Small Molecule Factor D Inhibitors Block Complement Activation in Paroxysmal
Nocturnal Hemoglobinuria and Atypical Hemolytic Uremic Syndrome
Eleni Gavriilaki1, Jane Thanassi2, Guangwei Yang2, Xuan Yuan1, Mingjun Huang2, Robert A. Brodsky1
1Division of Hematology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD
2Achillion Pharmaceuticals, New Haven, CT
PNH and aHUS Are Complement-mediated Hemolytic Anemias
• Paroxysmal nocturnal hemoglobinuria (PNH): – Caused by PIGA mutations
• Lead to absence of GPI-anchored complement regulatory proteins CD55 and CD59
• Atypical hemolytic uremic syndrome (aHUS): – Caused by mutations or autoantibodies that
activate the alternative pathway of complement (APC)
• Leads to endothelial damage, platelet activation, and end-organ damage to multiple organs
2 Brodsky RA. Blood 2014; Cataland SR, Wu HM. Blood Rev 2014
PNH and aHUS Treated by Terminal Complement Blockade
• Eculizumab: Monoclonal Ab that binds to C5 and blocks terminal complement activation
• FDA approved for PNH in 2007
• FDA approved for aHUS in 2011
3 Hilmen P, et al. N Engl J Med 2006.; Legendre CM, et al. N Engl J Med 2013
Limitations of Eculizumab
• Intravenous administration
• ~ 25% of PNH patients have suboptimal response – Symptomatic extravascular hemolysis due to
accumulation of C3 fragments on PNH red cells
– Mutations that prevent binding of eculizumab to C5
4 Risitano AM. Blood 2009; Nishimura J, et al. N Engl J Med 2014
PNH patients on eculizumab have continued hemolysis due to C3 fragment accumulation
A PNH red cell is protected from intravascular hemolysis
5 Revised from Luzzatto L, Risitano AM, Notaro R. Haematologica 2010;95(4):523–526
PNH: on eculizumab
C5 convertase
C6 C7 C8 C9 MAC
C5
C5b
C3b
Eculizumab Alternative
pathway
Classical pathway
Lectin pathway
Physiological C3 tick-over
+
C3
C3b
FD
FD
RES macrophages (liver, spleen)
C3 fragment C3 fragment opsonization
C3 fragment
but becomes susceptible to extravascular hemolysis
CD59 CD55
Rationale for Development of Small Molecule Factor D Inhibitor
• Factor D – Lowest abundance of all complement proteins in the serum – Factor B is its only substrate – Rate-limiting step of APC
• Oral bioavailability
– Able to achieve systemic concentration necessary to inhibit factor D
6
Factor D Inhibitors
7 Revised from Luzzatto L, Risitano AM, Notaro R. Haematologica 2010;95(4):523–526
Alternative pathway
Classical pathway
Lectin pathway
Physiological C3 tick-over
C3
C3b
FD
FD
C3 fragment
PNH: on factor D inhibitor
A PNH red cell will be protected from both intravascular and extravascular hemolysis
Aims
To demonstrate that:
1. Small molecule factor D inhibitors specifically block the APC in PNH and aHUS
2. To develop a reliable human in vitro model for aHUS
8
Methods
• Three APC-specific, reversible inhibitors of factor D developed for oral administration were assessed:
– ACH-4471 – ACH-4100 – ACH-3856
• Erythrocytes and serum from 3 PNH (on or off
eculizumab) • Serum from 4 aHUS patients (on or off eculizumab)
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Factor D Inhibitors Show High Binding Affinity to Human Factor D
Binding affinity
ka= association rate constant; kd = dissociation rate constant; KD=dissociation equilibrium constant
Compound ID
On rate ka (M-1s-1)
Off rate kd (s-1)
KD (nM)
ACH-4471 2.9e6 0.0016 0.54
ACH-4100 8.2e6 0.0022 0.27
ACH-3856 1.2e7 0.0043 0.36
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Factor D Inhibitors Inhibit Factor D Proteolytic Activity
Blockade of Factor B Cleavage
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Mean IC50
0.1 1 10 100 1000 0.0
0.2
0.4
0.6
0.8
Bb
(µg/
mL)
ACH-3856 = 9.8 nM ACH-4100 = 17 nM ACH-4471 = 20 nM
Concentration (nM)
Dose-dependent Inhibition of Hemolysis and C3 Deposition on Rabbit Erythrocytes
Hemolysis1
Compound ID
IC50 ± SD (nM)
IC90 ± SD (nM) N
ACH-4471 17 ± 11 70 ± 23 6
ACH-4100 17 ± 6.4 45 ± 15 8
ACH-3856 8.7 ± 3.9 22 ± 8.9 40
C3 Deposition2
12
0.1 1 10 100 100000 1000 10000
125
% o
f C3
frag
men
t pos
itive
cel
ls
Concentration (nM)
100
75
50
25
0
IC50 ACH-3856 = 50 nM ACH-4100 = 56 nM ACH-4471 = 96 nM
1. Incubation with Normal Human Serum (NHS) 2. Incubation with C5-depleted NHS
Patient 2
% h
emol
ysis
chan
ge
20
40
60
80
100
120
0
ACH-3856 ACH-4471
0.1 1 10 100 1000 nM
13
Patient 1
Patient 3
% h
emol
ysis
chan
ge
20
40
60
80
100
120
0
ACH-3856 ACH-4471
ECU treated
Patient 1 IC50 (nM) IC90 (nM)
ACH-4471 27 110
ACH-3856 17 56
Patient 3 IC50 (nM) IC90 (nM)
ACH-4471 14 26
ACH-3856 10 45 20
40
60
80
100
0
ACH-3856 ACH-4471
Dose-dependent Reduction of Hemolysis on PNH Erythrocytes (Ham Test)
67% Type III RBCs
15% Type III RBCs
0.1 1 10 100 1000
Patient 2 IC50 (nM) IC90 (nM)
ACH-4471 4 14
ACH-3856 3 6
97% Type III RBCs
0.1 1 10 100 1000
% h
emol
ysis
chan
ge
120
nM
nM
ECU treated
Treatment naive
Factor D Inhibitors Significantly Reduce C3 Fragment Deposition on PNH Erythrocytes
1 nM 300 nM 30 nM
14 0.01 0.1 1 10 100 1000
% C
3 po
sitiv
e ce
lls
0
10
20
30
40
50
ACH-4471 concentration (nM)
43.8% 22.4% 1.5%
IC50 = 32 nM
Ham and Modified Ham Test: Models of PNH and aHUS
15 Gavriilaki E, et al. Blood 2015
HYPOTHESIS
Modified Ham test as a reliable human in
vitro model for aHUS
aHUS Patients: Clinical Characteristics
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Age, gender
ADAMTS13 (%)
PLT count (x103/μL) LDH (U/L) Cr (mg/dL) Studied
47, F 75 9 2,505 2.3 Off eculizumab
59, M 15 39 676 2.3 On eculizumab
35, F 18 10 1,270 2.4 On and off eculizumab
23, F 102 62 2,820 6.3 Off eculizumab
Factor D Inhibitors Significantly Reduce Cell Killing Caused by aHUS Serum (Modified Ham Test)
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ACH-4471 concentration (nM)
% m
ean
non-
viab
le c
ells
0
20
40
60
80
1 10 100 10000 0 1000
Conclusions • Factor D inhibitors efficiently block APC activation
in PNH and aHUS
• Factor D inhibitors prevent accumulation of C3 fragments in vitro on PNH red cells
• The modified Ham test may be useful to test complement inhibitors in aHUS
• ACH-4471 is being advanced into clinical development as a potential oral treatment for PNH and other rare diseases
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