Post on 19-Jul-2018
transcript
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
ed.conway@ubc.ca
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
In pursuit of health through research in blood and blood related processes
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
In pursuit of health through research in blood and blood related processes
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
In pursuit of health through research in blood and blood related processes
Studies to evaluate the role of
PolyP in regulating complement activation
In pursuit of health through research in blood and blood related processes
• 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)
In pursuit of health through research in blood and blood related processes
Hemolytic Assay to measure terminal pathway
GVB-EDTA
Diluted human serum
Chicken RBCs (cRBC)
+ PolyP + C5b,6
37oC, 30' & spin
A405
In pursuit of health through research in blood and blood related processes
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
In pursuit of health through research in blood and blood related processes
*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