What does the best NAFLD/NASH clinical trial protocol look like?  That’s a difficult question to answer because there is no one right protocol.  Each study is designed with specific objectives and end points utilizing a mixture of serum and imaging biomarkers along with functional tests and patient-reported outcomes.  However, there may be common measurements that are useful in comparing results as a compound advances through its development or when comparing one asset to another across mechanisms.  Since the mechanisms can be different, and we expect different metabolic, inflammatory, and fibrotic pathways to be targeted (whether as an agonist or an inhibitor), we may use the similar tests outside of imaging.

I’ve written previous posts on the different imaging tests and now want to look at those blood/serum tests commonly available and used in NAFLD/NASH trials.  Each of the biomarkers listed below we’ve seen used in NAFLD/NASH protocols.  Some of these tests are sensitive, but not necessarily specific, and that’s commonly a limitation to blood- and urine-based samples.  This means their interpretation can encounter some bias, as we are always hopeful in our candidates and studies.

Since we’re interested in the liver, functional tests here usually related to safety but can be combined for efficacy in NASH.  These lists are arranged by assay, and it’s possible there’s some repetition. Common functional liver tests include:
•    Alanine Transaminase (ALT)
•    Aspartate Transaminase (AST)
•    Alkaline Phosphatase (ALP)
•    Albumin
•    Total Protein
•    Total Bilirubin
•    Direct Bilirubin
•    Indirect Bilirubin
•    Urate
•    Gamma-Glutamyltransferase (GGT)
•    L-Lactate Dehydrogenase (LD)
•    Prothrombin Time (PT)
•    International Normalized Ratio (INR)
•    Haptoglobin
•    ApoA1
•    α2-Macroglobulin
•    Serum Bile Acids
•    Steroid Hormone Binding Globulin (SHBG)
•    Creatinine Kinase
•    Creatinine Phosphatase
•    Lactate
•    Ketones
•    Lactate Dehydrogenase (LDH)
•    Ferritin
•    13C Methacetin Breath Test
•    Non-Esterified Fatty Acids (NEFA)
•    Beta-Hydroxybutyrate (BHB)

Blood lipids, usually as a measure of pharmacodynamic effects of altering lipids are in some combination of the follow tests or markers:
•    Triglycerides
•    Non-Esterified Fatty Acids (NEFA)
•    Total Cholesterol
•    Low Density Lipoprotein Cholesterol (LDL-C)
•    High Density Lipoprotein Cholesterol (HDL)
•    Non-High-Density Lipoprotein Cholesterol (Non-HDL-C)
•    Very Low-Density Lipoprotein Cholesterol (VLDL-C)
•    Apolipoprotein B (Apo B)
•    Lipoprotein (a) [Lp(a)]
•    Metabolic Syndrome Definition Modified from the NCEP ATP III Guidelines
•    7α-Hydroxy-4-Cholesten-3-One (or 7αC4)
•    Leptin
•    Soluble Leptin Receptor (sLEPR)
•    Angiopoietin-Like 3 (ANGPTL3)
•    Angiopoietin-Like 4 (ANGPTL4)
•    Angiopoietin-Like 8 (ANGPTL8)
•    Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)
•    Apolipoprotein CII
•    Apolipoprotein CIII
•    Adipsin

For those looking at cirrhotic livers (or those at risk of progressing) and biliary tree diseases, there are several serum bile acids that could be assayed:
•    Cholic Acid
•    Chenodeoxycholic Acid
•    Deoxycholic Acid
•    Lithocholic Acid
•    Ursodeoxycholic Acid
•    Collagen Proportionate Area (CPA)
•    Cholesterol Panel

With targets in the thyroid pathway, here’s assays that can be used to assess thyroid function:
•    Thyroid-Stimulating Hormone (TSH)
•    Total T3
•    Free 3,3’, 5-triiodo-L-thyronine (T3)
•    Total T4
•    Free Thyroxine (T4)

I’m a big advocate of inflammation markers; however, inflammation is not always specific to the liver.  However, significant reductions in inflammation markers as a result of drug exposure may be the confidence needed to pursue other doses or durations in development:
•    Adiponectin
•    HMW Adiponectin
•    Plasminogen Activator Inhibitor-1 (PAI1)
•    CK7
•    Cytokeratin 18/M30 Protein
•    Cytokeratin 18/M65 Protein
•    Monocyte Chemoattractant Protein-1 (MCP-1/CCL2)
•    Erythrocyte Sedimentation Rate (ESR)
•    C-reactive Protein (CRP)
•    High Sensitivity C-Reactive Protein (hsCRP)
•    Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)
•    N-Terminal Type III Collagen Propeptide (Pro-C3)
•    Amino terminal peptide of type III procollagen (PIIINP)
•    C1 Complement
•    C3 Complement
•    C4 Complement
•    Total gamma globulin
•    Total IGG Levels
•    P4NP7S
•    Glucagon-like Peptide-1
•    Fibrinogen
•    CK48
•    Serum Amyloid A1 (SAA1)
•    Phosphorylated Mitogen‑Activated Protein Kinase (p-p38)
•    YKL40
•    Fibroblast activation protein (FAP)
•    Anti-Smooth Muscle Antibodies (ASMA)

With inflammation comes cytokines.  Here is a target list of cytokines which could be used in your design:
•    IL-1β
•    IL-6
•    IL-8
•    IL-12
•    IL-17
•    IL-18
•    Tumor Necrosis Factor Alpha (TNFα)
•    FGF-19
•    FGF-21
•    embryonic liver beta-fodrin (ELF, not to be confused with the ELF test)
•    TGF-β

Depending on your subject composition, markers of insulin resistance and/or glycemic index may be of use, and several are integrated/calculated tests:
•    HOMA-IR
•    ADIPO-IR
•    Insulin
•    Pro-Insulin
•    Beta-hydroxybutyrate
•    C-peptide
•    Fasting Plasma Glucose
•    HbA1c
•    Adipose Tissue Insulin Resistance
•    Oral Glucose Tolerance Testing
•    De novo Lipogenesis Assessment

From safety perspective, you may be looking for coagulation function, and here are common coagulation tests that can be used:
•    INR
•    PT
•    Activated Partial Thromboplastin Time (APTT)
•    Factors II
•    Factor V
•    Factor VII
•    Factor VIIa
•    Factor VIII
•    Factor IX
•    Fibrinogen
•    Platelets
•    D-Dimers
•    Protein C
•    Protein S
•    Anti-Thrombin III
•    Thrombin – Antithrombin Complex (TAT)
•    Complement Component 3 Factor a (C3a)

While this list is extensive, it is by no means represents the totality of biomarkers that can be used in trials.  New assays and tests combining currently available biomarkers are being qualified and validated every day.  We understand that crafting your protocol objectives and end points are important parts of investigating a candidate’s pharmacology and pharmacodynamics.  We hope that you find this list a useful starting point or an expansion of markers that can be used in designing your next NAFLD/NASH clinical trial.