study following the progression of COPD in over 2000 patients. The study is actively looking for biomarkers that may help to improve our ability to diagnose COPD, define its severity and predict its progression over time, and ones that can eventually be used in drug development. We don‘t currently have many good biomarkers in respiratory medicine. The reality is, there‘s a disconnect between the science, and getting that science into the clinical arena. This need for biomarkers is not just restricted to respiratory medicine. ―There are substantial needs in cardiology. We need to develop drugs in practically every area, from congenital disease to hypertension, stroke and myocardial infarction. The difficulty is in understanding the disease process, and identifying some of the players in these pathways that will play a role in identifying whether your treatment is useful or not.‖ Drug development is based on different types of biomarker in the context of drug development. Diagnostic biomarkers provide the means to define a population with a specific disease. Prognostic

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biomarkers correlate with outcomes. For example, overexpression of Her-2/neu in breast cancer or EGFR expression in colorectal cancer indicates poor prognoses. Such prognostic markers are frequently the basis for establishing inclusion criteria for a clinical trial or for defining a patient population. Predictive biomarkers define populations that might respond more favourably to a particular intervention from an efficacy or safety perspective. They can be used to stratify patients for subgroup analyses. Surrogates are biomarkers that correlate with clinical benefit and changes in the marker correlate with alterations in outcome. Examples include response rate or Progression free survival in oncology or bone mineral density in osteoporosis prevention and treatment. If validated, a surrogate may serve as a primary endpoint in a pivotal registration study and could support approval of an anticancer agent. The Critical Path Initiative (Fig; 8) of the FDA aims to stimulate and facilitate a national effort to modernize the scientific process through which a potential human drug, biological product or medical device is transformed from the discovery or proof-of-concept stage into a standard therapeutic or diagnostic product. The focus of this initiative is to update the evaluative tools currently used to assess the safety and efficacy of new medical products, including the validation and use of biomarkers in clinical trial patient selection and as surrogate endpoints. From a regulatory point of view, biomarkers can be an indicator of efficacy as a surrogate marker but cannot serve as a marker of clinical benefit per se. ideally, validation should be carried out prospectively, although initially, retrospective studies undertaken to identify surrogate markers. Validation should be carried out in another, complementary population that does not include the original subpopulation found to be positive for the biomarker. The best sort of trial would be a may be

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randomized prospective trial which provides information not only on true endpoint differences between the two treatment arms, but also on the degree of correlation between surrogate endpoint and true endpoint.(U.S. Food and Drug Administration. Innovation or stagnation, 2004).

Fig: 8

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