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Platelet Aggregation Inhibiting and Anticoagulant Effects of Oligoamines XXVIAntiplatelet and Antithrombotic Effects of the Oligoamine RE 1492 in Combination with Standard and Future Antithrombotic Drugs.

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333
Antithrombotic Effects of RE 1492
Platelet Aggregation Inhibiting and Anticoagulant Effects of Oligoamines, XXVI’):
Antiplatelet and Antithrombotic Effects of the Oligoamine RE 1492 in
Combination with Standard and Future Antithrombotic Drugs
Klaus Rehse* and Thomas Ciborski+)
lnstitut fur Pharmazie der Freien Universitiit Berlin, Konigin-Luise-Str. 2+4. 14195 Berlin
Received May 4, 1994
The combined effects of the oligoamine RE 1492 with the NO-donor RE
2047 or ASA, ticlopidine, pentoxifylline and BM 14515 were determined
iri virm (Born-test) and iri vivo (rat thrombosis model). The effects in v i m
were supra additive but over independent. / r I vivo all combinations showed
over additive and over independent inhibition of thrombus formation. The
best results were obtained with a combination of 10 mg/kg RE 1492C and
10 nig/k,o RE 2047. It inhibited thrombus formation in arterioles (A) by
79 8 and 36 Q in venoles (V). The combined effect of RE 1492C and
pentoxifylline 10 mg/kg each was 67-56 (A) or 32% (V), respectively.
The prevention of thrombosis is still unsatisfactorily
achieved by drugs. One reason is that thrombus formation
is a multifactorial event involving several pathways of
fibrin formation and platelet aggregation. It has become
clear in the past decades that influencing only one of them
is unsuitable to improve sufficiently the clinical situation.
One rationale, therefore, has been to use a combination of
antithrombotic drugs with different mechanisms of actions.
As early as in 1977 Asasantin3, a mixture of acetylsalicylic
acid (ASA) and dipyridaniole became commercially available. Since then a rising number of papers reflect increasing
efforts to establish useful combinations of antithrombotic
drugs most of them involving ASA, pentoxifylline, TxA2
antagonists, ticlopidine, prostacyclin derivatives, and NOdonors’-’). This prompted us to evaluate the potential of R E
1492C (N,N’,N”-4-phenylbutyl- 1,3,5-trimethanamineN,N’,N”-ethyltricarbamate)
with respect to its combination
with commercially available drugs as well as with antithrombotic compounds which are still in development.
RE 1492C was chosen because of its unique mechanism
and its fit with many criteria which are regarded as essential
for an ideal antithrombotic drug6.’), inter alia interference
with all pathways of aggregation, slight interference with
fibrin formation, no bleeding, no interference with fibrinolytic activity, no acute toxicity, oral route of administration,
long half-live and rather easy synthesis8-”). For in vitro
experiments is used RE 1492 which is the active metabolite
of RE 1492C’O).
+I
Pan of the PhD thesis T. Ciborski. FU Berlin 1991
Arch. Pharm. (Weinheim)328, 333-337 (1995)
Antiaggregatorische und anticoagulante Eigenschaften von Oligoaminen?
Antiaggregatorische und antithrombotische Wirkungen des Oligramins RE 1492 in Kombination mit bewahrten Antithrombotika und
Entwicklungssubstanzen
Die kombinierten Wirkungen des Oligoamins RE 1492 mit dem NODonor RE 2047 bzw. ASA, Ticlopidin. Pentoxifyllin oder BM 14515 wurden it1 virro (Born-Test) und it1 vivo (Thrombose Modell. Ratte) bestimnit.
Die in virro-Effekte waren supraadditiv, jedoch iiberunabhhgig. hi i i v o
zeigten alle Kombinationen iiberadditive und uberunabhiingige Effekte.
Die besten Ergebnisse wurden mit der Kombination von je 10 mg/kg RE
1497-(3 und RE 2047 erzielt. Die Thrombusbildung i n Arteriolen ( A )
wurde hiermit zu 79%. in Venolen (V) zu 36% gehemmt. Die Kombination von je 10 mgkg RE 1492C und Pentoxifyllin zeigte eine 62proz. (A)
bzw. 32prOZ. (V) Hemmung.
RE 1492: R’ = H
RE 1492 C: R’ = COOCzH,
BM 14515
Scheme 1: Chemical structures of antithrombotic compounds.
I n vitro testing of anriplatelet drugs
The structures of the compounds comprised in this study
are given in Scheme 1 . It1 vitro only antiplatelet (aggregation) or anticoagulant (fibrin formation) effects can be measured. The terminus an tithrombotic, however, can be derived only from iw vivo experiments because the flow con-
0VCH Verlagsgesellschaft mbH, D-6945 1 Weinheim, 1995
0365-6233/95/0404-0333 $5.00
+ .25/0
334
Rehse and Ciborski
ditions in the blood vessels and the cooperative effects of
their endothelial cells strongly influence thrombus formation. The results obtained in the Born-test which is suitable
to demonstrate direct antiplatelet actions are summarized in
Tab. 1.
In exp. 1-5 the effects of the single compounds on the
platelet aggregation induced by collagen in platelet rich
plasma (PRP) are stated. Experiments 1-3 show that the oligoamine RE 1492 and the NO-donor RE 2047 are potent
inhibitors of the platelet aggregation. This effect is a clear
function of their concentration in PRP. The most throughly
investigated drug ASA was chosen for comparison. Its
effect in the Born-test is smaller by more than one order of
magnitude. Pentoxifylline and ticlopidine are weak anti% inhibition of platelet aggregation
J
a0
-
60
-
40
-
21
I
0
3,s
5.85
6.03
7,81
IE 1492 kg c [m/L]
Fig. 1: Classification of the type of synergism between a bioequivalent
concentration (2.92pmoliL) of RE 2047 (18% inhibition, see dotted line)
and increasing concentrations of RE 1492.
No. exp
1
Compound
RE 1492
RE 2047
ASA
Pentoxyphylliie (P)
Ticlopidine0
8
9
10
RE 1 4 9 m 2047
[2.92pmol/L]
RE 1492P
P [625pmoUl.1
RE 1492JASA
[125pmOl/L]
RE 1492
T [468p o V L ]
RE 149yRE2047
12.92CIIlloVLl
P [625pmol/L]
platelet drugs in v i m but have been included for well documented antithrombotic effects in ~ i v o ' . ' ~For
) . the combination experiments with RE 1492 threshold concentrations
(- IC20) of RE 2047, ASA, P or T were chosen.
For classifying the type of synergism observed in these
experiments, the data were processed as examplified in Fig.
1 for the combination of RE 2047 with RE 1492. Trace a
represents the data of exp. 1. The data for the theoretical
independent behaviour (different mechanism!) of the two
drugs were calculated using an equation suggested by
Ariend3) (trace b). The theoretical additive effect (trace d)
was calculated by the method of Piich and H o l ~ m a n n ' ~ ) .
The concentration of 2.92 pmol/l RE 2047 is equieffective
to 6 pmol/L RE 1492. The expected additive effect even
at the lowest concentration of RE 1492 (Zc = 3.9 + 6 = 9.9
pmolk) would be a 100% inhibition of platelet aggregation. Trace c represents the data of the combination exp. 6.
These effects, therefore, are supraadditive but over independent. This suggests a complex mechanism of interaction.
The same is true for the combination with P (625 pmoVL,
exp. 7) and ASA (125 pmoVL, exp. 8) at least with medium
concentrations of RE 1492.
The type of interaction with T (468 pmol/L, exp. 9) varies
with the concentration of RE 1492 and approaches an independent and additive mechanism at 6.83 pmoVL of the oligoamine. Exp. 10 was undertaken to evaluate the combination of three drugs including pentoxifylline. Even at the
lowest concentration of RE 1492 a 100% inhibition of the
platelet aggregation was observed. This indicates a remarkable improvement representing now an additive type of
effect.
In summary the results of Tab. 1 show, that the combination of small concentrations of antithrombotic drugs with
-
c[pmol/L] (% inhibition)
3.9 (0) 5.85 (6) 6.83 (91) 7.81 (100)
0.976 (0) 1.46 (5) 2.92 (18) 3.9 (32) 5.85 (50)
7.81 (82) 11.7 (100)
62.5 (0) 93.7 (6) 125 (18) 187 (40) 250 (100)
234 (7) 625 (13) 1250 (44) 1870 (88) 2500 (100)
312 (0) 468 (13) 625 (20) 937 (30)
1250(51) 1870 (83) 2500 (100)
3.9 (33) 5.85 (94) 6.83 (100) 7.81 (100)
++**
++.
++
n.m
3.9 (18) 5.85 (91) 6.83 (100) 7.81 (100)
++.
"
n.m.
3.9 (21) 5.85 (71) 6.83 (93) 7.81 (100)
+
++.
n.m.
n.m.
3.9 (54) 5.85 (100)6.83 (100) 7.81 (100)
..
++-
++*
+.
n.m.
3.9 (100) 5.85 (100) 6.83 (100) 7.81 (100)
n.m.
n.m.
n.m.
..
Arch. Pharm. (Weinheim) 328, 333-337 (1995)
335
Antithrombotic Effects of RE 1492
Tab. 2: In vivo inhibition of thrombus formation in rats after oral administration of the drugs stated
(m-THFT = medium thrombus formation index); control m-TFI = 1.28 (V); 1.96 (A); n = 5; p.0.;
U-test a*= 0.1-0.5;a**= 0.01-0.05; a***
= 0.001-0.005
venoles
arterioles
mglkg
ASA(1 h)m-TFI
% inhibition
a
RE 1492<3(4h)m-TFI
96 inhibition
a
RE2047 (2 h) m-TFI
% inhibition
0.5 2
5 10 3 0
1.96 2.13 2.37 2.7 3.5
0
4
10 18 38
n.s. n.s. * ** ***
-
a
Pentoxiph. (1h)m-TFI
% inhibition
a
BM 14515(1h)m-TFI
% inhibition
a
Ticlopidine(3h)m-TFI
% inhibition
a
1.97 2.1
0
3
n.s. n.s.
1.97 2.57
0
15
n*s* **
-
-
-
-
-
-
-
-
-
2.43 3.1 4.13
12 28 54
** ** ***
3.13 4.17 4.73
29 55 69
** *** ***
2 2.3 2.7 3.07
1
8 18 27
n.s. n.s. *
**
2.5 2.93 3.37
13 24 35
-
*
-
-
-
-
** ***
- 2.33
-
-
3.4
- 9 3 . 6
- n.s. ***
different mechanisms of action leads to a complete inhibition of platelet aggregation. As the side effects of these
compounds differ, we were encouraged to test these combinations in vivo.
In vivo inhibition of thrombusf o m t i o n in rats by RE
1492C and combinations with several other antithrombotic
drugs after oral administration
The oligoamine RE 1492 itself is not suitable for the oral
route of drug application because it is only poorly absorbed
from the gastrointestinal tract15).Therefore, its tricarbamate
derivative was designed as a prodrug (RE 1492C). Preliminary investigations had revealed its suitability for oral
administrations’0).The drugs were assayed as usual in an in
vivo thrombosis modello).
Briefly thrombi were induced in the mesenteric vessels of rats by means
of a laser beam. The number of expositions necessary to form a thrombus
were determined. If a compound is antithrombotic a higher number of laser
“shots” is needed for thrombi formation. From the average number of
expositions the % of inhibition of thrombus formation, which is a more
descriptive parameter, can be calculated (Experim. Part). The results
obtained with single drugs are summarized in Tab. 2. In preceding experiments we determined the time after oral administration which gave us the
peak effects (data not shown, results see first column).
In all cases the inhibition of thrombus formation was dose
dependent. ASA was used as a reference drug. Here as in
general the inhibition in arterioles was stronger than in
venoles.
This represents the different flow conditions in these vessels. In venoles it is much easier to induce a thrombus
(m-TFI = 1.28) than in arterioles (m-TFI = 1.96). It, therefore, is more difficult to inhibit thrombus formation in
Arch. Pharm. (Weinheim) 328, 333-337 (1995)
5
10 30 60
6 0 0.5 2
3.9 1.27 1.3 1.17 1.43 1.8 2.23
0
0
0
3
1120
48
*** n.s. ns. n.s. n.s. n.s. ***
-
-
-
1.2
0
n.s.
1.3
0
n.s.
-
-
-
-
-
_
’ -
_
_
-
1.17 1.37 1.63 2.03
0 2 7 1 6
n.s. n.s. * ***
1.23 1.37 2.2 3.3
0
2 1 9 4 1
n.s. n.s. *** ***
1.17 1.47 1.67 1.97
0 4 8 1 5
n.s. n.s. *
*
1.27 1.43 1.86 0
3 1 2 n.s. n.s. *** -
-
-
-
1.73 2.33
lo 22
*
**
venoles. On the other hand, in venoles fibrin formation plays
a more important role than in arterioles, so that antiplatelet
drugs should exhibit more prominent effects in arterioles.
Thus ASA significantly inhibits the thrombus formation in
arterioles at 5 mgkg and higher doses while in venoles this
only was significant with 60 mgkg. The oligoamine RE
1492C and BM 14515 have similar effects while the NOdonor RE 2047 is somewhat more active. Pentoxifylline and
especially ticlopidine show smaller inhibitory effects.
For the sequential application firstly doses of the drugs
were chosen which solely only produced small or marginal
inhibition. The results are compiled in Tab. 3. The data in
general show a dramatic increase of the antithrombotic
effect. For example: field l a represents the combination of
RE 1492/RE 2047,5 mgkg of each compound. In arterioles
41% inhibition of thrombus formation are observed. With
the single drugs such an effect can only be seen with doses
between 30 and 60 mgkg
1492C) or 10-30 mgkg (RE
2047). In venoles the inhibition of thrombus formation is
22%. This effect cannot be achieved with RE 1492C alone
up to 60 mgkg while a dose of RE 2047 between 30 and 60
mgkg would be necessary. Similar effects were obtained
combining 5 mgkg RE 1492C with 15 mgkg ticlopidine
(field 3a) or 10 mgkg ASA (7a) while 5 m&g BM 14515
(5a) or pentoxifylline (8a) were somewhat weaker.
Combining 10 mgkg RE 1492C with 10 mgkg RE 2047
affords a 79% inhibition in arterioles and 36% in venoles.
Such a strong effect cannot be reached with RE 1492C alone
( S 60 mgkg), while a 60 mgkg dose of RE 2047 must be
applied to get similar effects. An as well similar inhibition of
thrombus formation is seen with RE 1492C/pentoxifylline
10 mgkg each (see field 9b). The combination of RE 1492C
336
Rehse and Ciborski
Tab. 3: Antithrombotic effects of drug combinations. RE 1492C was given at time zero,
ticlopidine I h. RE 2047 2 h and ASA or BM 145 I5 3 h afterwards to rats in order to measure
the combined peak activities.
I
:xp. no.
l
Combination
I
a
RE 1492 C
;
"dkpv
1
RE2047
I5mgAg
h
m-TFI
sinhib.1
3.6
4:
I
2.3
:2
RE 2047
2
10 m i m
4
A
V
3.73
***
44
2.5
***
26
5.17
79
3
36
***
15 m&g
3.13
29
2.17
19
Ticlopidine
3.97
2.7
30
Ticlopidine
3
I
b
RE 1492 C
10 mg/kg
**
I
I
C
RE 1492 C
30 mg/kg
A
V
4.43
***
61
2.93
***
35
I
***
***
- I -
- I
BM 14515
n.s.
BM 14515
1.93
14
33
**
***
I
8
ASA
lom@g
Pentoxiphylline
5 mgncg
9
Pentoxiphylline
10
RE20475mgkg+
Pentoxiphylline
5 m@g
II
I5
3.54
2
***
3
9 **1
3.07
27
**
]
**
-
I1
-
- 1I
1.17
0
n.s.
4.47
4.03
51
-
2.77
2.73
31
***
with BM 14515 appear! o be less favorable (field 6b). The
fields la- Ic show how an ineffective dose of RE 2047 is
able to synergize the effect of RE 1492C. In combination
with 30 mg/kg RE 1492C the effect equals the maximum
inhibition seen in field 2b or 9b. Finally we tested a combination of RE 3492C with RE 2047 arid pentoxifylline giving
the rats a dose of 5 mg/kg of each compound. The results in
field IOa show a good inhibition of thrombus formation
which, however, is not so much superior to the result
obtained with the combination of two drugs only. With
respect to possible pharmacokinetic problems of such a
combination it was not regarded worth-while to spend further efforts on optimizing this combination.
In order to characterize the type of interaction the theoretical additive and independent effects were calculated and
compared to the experimental results. An example is given
for the combinations of RE 1192C and RE 2047 in Fig. 2. In
all cases the combined effect is over additive and over independent suggesting a real potentiation. This is especially
impressive in venoles where with 5 m g k g of each compound
a more than 20% inhibition is observed while the single
drugs show no effect at all at this dos The same was true for
all other combinations. This again is indicative for different
mechanisms of action which of course is not surprising as
this had been the criterion of choice in the combinations tested.
Experimental Part
The Borri-test was carried out as reported'@.
The animal experiments were performed as usuallo'. Statistics: U-test. % of inhibition of thrombus formation was
calculated from the medium thrombus formation index
(m-TFI) according to the equation
(m-TFI,,,, - m-TFI,,,,,,I)
o/c
inhibition =
. 100
6 - m-TFIc,"lrol
If after five expositions to the laser beam no thrombus
was formed the average "shot" number was put to six. A
TFI = 6, therefore, means that no thrombus formation at all
had taken place and represents the maximum effect which
can be observed in this model.
Arch. Pharm. (Weinheim)328. 333-337 (1995)
Antithrombotic Effects of RE 1492
337
% inhibition of thrombus formation
References
I
60
1
l
*)
1
arterioles
2
3
40
4
5
20
6
0
7
8
mo/kg R 1492C/k 2047
9
10
I1
12
13
14
15
5/5
10/5
30/5
mg/kg E 1492C/RE 2047
1O/l 0
16
17
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I
Fig. 2: Inhibition of thrombus formation in rats by RE 1492C and RE
2047. Comparison with the calculated theoretical independent and additive
data.
Arch. Pharm. (Weinheim) 328, 333-337 (1995)
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