Review Safety biomarker applications in drug development

Correspondence: John Burkhardt (E-mail: [email protected])

Safety biomarker applications in drug developmentShelli Schomaker, Shashi Ramaiah, Nasir Khan and John Burkhardt

Drug Safety R&D, Pfizer, Inc, Groton, CT, USA

[Recommended by Ikuo Horii]

(Received January 29, 2019; Accepted February 5, 2019)

ABSTRACT — Biomarkers are invaluable drug development tools to assess and monitor safety in early clinical trials especially when exposure margins are limiting for promising therapeutics. Although progress has been made towards identifying and implementing translational safety biomarkers for a number of organ toxicities such as kidney and liver, significant biomarker gaps still exist to monitor toxicities for testis, pancreas, etc. Several precompetitive consortia [e.g., Predictive Safety Testing Consortia (PSTC), Innovative Medicines Initiative (IMI)] are working with industry, academia, government, patient advo-cacy groups and foundations with a goal to qualify biomarkers such that they can be used in preclinical studies and clinical trials to accelerate drug development. This manuscript discusses the complexities of novel biomarker discovery, validation and international regulatory qualifications intended for clinical trial applications and shares specific examples from Pfizer Research and Development. As safety biomarkers become widely accepted and qualified by the regulatory agencies, they will increasingly be implemented in early clinical trials, play a key role in decision making and facilitate the progression of promising ther-apeutics from preclinical through clinical development. Key words: Safety biomarker, Clinical translation, Biomarker qualification

INTRODUCTION

The pharmaceutical industry continues to face chal-lenges in terms of declining productivity. The overall cost of drug development and time for most drugs to reach market has been increasing over the past several dec-ades. From 1975 to 2015, the total expenditures per year for drug discovery and development rose approximately 5-fold while the number of United States Food and Drug Administration (FDA) new drug registrations per year remained relatively flat (Boyer et al., 2016). Substantial resources are being invested in research and development (R&D) across the industry into compounds that eventual-ly fail. The current pharmaceutical R&D process results in only ~10% of the molecules entering phase 1 clinical tri-als reaching full approval by the FDA (Hay et al., 2014). This high rate of attrition is a key driver in reducing pro-ductivity and a significant challenge to the industry. There are a number of explanations for the decrease in produc-tivity including a focus of the industry in areas of unmet medical need and novel biological mechanisms with high risk of failure, a higher entry bar for new drugs due to a competition with enhanced standard of care, higher reg-ulatory hurdles, commercial and financial portfolio deci-

sions and an increase in the complexity and cost of clini-cal trials (Roberts et al., 2014; Hay et al., 2014); however when a root-cause analysis was completed on 359 phase 3 and 95 new molecular entities and biologic license appli-cations, safety and efficacy were shown to be the two pri-mary causes for compound suspension (Hay et al., 2014).

The standard preclinical testing paradigm has marked-ly improved drug safety over the past 30 years. In 2000, the results from a multinational pharmaceutical survey and the outcome of an International Life Sciences Insti-tute (ILSI) Workshop were reported and showed that 70% of human toxicity observed during clinical trials is pre-dicted by preclinical studies (Olson et al., 2000). In addi-tion, a 2013 study in Japan reported that 48% of adverse drug reactions observed in clinical trials were predict-ed by a comprehensive preclinical safety assessment (Ahuja and Sharma, 2014); and in 2017, the IQ Consor-tium created and utilized an industry-wide nonclinical to clinical translational database to report that animal stud-ies not only have value in predicting human toxicities but also that an absence of toxicity in nonclinical stud-ies predicts a similar outcome in the clinic (Monticello et al., 2017). However, the inability to predict failures before or early in clinical trials remains a main cause of

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attrition (Ahuja and Sharma, 2014) as failures in clini-cal safety not predicted in preclinical studies continue to be a major cause of compound termination (Clark and Steger-Hartmann, 2018). In addition, clinical failures due to toxicity are not limited to the early clinical portfolio but are also evident in late stage development and post-marketing surveillance (Waring et al., 2015). Thus while preclinical data has had a significant impact on improving clinical drug safety, failures due to toxicity remain a key challenge facing the industry.

The high rate of attrition and cost of drug development has prompted a surge in research in the area of biomar-kers. According to the FDA/National Institute of Health (NIH)’s BEST (Biomarkers, Endpoints, and other Tools Resource) guide, a biomarker is a ‘defined characteristic that is measured as an indicator of normal biologic proc-esses, pathogenic processes, or responses to an expo-sure or intervention, including therapeutic interventions (FDA, 2016a).’ Biomarkers are invaluable drug develop-ment tools utilized to better understand the translatabili-ty of preclinical findings into human adverse events and to better assess and monitor these findings in the clinic especially when exposure margins are limiting for prom-ising therapeutics. Although progress has been made towards identifying and implementing translational safety biomarkers for a number of organ toxicities such as kid-ney and liver, significant biomarker gaps still exist. Sever-al precompetitive consortia (e.g., PSTC, IMI) are working with industry, academia, government, patient advocacy groups and foundations with a goal to qualify biomark-ers such that they can be implemented in preclinical stud-ies and clinical trials to accelerate drug development. This paper discusses the complexities of discovering, val-idating and qualifying novel biomarkers to understand organ toxicities and to accelerate drug development deci-sions. Specific biomarker case examples are highlighted to describe their implementation in preclinical and early clinical development to characterize safety issues, under-stand mechanisms and monitor clinical safety. As safety biomarker assays are validated and qualified by the reg-ulatory agencies, they will increasingly play a key role in decision making and facilitate the progression of promising therapeutics from preclinical through clinical development. This, however, is not a trivial endeavor and the path to suc-cess may best be achieved through a collaborative effort among industry, academia and regulatory partners.

BIOMARKERS

In 2004, the FDA’s Critical Path Initiative emphasized the need for innovation in drug development and sug-

gested the use of biomarkers to evaluate and predict safe-ty and effectiveness, provide informative links between mechanism of action and clinical utility, understand the translatability between preclinical species and humans, and serve as surrogate endpoints. With a focus on gen-erating and implementing translatable biomarkers ear-ly in the drug development process, the goal was for the pharmaceutical industry to improve the high attrition rates and process inefficiencies in terms of cost and time observed across the industry (Haim, 2011; Woodcock and Woosley, 2008). In drug development, biomarkers catego-rized as target biomarkers assess the ability of the drug to reach and engage the target while mechanism biomarkers test the ability of the drug to induce the expected meas-urable molecular or cellular pharmacodynamics response. Finally, the linkage of the mechanism to the clinical response or efficacy is assessed by disease biomarkers or clinical activity scores. Ideally, safety biomarkers inform the presence or extent of toxicity and thus increase confi-dence in safety and the ability to predict, detect and moni-tor the progression of drug-induced toxicity. The ability of safety biomarkers to detect early toxicity, monitor onset and reversibility, and manage adverse effects observed in the clinic will determine its overall utility and impact in preclinical and clinical drug development. With the emer-gence of innovative technologies and a diverse set of in vitro and in vivo models, the development of novel safety biomarkers has evolved to include not only the singular measurement of circulating proteins but also expression profile signatures in blood/tissue, noninvasive imaging and genetic variations in DNA (Currid and Gallagher, 2008). Assays for these biomarkers range from explora-tory fit for purpose to fully validated and qualified by the regulators for a specific context of use and the process of assay validation should be regarded as being continuous and evolving over-time. Pfizer has a strong commitment to developing and implementing translatable biomark-er strategies within drug development including applying protein biomarkers for drug-induced toxicity in the stom-ach, kidney, skeletal muscle and liver and small mole-cules/metabolites as biomarkers for drug-induced toxici-ty to the liver and heart. Several biomarker case examples from Pfizer R&D efforts are included in this review. All procedures performed on animals used in Pfizer exam-ples were in accordance with regulations and established guidelines and were reviewed and approved by an Institu-tional Animal Care and Use Committee.

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APPLICATION OF SAFETY BIOMARKERS

Example 1: Translational kidney biomarker panel as a preclinical screening tool

A number of emerging urinary kidney markers includ-ing kidney injury molecule 1 (KIM-1), clusterin, micro-albumin, trefoil factor 3, α-glutathione S-transferase, N-acetyl-β- D-glucosaminidase (NAG), neutrophil gela-tinase-associated protein (NGAL) and osteopontin are currently being evaluated across the industry. Published studies of emerging markers show promising results in terms of increased sensitivity and specificity in com-parison to the standard serum markers, serum creatinine (sCr) and blood urea nitrogen (BUN) (Chen et al., 2017; Dieterle et al., 2010a; Fuchs and Hewitt, 2011; Ozer et al., 2010; Koyner et al., 2010). Although sCr and BUN continue to be utilized as primary renal markers in clinical practice, they are considered poor indicators of early renal dysfunction due to limited sensitivity, remaining relative-ly unchanged until significant renal damage occurs. Con-sequently, further evaluation of novel urinary biomarkers of acute kidney injury (AKI) is warranted both in pre-clinical species and in humans. In order to better under-stand the performance of KIM-1, NGAL, and NAG in the rat and to provide foundational information for potential clinical studies, a rat study was conducted to compare the performance of these urinary biomarkers to the standard serum markers of AKI for the detection of nephrotoxicity (Burt et al., 2014). These 3 markers were selected after a prescreening of a larger panel based on performance after administration of polymyxin B, a polypeptide antibiotic used for the treatment of life-threatening Gram-negative bacterial infections. Use of these antibiotics in the clin-ic has been drastically limited due to drug-induced AKI and thus the further development of more-sensitive renal markers has the potential to not only impact drug devel-opment but also patient care in the clinic. In this study, rats (5/group) were dosed with 0.1, 0.4, 1, 4 or 10 mg/kg polymyxin B for 2 or 14 days and then necropsied. The highest tolerated dose was 4 mg/kg. Histopathological examination of kidneys from the treated rats showed min-imal chronic progressive nephropathy (CPN) in a sin-gle rat at the 3 lowest doses, minimal to moderate tubu-lar degeneration/regeneration in 4 animals dosed at 4 mg/kg/day and minimal tubular necrosis in a single rat at 10/mg/kg/day, indicative of drug-induced kidney inju-ry. Of the biomarkers tested, only NGAL and KIM-1 produced dose-dependent statistically significant eleva-tions, 13-fold and 3-fold respectively, as early as 48 hr post-dose. In comparison, sCr was not affected by poly-myxin treatment and increases in BUN were small (1.3-

fold) and were not dose-dependent. Urinary NGAL, how-ever, was the most sensitive biomarker of AKI in this rat model, responding to the early onset of kidney inju-ry observed with polymyxin. Since urinary NGAL levels reached a maximum increase at 48 hr post-dose (Fig. 1) that correlated with kidney histopathology, a 2-day study design was implemented for screening and rank ordering potential drug candidates utilizing NGAL as the biomar-ker for AKI. This design provided an important tool for selecting novel polymyxin B analogs with an improved kidney safety profile, affording an efficient, cost-effective drug development biomarker-driven strategy. This work also provided a basis for potential clinical studies to fur-ther evaluate the diagnostic utility of these urinary kidney biomarkers in patients (Burt et al., 2014).

Example 2: Stress-associated hormones as mechanism-based biomarkers utilized to mitigate safety risk

Spontaneous histologic changes in laboratory ani-mal species including rabbits can hamper accurate toxi-cologic interpretation in preclinical safety studies, espe-cially if detailed study procedures are included. As early as 1924, publications described inflammatory heart find-ings with multifocal myocardial infiltrates of lymphocytes and/or macrophages in otherwise healthy rabbits (Sellers et al., 2017). In order to better characterize these myo-cardial findings and further understand the impact of study-related procedures on these findings, a large study

Fig. 1. NGAL response in rats to polymyxin B treatment. Urine was collected at intervals throughout the study with dosing on Day 0. Group mean values from the treated group were compared with the mean of the sa-line control group (* p < 0.05; ** p < 0.01).

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was designed in New Zealand White female rabbits with either an increase (Group 1) or decrease (Group 2) in the number of study-related procedures and animal han-dling. Blood was collected for coagulation, hematology and clinical chemistry analysis including stress-associated serum biomarkers (epinephrine, norepinephrine, cortisol, and corticosterone) at various time points throughout the study for Group 1 and only at baseline and necropsy for Group 2 to minimize handling and procedures (Sellers et al., 2017). Group 1 animals with the increased procedures had a higher incidence of inflammation with degenera-tion/necrosis in cardiac myocytes than the animals from the minimal procedure group (Group 2). Serum stress markers including cortisol and norepinephrine showed the greatest response in Group 1 animals with peak values usu-ally occurring 4 hr and 48 hr post-dose respectively (Fig. 2). This study provided further evidence that increased pro-cedures and handling during study conduct exacerbates the frequency and severity of myocardial inflammatory find-ings and may be mediated by a stress response. Based on these findings, it was proposed that stress hormones, in particular norepinephrine and cortisol, could be utilized to assess the risk of the myocardial findings observed in rab-bit toxicology studies (Sellers et al., 2017).

Example 3: Hyaluronic acid as a translational mechanism-based marker of liver microvascular injury

Hyaluronic Acid (HA) is a polysaccharide located in the extracellular matrix. It is synthesized by mesenchy-mal cells and cleared almost exclusively by liver sinusoi-dal endothelial cells (SECs). Circulating HA levels have been shown to be elevated with structural and/or func-tional damage to the liver SEC and have shown prom-ise in the clinic when studied for the noninvasive detec-tion of sinusoidal obstruction syndrome (SOS). Currently the diagnosis of SOS in the clinic relies on nonspecif-ic clinical and laboratory measures and/or events occur-ring late in the development of the disease including jaundice, painful hepatomegaly, weight gain and ascites. SOS has been reported in patients treated with antibody-calicheamicin conjugates including gemtuzumab ozo-gamicin and inotuzumab developed for acute myeloid leukemia and acute lymphoblastic leukemia, respectively (Guffroy et al., 2017). Liver toxicity based on elevat-ed aspartate aminotransferases (AST) and bilirubin lev-els was observed with occasional hepatic SOS following treatment with these antibody-drug conjugates (ADCs). While these two ADCs have different monoclonal anti-bodies, they are composed of the same linker and cali-cheamicin payload. To further evaluate the mechanism of the adverse event and to identify potential safety biomar-ker to detect the damage throughout the disease course, an experiment was initiated to characterize the liver inju-ry observed with the ADCs. Cynomologous monkeys were dosed with an antibody-calicheamicin conjugate containing the same linker-payload as gemtuzumab ozo-gamicin and inotuzumab. Monkeys were dosed with up to 3 intravenous bolus injections 3 weeks apart and were necropsied 48 hr following the first dose on day 3 and 3 weeks after the third administration on day 63. Liver histopathology showed midzonal degeneration and loss of SECs on day 3 and variable endothelial recovery and progression to a combination of sinusoidal capillariza-tion and sinusoidal dilation/hepatocellular atrophy, con-sistent with early SOS. Minimal increases in AST levels (up to 3.1x) were observed on day 4 and remained elevat-ed over the duration of the study. HA levels (up to 8.5x) were elevated on day 3 and were sustained thru day 63 in all treated monkeys (Fig. 3) demonstrating the ability of this marker to detect early structural damage (day 3) and later functional impairment (day 63) of SECs. HA also showed good correlation to AST levels and microscop-ic liver findings throughout the study. Since HA, unlike AST, is linked mechanistically to SOS, it was proposed as a sensitive exploratory diagnostic marker of liver micro-

Fig. 2. Norepinephrine and Cortisol levels in rabbits following dosing with saline on Days 29, 43 and 57 with blood collections pre-dose (vertical lines) and at 4, 24 and 48 hr post-dose. Data represents group means ± SEM.

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vascular injury capable of non-invasive detection of SOS in the clinic (Guffroy et al., 2017).

Example 4: Gastrin as a translational safety biomarker for drug-induced stomach toxicity

Gastrin is secreted from the stomach and plays a key role in the regulation of gastric acid secretion. Gastrin is synthesized in special endocrine cells (G cells) primarily in the antral region of the gastric mucosa and binds recep-tors found predominantly on parietal cells stimulating gastric juice secretion, the best-known of which is hydro-chloric acid (HCl) (Henderson, 2001). The capacity of the stomach to secrete HCl normally is directly proportional to the parietal cell number. High levels of circulating gas-trin can occur when the pH of the stomach is high while gastrin secretion by antral G cells is inhibited by the direct action of acid on the G cells. When the stomach lining is damaged and unable to produce and release acid, gastrin continues to be secreted from the fundus and circulating levels rise. Changes in serum gastrin have been shown to be predictive of the functional status of the antral mucosa, making it attractive as a potential biomarker of drug-in-duced stomach toxicity (Graham et al., 2006; Nicolaou et al., 2014). To further evaluate gastrin as a potential mark-er of stomach toxicity, a 28-day dog study was conducted with a compound associated with gastric effects. Minimal

to moderate atrophy and minimal degeneration of the fun-dic mucosa were seen at histologic examinations. Mean serum gastrin levels in the treated animals were 23x high-er than concurrent controls (Fig. 4) and were attributed to the lack of acid production by the damaged parietal cells, indicating a failure of the feedback mechanism that con-trols the acid output in the stomach. In individual dogs, serum gastrin levels were elevated (up to 52x) compared to concurrent controls and these levels correlated with the severity of the adverse microscopic stomach find-ings. Based on these data, serum gastrin was proposed as a non-invasive stomach-specific biomarker to monitor for stomach toxicity in the clinic.

PATHWAYS TO BIOMARKER ACCEPTANCE

Many of the safety biomarkers currently considered conventional and measured routinely in both preclinical and clinical drug development as well as in clinical prac-tice were established prior to the implementation of a for-mal regulatory qualification process. They were accepted based on scientific community consensus, i.e. review of datasets published in peer reviewed journals, experience in clinical practice, and recommendations from profes-sional medical associations. For example, cardiac tropon-in (cTn) was recognized as a clinical biomarker of acute myocardial infarction by the American College of Cardi-ology and the European Society of Cardiology in 2000 (Jaffe, 2001) just thirteen years after the development of the first troponin assay (Danese and Montagnana, 2016). Diagnostic criteria were recommended based on pub-

Fig. 3. Hyaluronic Acid (HA) and Aspartate Aminotransferase (AST) levels in monkeys following dosing (vertical lines) with an antibody-calicheamicin conjugate and linker. Data represents group means. Statistically sig-nificant changes were observed for both HA and AST when compared to vehicle controls at all time points following dosing.

Fig. 4. Gastrin concentrations in a 28 Day Dog Study fol-lowing treatment with vehicle or a compound as-sociated with gastric effects. Data represents group means ± SD.

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lished literature and clinical experience with an acknowl-edgement that the criteria should continue to evolve as additional knowledge was gained (Jaffe, 2001). Subse-quent to clinical acceptance, cTn was evaluated as a pre-clinical marker by the Health and Environmental Sci-ences Institute (HESI) Cardiac Troponins Biomarker Working Group (Reagan, 2010) and received full quali-fication from the FDA in 2012 based solely on evidence from peer reviewed scientific literature (FDA, 2012).

Another pathway to biomarker approval is through biomarker evaluation for a drug-specific application. In this case, the biomarker may only be used in a single drug development program and the data required to support the use of the biomarker in the program is determined by the sponsor communicating directly with the regulatory reviewing division accountable for the program (Mattes and Goodsaid, 2018). Utilization of this pathway lim-its the knowledge of the biomarker’s performance and intended use for the specified program but may enable time saving and efficient progression of a promising com-pound into clinical development.

In order for a biomarker to be approved for multiple drug development programs, it must pass the rigor of the full regulatory qualification process. Qualification is defined by the FDA as ‘a conclusion that within the stat-ed context of use, the results of assessment with a drug development tool (biomarker) can be relied upon to have a specific interpretation and application in drug devel-opment and regulatory review (FDA, 2014).’ Biomark-ers reaching this milestone can support regulatory deci-sions in drug development programs for the approved context of use (COU) in clinical trials. According to the FDA, the COU is ‘a comprehensive and clear statement that describes the manner of use, interpretation, and pur-pose of use of the biomarker in drug development (FDA, 2016b).’ With the enactment of the 21st Century Cures Act in December 2016, an updated multi-stage biomar-ker qualification process was established which includ-ed three submission stages: the Letter of Intent, the Qualification Plan and the Full Qualification Package. The Biomarker Qualification Program is one of the Drug Development Tools created by the Center of Drug Evalu-ation and Research to provide a framework for develop-ment and regulatory acceptance of biomarkers for use in drug development programs. Similar programs outlining processes for the submission and review of data support-ing the approval of new biomarkers are also in place at the European Medicines Agency (EMA) and the Japanese Pharmaceutical and Medical Devices Agency (PDMA). While the agencies work closely on qualification efforts, a fully harmonized approach has yet to be established.

However, these regulatory pathways provide a process to review, evaluate and adopt new tools into regulatory deci-sion making in drug development and facilitate consensus science and acceptance of the biomarker’s proposed COU in drug development (Dennis et al., 2013).

To better understand the elements of biomarker qualifi-cation, a framework for evidentiary standards for biomar-ker qualification was recently proposed under the auspic-es of the Foundation for the National Institute of Health (FNIH) Biomarkers Consortium with representatives from FDA, NIH, industry, patient groups and academia. Five components were incorporated into the framework and included: 1) defining a statement of need (knowl-edge gap or drug development need) that the biomarker intends to address; 2) defining the COU; 3) assessing the benefits in light of the COU; 4) assessing the risks with regards to the COU and 5) defining the evidentiary crite-ria required to support the COU (Leptak et al., 2017). The COU determines the level of evidence needed both for the validation of analytical technology employed and for the qualification of the biomarker. The greater the risk to human health of an incorrect decision based on the use of the biomarker, the greater the level of evidence required for the qualifying the biomarker for that COU (Dennis et al., 2013). The assessment of the evidentiary criteria is intended to be utilized as a ‘communication tool for gain-ing alignment between submitters and FDA reviewers at several key milestones for a biomarker development plan: (i) initial discussions to align expectations; (ii) purposeful interim progress updates to ensure that evidence expecta-tions have been met before proceeding further; and (iii) review evaluation to support the qualification outcome (Leptak et al., 2017).’ The ultimate goal of this effort was to improve the quality of submissions to the FDA, facil-itate a level of predictability in the qualification proc-ess, and provide clarity as to the type and level of evi-dence needed to support a biomarker’s COU (Leptak et al., 2017).

Due to the complexity of the qualification process and considerable resources required to reach full qualification of a biomarker, efforts are primarily focused in consortia such the HESI, the PSTC, the IMI Safer and Faster Evi-dence-based Translation (SAFE-T) consortium and more recently the IMI Translational Safety Biomarker Pipeline (TransBioLine) consortium. To date, 4 safety biomarker submissions have been successful. This included 2 pre-clinical qualifications for a number of emerging urinary nephrotoxicity biomarkers and one nonclinical qualifica-tion of circulating cardiac troponins as indicators of cardi-otoxicity. Earlier this year, the first clinical safety biomar-ker submission reached approval for a panel of urinary

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biomarkers to aid in the detection of kidney tubular injury in phase 1 trials in healthy volunteers (FDA, 2018b).

BIOMARKER QUALIFICATION EFFORTS

Serum glutamate dehydrogenase (GLDH) as a specific biomarker for hepatocellular injury

GLDH has been shown to be a sensitive measure of hepatotoxicity in preclinical species and in humans (Giffen et al., 2003; Schomaker et al., 2013) and is cur-rently going through the regulatory qualification proc-ess sponsored by the PSTC and the Duchenne Regulatory Science Consortia (D-RSC). The gold standard biomark-er for the diagnosis of liver injury is alanine aminotrans-ferase (ALT). However since ALT is also present in myo-cytes, serum ALT activities can increase with muscle injury; thus, the development of more specific biomarkers for drug-induced liver injury (DILI) is needed. For this qualification, the proposed COU for GLDH is that ‘ele-vated serum GLDH activity is a measure of hepatocellu-lar injury, and can be used in healthy subjects and patients as an adjunct to ALT, the current standard biomarker used to assess hepatocellular injury, in all stages of drug devel-opment. In a clinical situation when ALT increases are observed, GLDH can lend weight of evidence to confirm or rule out hepatocellular injury (EMA, 2017).’ The quali-fication submission will include a full technical validation of the assay and an evaluation of the clinical relevance of the biomarker, e.g., added value relative to aminotrans-ferase activity, correlation to histopathology in a preclin-ical species and exploratory and confirmatory analyses in clinical subjects with regulatory guidance. The eval-uation of clinical relevance includes establishing refer-ence ranges for healthy subjects and evaluating the influ-ence of gender and age, confirming GLDH as a sensitive biomarker of liver injury and establishing medically rel-evant cutoffs for DILI, and establishing GLDH as a spe-cific biomarker of hepatocellular injury in comparison to ALT. In March of 2017, a joint FDA and EMA biomark-er qualification consultation meeting was held for GLDH. This meeting provided regulatory support for using organ injury induced by diseases with a wide range of etiologies as approximation of chemical-induced organ injury for evaluation of performance of novel biomarkers. This was a paradigm shift in the development of safety biomark-ers, since it eliminates the need for lengthy clinical tri-als and improves feasibility and efficiency of biomarker research. The EMA issued a Letter of Support (LoS) in November 2017 demonstrating the Agency’s support of the qualification and provided additional feedback regard-ing data needed for achieving full qualification of GDLH

as a “Drug Development Tool”. This LoS not only rec-ognized the potential value of GLDH as a liver specific biomarker of hepatocellular injury to address important unmet medical need but also endorsed data interpretation including medically relevant levels of GLDH (2.5x and 5x above upper limit of normal) that were established as part of the qualification effort (EMA, 2017). This qualifi-cation effort, initiated in 2015 supported by Pfizer inter-nal exploratory data, is expected to reach fruition in 2019 with the submission of the full qualification package. The formal qualification of GLDH as a liver specific biomar-ker of hepatocellular injury will not only allow for the broad application of GLDH across programs, but will also enable the diagnosis of the onset of liver disease in subjects with underlying muscle impairments, which is an important unmet medical need widely recognized by the medical community. To this end, Pfizer has partnered with the PSTC, the Duchenne Regulatory Science Consortium (D-RSC), Roche, the maker of the research grade GLDH assay, and FDA to validate the GLDH assay as an in vitro diagnostic (IVD) which would allow the assay to be uti-lized in clinical practice and lead to an improved standard of care for patients living with muscle disease.

Next generation biomarkers for skeletal muscle degeneration

Serum AST and creatine kinase (CK) have been used for decades as the primary biomarkers for skeletal mus-cle (SKM) injury as a measure of myocyte degeneration/necrosis. However, these markers lack tissue specifici-ty for SKM and sensitivity for SKM degeneration/necro-sis in both rats and humans. In 2010, the PSTC’s Skel-etal Muscle Working Group was formed with a goal to identify and qualify novel safety biomarkers of drug-in-duced SKM injury that would add value to the current markers, CK and AST, for monitoring SKM injury. The muscle injury panel (MIP) selected for evaluation includ-ed skeletal troponin I, myosin light chain, fatty acid-bind-ing protein and creatine kinase measured by a mass assay. These markers were assessed in 34 rat studies and were shown to outperform AST and CK (enzymatic assay) individually and as a panel in terms of sensitivity and specificity and/or added value for the diagnosis of drug-induced SKM injury defined as myocyte degeneration/necrosis (Burch et al., 2016). Based on this data, the FDA and EMA issued Letters of Support (EMA, 2015; FDA, 2015) for the use of these markers in preclinical devel-opment and encouraged their use in early clinical trials in an exploratory context. This endorsement encouraged Burch et al. (Burch et al., 2015) to evaluate the translat-ability of these markers in patients with DMD and other

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muscular diseases and their ability to monitor disease pro-gression and the response to treatment. In this study, the MIP biomarker responses was compared to current clini-cal assessments including CK activity, ambulatory status and cardiac function and were shown to not only better reflect the patients’ disease state compared to CK activity but also correlate with clinical endpoints in patients with DMD and other muscular diseases. These preclinical and clinical evaluations provided support for a qualification submission which is currently in progress. This qualifica-tion, sponsored by the PSTC, will evaluate the marker’s ability to monitor SKM degeneration/necrosis in conjunc-tion with AST and CK enzymatic activity in early clinical trials and encourage the utilization of the MIP markers throughout drug development to improve patient safety in clinical trials (Burch et al., 2016). In support of the qual-ification effort, Pfizer has included the MIP markers on a number of clinical trials as exploratory biomarkers; how-ever, the utility of the markers as SKM safety or efficacy end points to these therapeutic interventions has yet to be determined (Goldstein, 2017).

Kidney safety urine biomarker qualificationsIn 2008 the first formal qualification of preclinical safe-

ty biomarkers was granted by the FDA (FDA, 2008) and EMA (EMA, 2008a) followed by PMDA in 2010 (PMDA, 2010) for seven urinary safety biomarkers submitted by the PSTC. In 2010, a qualification was rendered by the FDA for the two preclinical urinary biomarkers, clusterin, renal papillary antigen-1 (RPA-1), submitted by HESI and in 2018 the FDA qualified the first clinical safety biomar-kers, a set of six urinary markers interpreted as a Com-posite Measure (CM) (FDA, 2018a) submitted by PSTC. In all cases, the biomarkers are to be used in conjunction with the traditional measures, serum creatinine (sCr) and blood nitrogen urea (BUN), for the evaluation of neph-rotoxicity. sCr and BUN are both insensitive and non-specific, changing only after significant injury and with a time delay relative to the onset of injury (Vaidya et al., 2008) limiting their ability to accurately estimate injury onset and the severity of the dysfunction following injury (Ferguson et al., 2008).

The seven biomarkers included in the PSTC preclin-ical submission included KIM-1, clusterin (CLU), albu-min, total protein, β2-microglobulin, cystatin C and tre-foil factor 3 (TFF3) in urine. The submission contained data and data interpretation from a number of rat studies, a review of the scientific literature in humans (KIM-1, albumin, total protein, cystatin C, and β2-microglobulin), COUs for each biomarker (Dieterle et al., 2010b) and key conclusions. The PSTC put forth three specific biomarker

claims in the submission. First, urinary KIM-1, CLU, and albumin can individually outperform and add information to BUN and sCr assays as early diagnostic biomarkers of drug-induced kidney tubular alterations in rat toxicolo-gy studies. Second, urinary TFF3 can add information to BUN and sCr assays in rat toxicology studies as an early diagnostic biomarker of drug-induced acute kidney inju-ry tubular alterations. Third, total urinary protein, cysta-tin C, and β2-microglobulin can individually outperform sCr assays and add information to BUN and sCr assays as early diagnostic biomarkers in rat toxicology studies of acute drug-induced glomerular alterations or damage resulting in impairment of kidney tubular reabsorption (Dieterle et al., 2010b). In addition, the PSTC claimed that this rat data taken together with the published peer-re-viewed clinical data supported the voluntary use of KIM-1, albumin, total protein, cystatin C, and β2-microglobulin as bridging markers for early clinical trials on a case-by-case basis when concerns are generated in GLP animal toxicology studies. During the qualification effort, the FDA and EMA provided feedback regarding submission gaps, statistical considerations and preliminary conclu-sion statements (EMA, 2008b) which culminated in let-ters of acceptance for the preclinical qualifications from both agencies. In 2008, the FDA and EMA concluded that the urinary kidney biomarkers KIM-1, CLU, albu-min, total protein, β2-microglobulin, cystatin C and TFF3 were acceptable for the detection of acute drug-induced kidney injury in rats to be included along with traditional clinical chemistry markers and histopathology in toxicol-ogy studies. The EMA also stated that while it was worth-while exploring their utility in early clinical trials as clin-ical biomarkers, until additional data was available “to correlate the biomarkers with the evolution of the neph-rotoxic alternations, and their reversibility, their general use for monitoring nephrotoxicity in clinical setting can-not be recommended (EMA, 2008b).” Based on this data, in 2010 the PDMA announced the first biomarker quali-fication decision under the new consultation process on pharmacogenomics/biomarkers use in Japan. These rec-ommendations, consistent with the concept of a progres-sive biomarker qualification, prompted the generation and submission of additional preclinical and clinical data to expand the COU.

In an expanded effort, the SAFE-T and PSTC consortia continued the work on urinary kidney injury biomarkers by evaluating the performance of KIM-1, CLU, NGAL, albumin, total protein, cystatin C, α -glutatione S-trans-ferase and urinary osteopontin in clinical trials. The tri-als included a study in healthy volunteers, an explorato-ry study with cisplatin-treated cancer patients, and a study

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in patients undergoing coronary angiography. The mark-ers selected are localized in different regions of the neph-ron thus the panel was expected to respond to a variety of nephrotoxicants. Based on the readout from these stud-ies, both the FDA and EMA issued Letters of Support in 2016 encouraging the exploratory use of these markers as biomarkers of renal tubular injury in early clinical tri-als to be used in conjunction with traditional biomarkers and clinical and nonclinical findings (FDA, 2016c; EMA, 2016).

Also in 2016, the FNIH Biomarker Consortium and the PSTC submitted a qualification submission for a kid-ney injury biomarker panel to be interpreted as a Com-posite Measure (the geometric mean of the fold change from baseline of the six urine biomarkers normalized to urine creatinine) of the following six biomarkers: KIM-1, CLU, cystatin C, NAG, NGAL and urinary osteopontin. According to the COU, “the safety composite biomarker panel is to be used in conjunction with traditional meas-ures to aid in the detection of kidney tubular injury in phase 1 trials in healthy volunteers when there is an a pri-ori concern that a drug may cause renal tubular injury in humans (FDA, 2018a).” The qualification strategy includ-ed a nonclinical phase, a clinical exploratory phase and a clinical confirmatory phase with a cisplatin study in can-cer patients and an aminoglycoside study in cystic fibro-sis patients. Based on this data, this panel, interpreted as a CM, was the first clinical safety biomarker to be quali-fied by the FDA. Following on the success of this qualifi-cation, the FNIH and PSTC have continued their partner-ship and have submitted a letter of intent to FDA, EMA and PDMA to further qualify the panel of biomarkers based on the individual biomarker response thus expand-ing on the qualified CM COU.

CURRENT STATE OF TRANSLATIONAL SAFETY BIOMARKER DEVELOPMENT

Significant progress has been made towards imple-menting translational safety biomarker strategies into the drug development portfolio. Several pharmaceutical com-panies have been adopting exploratory biomarkers in their preclinical and clinical drug development programs and contributing to consortia led qualification efforts. While much progress has been made towards the qualification of biomarkers for some organ injures including liver, kid-ney, and muscle, significant gaps remain for other target organs such as biomarkers of vascular injury in humans, sensitive biomarkers of pancreatic injury, circulating biomarkers of testicular toxicity, and biomarkers of inju-ry to the central nervous system. Because the process of

biomarker development is complex and requires consid-erable resources (Gerlach et al., 2018), these efforts will best be pursued by consortia such as PSTC and IMI. Pri-or to widespread acceptance, each biomarker must under-go a rigorous assay validation; demonstrate relevance to humans and an association with clinical endpoints repro-ducibly in multiple studies and gain consensus regard-ing the level of evidence needed to support a qualification for the stated COU. Due to the complexity and feasibil-ity challenges of prospective randomized drug interven-tion clinical trials, alternative sample collection strate-gies like the prospective collection of samples of organ damage etiologies from patients undergoing hospital vis-its employed in the GLDH qualification effort, need to be considered. These disease patient populations, once iden-tified, can serve as surrogates for drug-induced organ inju-ry to evaluate biomarker performance as long as common molecular and mechanistic pathways are shared between the disease and organ toxicity of interest. The prospective collection and storage of samples from clinical trials con-ducted for drug development is also an option (Aubrecht et al., 2013). As safety biomarkers become accepted and qualified by the regulatory agencies, they will increas-ingly play a key role in decision making and facilitate the progression of promising therapeutics from preclini-cal through clinical development, leading to the realiza-tion of our most critical goal: delivering the right dose of the right medicines to the right patients at the right time (Gerlach et al., 2018).

SUMMARY AND CONCLUSIONS

Safety and tolerability of newly approved drug can-didates still remains a key concern in drug development leading to black box warnings and withdrawals of prom-ising therapeutics. A major hurdle is the lack of validated and qualified safety biomarkers that accurately diagnose, predict and inform mechanism of organ toxicities. Reg-ulatory qualification of a safety biomarker is critical for routine application in clinical development. To address this hurdle and the complexities in biomarker develop-ment, considerable resources and partnerships across industry, academia, and regulators is needed. Although tremendous progress has been made towards identify-ing and implementing translational safety biomarkers for organ toxicities such as kidney and liver, significant gaps still exist to monitor toxicities for other common tar-get organs such as pancreas, central nervous system, tes-tis, and skeletal muscle. With the enactment of 21st Cen-tury Cures Act, the FDA biomarker qualification process has become more streamlined. In addition to developing

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a clear frame work for regulatory qualification, there is optimism towards expediting the qualification process so that promising therapeutics can be safely tested in the clinic and ultimately provide patients access to new med-icines.

Conflict of interest---- The authors declare that there is no conflict of interest.

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Review Safety biomarker applications in drug development