Recombinant Human Annexin A5 Therapy – Inhibition of Proinflammatory Response with Improved Cardiac Function and Survival Rate

in Mice with Endotoxemia

Ashley DavisUniversity of North Florida

Collaborative Research• Paul Arnold & Fatemeh Armirahmadi – Department of

Physiology and Pharmacology at the University of Western Ontario, London, Canada

• Xiangru Lu MD - Center of Critical Illness Research at the Lawson Health Research Institute ON, Canada

• Katharina Brandl PhD – Department of Genetics at the The Scripps Research Institute La Jolla, California

• Qingpin Feng MD, PhD – Department of Medicine at the University of Western Ontario, London, Canada

• Dr. Feng is a career investigator at Heart and Stroke Foundation of Canada

Endotoxemia✧ Bacteria enter bloodstream and release endotoxins

✧ WBCs react to the presence of bacteria and release inflammatory substances – excessive cytokine production

✧ 18 million cases annually

✧ Increased endothelial permeability• Stage (II) - Severe Sepsis

• Organ dysfunction

• Mortality rate: 30-40%

• Stage (III) - Septic shock

• Myocardial depression

• Mortality rate: 40-80%

NIAID/RML, Science 2002

Host’s Immunity✧ Host’s innate immunity activated

✧ TLR2 = Gram-positive

✧ TLR4 = Gram-negative

✧ Activation of TLR4 via LPS binding

✧ Initiates signaling pathway that leads to

✧ activation of MAPKs (mitogen-activated protein kinases)

✧ production of inflammatory cytokines - tumor necrosis factor (TNF-α) and Interleukin (IL-β)

Celeste Thorpe MD 2004

Roll of Toll-like Receptors

Modlin, Nature 2000

LPS• Lipopolysaccharide

• From Gram-negative bacteria

• LPS binds to TLR4 in CD14

• Binding of LPS activations signaling pathway that leads to activation of MAPKs and production of inflammatory cytokines

• Necrosis factor alpha (TNF-α)

• Leading cause of cardiac dysfunction

• Interleukin-1 beta (IL-β)

Feng et al. Crit Care Med, 2014

Previous Treatment• Xigris = Activated Protein C

• Indications: for severe sepsis & septic shock

• Acute organ failure

• Properties: Anticoagulant, Anti-inflammatory, Profibrinolytic

• Side Effect: Serious Bleeding

• Was the only approved therapy by the U.S Food and Drug Administration

• No longer used due to severe bleeding risk

• Results from trial showed no survival benefit over placebo

• Xigris was voluntarily withdrawn from market in 2011

FDA Medwatch, 2011

Annexin A5• A 35-kDa phospholipid binding protein

• High affinity binding to negatively charged phospholipids

• Has antiapoptotic and anticoagulant properties – by forming 2D crystallized shield over the surface of cells

• Shield decreases ability to initiate phagocytosis or thrombosis

• Anx5 interacts with cell receptors to inhibit their function in binding to leucine-rich repeats

• LPS binds to extracellular domain of TLR4 at leucine-rich repeats

• Anx5 inhibits LPS binding to TLR4 and thus decreases downstream MAPK signaling

HTI Inc, 2009

Results• Anx5 improved cardiac function and survival rate during

endotoxemia by inhibiting LPS binding to TLR4/myeloid differentiation factor 2 (MD-2) receptor complex

• Anx5 decreased myocardial TNF-α and IL-βexpression

• Anx5 restored cardiac function to control levels

• via the inhibition of activation of NF-kB

Feng et al. Crit Care Med, 2014

Anx5 decreases myocardial MAPKs and NK-κB

Feng et al. Crit Care Med, 2014

CardiacTissue

MAPK

Side Pathway

Treatment with Anx5 significantly decreased myocardial TNF-αand IL-1β

Feng et al. Crit Care Med, 2014

mRNA

Protein

Plasma

Anx5 Improves Cardiac Function

Feng et al. Crit Care Med, 2014

Measuring meanarterial pressure

Improved Survival Rates5 day animal survival was significantly improved by either the immediate or 4-hour delayed

Anx5 treatment following LPS challenge.

Feng et al. Crit Care Med, 2014

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