Mechanistic Understanding of Disease Biomarkers Through Metabolomics

1
Mechanistic Understanding of Disease Biomarkers
Through Metabolomics Edward D. Karoly, Ph.D.
2
Metabolon technology and business model
mVision Commercial Service Global Biochemical Pr
ofiling Service
Metabolomics Platform Technology LC-MS GC-M
S Informatics Interpretation

• Mechanistic insight
• Biomarkers

Drug Safety And Efficacy
Bioprocess Optimization
Disease Biomarkers
Over 400 studies, 100 patent applications, 4
issued patents
3
Endogenous Constituents of Phenotype
3
4
(No Transcript)
5
Metabolon Process
Non-targeted Metabolomics (50-1000 m/z)
Metabolyzer
UHPLC-MS/MS (ESI)

Instrumentation
Peak Detection
Peak Integration
Biochemical Extraction
UHPLC-MS/MS (-ESI)
Library Search RT, Mass, MS/MS
Biochemical Analysis
QA/QC
GC-MS (EI)
6
Library Search for Biochemical ID
Biochemical Amount
Metabolyzer Software
14.43
4.01
5.84
4.38
10.66
8.46
10.18
11.76
4.55
6.52
6.73
7.74
9.34
8.01
11.03
11.79
13.05
9.47
7.50
5.34
11.21
12.89
3.17
13.30
4
5
6
7
8
9
10
11
12
13
14
Time (min)
6
7
Metabolyzer
UHPLC-MS/MS (ESI)

Peak Detection
UHPLC-MS/MS (-ESI)
Peak Integration
Biochemical Extraction
Library Search RT, Mass, MS/MS
QA/QC
GC-MS (EI)
Metabolon Platform Technology

• Biochemical
• Interpretation
• Pathway analysis
• Literature

Global Biochemical Pathway Changes Disease
Biomarkers Mechanistic Toxicology Drug
MOA Cellular Characteristics
Heat Maps by Pathway
8
Metabolomics Reveals Sarcosine as a Biomarker of
Prostate Cancer Aggressivness
9
Overview of Study

• Background
• PSA levels correlate weakly with prostate
  malignancy
• Finding markers which discriminate aggressive
  from non-malignant tumors would aid in prostate
  cancer treatment
• 42 prostate tissue samples from
• Benign adjacent prostate tissue (Benign)
• Localized prostate cancers (PCA)
• Metastatic prostate cancers (Mets)
• Study Objective
• Identify biochemical biomarkers for prostate
  cancer progression to metastasis
• Nature, 2009, 457(7231)799-800. (Arul
  Chinnaiyan, U. Michigan Metabolon, Inc.)

10
Global Biochemical Changes
gt600 Biochemicals Measured
11
Sarcosine Levels as Measured by Independent
Isotope Dilution GC/MS Assay

• Is Sarcosine Associated with Cancer Cell
  Aggressiveness?

12
Sarcosine correlated with aggressivity

• Assay invasive and non-invasive cell lines

Sarcosine Invaded cells
Sarcosine increase part of cancer biology or is
it an epiphenomenon?
13
Sarcosine Induces Invasiveness
Addition of Sarcosine to Benign Cells Induces
Invasiveness
13
14
Metabolism of sarcosine
14
15
Inhibiting Synthesis and Degradation of Sarcosine
16
Regulation by Androgen Signaling Genes
Androgen signaling (ETS family of genes ERG,
ETV1) key prostate cancer modulators of
progression
17
Impact of Prostate Biomarker Study

• Identified four biochemical markers which
  differentiate metastatic from non-metastatic
  tumors
• Sarcosine identified as a potentially important
  metabolic intermediary of cancer cell
  aggressiveness
• Diagnostic model well-defined (RUO test)
• Manuscript published in Nature

18
Identification of Novel Insulin Resistance
Metabolites in a non-Diabetic Population by
Metabolomics
19
Insulin Resistance (IR) Type 2 Diabetes
Diabetic
Normal
Pre-Diabetic
?-cell Function
80
Vascular Damage
Measuring the Key Mechanism of Diabetes
96 Pre-Diabetes Undiagnosed (55 M)
Insulin Resistance/Production
25 T2D Undiagnosed (6 M)
Insulin Resistance
Insulin Production
Diabetic
125 mg/dl
Fasting Plasma Glucose
Pre-Diabetic
100 mg/dl
Normal
Disease Progression
20
EGIR-RISC cohort biochemical profiling

• Objective Discover novel biomarkers of insulin
  sensitivity
• 399 non-diabetic subjects
• NGT-IS, NGT-IR, IGT, IFG
• All subjects have hyperinsulinemic euglycemic
  clamp samples

21
Markers Segregating Insulin Sensitivities

• Several biochemical contribute to separating high
  and low insulin sensitivities with
  a-hydroxybutyrate (AHB) the top marker

22
AHB is top-ranking IR metabolite
23
HI Clamp pre- and post-Intervention Study
(DeFronzo and UTHSCSA colleagues)
Glucose disposal (M)
a-HB

• AHB reduced with intervention correlates with
  improved glycemic status and clamp values
• BMI unchanged
• Muraglitazar drug treatment group

24
AHB in Bariatric Surgery Intervention

• a-hydroxybutyrate is reduced with intervention
  with improved clamp values

25
a-Hydroxybutyrate (AHB) Metabolism
?

• a-hydroxybutyrate, marker of energy stress

26
Summary IR Dx study

• AHB is an important IR metabolite
• Earliest IR biomarker in asymptomatic,
  non-diabetic population
• Other metabolites including a-ketoacids,
  creatine, and various lipid species also
  contribute to distinguishing IR subjects
• Bariatric/Drug data intervention studies help
  validate these IR markers
• Future studies will aim at mechanistic
  experiments to determine effect of these
  metabolites on muscle, fat, and liver tissue, as
  well as beta cells

27
Acknowledgments
Markers of prostate cancer Agressiveness Univ
ersity of Michigan Arul Chinnaiyan Arun
Sreekumar Metabolon, Inc. Jeff Shuster Danny
Alexander Alvin Berger

• Markers of insulin resistance
• EGIR-RISC consortium and investigators
• www.egir.org
• Università di Pisa
• Ele Ferrannini, M.D.
• Stefania Camastra, M.D.
• Andrea Natalia, M.D.
• Monica Nannipieri, M.D.
• M. Anselmino, M.D.
• Mauro Rossi, M.D
• UTHSC at San Antonio
• Ralph DeFronzo, M.D.
• Muhammad Abdul-Ghani, M.D., Ph.D.
• Eugenio Cersosimo, M.D., Ph.D.
• Nicholas Musi, M.D.

Metabolon, Inc. Walt Gall, Ph.D. Kirk Beebe,
Ph.D. Yun Fu Hu, Ph.D. DVM Costel Chirila,
Ph.D. Klaus-Peter Adam, Ph.D. Mike Milburn,
Ph.D. John Ryals, Ph.D
28
Take-Home
Metabolomics - A core and information-rich
component of systems biology - Metabolic changes
can related back to phenotype A non-hypothesis
driven, discovery tool - global approach is the
standard Biomarkers, discoveries, and
mechanistic insight can be gleaned from
-Human, animal and in vitro studies - Tissues -
Cells (microbial, plant and mammalian) - Fluids
(urine, plasma, medium, etc.)
29
Case Study snap-shots
29
30
Drug Mechanism of Action
Study Rationale Identify oncology drug MOA
31
Metabolomics-Derived Biomarkers for Early
Detection of Nephrotoxicity
Study Rationale Many drugs fail due to
nephrotoxicity Traditional markers not sensitive

• T

Toxicological Pathology Volume 37, Number 3
32
Metabolomics reveals sarcosine as a marker of
prostate cancer aggressiveness
Study Rationale PSA levels correlate weakly with
prostate malignancy
33
Identification of Biomarkers for Outcome in Sepsis
Study Rationale Leading cause of morbidity and
mortality (35-50) among critically ill patients
(750,000 cases per year)
Markers of Death and progression discovered
34
Biomarkers of Latent vs. Active Tuberculosis
Study Rationale Establish markers for monitoring
latent TB population
Markers of Death and progression discovered
35
Insight into Drug Mechanisms of ActionGemin X
Pharmaceuticals
36
Background and Study Design

• Background
• In-licensed by Gemin X from Leo Pharma in 2006.
• Broad spectrum antitumor activity in several
  tumor types
• Mechanism of action uncertain but thought to
  involve inhibition of NF-?B.
• Experimental Details
• Multiple myeloma line IM-9
• Dosing as shown (cultures per group)

GMX 1778
37
Changes in Global Metabolic Profile Six hours
after GMX1778 Treatment

• Four biochemicals showed greatest change after 6
  hours of drug treatment
• Largest Increase
• Glutamine (2.7 fold)
• ?-Glutamylglutamine (2.8 fold)
• Largest Decrease
• Nicotinamide (0.7 fold)
• NAD (0.38 fold)

38
Mapping Changes to PathwaysTwo Biosynthetic
Pathways for NAD
Nicotinic acid
NAPRT
Nicotinamide
PARP, SIRT1 etc.
NAMN
NAMPRT
NaMNAT
NAD Synthetase
NAAD
NMN
NMNAT
Glutamine
Glutamate
redox reactions
?-Glutamylglutamine
ATP
NAD decline was found to precede a loss of
cellular ATP
39
Cell Rescue Experiments
A. Add Nicotinic Acid
Cell Rescue
100
75
( ATP levels)
Relative Viability
50
Nicotinic acid
25
0
0.001
0.01
0.1
1
10
100
1000
10000
Concentration Nicotinic acid or Nicotinamide (uM)
Assay performed in presence of 10 µM GMX1778
40
Focus on Nicotinamide PathwayTwo Possible
Targets for NAD Biosynthesis
Isolation of each enzyme rNAMPRT and rNMNAT
Nicotinamide
PARP, SIRT1 etc.
NAMPRT
NMNAT No Effect
NAD
NMN
NMNAT
NAMPRT Inhibited
Differential inhibition of recombinant enzymes
41
NAMPRT Confirmed as Drug Target
42
Impact of Biochemical Profiling Study

• Defined GMX1778 action at a novel metabolism
  target (NAD biosynthesis)
• Proposed nicotinic acid/niacin as overdose
  antidote
• Identified biochemical markers for tracking
  pharmacodynamics
• Gives rationale for using GMX1778 as stand alone
  or combination therapy (alkylating/methylating
  agents)
• Manuscript in print

43
Insulin Resistance
Case Study
44
Insulin Resistance (IR)

• Importance of IR
• Clear mechanism for development of diabetes
• Associated with a number of other diseases
• Current Methodology for Measuring IR
• Hyperinsulinemic Euglycemic Clamp
• Gold Standard
• Advantage
• Direct measure of IR
• Disadvantage
• Expensive
• Difficult to get repeat measurements

45
Type 2 Diabetes
Vascular Damage
Diabetic
125 mg/dl
Fasting Plasma Glucose
Pre-Diabetic
100 mg/dl
Normal
Disease Progression
46
Type 2 Diabetes
Diabetic
Normal
Pre-Diabetic
?-cell Function
80
Vascular Damage
Measuring IR The Key Mechanism Of Diabetes
Insulin Resistance/Production
Insulin Resistance
Insulin Production
Diabetic
125 mg/dl
Fasting Plasma Glucose
Pre-Diabetic
100 mg/dl
Normal
Disease Progression
47
Biomarker Discovery Process
Global Biochemical Profiling Statistical
Analysis Biochemical Interpretation
Clinical Samples from Collaborators Disease vs.
Normal
Candidate Biomarkers Mechanistic Insight
Quantitative Assay Mechanistic Studies
Mechanistic Markers
48
EGIR-RISC Cohort Biochemical Profiling

• Objective Discover novel biomarkers of insulin
  sensitivity
• 399 subjects (non diabetic)
• NGT-IS, NGT-IR, IGT, IFG
• Baseline hyperinsulinemic euglycemic clamp
  samples

lt45 M-ffm
HI Clamp
gt45 M-ffm
lt140 mg/dL
FPG (gt100 mg/dL)
OGTT
gt140 mg/dL
49
Peak Detection
Peak Integration
Library Search Retention Time, Mass, MS/MS
QA/QC
Platform Technology

• Biochemical
• Interpretation
• Pathway analysis
• Literature

Mechanism-Based Results Disease
Biomarkers Mechanistic Toxicology Drug
MOA Cellular Changes
Heat Maps
50
Markers Segregating Insulin Sensitivities

• Several biochemicals contribute to separating
  high and low insulin sensitivities with
  alpha-hydroxybutyrate the top marker

51
a-hydroxybutyrate metabolism

• a-hydroxybutyrate, marker of energy stress

52
Drug Human Intervention Study with Diabetic
Subjects
a-HB
Placebo Mura (pre, post)

• a-hydroxybutyrate is reduced with intervention
  correlates with improved glycemic status and
  clamp values in diabetic subjects
• Mura-Muraglitazar drug treatment group

53
a-HB in Bariatric Surgery Intervention
glucose disposal rate
a-hydroxybutyrate

• a-hydroxybutyrate is reduced with intervention
  with improved clamp values

54
Summary

• IR markers such as a-hydroxybutyrate are
  important IR metabolites identified in the
  EGIR-RISC cohort
• Internal validation We have run several studies
  that provide initial confirmation of such IR
  markers (including 2 non-primate species)
• Clinical validation studies already sourced for
  FDA regulatory submission