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

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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)

9

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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