1. NCX-1000 is a nitric oxide-donating derivative of ursodeoxycholic acid that selectively delivers nitric oxide to the liver, reducing intrahepatic resistance without causing systemic hypotension. 2. In animal models of cirrhosis and portal hypertension, NCX-1000 lowers portal pressure and total intrahepatic resistance more effectively than ursodeoxycholic acid alone by increasing hepatic nitric oxide levels and preventing hepatic stellate cell contraction. 3. Clinical studies in healthy volunteers found NCX-1000 to have no effect on blood pressure or heart rate at doses up to 3000 mg, demonstrating good safety and tolerability without systemic side effects.

1. Liver selective NO donors
Portal hypertension
Fiorucci Stefano
GI and Liver Unit
Department of Internal Medicine
University of Perugia
Italy

2. Portal hypertension
Increased inflow (hyperdynamic
splanchnic circulation)
Increased intrahepatic resistance
(structural and dynamic-reversible-
component)
Wiest R, Groszmann RJ. Hepatology. 2002;35:478-91

3. Dynamic component of portal
hypertension
• reduced production of NO from SEC
• increased production of endogenous
vasoconstrictors (ETs, angiotensin II, NE,
vasopressin, 5-LOX-derived eicosanoids)
• increased HSC contraction
Rockey DC et al. Gastroenterology. 2000;118:1261-5.

4. The deficient intrahepatic NO production
is the result of an endothelial dysfunction
in the liver microvascultature
It provides a rational basis for using NO-
based therapies and other vasodilators in
the treatment of portal hypertension
Rockey DC Gastroenterology 1998;114:344–351
Gupta TK. Hepatology 1998;28:926–931.

5. How to compensate for
hepatic defective eNOS activity
- NO donating drugs (NODD):
- conventional NO donors
- liver selective molecules
(NO-UDCA, V-PYRRO/NO
nitric oxide-donating prodrug)

6. NODD: Nitrates
Act on the systemic circulation by
causing a systemic vasodilation and
reducing mean arterial pressures
Lebrec D. Gut. 2001;49:441-2.

7. NODD: Nitrates
Nitrates reduce portal blood flow
by causing a reflex splanchnic
vasoconstriction
by decreasing the vascular
resistance to portal-collateral
blood flow

8. NODD: Nitrates
In patients with cirrhosis that cannot
be treated with β-blockers
isosorbide mononitrate ( Is-MN) does
not have any beneficial effect in
preventing first variceal bleeding
Garcia-Pagan JC. Gastroenterology 2001;121:908–914.

9. NODD: Nitrates
Is-MN administered alone may even
promote a slight increase in the risk of
bleeding
Despite the lack of clinical efficacy, Is-
MN,reduces mean arterial pressure
(pharmacologically active drug)
Garcia-Pagan JC. Gastroenterology 2001;121:908–914.

10. NODD: Nitrates
Effect on first variceal bleeding
Garcia-Pagan JC. Gastroenterology 2001;121:908–914

11. Liver specific NODD
An optimal therapy for the treatment
of portal hypertension should aim at
specific intrahepatic vasodilatation
by selectively targeting NO
delivery to the liver

12. Liver specific NODD:NCX-1000
NCX-1000 is a chemical entity obtained by adding an
NO-releasing moiety to ursodeoxycholic acid (UDCA)
CO 2(CH 2) 4ONO 2
O
H 3C
O
CH 3 H
OMe
CH 3 H
H
HO OH NO
H
Ferulic acid
UDCA

13. Liver specific NODD:NCX-1000
Effects on portal pressure

14. Liver specific NODD:NCX-1000
Prevention of portal hypertension in
CCL4-treated rats
CCL4 NCX-1000 UDCA * Control
15
pressure (mmHg)
Mean perfusion
*
10 *
5
0
20 30 40 60
Perfusion flow
(ml/min)
Fiorucci S et al. Proc Natl Acad Sci U S A. 2001;98:8897-902

15. Liver specific NODD:NCX-1000
pM L-citrulline/mg tissue
(wet weight) 30
*
eNOS activity
20
10
0
Control CCL4
Alone UDCA NCX-1000
Fiorucci S et al. Proc Natl Acad Sci U S A. 2001;98:8897-902

16. NCX-1000 reduces intra-hepatic
resistance induced by NE in normal rats
NE NE + UDCA NE + NCX-1000
700
600
(% of ∆ increase)
Portal pressure
500
400
300
P<0.001
200
100
0
0 -8 -7 -6 -5
NE (log M)
Fiorucci S. et al., J Hepatol. 2003;39:932-9.

17. NCX-1000 reduces intra-hepatic
resistance induced by NE in BDL rats
Control UDCA 100µM NCX-1000 100 µM
600
(% of ∆ increase)
500
Portal pressure
400
P<0.001
300
200
100
0
0 -8 -7 -6 -5
NE (log M)
Fiorucci S. et al., J Hepatol. 2003;39:932-9.

18. Effect of NCX-1000 in liver cGMP
of BDL rats
2000
*
1500
Liver cGMP
(fmol/mg)
1000
500
0
CTRL UDCA
BDL NCX
Alone UDCA NCX-1000
Fiorucci S. et al., J Hepatol. 2003;39:932-9.

19. NCX-1000
NCX-1000 causes a similar reduction
of vasoconstriction induced by the
maximally effective concentration of
NE in sham operated and cirrhotic
animals

20. 0.5 1 2 4 6 16 32
ml/kg b.w.
0.8
0.5 1 2 4 6 16 32
PP increase (mmHg)
ml/kg b.w.
0
-0.8
-1.6
NCX-1000 UDCA
Loureiro-Silva MR., et al.,.J Hepatol. 2003;39:940-6.

21. (cmH20/ml/min)
∆ Resistance
P<0.16
0.4
UDCA
NCX-1000
0.2
0
-6 -5.5 -5 -4.5 -4 -3.5
Loureiro-Silva MR. et al., J Hepatol. 2003;39:940-6.

22. NCX-1000 is absorbed and metabolized
In cirrhotic liver
NCX-1000 is capable of increase the concentration
of tauro-conjugate of UDCA
in the bile of cirrhotic rats

23. Hypothetical metabolic pathway of
NCX-1000
CO 2(CH 2) 4ONO 2
O
O
H OMe
H
H
HO OH
H NCX 1000
O
O
O(CH2)4ONO 2 NCX 2057
OH
UDCA
H
+ HO
OMe
H O
H O(CH 2) 4OH
HO OH HO
H O
OMe
Denitrated-NCX 2057
Glycine OH
+ NO
HO
Taurine OMe Ferulic acid
Conjugation
Hydroxy-butyl
+ HO ONO 2
nitrate
HO OH 1,4 butandiol
T-UDCA
G-UDCA
+ NO
Fiorucci et al., Figure 3

24. Nitrates versus NCX-1000 for the
treatment of portal hypertension
NCX- 1000 is devoid by hypotensive
systemic effect
Tachyphylaxia is not observed after
NCX-1000, being it capable of produce
the same anti-hypertensive effect in the
liver after short (5 days) and long
treatment (7 weeks)

25. Liver specific NODD:NCX-1000
Cellular effects

26. NCX-1000 and UDCA
transport
Portal Blood
NO
Portal Blood
MR
P2
Bile
NTCP UDCA BSEP
canaliculus NCX-1000 NTCP
3
MDR
Taurine Taurine UDCA
Glucuronoconjugation
Solfatation

27. NODD: In vitro protection against apoptosis
hepatocytes
Control Jo2 60
UDCA
Percent of apoptotic
50
NCX-1000
40
cells
30 *
Jo2-NCX-1000 Jo2-UDCA 20
Control
10
* *
0
0 0.01 0.1 1.0 10
UDCA or NCX-1000 (µM)
Fiorucci S,et al . Proc Natl Acad Sci U S A. 2001;98:2652-7

28. Liver specific NODD:NCX-1000
SNAP NCX-1000 UDCA Control
100
*
* * *
Nitrite/nitrate
(nM/10 cells)
75
6 50 *
*
*
25 *
*
0
0 30 60 90 120 150 180 210 240
Time (min)
Fiorucci S et al. Proc Natl Acad Sci U S A. 2001;98:8897-902

29. NCX-1000 prevents HSC contraction
Control NCX-1000 UDCA SNAP
100
90
(percent of basal area)
HSC contraction *
80
70
60
50
40
40
0
0 4 8 12 16 20 24
Time (hr)
Fiorucci S., et al. Proc Natl Acad Sci U S A. 2001;98:8897-902

30. Liver specific NODD:NCX-1000
Clinical study
In healthy volunteers no toxicity
was observed after
a single or multiple administrations of
NCX 1000
Nicox-Axcan, data on file

31. Liver specific NODD:NCX-1000
No effect on:
sitting systolic and diastolic blood
pressure values
standing systolic and diastolic blood
pressure values
sitting and standing heart rate

32. Liver specific NODD:NCX-1000
Standing systolic blood pressure
130
120
110
100
NCX 500
NCX 1000
NCX 2000
90
NCX 3000
placebo
80
0 4 8 12

33. Liver specific NODD:NCX-1000
Standing diastolic blood pressure
90
80
Arterial pressure
70
mmHg
60
NCX 500
NCX 1000
NCX 2000
50
NCX 3000
placebo
40
0 4 8 12
hours

34. A second generation NO-UDCA:
NCX-999
O
ONO2
O
H
NO
H
OH
HO
H
UDCA

35. A second generation NO-UDCA:
NCX-999
Advantages
A better hydrosolubility
than NCX-1000
High capability of circulating into the
sinusoids and delivering NO into the
hepatic microcirculation, instead
being stored into the hepatocytes

36. NCX-999: effect on portal pressure
Normal rat liver
15 NE alone NE + NCX-999 100 µM
Portal pressure (mmHg)
10
*
5 *
* * *
0
0 -7 -6 -5 -4
Norepinephrine (log M)

37. Liver specific NODD:V-PYRRO/NO
Is metabolized by hepatocytes in vitro
Protects the hepatocytes from apoptotic cell
death induced by TNFα
Increased liver cGMP levels in vivo
Reduces hepatic resistance of the liver both
before and after ischemia /riperfusion in pigs
Saavedra et al. J Med Chem. 1997 Jun 20;40(13):1947-54.

38. Liver specific NODD:V-PYRRO/NO

39. Liver specific NODD:V-PYRRO/NO
150
MAP (mm Hg)
100
50
0
Sham Sham V-P BDL
Alone Y-P
Moal F, et al. Gastroenterology 2000, 118,4, ABS 1082

40. Liver specific NODD
The development of NCX-1000
indicates a strategy for development of
compounds that selectively deliver
NO to the liver
minimizing the side effects
of organic nitrates
Hepatic selectivity might be further
increased by targeting
specific molecular determinant in the
liver microcirculation

42. In CCL4 induced cirrhosis NCX-1000,
but not UDCA, reduces portal
pressure changes induced by
volume expansion
Loureiro-Silva MR.J Hepatol. 2003;39:940-6.

43. NCX-1000, but not UDCA, lowers
total intrahepatic resistance induced
by methoxamine in CCL4 cirrhotic
rats

44. Attempts to pharmacologically
augment the NOS system in liver to
correct hepatic vasoconstriction have
been largely impaired by the inability
to selectively deliver NO donor
agents to the desired site of action

45. NO NCX-1000
Systemic
circulation Systemic
circulation
Liver Liver
Vasodilation
Intrahepatic
resistance Selective liver
metabolism
Hypotension
Splancnic
vasocostriction
Intrahepatic
Reduced hepatic resistance
bolood flow

46. How to compensate for
hepatic defective eNOS activity
- gene therapy (eNOS, nNOS)
- NO-donating drugs:
- conventional NO donors
- new liver selective molecules
(NO-UDCA, V-PYRRO/NO
nitric oxide-donating prodrug)

47. Liver specific NODD:NCX-1000
NCX 1000 is a well tolerated and
safe compound in animals and
humans
It has a favorable toxicological
profile even after prolonged
treatment periods