Br. J. exp. Path. (1977) 58, 474
THE EFFECTS OF TREATMENT WITH ASPIRIN AND AN ANTITHROMBOTIC AGENT SH1117 UPON PLATELET THROMBUS FORMATION IN LIVING BLOOD VESSELS A. J. HONOUR. R. D. CARTER -tr J. I. ANNFrom the Departm7ent of the Regius Professor of Mledicine, and the Departmett of Social and Comm unity Medicine. University of Oxford, and Radcliffe Infirmary, Oxford Received for publication April 14, 1977
Summary. The work reported here describes an in vivo study, over several days in each animal, of the formation and behaviour of platelet thrombi in injured living blood vessels in response to topically applied adenosine diphosphate in rabbits which have been treated with oral doses of SH1117 alone or together with acetyl-salicylic (ASA) before and after i.v. injection of alloxan. These two substances SH1117 and ASA when given together display a synergism which is similar to that described for dipyridamole and ASA, but the antithrombotic action of SH1117 and ASA seems to be more profound. It may be of significance that oral SHM117 given alone appears to confer a degree of insensitivity of the injured vessel in its response to ADP, as such an effect is not displayed by dipyridamole.
INTRA-ARTERIOLAR aggregation of platelets in living blood vessels shows a much increased sensitivity in rabbits previously injected i.v. with alloxan, but this sensitivitv can be completelv reversed by treatment with oral doses of dipyridamole (Persantin, Boehringer Ingelheim), such responses being replicable (Honour and Hockadav, 1976; Honour et al., (1976). Recent work has shown that the required oral dose of dipy-ridamole to produce these changes in sensitivity may be much reduced by giving tLogether oral doses of dipyridamole and acetyl salicvlic acid (ASA), as such agents apparentlv act synergistically at the dose levels used (Honour et al., 1977). Dipyridamole has some undesirable sideeffects upon the circulation, and so a new substance designated SH 117 (Boehringer Ingelheim) has been produced which, in animals, has little of the vasodilator effects of dipvridamole, but nevertheless shows much promise in preliminary clinicopharmacological studies as an antithrom-
botic agent (personal communication, Boehringer Ingelheim, 1976). The work reported here describes an in tivuo study of the formation and behaviour of platelet thrombi in injured living blood vessels in response to topicallv applied adenosine diphosphate (ADP), in rabbits which have been treated with oral doses of either SH1117 alone or together with ASA. MATERIALS AND METHODS
Male New Zealand white rabbits, 253.0 kg body wt. have been used throughout. The animals were fed on "Oxoid diet 18" and water, except during the first 24 h after i.v. injection of alloxan, 85 mg/kg body wt, when they were given 50% glucose in water to drink. Rabbits were anaesthetized with i.p.Nembutal (pentobarbitone sodium). Preparation of the cerebral cortex, microscopic observation of the pial arteries, and the application of a window to the skull in the form of a perspex capsule with a removable lid, have been described elsewhere (Honour and Ross Russell, 1962; Honour and Hockaday, 1976). The preparation can be re-used on a number of occasions and may be monitored throughout by inspection
EFFECTS OF TREATMENT WITH ASPIRIN ON PLATELET THROMBUS
through the lid. Mlembranes regrow after a few days, but can be removed to expose afresh the pial surface, the previous injury sites to the surface arteries showing as slight swellings which are easily recognized and avoided. The arteries are between 100 and 200 Fum in diameter, their number and distribution being very variable, and at times presenting a limiting factor in the number of serial observations that may be made in any one preparation. Electrical injuries were inflicted using a unipolar system as described by Honour et al. (1971). For each series of observations the animal is anaesthetized. 'With sterile precautions the capsule lid is removed whilst the brain surface is kept moist with a warm sterile saline drip. The unipolar electrode is applied directly to the artery wall and at a potential of 5 V with a DC current of 10-20 ±A for 45 sec; replicable injuries are made. Subsequent events are observed With a stereoscopic microscope. The platelet-aggregating agent used was adenosine disphosphate (ADP, Sigma), dissolved in sterile normal saline, the solution containing 1 mg/ml, 2-34 x 106 umol/l, which was further diluted 1: 1 serially with sterile normal saline to the required test concentrations. Solutions were freshly prepared for each set of tests and kept on ice until all the observations were completed. Application was dropWise to brain surface from sterile Pasteur pipettes, the saline drip being interrupted during each challenge with ADP. The end-point for sensitivity to ADP was the most dilute solution at which Visible platelet thrombi formed at the site of injury. The antithrombotic agent SH1117 (personal communication, Boehringer Ingelheim) is a fluor-biophenyl ethyl sulfonyl acetate acid methylester which is practically insoluble in water, but can be given orally in 2% tylose. This was done in the rabbit using a syringe and a wide bore ball-ended needle to inject the agent into the space between the teeth and the cheek. The oral dose given initially was 10 mg daily in 2 ml of 2% tylose, reducing to 7-5 mg and 5 mg subsequently. Acetyl salicylic acid (ASA, Sigma) was dissolved in 500% alochol to a concentration of 42 mg/ml, oral dose 1 ml daily. Blood was taken from a marginal vein of the ear in a conscious rabbit for measurement of blood glucose using the hexokinase method of Slein (1963). Alloxan was given i.v. dissolved in warm sterile saline, the dose for each rabbit being 85 mg/kg bod w-t. RESULTS
Effects of SH1117 given alone In Experiments 1-3 (Table I) we studied the effects (as determined by the
4,
_
concentration of ADP necessary to produce platelet thrombi) of daily oral doses of 10 or 7-5 mg SH1117. In Experiments 1 and 2, 10 mg/day SH1117 was given to the animals before the alloxan injection. This dose can be seen greatly to increase the concentration of ADP required to produce platelet thrombi. Experiment 3 confirms the increase sensitivity to ADP previously observed following an injection of alloxan (Honour and Hockaday, 1976), white bodies being obser-ved after the application of ADP in a concentration of 1/65,536 (35-7 nmol/l). All three experiments show that 10 mg/ day (Experiments 1 and 2) or 7-5 mg/day (Experiment 3) SH1117 reduce this increased sensitivity observed in the "diabetic" animal. WN-hen 10 mg is used, the sensitivity of ADP required to induce platelet thrombi formation is similar to that seen in the untreated, non-diabetic rabbit (1/4096-1/8192, 570-285 nmol/l). Withdrawal of treatment with SH1117 resulted, in all 3 animals, in a considerable increase in sensitivity to ADP.
Dose response to SI1117 Experiment 3 (Table I) shows that 7-5 mg/dav SHI 117 reduces the concentration of ADP necessary to produce platelet thrombi nearlv to the level seen in the non-diabetic animal. Experiments 4, 5, 6 and 7 (Table II), however, show that after further reduction of this daily oral dose to 5 mg there was no effect upon the increased sensitivity to ADP which had been induced by i.v. alloxan.
Response to SH117 and ASA Experiments 5, 6 and 7 (Table II) show clearlv that when 42 mg/day ASA was added to the 5-mg/day dose of SH1117 the ADP sensitivitv drops dramatically to below the level observed in a "nondiabetic" animal. Withdrawal of the two agents induces the sensitivity to ADP to rise again to the levels usually observed following i.v. alloxan (Experiments 6 and 7, Table II).
476
A. J.
HONOUR.
R. D. CARTER AND J. I. MANN
TABLE I.-Concentration of ADP Required to Induce Platelet Aggregation in Injured Pial Arteries in Rabbits Before and After (Experinments 1 and 2), and After (Experiment 3) an Intravenous Injection of Alloxan. Levels of Blood Glucose and Details of Antithronnbotic Agents Used are Shown ExpEriment no. 1
Date 26.2.76 * 11.3.76
f18.3.76
t \25.3.76 2
3
8.3.76 * 22.3.76 t 29.3.76 5.4.76 *4.1.77 7.1.77
Blood glucose mm 6-40 6-44 21-50 22 40
ADP A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
(conc.) 1/4096 1/64 1/8192 1/131072
(nmol/1)
or
SH1117 10 mg/day 570 36,800 10 mg/day 285 10 mg/dav 17-8
1/819-2 285 10mg/day 1/512 4,600 10 mg/day 1/2048-1,4096 1150-570 10 mg/day 1/16384-1/32768 143-72
6.9 7.2 18.4 17.6
stopped stopped started
stopped stopped
-
1165536
9-05
35.
8.1.77
*
started
t14.1.77 9-50 1/8192--1/16384 19.1.77 8-44 1/65536 Intravenous injection of alloxan given after these observations
7-5
285-143 35 were
7
started stopped
mg/day
mg/day
made.
t No treatment between these dates.
TABLE I.-Concentration of ADP Required to Induce Platelet Aggregation in Injured Arteries in Rabbits Before and After (Experiments 4, 6 and 7), and After (Experiment 5), Intravenous Injection of Alloxan. Levels of Blood Glucose and of Antithrombotic Agents Used are Shown ADP sensitivity
Blood
Experiment No.
4
-
a
Date *10.2.77 15.2.77 21.2.77
mM/l
*10.1. 77 13.1.77
6-4 16-5 13-87
19s.1.77
24.1.77 28.1.77
*3.2.77 7 2.77 6
17.2. f1l4.2.77 t{22.2.77 3.3.77 77
*3.3.77 7.3.77 7
Oral doses of A
glucose
tf14.3.77 t17.3.77
6-65 8-70 8-71
or (conc.) 1/4096-1/8192 1/6,5536-1/131072 1/131072
(nmol 1) 570-285 35-7-17-8 17-8
1/262144
8 -9 17-8-8 -9
1/131072-1/262144
SH1117
ASA
5 ig/dav
started stopped
5 mg/day started 5 mg/day 5 mg/day 42 mg/day started 5 mg/day- 42 mg/day stopped -
21-52 7 -70 12 -26 9- 78 8-56 8-02 6- 80 8- 80 8-40
1/512 1/4096 1/131072-1/262144
1/131072 1/1024
570 17 -8-8-9 17- 8
2300 35 7
1 /65536
1/8192
1/65536 1/65536-
4600
31072
started stopped started st-opped
5 mg/day 5 mg/day
started
-
-
-
285 35- 7 35 7-17- 8
stopped
mg/day 42 mg/dav started
t 21.3.77
8-29 1150 1/2048 25 3.77 8-58 1/65536 * Intravenous injection of alloxan given aft-r these obsqrvations t No treatment between thesa dates.
5 mg/lday 5 mg/day 5 mg/day- 42 mg/day 5 mg/dav 42 mg/day
stopped
35.7
DISCUSSION
In previous experiments in rabbits using the techniques described here we
were
made.
have been able to assess the effects of various agents either singly or in combination upon the formation of platelet thrombi within living blood vessels in response to
EFFECTS OF TREATMENT WITH ASPIRIN ON PLATELET THROMIBUS
injurv, both before and after i.v. alloxan, and over periods of several days in the same animal (Honour et al., 1976, 1977). In previous studies ASA 42 mg/day given orally was without effect, but a synergistic effect was observed when given with dipyridamole (Honour et al., 1976, 1977). A like synergistic effect has again been demonstrated with a new antithrombotic preparation, SH1117. These two agents given separately at dose levels of 5 mg and 42 mg/day respectively were without effect (Table II). We have also been able to study their combined effects and to establish that there is a restoration of the sensitivity to ADP of injured pial arteries following i.v. alloxan, upon withdrawal of all treatment with these two agents in combination (Table II, Rabbits 6 and 7). There is clearly a profound antithrombotic action of SH1117 and ASA when given together in this experimental model, and in this connection the apparent reduction in sensitivitv to ADP in rabbits before i.v. alloxan must play some part. We have no explanation for this, but the results are replicable and do not occur with dipyridamole. SH 1 17 is an entirelv new substance which has yet to be subject to controlled clinical trial in man. Trials in laboratory animals have been intensive and have shown differences as compared with dipyridamole and ASA, in particular the much reduced dose level of SHI 117, 1 / 1Oth of that of dipyridamole, necessary to restore the shortened life spaii of platelets in the baboon to the control level (Harker, 1975). Of particular importance is the almost total absence of a cardiovascular effect as compared with
477
dipyridamole. The mode of action of SH 1117 is not clear. It undoubtedlv differs from both dipvridamole and ASA although there are certain unmistakable similarities (personal communication on SH1117, Boehringer Ingelheim. M. 1023, 1976). SH 1117 is a new substance which shows considerable promise in acting as an antithrombotic agent in the experimental model described, in particular when used together with aspirin, and the results would seem to justifv further studies of its behaviour in man. We gratefully acknowledge the skilled technical assistance of MIr E. Bown. Financial support came from a Medical Research Council grant to A.J.H. Supplies of SH I 1 17 were generously provided by Boehringer Ingellieim. REFERENCES HARKER, L. A. (1975) In viro Evaluation of Antiplatelet Drugs (SH1117). Boehringer Ingelheim. HON-OU-R, A. J. & HOCKADAY, T. D. R. (1976) Increased Sensitivity of in riro Platelet Aggregation after Alloxan on Streptozotocin. Br. J. exp.
Path.,57, 1. HON-OuR, A. J., HOCKADAY, T. D. R. & MANN, J. I. (1976) Reversibility by Dipy-ridamole of the Incr.ased Sensiti'vity of in riro Platelet Aggregation in Rabbits after Alloxan. Br. J. exp. Path., 57, 11. HO-NouR, A. J., HocKADAY, T. D. R. & A-Nx-, J. I. (1977) The Synergistic Effect of Aspirin and Dipyridamole upon Platelet Thrombi in Living Blood Vessels. Br. J. exp. Path., 52, 268. HoN-OUR, A. J., PICKER-NG, G. W. & SHEPPARD, B. L. (1971) Ultrastructure and BehaViour of Platelet Thrombi in Injured Arteries. Br. J. exp. Path., 52, 482. HoN-ouR, A. J. & Ross RussELL, W. R. (1962) Experimental Platelet Embolism. Br. J. exp. Path., 43, 350. SLEI-N, M. W. (1963) In Methodsof Enzymatic Assay. Ed. H. U. Bergmeyer, London: Verlag Chemie and Academic Press. p. 1 17.
THE EFFECTS OF TREATMENT WITH ASPIRIN AND AN ANTITHROMBOTIC AGENT SH1117 UPON PLATELET THROMBUS FORMATION IN LIVING BLOOD VESSELS A. J. HONOUR. R. D. CARTER -tr J. I. ANNFrom the Departm7ent of the Regius Professor of Mledicine, and the Departmett of Social and Comm unity Medicine. University of Oxford, and Radcliffe Infirmary, Oxford Received for publication April 14, 1977
Summary. The work reported here describes an in vivo study, over several days in each animal, of the formation and behaviour of platelet thrombi in injured living blood vessels in response to topically applied adenosine diphosphate in rabbits which have been treated with oral doses of SH1117 alone or together with acetyl-salicylic (ASA) before and after i.v. injection of alloxan. These two substances SH1117 and ASA when given together display a synergism which is similar to that described for dipyridamole and ASA, but the antithrombotic action of SH1117 and ASA seems to be more profound. It may be of significance that oral SHM117 given alone appears to confer a degree of insensitivity of the injured vessel in its response to ADP, as such an effect is not displayed by dipyridamole.
INTRA-ARTERIOLAR aggregation of platelets in living blood vessels shows a much increased sensitivity in rabbits previously injected i.v. with alloxan, but this sensitivitv can be completelv reversed by treatment with oral doses of dipyridamole (Persantin, Boehringer Ingelheim), such responses being replicable (Honour and Hockadav, 1976; Honour et al., (1976). Recent work has shown that the required oral dose of dipy-ridamole to produce these changes in sensitivity may be much reduced by giving tLogether oral doses of dipyridamole and acetyl salicvlic acid (ASA), as such agents apparentlv act synergistically at the dose levels used (Honour et al., 1977). Dipyridamole has some undesirable sideeffects upon the circulation, and so a new substance designated SH 117 (Boehringer Ingelheim) has been produced which, in animals, has little of the vasodilator effects of dipvridamole, but nevertheless shows much promise in preliminary clinicopharmacological studies as an antithrom-
botic agent (personal communication, Boehringer Ingelheim, 1976). The work reported here describes an in tivuo study of the formation and behaviour of platelet thrombi in injured living blood vessels in response to topicallv applied adenosine diphosphate (ADP), in rabbits which have been treated with oral doses of either SH1117 alone or together with ASA. MATERIALS AND METHODS
Male New Zealand white rabbits, 253.0 kg body wt. have been used throughout. The animals were fed on "Oxoid diet 18" and water, except during the first 24 h after i.v. injection of alloxan, 85 mg/kg body wt, when they were given 50% glucose in water to drink. Rabbits were anaesthetized with i.p.Nembutal (pentobarbitone sodium). Preparation of the cerebral cortex, microscopic observation of the pial arteries, and the application of a window to the skull in the form of a perspex capsule with a removable lid, have been described elsewhere (Honour and Ross Russell, 1962; Honour and Hockaday, 1976). The preparation can be re-used on a number of occasions and may be monitored throughout by inspection
EFFECTS OF TREATMENT WITH ASPIRIN ON PLATELET THROMBUS
through the lid. Mlembranes regrow after a few days, but can be removed to expose afresh the pial surface, the previous injury sites to the surface arteries showing as slight swellings which are easily recognized and avoided. The arteries are between 100 and 200 Fum in diameter, their number and distribution being very variable, and at times presenting a limiting factor in the number of serial observations that may be made in any one preparation. Electrical injuries were inflicted using a unipolar system as described by Honour et al. (1971). For each series of observations the animal is anaesthetized. 'With sterile precautions the capsule lid is removed whilst the brain surface is kept moist with a warm sterile saline drip. The unipolar electrode is applied directly to the artery wall and at a potential of 5 V with a DC current of 10-20 ±A for 45 sec; replicable injuries are made. Subsequent events are observed With a stereoscopic microscope. The platelet-aggregating agent used was adenosine disphosphate (ADP, Sigma), dissolved in sterile normal saline, the solution containing 1 mg/ml, 2-34 x 106 umol/l, which was further diluted 1: 1 serially with sterile normal saline to the required test concentrations. Solutions were freshly prepared for each set of tests and kept on ice until all the observations were completed. Application was dropWise to brain surface from sterile Pasteur pipettes, the saline drip being interrupted during each challenge with ADP. The end-point for sensitivity to ADP was the most dilute solution at which Visible platelet thrombi formed at the site of injury. The antithrombotic agent SH1117 (personal communication, Boehringer Ingelheim) is a fluor-biophenyl ethyl sulfonyl acetate acid methylester which is practically insoluble in water, but can be given orally in 2% tylose. This was done in the rabbit using a syringe and a wide bore ball-ended needle to inject the agent into the space between the teeth and the cheek. The oral dose given initially was 10 mg daily in 2 ml of 2% tylose, reducing to 7-5 mg and 5 mg subsequently. Acetyl salicylic acid (ASA, Sigma) was dissolved in 500% alochol to a concentration of 42 mg/ml, oral dose 1 ml daily. Blood was taken from a marginal vein of the ear in a conscious rabbit for measurement of blood glucose using the hexokinase method of Slein (1963). Alloxan was given i.v. dissolved in warm sterile saline, the dose for each rabbit being 85 mg/kg bod w-t. RESULTS
Effects of SH1117 given alone In Experiments 1-3 (Table I) we studied the effects (as determined by the
4,
_
concentration of ADP necessary to produce platelet thrombi) of daily oral doses of 10 or 7-5 mg SH1117. In Experiments 1 and 2, 10 mg/day SH1117 was given to the animals before the alloxan injection. This dose can be seen greatly to increase the concentration of ADP required to produce platelet thrombi. Experiment 3 confirms the increase sensitivity to ADP previously observed following an injection of alloxan (Honour and Hockaday, 1976), white bodies being obser-ved after the application of ADP in a concentration of 1/65,536 (35-7 nmol/l). All three experiments show that 10 mg/ day (Experiments 1 and 2) or 7-5 mg/day (Experiment 3) SH1117 reduce this increased sensitivity observed in the "diabetic" animal. WN-hen 10 mg is used, the sensitivity of ADP required to induce platelet thrombi formation is similar to that seen in the untreated, non-diabetic rabbit (1/4096-1/8192, 570-285 nmol/l). Withdrawal of treatment with SH1117 resulted, in all 3 animals, in a considerable increase in sensitivity to ADP.
Dose response to SI1117 Experiment 3 (Table I) shows that 7-5 mg/dav SHI 117 reduces the concentration of ADP necessary to produce platelet thrombi nearlv to the level seen in the non-diabetic animal. Experiments 4, 5, 6 and 7 (Table II), however, show that after further reduction of this daily oral dose to 5 mg there was no effect upon the increased sensitivity to ADP which had been induced by i.v. alloxan.
Response to SH117 and ASA Experiments 5, 6 and 7 (Table II) show clearlv that when 42 mg/day ASA was added to the 5-mg/day dose of SH1117 the ADP sensitivitv drops dramatically to below the level observed in a "nondiabetic" animal. Withdrawal of the two agents induces the sensitivity to ADP to rise again to the levels usually observed following i.v. alloxan (Experiments 6 and 7, Table II).
476
A. J.
HONOUR.
R. D. CARTER AND J. I. MANN
TABLE I.-Concentration of ADP Required to Induce Platelet Aggregation in Injured Pial Arteries in Rabbits Before and After (Experinments 1 and 2), and After (Experiment 3) an Intravenous Injection of Alloxan. Levels of Blood Glucose and Details of Antithronnbotic Agents Used are Shown ExpEriment no. 1
Date 26.2.76 * 11.3.76
f18.3.76
t \25.3.76 2
3
8.3.76 * 22.3.76 t 29.3.76 5.4.76 *4.1.77 7.1.77
Blood glucose mm 6-40 6-44 21-50 22 40
ADP A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
(conc.) 1/4096 1/64 1/8192 1/131072
(nmol/1)
or
SH1117 10 mg/day 570 36,800 10 mg/day 285 10 mg/dav 17-8
1/819-2 285 10mg/day 1/512 4,600 10 mg/day 1/2048-1,4096 1150-570 10 mg/day 1/16384-1/32768 143-72
6.9 7.2 18.4 17.6
stopped stopped started
stopped stopped
-
1165536
9-05
35.
8.1.77
*
started
t14.1.77 9-50 1/8192--1/16384 19.1.77 8-44 1/65536 Intravenous injection of alloxan given after these observations
7-5
285-143 35 were
7
started stopped
mg/day
mg/day
made.
t No treatment between these dates.
TABLE I.-Concentration of ADP Required to Induce Platelet Aggregation in Injured Arteries in Rabbits Before and After (Experiments 4, 6 and 7), and After (Experiment 5), Intravenous Injection of Alloxan. Levels of Blood Glucose and of Antithrombotic Agents Used are Shown ADP sensitivity
Blood
Experiment No.
4
-
a
Date *10.2.77 15.2.77 21.2.77
mM/l
*10.1. 77 13.1.77
6-4 16-5 13-87
19s.1.77
24.1.77 28.1.77
*3.2.77 7 2.77 6
17.2. f1l4.2.77 t{22.2.77 3.3.77 77
*3.3.77 7.3.77 7
Oral doses of A
glucose
tf14.3.77 t17.3.77
6-65 8-70 8-71
or (conc.) 1/4096-1/8192 1/6,5536-1/131072 1/131072
(nmol 1) 570-285 35-7-17-8 17-8
1/262144
8 -9 17-8-8 -9
1/131072-1/262144
SH1117
ASA
5 ig/dav
started stopped
5 mg/day started 5 mg/day 5 mg/day 42 mg/day started 5 mg/day- 42 mg/day stopped -
21-52 7 -70 12 -26 9- 78 8-56 8-02 6- 80 8- 80 8-40
1/512 1/4096 1/131072-1/262144
1/131072 1/1024
570 17 -8-8-9 17- 8
2300 35 7
1 /65536
1/8192
1/65536 1/65536-
4600
31072
started stopped started st-opped
5 mg/day 5 mg/day
started
-
-
-
285 35- 7 35 7-17- 8
stopped
mg/day 42 mg/dav started
t 21.3.77
8-29 1150 1/2048 25 3.77 8-58 1/65536 * Intravenous injection of alloxan given aft-r these obsqrvations t No treatment between thesa dates.
5 mg/lday 5 mg/day 5 mg/day- 42 mg/day 5 mg/dav 42 mg/day
stopped
35.7
DISCUSSION
In previous experiments in rabbits using the techniques described here we
were
made.
have been able to assess the effects of various agents either singly or in combination upon the formation of platelet thrombi within living blood vessels in response to
EFFECTS OF TREATMENT WITH ASPIRIN ON PLATELET THROMIBUS
injurv, both before and after i.v. alloxan, and over periods of several days in the same animal (Honour et al., 1976, 1977). In previous studies ASA 42 mg/day given orally was without effect, but a synergistic effect was observed when given with dipyridamole (Honour et al., 1976, 1977). A like synergistic effect has again been demonstrated with a new antithrombotic preparation, SH1117. These two agents given separately at dose levels of 5 mg and 42 mg/day respectively were without effect (Table II). We have also been able to study their combined effects and to establish that there is a restoration of the sensitivity to ADP of injured pial arteries following i.v. alloxan, upon withdrawal of all treatment with these two agents in combination (Table II, Rabbits 6 and 7). There is clearly a profound antithrombotic action of SH1117 and ASA when given together in this experimental model, and in this connection the apparent reduction in sensitivitv to ADP in rabbits before i.v. alloxan must play some part. We have no explanation for this, but the results are replicable and do not occur with dipyridamole. SH 1 17 is an entirelv new substance which has yet to be subject to controlled clinical trial in man. Trials in laboratory animals have been intensive and have shown differences as compared with dipyridamole and ASA, in particular the much reduced dose level of SHI 117, 1 / 1Oth of that of dipyridamole, necessary to restore the shortened life spaii of platelets in the baboon to the control level (Harker, 1975). Of particular importance is the almost total absence of a cardiovascular effect as compared with
477
dipyridamole. The mode of action of SH 1117 is not clear. It undoubtedlv differs from both dipvridamole and ASA although there are certain unmistakable similarities (personal communication on SH1117, Boehringer Ingelheim. M. 1023, 1976). SH 1117 is a new substance which shows considerable promise in acting as an antithrombotic agent in the experimental model described, in particular when used together with aspirin, and the results would seem to justifv further studies of its behaviour in man. We gratefully acknowledge the skilled technical assistance of MIr E. Bown. Financial support came from a Medical Research Council grant to A.J.H. Supplies of SH I 1 17 were generously provided by Boehringer Ingellieim. REFERENCES HARKER, L. A. (1975) In viro Evaluation of Antiplatelet Drugs (SH1117). Boehringer Ingelheim. HON-OU-R, A. J. & HOCKADAY, T. D. R. (1976) Increased Sensitivity of in riro Platelet Aggregation after Alloxan on Streptozotocin. Br. J. exp.
Path.,57, 1. HON-OuR, A. J., HOCKADAY, T. D. R. & MANN, J. I. (1976) Reversibility by Dipy-ridamole of the Incr.ased Sensiti'vity of in riro Platelet Aggregation in Rabbits after Alloxan. Br. J. exp. Path., 57, 11. HO-NouR, A. J., HocKADAY, T. D. R. & A-Nx-, J. I. (1977) The Synergistic Effect of Aspirin and Dipyridamole upon Platelet Thrombi in Living Blood Vessels. Br. J. exp. Path., 52, 268. HoN-OUR, A. J., PICKER-NG, G. W. & SHEPPARD, B. L. (1971) Ultrastructure and BehaViour of Platelet Thrombi in Injured Arteries. Br. J. exp. Path., 52, 482. HoN-ouR, A. J. & Ross RussELL, W. R. (1962) Experimental Platelet Embolism. Br. J. exp. Path., 43, 350. SLEI-N, M. W. (1963) In Methodsof Enzymatic Assay. Ed. H. U. Bergmeyer, London: Verlag Chemie and Academic Press. p. 1 17.
