A Novel Oral Form of Pentamidine (OCZ103) Blocks TNFMediated Mouse Liver Injury from GalN/LPS
Enpeng Zhao1, Ghulam Ilyas1, Yu Lin1, Kathryn Tanaka1, François Ravenelle2, Mark J. Czaja1
1Albert Einstein College of Medicine, Bronx, NY, United States, 2Oncozyme Pharma Inc, Montreal, QC, Canada
1. ABSTRACT
Figure 2. OCZ103 decreases liver injury from GalN/LPS.
Serum ALT, histological injury grading and TUNEL staining for
untreated controls (Con), PBS alone injected mice (PBS) and the
treatment groups receiving GalN/LPS (pretreated with PBS, 25 or 40
mg/kg of OCZ103). Liver injury from GalN/LPS was markedly reduced
by OCZ103 as demonstrated by statistically significant decreases in
serum ALT (left panel, *P<0.01) and histological grade of liver injury
(center panel, **P<0.001) at 6 h. As a result, hepatocyte death as
detected by TUNEL staining was also significantly prevented (right
panel, *P<0.0002) (n=3-7).
Mitochondrial protein
PBS
G/L
-
+
OCZ40
+
+
PBS
+
G/L
−
+
OCZ25
+
+
OCZ40
+
+
+
Pro
Casp 3
Cyt ox
Cytosolic protein
PBS
-
-
+
+
+
p17
Pro
Casp 7
OCZ40
p19
p116
p85
+
tBid
PARP
Cyt c
Tubulin
Tubulin
25
0
1h
100
2h
50
25
1h
2h
0
100
1h
2h
IFNγ (pg/ml)
*
1h
2h
4h
*
100
1h
6
4
2
2h
2h
4h
PBS
OCZ
4h
O
1h
10000
2h
4h
PBS
OCZ
*
7500
5000
2500
0
O
1h
2h
1.Oral OCZ103 specifically distributes
to the liver.
2.OCZ103 blocks TNF-dependent liver
injury and mortality from GalN/LPSinduced liver injury.
3.OCZ103 improves survival in the
GalN/LPS model, reducing the risk of
death by ˃ 19-fold.
4.OCZ103 inhibits the TNF-dependent
mitochondrial cell death pathway,
partially as the result of alterations
in macrophage cytokine release, but
also likely secondary to additional
direct hepatoprotective effects of
the drug.
5.The combination of the liver
targeting biodistribution of oral
OCZ103, and its hepatoprotective
properties, suggest that OCZ103 may
be effective in the treatment of liver
diseases such as alcoholic hepatitis
and NAFLD.
5. REFERENCES
1. Corsini, E. et al. (1992) Immunol. Lett.
34, 303-308 & Int. J. Immunopharmacol. 14, 121-130.
2. Amir, M. et al. (2013) Cell Death
Differ. 20, 878-887.
3. Wang, Y. et al. (2006) J. Biol. Chem.
281, 15258-15267.
4. Wang, Y. et al. (2004) J. Biol. Chem.
279, 31089-31097.
200
25000
PBS
OCZ
1h
PBS
OCZ
50000
0
O
4h
300
0
4h
4h
8
0
2h
75000
*
PBS
OCZ
O
2h
300
10
0
1h
IL-6 (pg/ml)
TNF (pg/ml)
400
O
1h
PBS
OCZ
PBS
OCZ
30
20
0
10
500
0
500
20
4h
200
1000
4h
30
PBS
OCZ
75
IL-1β (pg/ml)
Figure 4. OCZ103 improves
survival in GalN/LPS model.
Based on the multiple comparison analysis of pooled
data, OCZ103 at the doses of
40 and 50 mg/kg significantly reduced mortality (by
> 19- and 37-fold, respectively). The effect at 25
mg/kg was not significant
(n=6-12).
*
PBS
OCZ
IL-10 (pg/ml)
OCZ 40 GalN/LPS
50
Relative Infγ Levels
PBS GalN/LPS
1500
PBS
OCZ
Relative Il-10 Levels
75
Relative Il-6 Levels
OCZ 25 GalN/LPS
Relative Tnf Levels
Figure 3. OCZ103
improves liver
histology.
Representative H&Estained livers sections
from mice treated as
indicated.
PBS
Figure 6. Effects of OCZ103 on cytokine production.
Effects of OCZ103 on hepatic cytokine mRNA levels by qRT-PCR (top
5 panels) and serum cytokine levels (bottom 5 panels).
Significant decreases in proinflammatory TNF and increases in
anti-inflammatory IL-10 occurred with OCZ103 treatment at the
mRNA and protein levels (*P<0.01; n=4-6).
0
Figure 1. Oral OCZ103 provides
a liver-targeted delivery of
pentamidine. Selected tissue
distribution
following
oral
OCZ103 administration. Concentrations reported at 12 h after
last dose of OCZ103.
-
Total liver protein
tBid
G/L
2. ORAL BIODISTRIBUTION
Injections of standard pentamidine provide a high systemic
exposure with associated potential pancreatitis and kidney
toxicity. The new oral form of OCZ103 provides a high
pentamidine liver exposure in dogs (Fig. 1), with a low
systemic exposure characterized by blood Cmax of 0.19 µM.
4. CONCLUSIONS
Figure 5. OCZ103 blocks activation of the mitochondrial cell
death pathway and prevents caspase 3 and 7 and PARP cleavage.
Immunoblots of mitochondrial, cytosolic and total liver protein
fractions from treated mice probed for the indicated antibodies.
Relative Il-1β Levels
A new oral form of pentamidine, OCZ103, has been developed
for the treatment of liver disease. Oral administration of
OCZ103 specifically targets the liver where drug
concentrations are 15- to 150-fold greater than in other
organs such as the pancreas and kidney. The known antiinflammatory effects of pentamidine, together with the
favorable hepatic bioavailability of OCZ103, suggested that
OCZ103 may be effective in the treatment of liver diseases
mediated by overactivation of the innate immune response.
We therefore tested the hypothesis that OCZ103 would
protect mice against TNF-dependent liver injury and mortality
induced by galactosamine/lipopolysaccharide (GalN/LPS).
Methods: Fasted male Beagle dogs were administered oral
OCZ103 twice daily (42.6 mg/kg 12 h apart) for 3 consecutive
days (n=9). C57BL/6 mice were pretreated with vehicle control
or OCZ103 IP 30 min prior to GalN/LPS administration and
evaluated for effects on liver injury and survival. Results: Liver
injury from GalN/LPS was markedly reduced by OCZ103 as
demonstrated by statistically significant decreases in serum
ALT (177 vs. 5,632 IU/L; P<0.002) and histological grade of liver
injury (1.7 vs. 3.7; P<0.001) at 6 h. As a result, hepatocyte
death as detected by TUNEL staining was also significantly
prevented (2.1 vs. 75.8 TUNEL positive cells/HPF; P<0.0002).
Survival after GalN/LPS administration was also markedly
improved in OCZ103-treated mice. Mechanistic studies
revealed that OCZ103 blocked mitochondrial cell death
pathway and caspase activation. Conclusions: A new oral form
of pentamidine, OCZ103, markedly decreased TNF-dependent
liver injury and mortality from GalN/LPS.
3. LIVER INJURY MODEL RESULTS
4h
6. DISCLOSURES
• François Ravenelle is an employee of
Oncozyme Pharma Inc.
• This work was funded by Oncozyme
Pharma Inc.