© 1996 American Heart Association, Inc.
Articles |
Inhibitory Effects of TA-993, a New 1,5-Benzothiazepine Derivative, on Platelet Aggregation
From the Pharmacological Research Laboratory, Tanabe Seiyaku Co, Ltd, Toda, Japan.
Correspondence to Dr Akio Odawara, Pharmacological Research Laboratory, Tanabe Seiyaku Co, Ltd, 2-2-50 Kawagishi, Toda, Saitama 335, Japan.
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Abstract |
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Abstract TA-993, an l-cis 4',8-dimethyl derivative of the Ca2+ antagonist diltiazem, and some of its metabolites inhibited platelet aggregation induced by collagen, ADP, epinephrine, platelet activating factor, arachidonic acid, and U-46619 in human platelets in vitro. Among the metabolites, MB3 was the most potent (IC50, <1 µmol/L; several hundred times more potent than the parent compound). The d isomer of MB3 was >100 times less potent than the l isomer. Unlike acetylsalicylic acid (ASA), TA-993 inhibited both primary and secondary phases of ADP-induced platelet aggregation and also exhibited a disaggregating effect on human platelet aggregates. The inhibitory effect of TA-993 was enhanced when used in combination with ASA. In ex vivo studies involving rats, TA-993 (
0.3 to 100 mg/kg PO) dose-dependently inhibited
collagen-induced platelet aggregation (ED50,
3 mg/kg PO). In the whole-blood platelet aggregation system in
rats, orally administered TA-993 was also inhibitory in
single (3 to 30 mg/kg) or repeated daily (10 mg/kg per day for 10 days)
dosage. Orally administered TA-993 dose-dependently inhibited
ADP-induced platelet aggregation ex vivo in dogs (0.3 to 10 mg/kg),
significantly protected mice against
collagen+epinephrine–induced thromboembolic death (10
mg/kg), and inhibited thrombus formation in an arteriovenous shunt in
rats (30 mg/kg). The Ca2+-antagonistic action
of TA-993 was very weak in depolarized canine basilar arteries: the
potency was 1/10 that of diltiazem (d-cis) and
d-TA-993. These results suggest that antiplatelet
action is more characteristic of the l-cis than
the d-cis 1,5-benzothiazepine structure and
that TA-993 may become a clinically useful antiplatelet agent
of this structure series.
Key Words: TA-993 • antiplatelet effect • ticlopidine • acetylsalicylic acid • diltiazem
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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It is generally accepted that platelets play a pivotal role in the progress and development of thrombotic disorders,1 2 3 4 especially cerebral vascular diseases such as transient ischemic attack,5 ischemic heart diseases such as myocardial infarction,6 7 8 and peripheral vascular diseases.9 Recently, the inhibition of platelet functions by drugs is thought to be a useful therapeutic means for the prophylaxis and treatment of these diseases. For this purpose, many inhibitors of platelet aggregation such as ASA, sulfinpyrazone, dipyridamole, and ticlopidine have been used clinically.10 11 12 13 14 15 16 17 18 19
Meanwhile, diltiazem, a Ca2+ antagonist of the 1,5-benzothiazepine structure, has been developed, and its efficacy on angina pectoris, hypertension, and myocardial infarction has been well established.20 It has already been reported that diltiazem shows antiplatelet action,21 22 23 24 25 and we have also reported that diltiazem,26 27 its derivative clentiazem, and their basic metabolites28 have inhibitory effects on platelet aggregation.
Among numerous 1,5-benzothiazepine derivatives tested, TA-993 (Fig
1
) was found to possess a highly potent
antiplatelet activity, whereas it shows only very weak
Ca2+-antagonistic
cardiovascular effects. In the present study, we
describe its in vitro, ex vivo, and in vivo antiaggregatory effects on
human, dog, and rodent platelets in comparison with those of ASA
and ticlopidine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Preparation of PRP
PRP was prepared from blood withdrawn with a plastic syringe from the forearm vein of healthy male volunteers or beagle dogs or from the abdominal aorta of rats under ether anesthesia. Blood samples from humans and dogs were instantly mixed with 1/10 vol of 3.8% trisodium citrate and centrifuged at 150g for 6 minutes, and those from rats were mixed with 1/10 vol of 2.2% trisodium citrate29 and centrifuged at 250g for 6 minutes to give PRP. The remaining blood was further centrifuged at 1000g for 10 minutes to give PPP. The platelet counts of human, dog, and rat PRP were adjusted to
4x105, 2x105, and
8x105 cells per microliter plasma, respectively, by
dilution with PPP.
Platelet Aggregation
In Vitro Studies
Platelet
aggregation was measured by the method of
Born30 as the optical density at 600 nm of PRP during
aggregation and expressed as the percentage of the difference in
optical density between PRP at maximum aggregation and untreated PRP.
PRP (200 µL) was preincubated with a 25-µL test compound solution
at 37°C for 2 minutes in the cell of an aggregometer (Hema tracer 1
[model pat-4A], Hema tracer 601, Niko Bio Science), and then with a
25-µL solution or suspension of various aggregating agents, such as
collagen (Hormon-Chemie), ADP (Sigma Chemical Co), epinephrine
(Bosmin Inj, Daiichi Seiyaku Co), platelet activating factor
(synthesized at organic Chemistry Research Laboratory of Tanabe Seiyaku
Co), arachidonic acid (Sigma), and U-46619 (Cayman
Chemical), was added to activate the platelets. The
concentrations of the inducers were adjusted so as to give a
concentration that would cause an aggregation of 70% upon addition
to PRP. Test compounds were dissolved in
physiological saline with ultrasonic treatment. The
disaggregating effect was investigated by adding test drugs to PRP 1
minute after platelet aggregation was induced by ADP.
Ex
Vivo Studies
Test compounds were dissolved or suspended in pure water
in a
volume of 10 mL/kg and then orally administered to rats (male, 6 weeks
old) fasted overnight. Three hours after the administration, blood (4.5
mL) was withdrawn from the abdominal aorta under ether
anesthesia with a plastic syringe containing 0.5 mL of
2.2% trisodium citrate solution and quickly centrifuged at
250g for 6 minutes to afford PRP. The remaining blood was
further centrifuged at 1000g for 10 minutes to yield
PPP. Platelet counts were adjusted to 8x105 cells per
microliter plasma by dilution with PPP. After the PRP (225 µL) was
preincubated at 37°C for 2 minutes in the cell of an aggregometer,
platelet aggregation was measured by adding 25 µL of collagen
suspension (final concentration, 3.6 to 5 µg/mL).
In the whole-blood platelet aggregation test, compounds were orally administered to rats (male, 5 to 6 weeks old) in a single dose (3 to 30 mg/kg) or in repeated daily dosage at 10 mg/kg per day for 10 consecutive days. Thirty minutes or 3 hours after the single dose or 3 hours after the last drug administration, blood (4.5 mL) was taken with a plastic syringe from the abdominal aorta of rats under ether anesthesia. Blood samples were instantly mixed with 1/10 vol of 3.13% trisodium citrate.31 Whole-blood platelet aggregation was measured by the impedance method of Cardinal and Flower.32 Whole blood (1 mL) was preincubated at 37°C for 3 minutes in the cell of a whole-blood platelet aggregometer (model C 560, Chrono-Log), and then 4 µL of a suspension of collagen (1 mg/mL, Chrono-Log) was added to activate the platelets.
Beagle dogs weighing 9 to 13 kg were used after overnight fasting. The animals were orally administered drugs in a capsule. Blood (4.5 mL) was taken from the forearm vein with a plastic syringe containing 0.5 mL of 3.8% trisodium citrate solution just before drug administration and 1, 3, 5, and 7 hours after. Platelet aggregation in PRP was measured by adding ADP (final concentration, 2 to 25 µmol/L) in the above in vitro studies.
In Vivo Antithrombotic Activity
Collagen+Epinephrine–Induced Pulmonary Thrombosis
in Mice
The experiment was performed according to the method of
Ortega.33 TA-993, ASA, and ticlopidine were dissolved in
0.25% carboxymethylcellulose and then orally administered to
overnight-fasted male mice weighing 21 to 28 g. Three hours after
the drug administration, pulmonary thrombosis was induced by
injection of a mixture of collagen suspension (900 µg/kg) and
epinephrine solution (70 µg/kg) into the tail vein, and
mortality from thromboembolic death 1 hour after the inducer injection
was observed.
Arteriovenous Shunt Model in Rats
The
experiment was performed according to the method of Paris et
al.34 TA-993, ASA, and ticlopidine were dissolved in
0.25% carboxymethylcellulose and then orally administered to
overnight-fasted male rats weighing 220 to 268 g. Three hours after
the administration, the rats were anesthetized by
intraperitoneal administration of sodium
pentobarbital (50 mg/kg). Two 12.5-cm pieces of polyethylene tubing,
one with an inner diameter of 0.5 mm and an external diameter of 1.0 mm
and the other with an inner diameter of 1.0 mm and an external diameter
of 2.0 mm (Hibiki Tube Nos. 3 and 6, Hibiki Co), were filled with
heparin and connected to each other with a small piece of silk thread
inserted between them. The thinner end was inserted into the right
carotid artery, and the thicker end was inserted into the left jugular
vein to make an extracorporeal loop shunt between the carotid artery
and the jugular vein. Thirty minutes after the start of blood
circulation, a thrombus produced on the thread was excised, and its dry
weight was measured.
Ca2+-Antagonistic Activity in Depolarized
Canine Basilar Arteries
Suppression of
Ca2+-Induced Contraction
The experiment was performed
according to the method of Kikkawa
et al.35 Mongrel dogs of either sex weighing 7.2 to 14.0
kg were anesthetized by intravenous injection of
sodium pentobarbital (30 mg/kg) and were sacrificed by exsanguination.
The basilar arteries (external diameter, 1.0 to 1.2 mm) were removed
immediately, cleaned of surrounding connective tissue, and cut into
ring segments 5 mm in length.
The ring segment was suspended in a buffer (see below for composition), which was maintained at 37°C and aerated with 95% O2/5% CO2. The resting tension was adjusted according to the size of the artery. The composition of the buffer was as follows (mmol/L): NaCl 72.6, KCl 80.0, MgCl2 1.0, NaHCO3 14.5, and glucose 5.4 (pH 7.3 to 7.4).
Isometric tension of the artery was measured by a strain-gauge transducer (UL-10, Minebea). Contraction was induced by the addition of Ca2+ (1 mmol/L) to the Ca2+-free K+-depolarizing medium, and the relaxing effect of drugs on the contraction was evaluated as Ca2+-antagonistic activity.
Animals and Drugs
Male rats (Sprague-Dawley strain) and male
mice (ddy
strain) were purchased from Japan SLC, Inc. Beagle dogs were purchased
from Yoshiki Farm. TA-993, its metabolites, and diltiazem were
synthesized at Organic Chemistry Research Laboratory of Tanabe Seiyaku
Co, Ltd. Ticlopidine was extracted from Panaldine (Daiichi Seiyaku Co,
Ltd) and purified at the above laboratory. ASA was obtained from
Nacalai Tesque, Inc.
Statistical Analysis
Data were expressed as the
mean±SEM and statistically
analyzed by one-way ANOVA or the Kruskal-Wallis test. When
any significant differences were found, Tukey's method was
applied.
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Results |
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Inhibition of Platelet Aggregation
In Vitro Studies
The Table
lists the antiaggregating activities of
TA-993 as IC50 or IC30 determined in in vitro
studies. TA-993 strongly inhibited platelet aggregation of human,
dog, and rat PRP preparations induced by various agonists. The
potencies of TA-993 were equivalent to those of ASA and greater than
those of ticlopidine. The metabolites of TA-993 (MB1, MB2, and MB3; Fig
1
) also showed inhibitory effects, and their activities
were greater than those of TA-993. In particular, MB3 was the most
potent and 20 to 800 times more potent than TA-993. The d
isomer of TA-993 was nearly equipotent to TA-993 in
collagen-induced platelet aggregation with human
platelets (data not shown). However, the d isomer of MB3
was 150 and 580 times less potent than the l isomer in
collagen- and ADP-induced platelet aggregation, respectively
(Table). TA-993 (300 µmol/L) and MB3 (1 µmol/L)
inhibited both
ADP-induced primary and secondary aggregation of human platelets
and further caused disaggregation to human platelet aggregates,
whereas ASA did not show the latter effect even at 1000 µmol/L (Fig
2). Although TA-993 alone at a concentration of 30
µg/mL did not show any inhibitory effect on
collagen-induced human platelet aggregation, the
inhibitory effect of TA-993 was enhanced in the presence of
ASA (10 µg/mL). A combination of ASA (10 µg/mL) and ticlopidine
(100 µg/mL), on the other hand, produced only a weak effect (Fig
3).
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P<.05 and
Ex Vivo Studies
When
administered orally to rats, TA-993 exhibited
dose-dependent inhibitory activity ex vivo on
collagen-induced platelet aggregation, with the
ED50 being 3 mg/kg, and its potency was greater than
that of ASA (ED50, 12 mg/kg), ticlopidine
(ED50, 119 mg/kg), and diltiazem
(ED50, >100 mg/kg) (Fig 4). In the
whole-blood platelet aggregation system, orally administered
TA-993 (3, 10, and 30 mg/kg) also inhibited collagen-induced
whole-blood platelet aggregation in rats in a
dose-dependent manner 30 minutes after drug administration (Fig
5a). Three hours after single oral administration of 30
mg/kg, only ASA exhibited depression of the whole-blood
platelet aggregation, whereas TA-993 and ticlopidine did not (Fig
5b). However, when these drugs were administered orally for 10
consecutive days at 10 mg/kg per day, all the drugs showed
inhibitory activity on the collagen-induced
whole-blood platelet aggregation 3 hours after the last drug
administration (Fig 5c).
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In beagle dogs, orally
administered TA-993 (0.3, 1, and 10 mg/kg)
exhibited dose-dependent inhibition of ADP-induced platelet
aggregation, which showed a peak effect 1 or 3 hours after
administration, and this effect persisted even at 7 hours, when the
dose was 10 mg/kg (Fig 6). Although a similar
dose-dependent effect was observed with ASA (1 and 10 mg/kg), this
activity was weaker than that of TA-993, and ticlopidine (10 mg/kg) was
ineffective under these experimental conditions.
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Antithrombotic Activity
Intravenous injection of a
mixture of collagen and
epinephrine into the tail vein of mice caused pulmonary
embolism resulting in death, giving a survival rate of 30% in the
control group. Previous oral administration of TA-993 (10, 30, and 100
mg/kg) exhibited a significant dose-dependent preventive effect
(Fig 7). Similarly, ASA and ticlopidine, given orally at
doses of 30 and 100 mg/kg, respectively, exhibited significant
preventive effects on pulmonary thromboembolic death.
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In the
atrioventricular shunt model in rats, TA-993
administered orally at 30 mg/kg significantly inhibited thrombus
formation, and so did oral administration of ticlopidine and ASA at the
same dose. However, statistical significance was not observed among
these three compounds (Fig 8).
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Inhibition of Ca2+-Induced Contraction in Depolarized
Canine Basilar Arteries
Typical tracings of the relaxing effects of
TA-993,
d-TA-993, and diltiazem on the sustained tonic contraction
induced by 1 mmol/L Ca2+ in depolarized canine basilar
arteries are shown in Fig 9A. TA-993 (3 µmol/L),
diltiazem (0.3 µmol/L), and d-TA-993 (0.3 µmol/L)
relaxed Ca2+-induced contraction in the artery, and the
relaxing actions were antagonized by further addition of
CaCl2 solution (3 or 10 mmol/L). The
Ca2+-antagonistic potency of TA-993 was 1/10
that of diltiazem, although d-TA-993 was almost as active as
diltiazem as a Ca2+-antagonist (Fig 9B).
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Among the numerous 1,5-benzothiazepine derivatives so far synthesized, TA-993 is one of the most potent antiplatelet agents when administered to experimental animals. In the ex vivo study, TA-993 was shown to be more potent than ASA and ticlopidine in dogs, and its oral administration at doses below 1 mg/kg was effective in inhibiting aggregation of the platelets from the blood withdrawn 3 hours after drug administration. There were also qualitative differences in the antiplatelet action of TA-993 compared with ASA. Unlike ASA, TA-993 inhibited ADP-induced primary aggregation and U-46619–induced aggregation of human platelets (Table
),
and it
showed a disaggregating effect on the human platelets aggregated by
ADP (Fig 2). Thus, the mechanism of antiplatelet action of
TA-993 must be different from that of ASA. This conclusion is supported
by the synergism observed between TA-993 and ASA (Fig 3). ASA is known to exert its antiplatelet effect through prevention of thromboxane A2 production by inhibiting cyclooxygenase activity in platelets.36 37 However, ASA at higher concentrations also inhibits the cyclooxygenase of vascular endothelial cells involved in prostaglandin I2 production.38 39 This inhibition may conversely enhance the progression of thrombosis or atherosclerosis.40 Therefore, if clinical doses of ASA could be lowered by combination with TA-993, the combination therapy would be advantageous over a therapy with each drug alone in terms of diminishing side effects as well as potentiating the efficacy. It has already been reported that dipyridamole or diltiazem in combination with ASA clinically augments their platelet aggregation–depressing activities.41 42 43 We have also reported that diltiazem and clentiazem, a derivative of diltiazem, shows synergistic inhibitory effects on platelet aggregation in combination with ASA or ticlopidine.28 However, any possible benefit of the combination therapy of TA-993 and ASA that has been suggested in the animal studies will need to be clarified in clinical studies. The reason for the presence of a synergistic effect by TA-993 and ASA is possibly derived from the difference in the mechanism of their antiplatelet actions.
Meanwhile, the antiplatelet action of ticlopidine has been reported to be mediated by elevation of cAMP levels in the platelets through activation of adenylate cyclase activity.44 However, the exact mechanism of the action of ticlopidine is still unknown. Chap et al45 have reported that ticlopidine only at high concentrations inhibited the intake of arachidonic acid into platelets. This agrees with our present finding that the IC50 or IC30 values were high with any of the inducers used.
In the case of oral administration, TA-993 is converted to MB3 via MB1
or MB2 (authors' unpublished data, 1995) and may exert its action
through its metabolites. In fact, the metabolites of TA-993, especially
MB3, showed much higher activities than the parent compound in vitro
(Table). Kiyomoto et al27 also found that some
metabolites
of diltiazem were stronger inhibitors of platelet
aggregation than diltiazem itself. There is an additional report that
much of the clinical antiplatelet effect of diltiazem depended
on the contribution of its metabolites.41 Such a
contribution of the metabolites to the antiplatelet effect of
orally administered TA-993 is also likely. The ex vivo potency of
TA-993, which was greater than expected from its in vitro activity,
supports this possibility (Figs 4 and 6). In the
whole-blood
platelet aggregation system, TA-993 was more effective by
consecutive administration than by single administration (Fig
5). This
augmented efficacy of consecutive administration may be attributable to
the elevated plasma levels of its active metabolites (data not
shown).
TA-993 also demonstrated remarkable inhibitory effects on
thrombus formation in vivo. In the mouse pulmonary embolization
model, death is considered to occur as a result of the embolization
caused by platelet aggregates that have been generated
intravascularly in the pulmonary microcirculation system after
intravenous injection of platelet aggregation
inducers.46 The induction of pulmonary embolism by
collagen+epinephrine is considered to occur by a synergistic
effect of these inducers.47 TA-993 and ASA appeared to be
equally effective in this system (Fig 7).
In the atrioventricular shunt model, the thrombi formed
within the atrioventricular shunt in rats
histologically consist of platelets.34
Since ticlopidine and ASA were effective in these in vivo
models,48 49 50 51 the
antithrombotic effects of TA-993 (Fig 8)
are also likely to be based on its antiplatelet activity.
Diltiazem is widely used in the treatment of
cardiovascular diseases, and its efficacy is based on
Ca2+-antagonist action.20 The
potency of TA-993 as a Ca2+ antagonist proved
to be 1/10 that of diltiazem (Fig 9). Doyle and
Ruegg52
have reported that the platelet lacks voltage-dependent
Ca2+ channels. Therefore, the antiplatelet action
of TA-993 must be based on mechanisms other than
Ca2+-antagonist action. Besides,
notwithstanding its 1,5-benzothiazepine skeleton, TA-993 is an
l isomer, and the d isomer of the most potent
metabolite MB3 was >100 times less potent than the l isomer
(Table), whereas the relationship is reversed in their
Ca2+-antagonist activities (Fig 9). In other
words, the optical resolution of 1,5-benzothiazepine derivatives could
result in partial dissociation between the
Ca2+-antagonistic and antiplatelet
actions. The mechanism of the antiplatelet action of TA-993 has
not been fully elucidated and is currently under investigation.
In conclusion, the above results indicate that TA-993 is the first selective antiplatelet agent of the 1,5-benzothiazepine structure, and it possesses not only a potent antiplatelet activity but also a unique mode of action. This compound may be useful in the prevention and/or treatment of thrombotic disorders.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We would like to thank Dr S. Murata and Dr T. Ikeo for their helpful discussions throughout this study, Dr I. Yamaguchi for his constant encouragement, Dr S. Takeyama for his valuable suggestions regarding the manuscript, and H. Okonogi for his excellent technical assistance.
Received February 27, 1995; accepted January 2, 1996.
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