polyphosphate: a novel therapy for age related macular ...lsi.ubc.ca/files/2014/09/amd.pdf ·...

Polyphosphate: A novel therapy for Age

Related Macular Degeneration (AMD)

Edward M. Conway, MD, PhD

Professor of Medicine, UBC

Canada Research Chair in Endothelial Cell Biololgy

Director, Centre for Blood Research

University of British Columbia, Vancouver, Canada

[email protected]

tel. 604 822 4252

In pursuit of health through research in blood and blood related processes

Polyphosphate (polyP)

Polyphosphate (PolyP)

• linear polymers of inorganic orthophosphate,

linked by high energy anhydride bonds

• strongly anionic

• ubiquitously expressed by all cells & oganisms

• varying length

• functions in bacteria

• pH control

• chelator of metal ions

• phosphate reserve

• chaperone for protein delivery

Morrissey


Polyphosphate in micro-organisms

PolyP in micro-organisms

• chains may be >10,000 units

• multiple functions in metabolism,

growth, biofilm formation

• used for immune evasion

• virulence factor

• H. pylori, P. aeruginosa, V.

cholera

• inhibits phagocytosis

• inhibits complement

• e.g. Neisseria meningitidis

Morrissey


Polyphosphate in humans

PolyP humans

• found in all cells

• 40-800 phosphate units long

• released upon activation

• role in coagulation

• found normally in blood (50-160 uM)

• likely protective function


Studies to evaluate the role of

PolyP in regulating complement activation


• Wat J, Foley JH, Krisinger MJ, Ocariza LM, Lei V, Wasney GA, Lameignere

E, Strynadka N, Smith SA, Morrissey JH, Conway EM. Polyphosphate

suppresses complement via the terminal pathway. Blood 2014;123:768-76.

• Provisional patent with UBC filed on Nov 8, 2013 (#61901759)

"Polyphosphate suppresses complement" (broad applications)

6

PolyP targets the Terminal Pathway

C5 convertase:

C3bBbC3b

C4b2aC3b C5b C5

C5a

7 8

9 9 9

Terminal Pathway

• After generation of C3a & C5a

• Spontaneous

• No enzymes involved

• Ion-independent

• Implicated in damage in AMD (wet and dry)


Hemolytic Assay to measure terminal pathway

GVB-EDTA

Diluted human serum

Chicken RBCs (cRBC)

+ PolyP + C5b,6

37oC, 30' & spin

A405


For studies of effect of polyP,

use C5b,6 concentration to

obtain ~75% lysis.

PolyP inhibits the terminal pathway in serum

• Monophosphate (P1) has no effect on complement (lysis)

• Polyphosphate (P>1000) suppresses complement (lysis)

* Performed by Dr. Stephanie

Smith (University of Illinois)

Chain-length dependent effects of PolyP

• Longer chains of PolyP have more anti-complement

activity

PolyP inhibits terminal pathway in purified system

At what step does PolyP suppress TP?

6 C5b 7 8

9 9 9


*Performed by Linnette Ocariza

and Greg Wasney

PolyP binds to C5b,6

by gel filtration

PolyP destabilizes C5b,6

More PolyP (20 uM-2 mM) causes less stable C5b,6

• measured by differential scanning fluorimetry (DSF) with C5b,6

Hypothesis

PolyP attenuate diseases associated

with excess complement activation

In vivo Validation

Many complement-mediated diseases e.g. Age-related macular degeneration

Source: Histology of normal retina.

http://www.sciencephoto.com/media/308887/view

Choroid

Outer nuclear layer

Bruch’s membrane

Rods and cones

Outer plexiform layer

Inner nuclear layer

Inner plexiform layer

Ganglion cell layer

Optic fibre layer

RPE layer

Age-related Macular Degeneration

Prevalence: Leading cause of blindness for people 50 yrs or older Two types: Dry: 90% of AMD Wet: 10% of AMD

Risk Factors: Increasing age Hereditary (complement system genetic defects) Smoking Poor diet

Costs: Economic burden for U.S. government ~ $30 billion annually

Laser-induced CNV in Rats

Oct 2013

Pilot in vivo Studies

• Two groups, 2 rats per group

Grp 1: Laser + 1 injection of polyP>1000 (final concentration 200 uM)

Grp 2: Laser + 1 injection of monophosphate (monoP) (200 uM)

• 5 lasers per eye (1 eye per rat)

• Intravitreal injections with Hamilton syringe at t=10' after laser

• At t=5 days, whole mounts of retinas from rats 5 days after laser

induction of choroidal neovascularization.

• Post-fixation staining for vascular endothelium (green, CD31) and

membrane attack complex (MAC) (red)

• Merged images are shown, all at same magnification.

Oct 2013

PolyP s vascular response and MAC

B

Flat mounts of

retina after

laser-induced

CNV in rats

PolyP PolyP

MonoP MonoP

Oct 2013

Immediate plans

Objectives

1. Dose-finding and validation of polyP in murine models of AMD (wet

and dry)

i. laser-induced CNV model of wet AMD

ii. carboxymethylpyrrole (CEP)-MSA model of dry AMD

iii. Ccl2:Cx3cr1 double ko mice + Crbrd8 background for dry AMD

2. Safety and toxicity studies with P>1000. Pharmacokinetics and

biodistribution of intravitreal biotin-P>1000

3. Reformulation of P>1000 with polymers, liposome nanoparticles, with

anti-VEGF antibodies and test in vitro and in vivo for efficacy and safety

Oct 2013

Advantages and Limitations

Polyphosphate

Lead compound P>1000 characterized in vitro and in vivo

Naturally occurring in blood; therefore likely not toxic

Inexpensive and easily prepared, highly soluble and stable in water

Uniquely targets Membrane Attack Complex (MAC, C5b-9) and not C3a or

C5a

No other targets of MAC in preclinical trials except solCD59

Not expected to adversely affect vasculature

PolyP able to be modified and/or reformulated

liposomes, polymers, anti-VEGF compounds

Available sensitive functional assays to monitor effects

Available expertise and animal models

Issues to address:

Safety and toxicity (not likely)

Stability (Functional t1/2 exceeds 2 weeks in serum)

Predictive value of dry AMD models

Oct 2013

$$$

Funding

Seeking partners/investors to strengthen IP

Proof-of-Principle Grant application submitted to CIHR

Other grants submitted

Foundation Fighting Blindness (submitted)

CNIB (submitted)

CIHR (submitted)

Genome Canada

Other discoveries in lab re: complement/coagulation

polyphosphate: a novel therapy for age related macular ...lsi.ubc.ca/files/2014/09/amd.pdf ·...

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