Connective Tissue Proteoglycan

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Hyaluronic Acid
The future of liver disease assessment
Denice Taylor March 2003
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Overview

• HA Market Potential
• Rheumatology Application
• Liver Disease Application
• Features of Corgenix HA Kit
• Sales tools
• Competition
• Benefits
• Sales targets

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Annual Market Potential for HA gt 65 M
Market Potential

• US EU ROW
• Liver 14 14 14
• RA/OA 4 4 1
• Misc 3 3 1
• Rx 3 3 1

JP
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Hyaluronic Acid (HA) Test Kit

• Unique Product
• Large Potential Undeveloped Market
• Chronic HCV 180 million patients (1999)
• Chronic HBV 350 million patients (2000)
• Successful in Asian Market since 1995
• Emerging market in Germany, Turkey
• and Scandinavia

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HA Test Kit

• Sponsored International Conference on Measurement
  of Fibrosis, AASLD, Boston 2002
• Currently in 4 major HCV trials COPILOT, Aegis,
  and 2 NIH funded studies
• Corgenix Product Line to be extended

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Hyaluronic Acid (Hyaluronate, Hyaluronan, HA)

• High molecular weight polysaccharide, widely
  distributed throughout body
• Produced mainly by fibroblasts and other
  specialized connective tissue cells
• Function
• Structural role connective tissue matrix
  proteoglycan
• Participates in cell-to-cell interactions
• Fluid retention and lubrication of joints

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Hyaluronic Acid (HA) Connective tissue
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Hyaluronic Acid (HA) Joint Production
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Hyaluronic Acid (HA) Liver Removal
HA is cleared from the blood by specific
receptors in liver sinusoidal endothelial cells
(SEC)
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Possible causes of elevated HA levels

• ? ? Increased HA production
• - inflammation such rheumatoid
  synovium
• ? Increased release of HA from tissues
• - cell damage
• ??? Decreased HA removal from plasma
• - decreased activity of SEC due to
  fibrosis or cirrhosis

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HA Applications

• Clinical use
• Rheumatology
• Liver disease, liver transplantation
• Commercial and research use
• Pharmaceutical industry
• Nutritional supplements
• Veterinary applications
• Research

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Rheumatology Application

• Synovitis is the hallmark of RA
• Increased production of HA is seen in RA, related
  to synovial involvement and the inflammatory
  process
• Plasma HA levels have been shown to correlate
  with disease activity in RA

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Rheumatology Application

• Mean plasma HA levels are 7-fold higher in RA
  patients with active disease and 2-fold higher in
  OA patients when compared to healthy controls
• HA can be used as a marker of synovitis in RA and
  osteoarthritis (OA)
• HA may be useful in monitoring efficacy of RA
  therapeutics including DMARDs and biologic
  response modifiers

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HA and Rheumatic Disease - RA and OA
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Liver Disease

• Serum HA levels ? with chronic injury (chronic
  hepatitis) to liver
• Chronic injury (chronic active hepatitis) causes
  histologic changes in the liver
• Fibrosis (infiltration of fibrous tissue) early
  histologic changes
• Treatable
• Reversible
• Various grades (eg. Metavir, Ishak staging
  scores)
• Cirrhosis advanced fibrosis and scarring
• Reversible in certain cases
• May lead to liver failure

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HA in Normal Liver
Sinusoidal Endothelial Cells (SEC)
HA Receptors in Fenestrae
Stellate Cells
Space of Disse
Hepatocytes
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HA in Liver Disease - Fibrosis
Kupffer Cell
Activated Stellate Cells
Fibrous (ECM) Proteins
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HA in Liver Fibrosis/Cirrhosis
Irreversible Damage to Hepatocytes
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HA Correlation with Fibrosis/Cirrhosis
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HA and Traditional Liver Markers
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Chronic Hepatitis

• Multiple etiologies
• Viral Hepatitis (80-90)
• Hepatitis C (HCV) 90 of cases chronic 30-40
  progress to cirrhosis small develop CA
• Hepatitis B (HBV) 5-10 develop chronic
  infection
• Chronic substance abuse (alcohol)
• Miscellaneous
• Autoimmune hepatitis Hemochromatosis
• Drug-induced hepatitis Primary Biliary
  Cirrhosis
• NASH

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Liver Biopsy

• The Gold Standard method to diagnosis and stage
  fibrosis
• Advantages
• Can accurately stage the extent of liver injury
  and fibrosis
• Is used to confirm the diagnosis and may lead to
  additional unsuspected diagnoses
• Provides important prognostic information

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Liver Biopsy

• Disadvantages
• Sampling error est. 24 false negative
• Costly requires hospitalization
• Risk of complications
• Pain
• Infection
• Bleeding
• Typically performed only every 2-5 years

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Serum Fibrosis Markers

• Advantages
• Reflect the overall status of the liver (no
  sampling error)
• Non-invasive
• Less painful, less resistance from patients
• Less costly (can be performed on outpatients)
• Negligible risk of complication
• Can be performed more frequently than liver
  biopsy

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Serum Fibrosis Markers

• Disadvantages
• Cannot use to diagnose liver disease
• Less sensitive and specific when compared with
  gold standard

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HA in Liver Disease
Cutoff
cutoff
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Utility of HA in Chronic Liver Disease

• Distinguishes between cirrhotic and non-cirrhotic
  liver, regardless of etiology
• Most sensitive serum indicator of changes in
  fibrosis and cirrhosis in CLD
• HA reflects change in fibrosis following
  ?-interferon therapy
• A sensitive marker for progressive liver
• damage in primary biliary cirrhosis

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HA Positioning in CLD

• Use liver biopsy to establish the diagnosis of
  chronic liver disease
• Measure baseline HA level at diagnosis
• Monitor disease status with serum HA levels every
  4-6 months
• Use HA to measure therapeutic response

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Transplantation

• Liver Transplant (approx. 5000/year in US)
• HA is a sensitive indicator of SEC injury
• Vascular damage prominent feature of graft
  rejection
• HA is an early marker of rejection
• Reflects viability of donor organ

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HA levels in Liver Transplant Patients
Adams DH, Wang L et al. Hepatic Endothelial
Cells.Transplantation 47479-482 (1988)
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HA in Liver Disease vs Bone Marrow Transplants
Serum HA in patients with veno-occlusive disease
following bone marrow transplantation
Fried MW, et.al. Bone Marrow Transplantation
27635 (2000)
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HA and Bone Marrow Transplants (VOD)
Serum HA vs Bilirubin in veno-occlusive disease
patients
Fried MW, et.al. Bone Marrow Transplantation
27635 (2000)
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Hyaluronic Acid (HA) Protein Binding Assay
(microwell format)
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Features of Corgenix HA

• Experts in ELISA technology
• 18 months shelf life
• Convenient procedure, total time 2.5 hours
• No known interference or cross-reactivity
• Proven track record since 1995

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HA competition

• Other HA kits
• Conventional Liver Function Tests (LFTs)
• Bilirubin ALT
• AST Alkaline Phosphatase
• GGT Albumin
• Prothrombin Time
• Other fibrosis markers
• ?-GST PIIINP
• Type IV Collagen Laminin
• TGF-?-1 ?-2 Macroglobulin
• TIMP-1
• Fibrosis Panels
• Prometheus
• Fibrotest

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Other HA methods

• Pharmacia RIA kit
• Echelon Competitive HA ELISA
• Wako LiBASys
• Mitsubishi LPIA

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Pharmacia HA Test (RIA)
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HA vs LFTs

• HA
• Reflects SEC function
• Useful in chronic liver disease
• Correlates with histological changes in liver
• Early, sensitive marker for toxic liver damage

• LFTs
• Reflects hepatocyte function
• Useful in acute liver disease
• Insensitive due to extensive reserve capacity of
  liver
• Do not reflect degree
• of fibrosis

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HA vs ALT as serum fibrosis markers
Serum HA is a useful marker for liver fibrosis in
chronic hepatitis C virus infection. Prospective
study n 130
Wong VS, et.al. J. Viral Hepatol 5 187 (1998)
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Serum Fibrosis Markers

• HA shows best sensitivity and specificity for
  cirrhosis
• One test may not be enough
• Additional novel markers (and therapeutics) may
  be discovered through basic research in fibrosis

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Fibrosis panels
FIBROSpect Prometheus Labs, US
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Fibrosis panels
Fibrotest T. Poynard et al., France
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HA Market Japan

• Market size (2002) 7 million
• Clinical application chronic hepatitis
• Disease groups
• Hepatitis C
• Hepatitis B
• Alcoholic liver disease

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HA Clinical Utility in Japan

• Baseline measure HA levels at diagnosis
• Monitor periodically thereafter (? 4 x per yr)
• With anti-fibrotic therapy
• Before treatment
• End of treatment
• 6-12 months post treatment
• Disease specific cutoffs
• Fibrosis 50 ng/mL
• Cirrhosis 130 ng/mL

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HA marketing challenges

• Physician Awareness of test
• Publications by opinion leaders
• Training workshops
• Advertising
• Trade shows
• Competition
• Positioning

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Sales targets

• Reference Laboratories
• Medical Centers
• RA clinics
• Hepatology clinics (with liver clearance)
• Transplant centers
• Hospital Laboratories
• Research Centers
• Pharmaceutical Companies
• Nutritional Companies

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510(k) status

• Liver Clinical trial protocol under review by FDA
• Three study sites identified
• Trials to begin spring 2003
• 510(k) submission late 2003