Discovery and Safety Profiling of a Potent Preclinical Candidate, (4-[4-[[(3 R)-3-(Hydroxycarbamoyl)-8-azaspiro[4.5]decan-3-yl]sulfonyl]phenoxy]- N -methylbenzamide) (CM-352), for the Prevention and Treatment of Hemorrhage

Article abstract of DOI:10.1021/jm501939z

Discovery of potent and safe therapeutics that improve upon currently available antifibrinolytics, e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix metalloproteinases (MMPs) participate in thrombus dissolution. Then we designed a

Full text of DOI:10.1021/jm501939z

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Drug Annotation
Discovery and safety profiling of a potent pre-clinical candidate, (4-[4-
[(3R)-3-(hydroxycarbamoyl)-8-azaspiro[4.5]decan-3-yl]sulfonyl]phenoxy]-N-
[
methyl-benzamide) (CM-352), for the prevention and treatment of hemorrhage.
Josune Orbe, Jose A Rodriguez, Juan A Sanchez-Arias, Agustina Salicio, Miriam Belzunce,
Ana Ugarte, Haisul C. Y. Chang, Obdulia Rabal, Julen Oyarzabal, and Jose A Paramo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501939z • Publication Date (Web): 16 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Discovery and safety profiling of a potent preꢀclinical candidate, (4ꢀ[4ꢀ
[[(3R)ꢀ3ꢀ(hydroxycarbamoyl)ꢀ8ꢀazaspiro[4.5]decanꢀ3ꢀyl]sulfonyl]phenoxy]ꢀ
Nꢀmethylꢀbenzamide) (CMꢀ352), for the prevention and treatment of
hemorrhage
.
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Authors:
1
1
2
1
Josune Orbe, José A. Rodríguez, Juan A. SánchezꢀArias, Agustina Salicio, Miriam
1
2
3
2
2,∗
Belzunce, Ana Ugarte, Haisul C.Y. Chang, Obdulia Rabal, Julen Oyarzabal and José A.
1,4,∗
Páramo,
1
2
Atherosclerosis Research Laboratory, Small Molecule Discovery Platform, Molecular
3
Therapeutics Program; Experimental Hepathology, Center for Applied Medical Research
4
(CIMA); Hematology Service, Clínica Universidad de Navarra. University of Navarra.
Pamplona, Spain.
∗
To whom correspondence should be addressed. For J.O.: phone, +34 948 194700 (ext
044); eꢀmail, julenoyarzabal@unav.es. For J.A.P.: +34 948 194700 (ext. 3015), eꢀmail,
japaramo@unav.es
2
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Abstract
Discovery of potent and safe therapeutics that improve upon currently available
antifibrinolytics; e.g., tranexamic acid (TXA, 1) and aprotinin, has been challenging. Matrix
metalloproteinases (MMP) participate in thrombus dissolution; then, we designed a novel
series of optimized MMP inhibitors that went through phenotypic screening consisting of
thromboelastometry and mouse tailꢀbleeding. Our optimized lead compound, CMꢀ352 (2),
inhibited fibrinolysis in human whole blood functional assays and was more effective than the
current standard of care, 1, in the tailꢀbleeding model using a 30,000 times lower dose.
Moreover, 2 reduced blood loss during liver hepatectomy, while 1 and aprotinin had no effect.
Molecule 2 displayed optimal pharmacokinetic and safety profiles with no evidence of
thrombosis or coagulation impairment. This novel mechanism of action, targeting MMP,
defines a new class of antihemorrhagic agents without interfering with normal hemostatic
function. Furthermore, 2 represents a preꢀclinical candidate for the acute treatment of
bleeding.
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Introduction
Bleeding represents a dangerous complication that can result in the need for blood transfusion
and is associated with substantial morbidity and mortality. Thus, this adverse event has
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significant health and economic repercussions. In trauma patients, severe hemorrhaging is
responsible for 50% of deaths within the first 24 h, with approximately 10% of military and
2
ꢀ5
3–5% of civilian trauma patients requiring massive blood transfusions.
Hyperfibrinolysis constitutes an important contributor to major bleeding events. Therefore,
antifibrinolytic agents have become indispensable in various clinical settings, including
surgery and trauma. In this regard, the synthetic lysine analogues, tranexamic acid (TXA, 1)
and εꢀaminocaproic acid (EACA, 3) (see Chart 1), delay fibrinolysis by competitively
6
reducing the binding of plasminogen to fibrin, thereby enhancing clot stability. Although
aprotinin, which is a bovineꢀderived protease inhibitor (monomeric globular polypeptide
derived from bovine lung tissue), was found to effectively reduce fibrinolysisꢀmediated blood
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loss during cardiac surgery, major safety issues led to its withdrawal from the market;
however, very recently the European Medicines Agency (EMA) has recommended that the
8
suspension be lifted for a restricted range of indications. Nevertheless, molecules 1 and 3 are
safer than aprotinin and appear to be effective for treating inherited and acquired clinical
conditions involving excessive fibrinolysis (e.g., cardiac or liver surgery, trauma, and
9ꢀ15
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postpartum hemorrhage).
Unfortunately, 1 has been associated with adverse side effects,
12,14
thromboembolic events and mortality in trauma and surgical patients;
while 3 is even less
17
effective.
The fibrinolytic and matrix metalloproteinase (MMP) systems cooperate to regulate thrombus
dissolution and extracellular matrix proteolysis. In fact, the plasminogen/plasmin system is
capable of activating specific MMPs (e.g., MMPꢀ1, ꢀ3, and ꢀ9), which in turn participate in
the dissolution of fibrin through direct fibrin/ogen proteolysis or by modifying plasmin
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activity. Indeed, previous studies from our group have revealed a role for MMPꢀ10
stromelysinꢀ2) in fibrinolysis. We have demonstrated that MMPꢀ10 was among the genes
induced by Cꢀreactive protein in endothelium and that thrombin, via proteaseꢀactivated
(
1
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receptorꢀ1, markedly enhanced endothelial MMPꢀ10 expression in vitro and in vivo.
In
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addition, we recently revealed an unexpected role for MMPꢀ10 in promoting the dissolution
of fibrin thrombi by enhancing tissue plasminogen activator (tPA)ꢀinduced fibrinolysis, which
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we observed both in vitro and in vivo using an experimental model of stroke. Importantly,
the role of MMPs in hemorrhaging is supported by the fact that increased levels of certain
MMPs are associated with prolonged bleeding in human and animal studies, whereas genetic
24,25
deletion of MMPs prevented bleeding in experimental models.
In this regard, fibrinolytic
–
/–
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activity and bleeding time were found to be markedly reduced in Mmp10 mice. Also,
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MMPꢀ3, which is a fibrinolytic MMP that is 82% homologous to MMPꢀ10, deserves
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particular attention as it has been consistently associated with intracranial hemorrhage.
This collective body of evidence led us to hypothesize that inhibition of MMPꢀ10 and other
MMPs might constitute a new, effective and safe therapeutic strategy for the prevention of
bleeding. Therefore, we initially analyzed the effect of GM6001 (Ilomastat, 4) (Chart 1), a
2
7
synthetic broadꢀrange MMP inhibitor, on fibrinolysis and bleeding. Then, we designed and
synthesized a novel series of optimized MMP inhibitors. Following initial biochemical
profiling, prioritized molecules underwent a phenotypic screening process consisting of
28
functional thromboelastometry assays in vitro and bleeding tests in vivo. Overall, these
analyses revealed that our optimized lead compound, 2, was significantly more effective than
the current standard of care, the antifrinolytic drug 1. Moreover, this inhibitor 2 exhibited
suitable ADME/toxicity and pharmacokinetic profiles, as well as cardiac safety profiling, as it
did not result in thrombus formation or other unwanted side effects. Here, we provide a
detailed characterization of this preꢀclinical candidate for the acute treatment of bleeding.
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Chart 1. Chemical structure of compounds 1, 3 and 4
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Results
Validation of MMPs as therapeutic targets in hemorrhage
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Due to the fact the MMPꢀ10 plays a role in fibrinolysis, we assessed whether antibodyꢀ
mediated inhibition of MMPꢀ10 would reduce fibrinolysis and prevent bleeding. We first
determined the concentration of antibody required for effective inhibition of MMPꢀ10
activity, IC50 of 2.5 nM; and, as expected, no inhibition of MMPꢀ3 was detected (Fig. 1A).
AntiꢀMMPꢀ10 prolonged clot lysis time (CLT) in the thromboelastometry assay, delaying
fibrinolysis by 50% (EC50) at a concentration of 2 nM, without effecting changes in
coagulation activation and clot firmness (Fig. 1B). In addition, we tested the ability of this
antibody to control tPAꢀinduced bleeding in a mouse tail transection model. We observed
significant reductions in bleeding time (Fig. 1C) and blood loss (Fig. 1D) upon intravenous
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(i.v.) treatment with 500 ng/kg antiꢀMMPꢀ10 as compared to isotype control (IgG2a).
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Administration of a very high dose of compound 1 (300 mg/kg; LD50=1350mg/kg) achieved
a similar effect on tailꢀbleeding time but with no reduction in blood loss (Fig. 1C, D). These
–
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23
results are consistent with reduced bleeding observed in Mmp10 mice and reveal a novel
mechanism for controlling hemorrhaging.
1
8
Since various MMPs within whole blood can enhance fibrin degradation, we investigated
whether inhibition of MMPs might reverse the fibrinolytic activity in whole blood by
performing thromboelastometric assays in the presence of a broadꢀspectrum MMP inhibitor.
Thus, compound 4 was utilized as pharmacological tool to validate, inꢀvitro proofꢀofꢀconcept,
proposed antifibrinolytic strategy. Interestingly, thromboelastometric assay showed that 4
effectively delayed CLT by 50% (EC ) at a concentration of 6.2 nM (Fig. 2A), without
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changes in coagulation activity (Fig. 2B,C). Notably, 4 was more effective at lower
concentrations than 1 (EC50=3 µM; Fig. 2EꢀG). Compound 4 also reduced inꢀvivo tailꢀ
bleeding time when compared to saline control (16.4 ± 3.9 min vs. 29.3 ± 0.4 min, p<0.0001)
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at 1 mg/kg, which is 300 times lower than the effective dose of 1 (Fig. 2D). These results
indicate that MMP inhibition may represent a useful approach for delaying fibrinolysis,
thereby reducing bleeding.
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a
Figure 1. Effect of MMPꢀ10 inhibition on fibrinolysis
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a
(
A) Doseꢀdependent inhibition of MMPꢀ10 using antiꢀMMPꢀ10 showed an IC50 of 2.5nM, whereas MMPꢀ3
activity was unaffected (n=2). (B) Thromboelastometry parameters after addition of antiꢀMMPꢀ10 (2.5 nM). For
activation, the InTEM reagent containing ellagic acid was used according to the manufacturer’s instructions.
Clotting time (CT), maximum clot firmness (MCF), and lysis time (LT) are shown as mean ± SEM (n=3
independent experiments, *p<0.05 vs. control). (C) Bleeding time and (D) blood loss following infusion of 1,
MMPꢀ10 antibody (MAb), or isotype control in the hyperfibrinolytic tailꢀbleeding model. Data are shown as
mean ± SEM (n=10 per group; ** p< 0.01 vs. saline; † p<0.05 vs. isotype control).
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a
Figure 2. Impact of known panꢀMMP inhibitor, 4, on fibrinolysis
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4, a broadꢀrange MMP inhibitor, reduces fibrinolysis in vitro and in vivo. Thromboelastometry parameters after
addition of 4: (A) lysis time (LT), (B) maximum clot firmness (MCF) and (C) clotting time (CT) are shown as
mean ± SEM (n=3 independent experiments; *p<0.05 vs. control). (D) Bleeding time after infusion of 1, 4, or
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vehicle (saline) in the inꢀvivo hyperfibrinolytic tailꢀbleeding model. Data are shown as mean ± SEM (n=10 per
group; *** p< 0.001; ** p< 0.01 and * p<0.05 vs. saline). Inhibition of fibrinolysis by 1 in thromboelastometric
assay: clotting time (E), maximum clot firmness (F), and lysis time (G). Data are shown as mean ± SEM (n=5
independent experiments; * p<0.05 vs. control).
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Discovery of an effective and safe antihemorrhagic agent: (4ꢀ[4ꢀ[[(3R)ꢀ3ꢀ
(hydroxycarbamoyl)ꢀ8ꢀazaspiro[4.5]decanꢀ3ꢀyl]sulfonyl]phenoxy]ꢀNꢀmethylꢀbenzamide),
2
3
0
(CMꢀ352)
Although 4 showed antihemorrhagic activity, it is known to display poor solubility and
31
bioavailability. Therefore, we set out to design a new series of small molecules with
improved pharmacokinetic properties that would be appropriate to achieve our goal.
In this regard, structureꢀ and knowledgeꢀbased approaches were employed to design and
synthesize a series of novel compounds, which proved to be active and soluble. We selected
compounds that were capable of inhibiting MMPꢀ10 and MMPꢀ3 activity in the nM range
(IC50<100 nM) and evaluated their functional response as antifibrinolytic agents using
28,30
thromboelastometry of human whole blood in the presence of tPA.
CLT delays above
50% were considered to represent antifibrinolytic activity; however, considering the current
standard of care (1 [EC50=3 µM]), only those molecules displaying an EC50 lower than 1 µM
were further tested in the inꢀvivo hyperfibrinolytic tailꢀbleeding model. We were able to
synthesize several small molecules that acted as antifibrinolytic agents, ultimately delivering
an optimized lead compound, 2 (4ꢀ[4ꢀ[[(3R)ꢀ3ꢀ(hydroxycarbamoyl)ꢀ8ꢀazaspiro[4.5]decanꢀ3ꢀ
2
8,30
yl]sulfonyl]phenoxy]ꢀNꢀmethylꢀbenzamide).
Scheme 1. The last step involved a chiral separation by supercritical fluid chromatography
SFC) to obtain an enantiomeric excess (ee) of 100% and +16.7 for the specific optical
rotation.
Molecule 2 was prepared as shown in
(
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To verify the structure of the optimized lead compound 2, pure enantiomer, a crystallographic
approach was adopted; however, it did not crystallize after several attempts (as free base or
HCl salt, using different solvent systems and temperature gradients). As alternative,
enantiomerically pure derivative 14 (Scheme 2) was crystallized and then analyzed by Xꢀray
single crystal diffraction (Figure 3) to confirm its structure and stereochemistry. Compound 2
was also achieved in 5 synthetic steps, from 8a without any need for chiral separation
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(Scheme 2). Molecule 2 was confirmed by its retention time (Rt), according to SFC
purification method, and optical rotation. On the other hand, the corresponding Sꢀenantiomer,
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from 8b, was also confirmed by crystallography.
a
Scheme 1. Synthesis of compound 2
c)
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(R)
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Conditions: (a) K
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3 3
CO (2 mol equiv), CH I (1.5 mol equiv) in DMF, RT, 5h; (b) LDA (1.25 M) in THF, 1 h at
ꢀ78 ºC; then, (4ꢀfluorobenzenesulfonyl fluoride, (15) (1.4 mol equiv), 1 h at ꢀ78 ºC, then RT overnight; (c)
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LiOH·H
equiv), THPONH
mol equiv), Cs CO
h; (g) EDC·HCl (2 mol equiv), HOBt (2 mol equiv), CH
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O (10 mol equiv) in THF/MeOH/H
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O (3:3:2), RT, 6h; (d) EDC·HCl (2 mol equiv), HOBt (2 mol
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(2 mol equiv), NMM (3 mol equiv) in DMF, RT, overnight; (e) methyl 4ꢀhydroxybenzoate (2
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(2 mol equiv), DMF, 12 h at 90 ºC; (f) LiOH·H
2 2
O (10 mol equiv) in THF/H O (3:2), RT,
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NH ·HCl (2 mol equiv), NMM (3 mol equiv) in DMF,
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RT, overnight; (h) HCl/Dioxane (2 N), RT for 1 h; (i) chiral separation (SFC).
a
Scheme 2. Structural characterization of compound 2
H O
H O
HO
HO
O
O
O
O
S
F
(
S)
S
F
(R)
S
F
(R)
S
F
a)
O
O
a)
b)
O
O
O
O
O
O
N
N
N
N
H
14
8b
O
8
8a
O
O
O
O
O
c)
H
N
O
H
N
O
THP
THP
O
O
(
R)
S
O
d)
(
R)
S
F
O
O
O O
N
N
O
O
10a
O
9a
O
O
O
e)
H
N
H
N
H
H O
N
O
O
THP
THP
O
O
O
(
R)
S
O
f)
(R)
S
O
g)
(R)
S
O
O
O
O
O
O
O
N
N
N
H
O H
N
N
O
O
O
O
2
O
O
11a
O
12a
a
Conditions: (a) chiral separation (SFC); (b) HCl/EtOAc (2 N), RT for 1 h; (c) EDC·HCl (2 mol equiv), HOBt (2
mol equiv), THPONH (2 mol equiv), NMM (3 mol equiv) in DMF, RT, overnight; (d) methyl 4ꢀ
2
hydroxybenzoate (2 mol equiv), Cs CO (2 mol equiv), DMF, 12 h at 90 ºC; (e) LiOH·H O (10 mol equiv) in
2
3
2
THF/H
2
O (3:2), RT, 4h; (f) EDC·HCl (2 mol equiv), HOBt (2 mol equiv), CH
3
NH
2
·HCl (2 mol equiv), NMM (3
mol equiv) in DMF, RT, overnight; (g) HCl/Dioxane (2 N), RT for 1 h.
a
Figure 3. Xꢀray structure of compound 14
A)
B)
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4
14
a
Deconvolution of the diffraction pattern gives an electron density map, from which molecular structure can be
determined. A) Here the structure is rendered as an ORTEP drawing (including its counterion, chlorine atom)
and B) as its corresponding colorꢀcoded stick style drawing (without couterion). Details about Xꢀray
crystallographic data of 14 and its corresponding coordinates are reported in the Supplementary Information.
Compound 2 was found to potently inhibit MMPꢀ10 and MMPꢀ3 (IC50: 12 nM and 15 nM,
respectively) (Fig. 4A, B). Moreover, at 10 ꢀM, 2 behaved as a panꢀMMP inhibitor,
displaying greater than 93% inhibition for eight different MMPs (MMPꢀ1, ꢀ2, ꢀ8, ꢀ9, ꢀ12, ꢀ13,
ꢀ
14, and ꢀ20) and greater than 73% for MMPꢀ7 (Table 1). Molecule 2 bears a hydroxamic acid
moiety, which has been described as an effective metalꢀbinding group for a variety of
2
+
metalloenzymes. For this reason, it is promiscuously effective against all of the Zn ionꢀ
containing MMPs. Therefore, in order to perform offꢀtarget selectivity profiling, 2 (10 µM)
was assayed against a panel of 84 different protein targets bearing metalꢀbinding sites (e.g.,
cathepsins, caspases, HDACs, etc.). Notably, ≤50% inhibition was observed for all of these
(
Table 1).
Antifibrinolytic properties of 2 were evaluated in vitro using confocal microscopy and
thromboelastometry. Molecule 2 (200 nM) delayed fibrinolysis front velocity induced by tPA
(
76% reduction, Fig. 4C) and exhibited higher antifibrinolytic activity than 1 in whole blood
(Fig. 4D). When compared to control, EC50 for 2 was 0.7 nM in human blood, without
changes in coagulation and clot firmness. Notably, this dose was 4,000 times lower than that
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required for 1 (EC50=3 µM; Fig. 4D). In contrast, the EC50 of 2 for mouse blood was 200 nM,
which was considered when planning our in vivo assays (Table 2).
0
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a
Figure 4. Chemical and functional characterization of 2 in vitro
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a
A) Structure of molecule 2. B) Inhibition of MMPꢀ10 and MMPꢀ3 by 2 (IC50: 12 nM and 15 nM, respectively).
C) Gold nanoparticleꢀlabeled fibrin plate showing a significant reduction in fibrinolytic activity after addition of
(left side). A series of micrographs showing dynamic fibrin lysis with tPA (2 U/ml, upper panels) or tPA (2
2
U/ml) plus 2 (200 nM, lower panels). The lysis front was visualized as a straight, sharp line in the clot, and was
scanned at different times. A lower velocity was observed for tPA plus compound 2 as compared with tPA alone.
D) Thromboelastometry parameters after addition of compound 1 (3 µM) or molecule 2 (0.7 nM). Clotting time
(CT), maximum clot firmness (MCF), and lysis time (LT) are presented. Data are shown as mean ± SEM from
five independent experiments (* p<0.05 vs. control).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Selectivity profile of compound 2 at 10 ꢀM against different protein targets. Heatꢀ
a
map colorꢀcoded according to corresponding percentages (%) of inhibition
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a
All assays were performed in duplicate at GenScript, except for cathepsin F, HDACs, DUBs and HDMs, that
were screened at BPS Bioscience (see Materials and Methods). Percentages of inhibition as the mean of two
independent experiments. (see Supplementary Information for additional details)
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
Table 2. Effective concentration to delay lysis time by 50% (EC50) in thromboelastometry
a
using human and mice blood samples
Compound
Human blood (µM)
Mice blood (µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
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8
9
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8
9
0
1
2
3
4
5
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8
9
0
1
Aprotinin
2
3
0.012
b
0.4
N.T.
0.0007
0.2
4
0.0062
0.0062
a
b
Experiments performed in triplicate. N.T. means Not Tested.
In order to achieve optimal drugꢀlike properties for MMP inhibitors, several limitations must
be overcome, including solubility, bioavailability and free drug concentration at target tissue.
32
For this reason, based on structural information [www.pdb.org; e.g., entry 1D8F], our
2
8
chemical series were designed to bear a basic nitrogen to improve solubility. Indeed,
compound 2 displays a solubility greater than 100 µg/ml. However, while this secondary
aliphatic amine makes 2 titratable at physiological pH, it may also increase the compound’s
+
33
affinity for the human etherꢀaꢀgoꢀgoꢀrelated gene (hERG) potassium (K ) channel. Since
use of the antifibrinolytic agent, aprotinin, was associated with a 55% increase in
3
4
+
cardiovascular risk, we assessed drug safety through analysis of hERG K channel binding
and electrophysiological function using manual patch clamp. In this regard, our preꢀclinical
candidate 2 displayed an optimal response (IC50: >100 µM and >30 µM, respectively).
For compound 2 to be efficacious, it must remain in the bloodstream. Therefore, to minimize
tissue distribution and avoid undesirable side effects, low permeability of the compound is
desired. Therefore, a Cacoꢀ2 assay was performed to assess diffusion of 2 in vitro. We found
ꢀ6
that the inhibitor showed very low permeability (0.16 x 10 cm/s) and an efflux rate >2.5,
suggesting that it likely represents an efflux pump substrate. Moreover, its remarkably low
plasma protein binding capacity (29.1% and 55.0% unbound fraction in mouse and human,
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respectively) could also contribute to the remarkable efficacy in vivo; higher free drug
concentration at bloodstream than usual. Thus, considering these findings, 2 should display
low volume of distribution.
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To explore drug–drug interactions, compound 2 was tested against major cytochrome P450
isoforms (1A2, 2D6, 3A4, 2C9, and 2C19), and inhibition at a single concentration (10 ꢀM)
was found to be < 50% in all cases (see Supplementary Information for details). In addition,
we tested its ability to activate human pregnane X receptor (hPXR), a key regulator of
CYP3A4 expression, observing no significant impact (EC50>100 µM).
For safety purposes, we performed an additional set of in vitro assays to assess the therapeutic
window (i.e., efficacy vs. safety) associated with 2 as an antihemorrhagic agent before
progressing to the in vivo models. An analysis of cytotoxicity against transformed human liver
35
epithelial cells (THLEꢀ2) and peripheral blood mononucleated cells (PBMC) revealed that
the lethal concentration (LC50) was higher than 100 µM. Also, the Ames test was employed to
confirm the safety of our preꢀclinical candidate 2, demonstrating that both directly and
indirectly (using the S9 fraction) the compound yielded no mutagenic response in two
different strains of Salmonella typhimurium.
After achieving satisfactory in vitro ADME/Tox profiling results, a detailed in vivo
pharmacokinetic study was performed following i.v. administration of 2 (1 mg/kg, details in
Table 3). This analysis revealed three key features of 2: short halfꢀlife (1.4 h), low volume of
distribution (0.94 L/kg), and maximal concentration (Cmax=3.3 ꢀM). Notably, this profile
was favorable for the intended use, as short halfꢀlife is desirable in the acute treatment of
bleeding.
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2
2
2
2
2
2
2
2
2
3
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4
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5
5
5
5
5
5
5
5
5
6
Table 3. Pharmacokinetic study for molecule 2
a
b
Compound 2 (nM)
Pharmacokinetic parameters
Time (h) Mean
SD
444
99
Mean value
1786
0
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0.0833
3035
1646
1176
438
AUC0ꢀinf (h*nM)
b
0
0
1
2
4
8
2
.25
t
½
(h)
1.40
.5
125
29
C
max (nM)
3241
Cl (L/h/Kg)
1.2
128
17
Norm. Vss (L/Kg)
0.94
25
4
c
c
N.D.
N.D.
N.D.
N.D.
c
c
4
a
Plasmatic concentrations after intravenous administration of molecule 2 (1 mg/kg; n=3 independent
b
experiments, measurements performed in duplicate). Compound 2 pharmacokinetic parameters estimated by
fitting the experimental data to a bicompartmental model using Winnonlin software for pharmacokinetic
analysis. The parameters are: Area Under the Curve (AUC); half life of the product (t
½
); maximum concentration
c
d
(
Cmax); clearance (Cl); volume of distribution (Vss). N.D. means Not Detected; Half life is BetaꢀHL
Preꢀclinical candidate 2 does not alter hemostasis
In order to assess whether 2 could affect hemostatic balance, we performed an exhaustive
screening using standard laboratory tests. Compound 2 (10 µM) did not modify kallikreins,
coagulation factors (fibrinogen, FVII, FVIII, FX, FXII, and FXIII), protein C, thrombin
generation, and Dꢀdimer. Moreover, it did not alter the activities of various fibrinolytic
proteins, including plasminogen activators (tPA, uPA), plasminogen activator inhibitorꢀ1, and
thrombin activatable fibrinolytic inhibitor (TAFI). Taken together, these findings suggested
that 2 did not impact global hemostasis, having no effect on coagulation pathways (extrinsic,
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intrinsic, or common) or fibrinolytic proteins (Fig. 5 and Table 4). Moreover, platelet
aggregation analysis confirmed that 2 did not affect hemostatic plug formation (Fig. 5B).
a
Table 4. Hemostatic parameters of compound 2 at 10 µM
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a
KLK= Kallikreins; PC= protein C; tPA= tissue plasminogen activator; uPA= urokinase; PAIꢀ1= plasminogen
activator inhibitorꢀ1; TAFI= thrombin activatable fibrinolytic inhibitor. Data are presented as percentage of
inhibition in different systems and pathways as the mean of three independent experiments (see Supplementary
Information for additional details)
Figure 5. Impact of preꢀclinical candidate, 2, on thrombin formation and platelet aggregation.
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Molecule 2 does not affect thrombin formation and platelet aggregation. A) Endogenous thrombin potential
(ETP) curves for control and 2 (10 µM). Comparison of thrombinꢀgeneration area under the curve (AUC) in
control and 2–treated samples (n=3 independent experiments). B) Platelet aggregation curves for 2 or control
showing parallel measurements after activation with thrombin receptorꢀactivating peptide 6 (TRAPꢀ6), collagen
(COL), adenosine diphoshate (ADP), and arachidonic acid (AA) as platelet agonists (data from a single
experiment, representative of three independent experiments).
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In vivo efficacy and safety
The antihemorrhagic effect of 2 was tested in vivo using a mouse tailꢀbleeding model (Fig.
A). Bleeding time, defined as the interval between the initial transection and the visual
6
cessation of hemorrhaging, was significantly prolonged (>30 min) after administration of 0.5
mg/kg tPA alone, with 189±26 µl of blood loss observed (i.e., 10% of the total volume).
However, while 1 (300 mg/kg) reduced bleeding time relative to control (14.2±3.1 vs.
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9.3±0.4 min, p<0.0001), it did not decrease blood loss (196±47 µl). Notably, 2 (1 mg/kg)
resulted in significant decreases in bleeding time (1.82±0.42 min; p<0.0001) and blood loss
63±25 µl; p<0.01) compared to vehicleꢀtreated controls, 1 (p<0.001 and p<0.05), and
(
molecule 4 (p<0.01 and p<0.05; Fig. 6B,C). In addition, 2 (1 mg/kg) not only led to significant
reduction in time bleeding in comparison to saline, 1 and 4, but also tends to a significant
2
8
reduction versus aprotinin (10 mg/kg, p=0.079) in the bleeding time test.
We found that the minimum effective dose was 10 µg/kg, which reduced bleeding time by
89% and blood loss by 61% (p<0.0001 and p<0.05). Therefore, 2 was highly efficient for
reducing bleeding in vivo in this mild model of hemorrhage.
We next tested the efficacy of 2 during murine hepatectomy, which represents a more severe
model of bleeding where approximately 28% of the total blood volume is lost (Fig. 6D).
Administration of 2 (1 mg/kg) reduced blood loss after liver lobes resection by 25%, while 1
or even aprotinin, which has higher antifibrinolytic efficiency, had no effect (Fig. 6E).
Notably, anatomopathological analysis in mice following injection of 2 with a 10ꢀtimes
higher dose (10 mg/kg) revealed no signs of toxicity or thrombosis in brain, liver, lung, and
kidneys (Fig. 6F). Similar assay was also performed with aprotinin and 1; and, no signs of
28
toxicity or thrombosis were identified.
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Finally, in order to assess toxicity of the compound and estimate its lethal dose, mice were
injected with increasing concentrations of 2. At a dose of 100 mg/kg, which is 10,000 times
higher than the minimum effective dose, four out of five mice (80%) died within 24 h of
injection. Notably, thrombosis was not observed in any of these animals.
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a
Figure 6. Antihemorrhagic effect of 2 in murine bleeding models
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a
A) A schematic representation of the tPAꢀinduced hyperfibrinolytic tailꢀbleeding model and infusion regime. B)
Bleeding time in the presence of 1 (300 mg/kg; n=10), 4 (1 mg/kg; n=10), and various doses of compound 2
0.01–1 mg/kg; n=9), compared with saline (n=10). C) Molecule 2 efficiently reduced blood loss at the three
(
concentrations tested (box plot shows median and interquartilic range). D) Schematic representation of the
murine hepatectomy model and infusion regime. E) Reduced blood loss was observed with 2 treatment (1
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mg/kg; n=10), but not with 1 (300 mg/kg; n=11) or aprotinin (10 mg/kg; n=9), when comparing with saline
(n=11). F) Anatomopathological images of mice treated with 2 (10 mg/kg). Hematoxylin and eosin staining of
lung tissue vessels (magnification x200), and kidney glomeruli and tubules (magnification x400) showing no
thrombosis after treatment. Data are shown as mean ± SEM (* p< 0.05, ** p<0.01 and *** p<0.001 vs. saline; †
p<0.05 and ††† p<0.001 vs. 1; $ p<0.05 and $$ p<0.01 vs. 4).
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Discussion
Bleeding represents a dangerous complication of surgery and trauma that can result in the
need for blood transfusion. It is associated with substantial morbidity and mortality, thereby
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producing significant health and economic ramifications.
The lethality of
hyperfibrinolysis emphasizes the importance of early administration of antifibrinolytic
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drugs.
Here, we have reported our discovery that fibrinolysis can be modulated by MMP
inhibitors. Moreover, we have synthesized the novel compound, 2, which is a safe and potent
antihemorrhagic agent that presents a novel mechanism of action and exhibits marked
therapeutic superiority when compared to current clinical antifibrinolytic agents.
Inactivation of MMPꢀ10 was shown to inhibit in vitro fibrinolysis and in vivo bleeding,
indicating that MMP inhibition represents an unexplored mechanism for fineꢀtuning the
18,23
fibrinolytic system.
blood (e.g., MMPꢀ3) suggests that these observed effects could be enhanced.
ꢀmediated effects on fibrinolysis and bleeding have provided further experimental evidence
However, the existence of other MMPs with fibrinolytic activity in
39,40
In addition,
4
that broadꢀrange MMP inhibition may constitute a useful therapeutic strategy for tight control
of fibrinolysis and hemorrhaging.
In addition to the lack of efficacy of hydroxamateꢀbased MMP inhibitors in human cancer
trials, their limited clinical success was also due to reports of side effects (i.e.,
4
1,42
musculoskeletal pain and inflammation), which occurred after 2ꢀ3 months of treatment
4
2
and subsided within 1ꢀ3 weeks of discontinuation. Notably, the doses of MMP inhibitors
required for reducing metastasis were much higher than those that we describe here for acute
43
use in antihemorrhagic treatment. Therefore, our proposed therapeutic strategy involving
shortꢀterm MMP inhibition for antifibrinolytic treatment should minimize the risk of possible
side effects.
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We produced novel spirocyclic molecules for inhibiting MMPꢀ10 and MMPꢀ3 activity and
phenotypically screened their antiꢀfibrinolytic activity in a functional assay using human
blood. Our optimized lead compound (2) showed higher in vitro antifibrinolytic activity than
current reference antifibrinolytic agent (1) at significantly lower concentrations. Furthermore,
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displayed optimal characteristics in terms of ADME/Tox profiling and a low risk for
+
cardiotoxicity, as determined through hERG K channel binding and an electrophysiological
assay. In addition, pharmacokinetic profiling revealed various functionally relevant
characteristics of 2, including a short halfꢀlife (allowing fast removal of the inhibitor
following therapy), low volume of distribution (restricting the compound to the bloodstream),
and Cmax of 3.3 ꢀM. Considering that Cmax corresponds to the optimal dose for treating
severe bleeding (1 mg/kg), we conclude that 2 displays an optimal therapeutic window.
Indeed, much higher concentrations of 2 were analyzed for potential offꢀtarget effects: 10 ꢀM
(selectivity profiling and P450 screening), 30 ꢀM (patch clamp), and 100 ꢀM (hERG binding,
cytotoxicity, and hPXR activation). Moreover, two important ADME parameters, optimal
solubility and large free drug concentration at bloodstream, may also contribute to the in vivo
efficacy of 2.
Notably, we demonstrated that 2 effectively inhibited fibrinolysis without interfering with
normal hemostatic function in traditional clotting and aggregometer assays.
Pathophysiological situations such as major bleeding or surgery, require efficient clot
formation as well as operative intra and extravascular fibrinolysis as part of the global process
44
of wound healing. Our observations support a role for MMPs as a new target beyond the
plasminogen/plasmin system to stop bleeding in a rapid an effective manner.
Given its desirable pharmacokinetic and safety profile, we assessed compound 2 for in vivo
efficacy using mouse models of mild and severe bleeding. We found that 2 was much more
1
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potent than 1 , reducing hemorrhaging in both models and resulting in significantly
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decreased blood loss under conditions where current standard of care, 1, and aprotinin had no
effect. In addition, anatomopathological analyses confirmed the safety profile of 2, as no signs
of toxicity or thrombosis were observed.
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Hyperfibrinolysis is an important contributor to increased bleeding in various clinical settings
4
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(
e.g., cardiac surgery, trauma and stroke after tPA treatment). However, since restricted
range of indications covered by aprotinin, the choice of antifibrinolytic agents has been
1
1
limited to 1 and 3. Recent trials involving large numbers of trauma and surgical patients
have demonstrated the efficacy of 1; nevertheless, its effect on mortality in such patients
12,14
remains uncertain.
To our knowledge, no clinical studies have reported the use of MMP
inhibitors to treat bleeding. In this regard, 2 represents a valid clinical candidate, as it was
more effective in human blood than murine blood.
Taken together, our findings suggest that MMP inhibition represents a new therapeutic
strategy for preventing hyperfibrinolysis. In fact, this novel mechanism of action defines a
new class of antihemorrhagic agents that might also lead to drug repositioning: known MMP
inhibitors that reached clinical phases for oncological indications. In this case, the required
acute treatment avoids any reversible musculoskeletal toxicity identified during the chronic
dosing regime. The corresponding drug repositioning study has been performed in our
laboratory, and results will be published shortly.
Notably, we have established the efficacy of 2 for reducing in vitro fibrinolysis and in vivo
bleeding, more effective than the current standard of care 1 and aprotinin, with no evidence of
thrombosis or coagulation impairment. In addition, this novel mechanism of action leads to a
new class of antihemorrhagic agents without interfering with normal hemostatic function. The
potency of 2 at low doses and the absence of side effects underscore its therapeutic potential
for use in surgery, trauma or intracerebral hemorrhage patients. In fact, compound 2 is preꢀ
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