AQX-1125, a First-In-Class Clinical-Stage SHIP1 Activator Small Molecule for the Treatment of
Pulmonary Diseases: Suppression of Chemotaxis In Vitro and Efficacy in Rodent Models of Asthma
and COPD In Vivo
C. Szabo, L.F. Mackenzie, G.R. Stenton, P. Tam, J.L. Cross, C. Harwig, J. Raymond, J. Toews, D. Chernoff, T. MacRury
Aquinox Pharmaceuticals Inc., Richmond, BC, Canada
Abstract
Background
Introduction
Targeting SHIP1
  PI3K pathway is one of the most active
areas in Biotech/Pharma
  SHIP1 is an ideal drug target
  PI3K/SHIP1 pathway plays a key role in
regulating cell migration and activation
  SHIP1
expression
restricted
to
hematopoietic derived cells - limits off target
toxicity
  SHIP1 is a novel target distinct from the
extensive PI3K investigations
  Redirects cellular PI3K signalling, rather than
prevent it
PI3K
All cells
PI-3,4,5-P3
PI-4,5-P2
PI-3,4-P2
AQX-1125 inhibits leukocyte chemotaxis
B cells:
Monocyte:
NPNY NPL
Y
hSHIP1
SH2
hsSHIP1
5-ptase
C2
NPNY NPLY
Proline rich
5-ptase
C2
Proline rich
hSHIP2
5-ptase
SH2
1188
C2
928
1258
Proline rich
Good homology between human and rodent
SHIP1
hSHIP2
mSHIP1
rSHIP1
hSHIP1
51%
92%
91%
hSHIP1
C2
domain
38%
91%
90%
Figure 2. SHIP enzymes – 51% homology between
hSHIP1 and hSHIP2, and 38% homology between
the hSHIP1C2 and hSHIP2C2 domains. This
reduced homology between SHIP1 and SHIP2
confers selectivity.
SHIP1
Knockout1
+/+
+/+
n
io
at )
tiv ors
Ac at
ic ctiv
r
e A
st
lo P1
A l SH I
(
-/-
+/+
+/+
• Viable & fertile, ~40%
-/--/-
D1, D2, D3
-/-/-
Aquinox Pharmaceuticals Inc.
Suite 430, 5600 Parkwood
Way, Richmond, BC,
V6V 2M2, Canada
T: 604.629.9223
F: 604.295.4748
aqxpharma.com
Figure 9. AQX-1125, but not dexamethasone
reduces neutrophil infiltration. Data are expressed
as mean+SEM of n=7-8 determinations.
D19 D20 D21 D22
C
• Increased GM progenitors
• Infiltration of lungs by macrophages
& neutrophils
survival by 14 weeks
OVA IP
Sensitization
B
PO Dosing
QD x 3
Sacrifice/
Lavage
Pharmacokinetics of AQX-1125 in rats
IV
PO
PO
1 mg/kg
10 mg/kg
30 mg/kg
0.083
1.00
1.17
Cmax (ng/mL)
144
267
1226
AUCinf /D
268
176
227
t ½ (hr)
5.6
5.2
4.5
F (%)
N/A
66
85
C
Tmax (hr)
Figure 3. Phenotype of the SHIP1 knockout mouse.
A) small stature, B) splenomegaly and C)
inflammatory infiltrate of the lungs.
SHIP1 Activation
Pelorol was the first generation SHIP1 activator
isolated. Analogues of Pelorol, AQX-016A and
AQX-MN100, were synthesized with greater
SHIP1-activating
properties2.
These
small
molecules activate SHIP1 through an allosteric
mechanism, via interaction with the C2 domain,
and are anti-inflammatory in cellular and murine
models2.
AQX-1125 is a small molecule next generation
SHIP1 activator. It has many of the biological
effects of the earlier generation SHIP1 activators,
but has more drug-like properties.
The compound has recently completed Phase I
safety testing in healthy human volunteers and is
progressing into Phase IIa proof-of-concept
clinical studies.
10000
30 mg/kg, Oral
10 mg/kg, Oral
Figure 6. (A) Male Brown Norway rats were
sensitized to OVA, followed by oral AQX-1125
administration and OVA challenge. BAL was
performed and resulting data shown expressed as
mean±SEM of (B) BAL leukocyte and (C)
eosinophil counts, *p<0.05 vs OVA.
1 mg/kg, IV
1000
100
10
1
0
AQX-1125
inhibits
OVA-induced
inflammatory mediator content in Brown
Norway rat BALF
B B
A
5
10
15
20
25
Time (h)
Figure 10. Pharmacokinetics of AQX-1125 in male
Sprague Dawley rats. AQX-1125 was administered
intravenously (IV) or by oral gavage. Plasma
concentrations of AQX-1125 were determined at
various times. Data are expressed as mean±SD.
Cellular and In Vivo Models
•  Akt phosphorylation in T cell lines – Western
blotting
•  Leukocyte chemotaxis in vitro
•  PMA-induced ear edema in mice
•  Ovalbumin-mediated
allergic
airway
inflammation in rats
•  Lung inflammation induced by intratracheal
administration of LPS in rats
•  Lung inflammation induced by cigarette smoke
inhalation in mice
Results
AQX-1125 inhibits Akt phosphorylation in
SHIP1-proficient MOLT-4, but not in SHIP1deficient Jurkats
MOLT-4 (PTEN-/SHIP1+)
Jurkat (PTEN-/SHIP1-)
IGF-1
)
(0.1
25
)
µM
)
)
µM
)
µM
µM
0 µM
(10
(10
(1
25
11
11
AQ
X-
%)
25
0 (1
02
11
(0.1
50
40
29
AQ
X-
AQ
X-
1
LY
AQ
X-
DM
SO
IG
F-
)
)
No
stim
ulat
ion
)
)
µM
)
µM
(0.1
25
11
µM
µM
0 µM
(10
(10
(1
25
11
AQ
X-
%)
25
0 (1
02
11
(0.1
50
40
29
AQ
X-
AQ
X-
AQ
X-
stim
ulat
ion
1
LY
DM
SO
IG
F-
No
Contact information
AQX-1125
inhibits
inflammatory
cell
accumulation in the BAL of BALB/c mice in a
steroid-insensitive
cigarette
smoke
inhalation model
OVA Chal
A
B
-/-
IGF-1
inflammation
Figure 5. Human blood leukocytes were treated
with AQX-1125 for 30 min, followed by induction of
chemotaxis with chemokines listed.
• Splenomegaly
Cell activation
and function in
Figure 1. SHIP1 and PI3K signalling. SHIP1
redirects PI3K signalling, PI3K inhibitors block
PI3K signalling.
Figure 8. AQX-1125, but not dexamethasone
reduces neutrophil infiltration in a rat model of lung
inflammation induced by intratracheal LPS
challenge. Data are expressed as mean+SEM of
n=6 determinations.
AQX-1125 inhibits OVA-induced allergic
airway inflammation in Brown Norway rats
A
Blood cells
All cells
AQX-1125
inhibits
inflammatory
cell
accumulation in the BAL of rats after LPS
NPAY
SHIP1
PTEN
Results
Results
SHIP enzymes
Concentration (ng/mL)
Pharmacological
modulation
of
the
phosphoinositide 3-kinase (PI3K)/Akt pathway is
emerging as a means to control inflammatory
disorders. The SH2-containing inositol-5'phosphatase 1 (SHIP1) metabolizes PI[3,4,5]P3
to PI[3,4]P2. SHIP1-deficient mice exhibit a
marked degree of inflammatory cell recruitment
into the lung, leading to pulmonary inflammation.
Pharmacological, allosteric activation of SHIP1
has been shown to enhance the degradation of
PI[3,4,5]P3
to PI[3,4]P2 and exerted antiinflammatory effects (Ong et al., Blood, 2008).
Here we overview the biological effects of
AQX-1125, a small molecule SHIP1 activator, a
current
clinical
development
candidate.
AQX-1125 induced a concentration-dependent
increase in the catalytic activity of human
recombinant SHIP1 enzyme.
AQX-1125
suppressed Akt phosphorylation in SHIP1proficient MOLT-4 cells, but not in SHIP1deficient Jurkat cells. AQX-1125 inhibited
degranulation of wild-type bone marrow-derived
mast cells, but not of SHIP1-deficient cells. As
phosphoinositide signaling plays a key role in
cytokinesis, the effect of AQX-1125 was tested
on leukocyte chemotaxis. AQX-1125 was found
to be a nanomolar, a pan-selective inhibitor of
leukocyte chemotaxis. AQX-1125 exhibited high
oral bioavailability (>80%) and long terminal
half-life (>5h) in rodents and dogs, and yielded
high concentrations in a number of parenchymal
tissues, including the lung. The efficacy of
AQX-1125 was next evaluated in (a) a rat
ovalbumin-mediated airway inflammation model:
(b) a rat pulmonary inflammation model induced
by intratracheal administration of LPS, and (c) in
a mouse model of cigarette smoke-induced
airway inflammation. AQX-1125 induced a dosedependent reduction in leukocyte infiltration into
the bronchoalveolar lavage fluid in all three
experimental models. AQX-1125 markedly
reduced the production of multiple chemokines,
cytokine and growth factors in the lung and
exhibited
a
unique
characteristic
antiinflammatory signature pattern distinct from
dexamethasone.
The current preclinical data
suggest that SHIP1 activation may have utility
for the treatment of respiratory disorders such
as chronic obstructive pulmonary disease and
asthma. The first-in-class SHIP1 activator
AQX-1125
demonstrates
ideal
drug-like
properties and has recently entered Phase I
clinical testing. Future clinical efficacy studies
will assess the utility of the SHIP1 activator
AQX-1125 in human pulmonary diseases.
SHIP1
SHIP1
pAkt (S473)
pAkt (S473)
Total Akt
Total Akt
Actin
Actin
C
Figure 7. Male Brown Norway rats were sensitized
to OVA, followed oral AQX-1125 (10 mg/kg)
administration and OVA challenge. BAL was
performed and resulting data shown expressed as
mean±SEM of (A) eotaxin, (B)
IL-1α and (C)
IL-11concentrations, *p<0.05 vs OVA.
Figure 11. Pharmacokinetics of AQX-1125 in male
Sprague Dawley rats after repeat dosing at 30 mg/
kg/day; comparison of plasma and pulmonary
concentrations. Please note the significant
pulmonary accumulation of the compound at all time
points. Data are expressed as mean±SD.
Summary and Future Directions
SHIP1 is a novel drug target which controls PI3Kdriven cellular migration and activation. SHIP1’s
restriction to hematopoietic cells and poor
homology with SHIP2 reduces the likelihood of
off-target, off-tissue toxicity. AQX-1125, a small
molecule with PK properties suited to once per
day dosing, inhibits the PI3K pathway through
activation
of
SH2-containing
inositol-5'phosphatase 1 (SHIP1), resulting in a reduction of
pAkt in T and B lymphocytes. AQX-1125 has
significant in vivo anti-inflammatory activity, in
models of allergic inflammation.
These data
indicate that AQX-1125 has significant clinical
potential in inflammatory disorders such as
asthma. Phase IIa proof-of-concept studies,
scheduled to start in 2011 will be instrumental in
determining the potential human clinical
therapeutic utility of the compound.
References
Figure 4. MOLT-4 and Jurkat cells were treated with
AQX-1125 for 30 min, followed by stimulation with
IGF-1 for 30 min.
1.  Helgason et al. Targeted disruption of SHIP leads to hemopoietic
perturbations, lung pathology, and a shortened life span. Genes & Dev.
1998;12:1610-1620.
2.  Ong et al. Small molecule agonists of SHIP1 inhibit the
phosphoinositide 3-kinase pathway in hematopoietic cells. Blood.
2007;110:1942-1949.
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