PROJECT TITLE
QUALIFICATION PLAN FOR GLUTAMATE DEHYDROGENASE
(GLDH) AS A BIOMARKER OF DRUG
INDUCED LIVER INJURY IN INDIVIDUALS WITH SKELETAL
MUSCLE DEGENERATION HISTORY
• Submitted Letter of Intent to FDA – November 2016
• Submitted Briefing Book to FDA and EMA – December 2016
• Held joint meeting with FDA and EMA on Briefing Book – March 2017
• Responded to questions and issues from FDA and EMA – Q2-3 2017
• Received Letter of Support for GLDH from EMA – November 2017
• Submitted Legacy Biomarker Qualification Project Status Update to FDA – May 2018
• Received Decision Letter on Legacy Biomarker Qualification Project Status Update from
FDA – October 2018
1.2 Background
Drug-induced liver injury (DILI) remains the single greatest cause for termination of development
of drug candidates and withdrawal of approved drugs from the market (Yuan and Kaplowitz, 2013;
Kaplowitz, 2005). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum
activity measurements are used as current standard biomarkers for the identification of liver injury
in clinical practice, and commonly used to assess risk of liver injury during drug development.
Hy’s Law and a recent definition by an international DILI Expert Working Group (EWG) provide
guidance to help differentiate mild or transient hepatocellular injury from more severe DILI and
functional hepatocellular damage (Temple, 2006; FDA, 2009; Aithal et al., 2011). A 3x and 5x
ALT elevation from the upper limit of normal (ULN) are commonly used thresholds of concern in
clinical trials, triggering confirmatory testing and close observation of an individual as suggested
in the Food and Drug Administration (FDA) Guidance for Industry: Drug-Induced Liver Injury:
Premarketing Clinical Evaluation (FDA, 2009). However, increases in ALT do not always signal
hepatocellular injury.
Although ALT and AST are sensitive markers of hepatocellular injury, both enzymes are also
expressed in other tissues such as muscle. This severely limits the utility of ALT and AST as
markers of liver damage in subjects with underlying muscle impairments, such as Duchenne
muscular dystrophy (DMD) or other neuromuscular diseases, in clinical trials with simultaneous
drug-induced liver and muscle injury or even in subjects engaging in strenuous exercise. In
addition, increased levels of ALT and AST due to underlying muscle damage may potentially
mask a hepatotoxic signal, creating a diagnostic challenge for clinicians. Therefore, the
development of additional biomarkers of DILI is warranted.
Glutamate Dehydrogenase (GLDH) is a mitochondrial enzyme that plays a role in amino acid
oxidation and urea production. GLDH is primarily found in the liver with only a trace amount in
skeletal muscle (Mastorodemos et al., 2005; Jaeschke and McGill, 2013). In humans, serum GLDH
CONFIDENTIAL 9
, activity is elevated in patients with hepatic ischemia (Kretzschmar et al., 2003), progressively
increasing with increased severity of disease (Schmidt and Schmidt, 1988). Furthermore, serum
GLDH activity has been shown to be a sensitive marker of a mild hepatocyte necrosis in patients
treated with heparin (Harrill et al., 2012). Like ALT, increases in GLDH within 8 hours of
acetaminophen (APAP) overdose predicted patients that proceeded to acute liver injury (Antoine
et al., 2013). GLDH has a half-life of 18 hours (Schmidt and Schmidt, 1988; Schmidt and Otto,
1967) whereas ALT has a half-life of 47 hours (Kim et al., 2008; Giannini, 2005), thus GLDH
levels would be expected to return to baseline more quickly than ALT levels upon resolution of
injury. However, unlike ALT and AST, GLDH activity does not increase following muscle injury
or degeneration (Thulin et al., 2014).
As part of the Critical Path Institute (C-Path) Predictive Safety Testing Consortium’s (PSTC)
ongoing efforts to augment translational biomarker tools for DILI, the Hepatotoxicity Working
Group (HWG) is proposing the qualification of serum GLDH activity as a biomarker of liver injury
to confer specificity to the liver in human subjects with underlying muscle injury or degeneration.
Serum GLDH is a safety biomarker capable of detecting drug-induced hepatocellular injury that
can be used in place of ALT in clinical trials for subjects and patients with elevated serum
transaminases due to muscle injury or degeneration and should be used in conjunction with
standard hepatic injury monitoring biomarkers, alkaline phosphatase (ALP) and total bilirubin
(TBil).
1.3 Biomarker Name, Type and Description
Glutamate dehydrogenase (GLDH), a mitochondrial enzyme expressed primarily in liver, is a
molecular biomarker whose activity can be measured in serum. The unique identifier for GLDH
is UniProt ID P00367. GLDH is a safety biomarker capable of sensitively and specifically
detecting drug-induced hepatocellular injury in humans and nonclinical species.
1.4 Context of Use (COU) Statement
Serum glutamate dehydrogenase (GLDH) is a safety biomarker capable of detecting drug-induced
hepatocellular injury that can be used in place of alanine aminotransferase (ALT) in clinical trials
for subjects and patients with elevated serum transaminases due to muscle injury or degeneration.
GLDH should be used in conjunction with standard hepatic injury monitoring biomarkers (e.g.
total bilirubin and alkaline phosphatase).
The current COU has evolved beyond its previous iteration, narrowing its focus to the utility of
GLDH in subjects and patients with muscle injury and degeneration.
1.5 Measurement Method
GLDH can be reliably measured in serum. The Randox GLDH assay utilizes the conversion of
αoxoglutarate to glutamate for detection of GLDH enzymatic activity as recommended by the
Deutsche Gesellschaft für Klinische Chemie (DGKC).
CONFIDENTIAL 10
QUALIFICATION PLAN FOR GLUTAMATE DEHYDROGENASE
(GLDH) AS A BIOMARKER OF DRUG
INDUCED LIVER INJURY IN INDIVIDUALS WITH SKELETAL
MUSCLE DEGENERATION HISTORY
• Submitted Letter of Intent to FDA – November 2016
• Submitted Briefing Book to FDA and EMA – December 2016
• Held joint meeting with FDA and EMA on Briefing Book – March 2017
• Responded to questions and issues from FDA and EMA – Q2-3 2017
• Received Letter of Support for GLDH from EMA – November 2017
• Submitted Legacy Biomarker Qualification Project Status Update to FDA – May 2018
• Received Decision Letter on Legacy Biomarker Qualification Project Status Update from
FDA – October 2018
1.2 Background
Drug-induced liver injury (DILI) remains the single greatest cause for termination of development
of drug candidates and withdrawal of approved drugs from the market (Yuan and Kaplowitz, 2013;
Kaplowitz, 2005). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum
activity measurements are used as current standard biomarkers for the identification of liver injury
in clinical practice, and commonly used to assess risk of liver injury during drug development.
Hy’s Law and a recent definition by an international DILI Expert Working Group (EWG) provide
guidance to help differentiate mild or transient hepatocellular injury from more severe DILI and
functional hepatocellular damage (Temple, 2006; FDA, 2009; Aithal et al., 2011). A 3x and 5x
ALT elevation from the upper limit of normal (ULN) are commonly used thresholds of concern in
clinical trials, triggering confirmatory testing and close observation of an individual as suggested
in the Food and Drug Administration (FDA) Guidance for Industry: Drug-Induced Liver Injury:
Premarketing Clinical Evaluation (FDA, 2009). However, increases in ALT do not always signal
hepatocellular injury.
Although ALT and AST are sensitive markers of hepatocellular injury, both enzymes are also
expressed in other tissues such as muscle. This severely limits the utility of ALT and AST as
markers of liver damage in subjects with underlying muscle impairments, such as Duchenne
muscular dystrophy (DMD) or other neuromuscular diseases, in clinical trials with simultaneous
drug-induced liver and muscle injury or even in subjects engaging in strenuous exercise. In
addition, increased levels of ALT and AST due to underlying muscle damage may potentially
mask a hepatotoxic signal, creating a diagnostic challenge for clinicians. Therefore, the
development of additional biomarkers of DILI is warranted.
Glutamate Dehydrogenase (GLDH) is a mitochondrial enzyme that plays a role in amino acid
oxidation and urea production. GLDH is primarily found in the liver with only a trace amount in
skeletal muscle (Mastorodemos et al., 2005; Jaeschke and McGill, 2013). In humans, serum GLDH
CONFIDENTIAL 9
, activity is elevated in patients with hepatic ischemia (Kretzschmar et al., 2003), progressively
increasing with increased severity of disease (Schmidt and Schmidt, 1988). Furthermore, serum
GLDH activity has been shown to be a sensitive marker of a mild hepatocyte necrosis in patients
treated with heparin (Harrill et al., 2012). Like ALT, increases in GLDH within 8 hours of
acetaminophen (APAP) overdose predicted patients that proceeded to acute liver injury (Antoine
et al., 2013). GLDH has a half-life of 18 hours (Schmidt and Schmidt, 1988; Schmidt and Otto,
1967) whereas ALT has a half-life of 47 hours (Kim et al., 2008; Giannini, 2005), thus GLDH
levels would be expected to return to baseline more quickly than ALT levels upon resolution of
injury. However, unlike ALT and AST, GLDH activity does not increase following muscle injury
or degeneration (Thulin et al., 2014).
As part of the Critical Path Institute (C-Path) Predictive Safety Testing Consortium’s (PSTC)
ongoing efforts to augment translational biomarker tools for DILI, the Hepatotoxicity Working
Group (HWG) is proposing the qualification of serum GLDH activity as a biomarker of liver injury
to confer specificity to the liver in human subjects with underlying muscle injury or degeneration.
Serum GLDH is a safety biomarker capable of detecting drug-induced hepatocellular injury that
can be used in place of ALT in clinical trials for subjects and patients with elevated serum
transaminases due to muscle injury or degeneration and should be used in conjunction with
standard hepatic injury monitoring biomarkers, alkaline phosphatase (ALP) and total bilirubin
(TBil).
1.3 Biomarker Name, Type and Description
Glutamate dehydrogenase (GLDH), a mitochondrial enzyme expressed primarily in liver, is a
molecular biomarker whose activity can be measured in serum. The unique identifier for GLDH
is UniProt ID P00367. GLDH is a safety biomarker capable of sensitively and specifically
detecting drug-induced hepatocellular injury in humans and nonclinical species.
1.4 Context of Use (COU) Statement
Serum glutamate dehydrogenase (GLDH) is a safety biomarker capable of detecting drug-induced
hepatocellular injury that can be used in place of alanine aminotransferase (ALT) in clinical trials
for subjects and patients with elevated serum transaminases due to muscle injury or degeneration.
GLDH should be used in conjunction with standard hepatic injury monitoring biomarkers (e.g.
total bilirubin and alkaline phosphatase).
The current COU has evolved beyond its previous iteration, narrowing its focus to the utility of
GLDH in subjects and patients with muscle injury and degeneration.
1.5 Measurement Method
GLDH can be reliably measured in serum. The Randox GLDH assay utilizes the conversion of
αoxoglutarate to glutamate for detection of GLDH enzymatic activity as recommended by the
Deutsche Gesellschaft für Klinische Chemie (DGKC).
CONFIDENTIAL 10
