1. Macitentan – A novel sulfamide
Starting from the structure of bosentan, a medicinal chemistry program was carried out that led to the development of the novel highly potent dual ERA macitentan. The following changes were made to the structure of bosentan during the discovery of macitentan:
Purple group: Bosentan is a sulfonamide and macitentan is a sulfamide. Sulfamide derivatives have a higher affinity for ETB compared with the corresponding sulfonamide analogues.
Green/purple group: The bulky 4-tert-butyl benzene sulfonamide in bosentan can be replaced by an alkyl-sulfamide group to reduce the size of the molecule, without affecting affinity for the ET receptors. Molecules with a smaller alkyl-sulfamide (such as macitentan, which has a propyl-sulfamide group) had better in vivo activity in a rat model of hypertension. Macitentan was of particular interest as it had excellent in vivo activity and high potency on the ET receptors.
Blue group: The bulky substituent in bosentan is not mandatory to obtain potent ET antagonism. To reduce the molecular size, this has been removed in macitentan.
Yellow group: In macitentan, a 5-bromopyrimidine ring has been added to the ethylene glycol group. This has been shown to improve affinity for both ET receptors significantly.
Red group: Replacement of the group in bosentan with a 4-bromophenyl residue (as seen for macitentan) leads to potent ET receptor antagonism.
Bosentan
Macitentan
Bolli M, et al. J Med Chem 2012; 55:
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2. Tissue penetration requires drugs to cross from the bloodstream through the lipophilic cell membrane
The ability of a drug to cross the bilipid membrane depends on
Ionisation properties - drugs cross the membrane in the non-ionised, lipophilic form1
Affinity for the lipid vs the aqueous phase1
Size of the molecule1
Since ET-1 is localised to the tissue, an ERA that can penetrate into the tissue is desirable.
In order for a drug to effectively penetrate tissues, e.g. into pulmonary smooth muscle, it must first cross from the bloodstream through the lipophilic cell membrane of endothelial cells.
The ability of a drug to cross lipid membranes depends on its ionisation properties (i.e. drugs cross the lipid membrane in the non-ionised, lipophilic form) and the drug’s affinity for the lipid versus the aqueous phase.
In the case of macitentan, the discovery process focused on the optimisation of molecules with enhanced tissue penetrating properties (a high ionisation dissociation constant reflecting a high non-ionised fraction and a high distribution coefficient favouring distribution to the tissues). .
1. Rowland M and Tozer TN. Clinical Pharmacokinetics: Concepts and applications. Lippincott Williams & Wilkins, 1995.
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3. Optimisation of the physicochemical properties compared to Bosentan may favour macitentan’s penetration into tissues: pKa
Macitentan, a novel pyrimidin-propylsulfamide ET receptor antagonist has been selected based on its inhibitory potency of both ET receptors and optimised physicochemical properties to achieve high affinity for the lipophilic milieu.
Physiochemical properties (that favour tissue penetration) can be investigated using well-established laboratory parameters such as the ionisation dissociation constant (pKa) and the distribution coefficient (Log D) in the lipid or aqueous phase.
The pKa is a measure of the drug’s ionisation properties (higher pKa values correspond to a greater percentage of non-ionised form and thus greater lipophilicity and greater tissue penetration potential).
Macitentan has a pKa value of 6.2 compared with values of 5.1 and 3.5 for bosentan and ambrisentan, respectively. Consequently, a higher percentage of macitentan (6%) exists in the non-ionised form compared with bosentan (1%) or ambrisentan (0.01%) at physiological pH (7.4).
A higher pKa corresponds to greater lipophilicity and thus greater tissue targeting potential
pKa: ionisation dissociation constant
Adapted from Iglarz M, et al. J Pharmacol Exp Ther 2008; 327:
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4. Log D (Distribution coefficient)
Optimisation of the physicochemical properties compared to Bosentan may favour macitentan’s penetration into tissues: Log D
In vitro data
Log D (Distribution coefficient)
Lipid:Aqueous
Macitentan
800:1
Bosentan
20:1
Ambrisentan
1:2.5
Blood
Membrane
Tissue
The distribution coefficient defines the distribution of a compound between the aqueous (buffer pH 7.4, blood) and lipid (n-octanol, tissue) phase. Higher distribution coefficients correspond to greater lipophilicity and thus greater tissue penetration potential.
For macitentan, the ratio is 800 to 1 (lipid phase: aqueous phase) compared with 20 to 1 and 1 to 2.5 for bosentan and ambrisentan, respectively.
This corresponds to a 40- and 2000-fold increased affinity for the lipid phase with macitentan compared with bosentan and ambrisentan, respectively.
Ambrisentan has more affinity for the aqueous milieu than for lipids.
The physicochemical properties of macitentan favour tissue penetration.
The distribution coefficient (Log D) defines the distribution of a compound between an aqueous and a lipid phase
A greater affinity for the lipid phase may favour tissue penetration
Macitentan may have a greater affinity for the lipid phase compared with other ERAs*
*To date, no head-to-head trials in humans have been performed.
Iglarz M, et al. J Pharmacol Exp Ther 2008; 327:
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5. Macitentan displays sustained binding to its target receptors
Experimental system:
Determination of changes in inhibitory potency after antagonist wash-out (calcium flux in PASMC)
In vitro data
Bosentan 20 40 60 *
Ambrisentan
t1/2~40 sec
Time after wash-out (minutes) *
Macitentan 60 40 20
t1/2~17 min
Time after wash-out (minutes)
1000
Kb after washout (nM) * * * *
100 * * 10
t1/2~70 sec * 1
Bosentan and ambrisentan displayed relatively short receptor occupancy times (t1/2; bosentan t1/2 ~70 seconds and ambrisentan t1/2 ~40 seconds), i.e., they quickly dissociate from ET receptors.
In contrast, inhibition of ET signaling was maintained in the macitentan-treated cells (t1/2 ~17 minutes); macitentan therefore has a relatively long receptor occupancy time as a result of slow receptor dissociation.
Since macitentan displays a 15-fold increased receptor occupancy half-life compared with ambrisentan and bosentan, this leads to a sustained blockade of ET signaling in comparison with ambrisentan and bosentan.
0.1 20 40 60
Time after wash-out (minutes)
Macitentan displays a 15-fold increased receptor residence time (t1/2) compared to ambrisentan and bosentan
*p < 0.05
Gatfield J, et al. PloS One 2012; 7(10):e47662.
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6. Protection against high ET-1
In contrast to ambrisentan and bosentan, macitentan remains potent at elevated ET-1 concentrations
Experimental system:
ET-1-induced signalling in human PASMCs measuring IP1
ET-1 concentration-response curves in presence of different concentrations of antagonists
In vitro data X
Protection against high ET-1
Bosentan
10000 nM
1000 nM
100 nM
10 nM
1 nM
0 nM
Normalised IP1 conc. (%) 20 40 60 80
100
120
-11
-10 -9 -8 -7 -6 -5
ET-1 (log M)
20 nM
3000 nM
Inactive
IC50 x X
Ambrisentan
-11
-10 -9 -8 -7 -6 -5
ET-1 (log M)
2 nM
500 nM
Inactive x 
Macitentan
-11
-10 -9 -8 -7 -6 -5
ET-1 (log M)
1 nM
5 nM x
The effect of bosentan, ambrisentan and macitentan on ET-induced IP1 accumulation was investigated in vitro. The graphs show concentration response curves.
Human PASMCs were pre-incubated with different dilutions of the three ERAs (bosentan, ambrisentan or macitentan) for 120 minutes followed by the addition of different dilutions of ET.
After 20 minutes of ET stimulation, IP1 accumulation was determined (as an indicator of the amount of ET signaling that occurred).
All three antagonists blocked ET-induced IP1 accumulation in PASMC, in a concentration-dependent manner.
However, while ambrisentan and bosentan antagonism could be overcome by increased concentrations of ET (i.e., both became inactive at higher concentrations of ET-1, as indicated by the normalised IP1 concentration), macitentan antagonism of ET was demonstrated even in the presence of high concentrations of ET-1 (i.e., macitentan remained active even at the highest concentration of ET-1).
These data indicate that macitentan (in contrast to ambrisentan and bosentan) remains potent at elevated ET-1 concentrations.
Gatfield J, et al. PloS One 2012; 7(10):e47662.
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7. Chronic† macitentan administration reduced mPAP at a dose 10-fold lower than bosentan in the MCT rat model
Animal model
+++
+++ p < vs. control
** p < 0.01 vs. MCT + vehicle
* p < 0.05 vs. MCT + vehicle
*** p < vs. MCT + vehicle
Veh
0.3 3 10 30
100
300
Dose (mg/kg/day) 40 20
mPAP (mmHg) *
*** **
***
MCT + bosentan
MCT + macitentan
MCT (monocrotaline)
Control
***
***
***
In the MCT rat model of PAH, chronic oral dosing with macitentan for 4 weeks dose-dependently reduced mPAP at a 10-fold lower dose than bosentan.
Maximal efficacious dose macitentan: 30 mg/kg/day vs bosentan: 300mg/kg/day.
Control rats received normal rat chow.
†Administered for 4 weeks
Iglarz M, et al. J Pharmacol Exp Ther 2008; 327:
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8. Chronic† macitentan administration reduced RV hypertrophy at a dose 10-fold lower than bosentan in the MCT rat model
Animal model
+++ p < vs. control
** p < 0.01 vs. MCT + vehicle
* p < 0.05 vs. MCT + vehicle
*** p < vs. MCT + vehicle
Veh
0.3 3 10 30
100
300
Dose (mg/kg/day)
0.5
0.4
0.2
RV/LV+S
+++ * ** **
MCT + bosentan
MCT + macitentan
MCT (monocrotaline)
Control
***
***
In the MCT rat model of PAH, chronic oral dosing with macitentan for 4 weeks dose-dependently reduced right ventricular (RV) hypertrophy at a 10-fold lower dose than bosentan.
The ratio of the right ventricle to left ventricle plus septum weight (RV/LV+S) was calculated as an index of right ventricular hypertrophy.
Control rats received normal rat chow.
***
RV: right ventricular †Administered for 4 weeks
Iglarz M, et al. J Pharmacol Exp Ther 2008; 327:
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9. Effect of macitentan on survival in the MCT rat model of PAH
Animal model 20 40 60 80
100
Survival (%) 7 14 21 28 35 42
Time (days)
MCT + macitentan*
83%
p = 0.002
Monocrotaline (MCT) + vehicle
50%
Control
The effects of macitentan on survival were investigated using the monocrotaline (MCT) rat model of PAH.
At the end of the study, the MCT rats receiving 30 mg/kg/day macitentan demonstrated significantly higher rates of long-term survival (83%) than untreated MCT rats (50%).
66% reduction in mortality at day 42.
The first death was delayed from 25 days to 38 days with macitentan treatment, supporting the potential of macitentan to improve long-term PAH outcomes.
Control (n = 10), monocrotaline + vehicle (n = 30), macitentan (n = 30).
Iglarz M, et al. J Pharmacol Exp Ther 2008; 327:
Raja SG. Curr Opin Investig Drugs 2010;11:
*macitentan was administered at 30 mg/kg/day
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10. Macitentan added to ambrisentan leads to a further reduction in MAP
Animal model
**p < 0.05 vs. Ambrisentan then vehicle
Ambrisentan
then
vehicle
Macitentan
-50
-40
-30
-20
-10
Delta MAP (mmHg) **
p = ns
In a rat model of systemic hypertension, macitentan was administered on top of the maximally effective dose of ambrisentan to investigate whether macitentan provides a more complete blockade of the ET receptors.
A 30 mg/kg dose of macitentan caused a statistically significant decrease in mean arterial blood pressure (MAP) in animals pre-treated with a maximally effective dose of the selective ETA antagonist ambrisentan.
Dahl-S rats (n = 5), telemetry equipped
Dose: 30 mg/kg (ambrisentan or macitentan) by gavage
Iglarz M, et al. Eur Respir J 2012; 40: Suppl. 56:717s.
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11. Addition of ambrisentan to macitentan did not lead to further reductions in MAP
Animal model
Macitentan
then
vehicle
Ambrisentan
-50
-40
-30
-20
-10
Delta MAP (mmHg)
p = ns
However, in the converse experiment, ambrisentan did not further reduce MAP in rats pre-treated with the maximally effective dose of macitentan.
This implies that macitentan is able to achieve a more complete blockade of ET receptors than ambrisentan and may suggest the potential for improved efficacy.
Dahl-S rats (n = 5), telemetry equipped
Dose: 30 mg/kg (ambrisentan or macitentan) by gavage
Iglarz M, et al. Eur Respir J 2012; 40: Suppl. 56:717s.
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12. Macitentan added to bosentan leads to a further reduction in MAP
Animal model
**p < 0.05 vs. Bosentan then vehicle
Bosentan
then
vehicle
Bosentan
then
bosentan
Bosentan
then
macitentan
-10
-20
Delta MAP (mmHg)
p = ns
-30
In the Dahl-salt rat model of systemic hypertension, macitentan was administered on top of the maximally effective dose of the dual ETA/ETB receptor antagonist bosentan to investigate whether macitentan provides a more complete blockade of the ET receptors due to an additive effect on MAP.
The maximal effective dose of bosentan was determined to be 100 mg/kg. Addition of bosentan 100 mg/kg on top of bosentan 100 mg/kg had no additional effect on MAP (p = not significant), confirming the use of the maximal effective dose of bosentan.
A 30 mg/kg dose of macitentan led to a statistically significant decrease in MAP in animals pre-treated with a maximally effective dose of bosentan.
A further decrease in MAP by approximately 21 mmHg (p < 0.02 vs. vehicle) was seen when macitentan was given on top of bosentan.
-40 **
-50
Dahl-Salt rat model of systemic hypertension (n = 4 to 9)
Measurements in conscious animals (telemetry)
Dose (by gavage): bosentan 100 mg/kg; macitentan 30 mg/kg
Iglarz M, et al. Eur Respir J 2012; 40: Suppl. 56:717s.
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13. Addition of bosentan to macitentan did not lead to further reductions in MAP
Animal model
Macitentan
then
vehicle
Macitentan
then
bosentan
-10
-20
Delta MAP (mmHg)
-30
In contrast, administration of bosentan (100 mg/kg) on top of the maximal effective dose of macitentan failed to elicit any further decrease in MAP.
The add-on effect of macitentan on top of ambrisentan or bosentan confirms that this novel compound is able to achieve a more complete blockade of ET receptors and may suggest the potential for improved efficacy.
-40
p = ns
-50
Dahl-Salt rat model of systemic hypertension (n = 4 to 9)
Measurements in conscious animals (telemetry)
Dose (by gavage): bosentan 100 mg/kg; macitentan 30 mg/kg
Iglarz M, et al. Eur Respir J 2012; 40: Suppl. 56:717s.
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14. Pre-clinical, in vitro data and hepatic safety for macitentan
Inhibition of BSEP, resulting in the disruption of bile salt homeostasis, is a key mechanism that may cause hepatic cholestasis and liver damage1
Macitentan and its active metabolite do not have significant inhibitory effects on bile salt transport2,3
In vitro studies suggest that hepatic disposition of macitentan is mainly driven by passive diffusion rather than OATP-mediated uptake4
Fattinger K, et al. Clin Pharmacol Ther 2001; 69:
2. Raja SG. Curr Opin Investig Drugs 2010; 11: Bolli M, et al. J Med Chem 2012; 55:
4. Bruderer S, et al. AAPS 2012; 14:68-78.
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15. Macitentan – Summary
Macitentan is a newly developed molecule displaying:
Optimised physicochemical properties that facilitate penetration into the tissue
High affinity for the ET receptors and sustained receptor binding resulting in more effective receptor antagonism
Pre-clinical data suggest that macitentan may have the potential for favourable potency and efficacy
In vitro and pre-clinical data suggests that macitentan has the potential for favourable safety and tolerability
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