ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1978, p. 881-883 0066-4804/78/0013-0881$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 13, No. 5
Printed in U.S.A.
NOTES Inhibition of Platelet Function by Sulbenicillin and Its Metabolite YASUO
IKEDA,`*
MASAO KIKUCHI,' SHIGEYOSHI MATSUDA,' KEISUKE TOYAMA,' MITSUTO HASEGAWA,' KIYOAKI WATANABE,2 AND YASUHIKO ANDO2
Department of Hematology' and Department of Clinical Pathology,2 School of Medicine, Keio University,
Tokyo, Japan
Received for publication 14 October 1977
The effect of sulbenicillin and its major metabolite, a-sulfobenzylpenicilloic acid, on platelet function was investigated. Sulbenicillin caused inhibition of platelet aggregation and release reaction in the same manner as carbenicillin. aSulfobenzylpenicilloic acid was found to cause much stronger inhibition of platelet function. The results indicate that the strong inhibitory action of a-sulfobenzylpenicilloic acid may also take part in impaired platelet functions following administration of sulbenicillin to humans. There have been a number of reports of inpaired platelet response to aggregating agents when penicillin G (Pc-G) and related antibiotics (carbenicillin, ticarcillin) have been added to platelet-rich plasma (PRP) in vitro or when patients or volunteers received large doses of these antibiotics (2, 3, 6). Reviewing these studies, we realized that the minimal concentration of the drugs that would show their inhibitory effects in vitro far exceeded the blood level of the antibiotics reached during treatment of severe gram-negative infection, but the reason for this discrepancy has not been clarified yet. Sulbenicillin (SB-PC), a newly developed semisynthetic penicillin, was found to cause impaired platelet functions in vitro, and a similar discrepancy of in vivo and in vitro inhibitory effect was also observed. This prompted us to investigate the effect of a-sulfobenzylpenicilloic acid (SB-PA), the major metabolite of SB-PC, on platelet function in comparison to SB-PC. Platelet aggregation was studied by a turbidimetric method according to Born et al. (1). Blood was obtained from normal healthy subjects who had not taken any drugs for at least 10 days. The anticoagulant was 3.8% trisodium citrate dihydrate, 1 part to 9 parts of blood. PRP was prepared by centrifugation at 77 x g for 15 min at room temperature. PRP was incubated with 0.1 volume of SB-PC or SB-PA (Takeda Pharmaceutical Co., Japan) for 1 min before the addition of aggregating agents. Adenosine 5'-diphosphate (ADP) was obtained from Sigma Chemical Co., St. Louis, Mo., epinephrine from
Daiichi Pharmaceutical Co., Japan, and collagen from Hormon-Chemic, Germany. Maximal change in light transmission in control PRP was assigned 100% and in PRP treated with antibiotics was expressed as percentages of the control value. In collagen-induced platelet aggregation, aggregation time was also used as an indicator of platelet response to collagen. Serotonin release was monitored by measuring the release of ['4C]serotonin from platelets prelabeled with this amine according to the method of Mills et al. (7). PRP was incubated with 0.4 ,uM [14C]serotonin (specific activity, 52 mCi/mmol; Radiochemical Centre, Amersham, England) for 60 min at 37°C. Collagen (x32 dilution) or epinephrine (5 ,g/ml) was used to induce release of serotonin. Release was terminated after constant stirring for 5 min at 37°C by immediate centrifugation at 7,000 x g for 1 min. The radioactivity in the supernatant was counted in a liquid scintillation counter. The release was expressed as a percentage of the total amount of radioactivity originally contained in the platelets. The effect of SB-PC and SB-PA on ADPinduced platelet aggregation is shown in Fig. 1. High concentrations of SB-PC or SB-PA (25 mM) grossly inhibited the primary aggregation induced by ADP; the latter showed more pronounced inhibitory effect. In the presence of 10 mM SB-PC or 2 mM SB-PA, nearly normal primary aggregation was observed with subsequent rapid disaggregation. The minimum concentration of SB-PC that inhibited the second-
881
882
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
ary aggregation was found to be 5 mM, whereas that of SB-PA was much lower (less than 1 mM). Similar results were obtained with epinephrine-induced platelet aggregation (Fig. 2). The effect of several concentrations of SB-PC and SB-PA on collagen-induced platelet aggregation is shown in Table 1. Maximal aggregability as well as aggregation time of collagen-induced aggregation was markedly affected both by SB-PC and SB-PA. Again SB-PA showed more pronounced inhibitory effect on collageninduced aggregation as compared with SB-PC. The effect of SB-PC or SB-PA on ['4C]serotonin release induced by epinephrine and collagen is summarized in Table 2. SB-PC at a concentration of 25 mM grossly inhibited epinephrine- or collagen-induced serotonin release from human platelets in vitro at 5 min, whereas SBPA exhibited similar inhibitory effects at 5 mM. Although there are no reports regarding the SB
-
effect of SB-PC on platelet function, it is ekpected that SB-PC would show significant inhibitory effect on platelet function because of their TABLE 1. Effect of SB-PC and SB-PA on collageninduced aggregation of human platelets Concn Concn Agent (M AgeM)
Control SB-PC
0 0.2 1.2 2.5 5.0 10.0 0.2 1.0 2.0 5.0 10.0
SB-PA
aggregability of control) (%
Maximum
ti Agtime (mi) Aggregation)
100 100 100 95 54.5 25 100 75 60.5 12.5
2.0 2.0 2.0 2.2 2.8 3.2 2.0 3.0 3.0 3.2
0
SB - PA
PC
N
"I
C I._
a
._
-E01 ;i
imM 0
1
2
3
4
min .
FIG. 1. The effect of SB-PC and SB-PA on ADP-induced platelet aggregation. Platelet aggregation studies were carried out in the presence of various concentration of SB-PC and SB-PA as described in the text. SB- PA
SB- PC
l.oOr
.E C 50 Epin.phrine 2pg/m!, -
-J
0
1
2
3
4
0
1
2
3
4 min.
FIG. 2. The effect of SB-PC and SB-PA on epinephrine-induced platelet aggregation. PRP was preincubated with various concentrations of SB-PC and SB-PA for 5 min. Platelet aggregation was determined as described in the text.
VOL. 13, 1978
TABLE 2. Effect of SB-PC or SB-PA on serotonin release from human platelets Agent
Control SB-PC SB-PA
x32
Concn
(mM)
Epinephrine, 5 ytg/ml (%)
Coagen
5 25
32.6 30.4 13.0 14.2
50.4 48.6 22.8
5 25
4.5
27.6 9.5
close structural similarities with carbenicillin. The mechanism of Pc-G-induced platelet dysfunction was studied in detail by Cazenave et al. (4), who showed that Pc-G may cause impairment of platelet function by coating the platelet surface, since it inhibited various aspects of platelet function. SB-PC and SB-PA also inhibited primary aggregation as well as secondary aggregation of platelet in the same manner as carbenicillin or Pc-G. SB-PC was also found to inhibit the ability of platelets to adhere to everted rabbit aorta (data not shown). Therefore, the mechanism of SB-PC- and SB-PA-induced inhibition of platelet aggregation appears to be identical to that of carbenicillin or Pc-G. Our interesting finding was that SB-PA, the major metabolite of SB-PC, inhibited platelet aggregation and release of serotonin from platelets in vitro. The inhibitory effect of SB-PC and SB-PA was compared on a molar basis, and SBPA was found to be much stronger than SB-PC. Following administration of high doses to human volunteers, carbenicillin was found to inhibit platelet function (3). Maximal concentration of carbenicillin reached during treatment was estimated to be 100 to 150 ,ug/ml (5). On the other hand, carbenicillin at a concentration of 1,000 ,g/ml showed no inhibitory effect on platelet aggregation in vitro (1). A similar discrepancy of in vivo and in vitro results was observed in SB-PC. The minimal concentration of SB-PC that inhibited platelet aggregation in vitro was 5 mM (2,070 ,g/ml), whereas patients receiving
883 NOTES 32 g of SB-PC per day showed markedly decreased platelet aggregation. (Maximal concentration of SB-PC in blood was approximately 100 to 150 ,ug/ml.) SB-PA, the major metabolite of SB-PC, showed marked inhibitory effect. Pc-G and carbenicillin were also known to have their penicilloic acid derivatives, although the effect of these compounds on platelet function was not examined. Data are currently not available to show the concentration of SB-PA in circulating blood in humans. In rats, however, approximately 10 to 20% of SB-PC was found to be excreted into urine as SB-PA. The fact that SB-PA showed stronger inhibition of platelet function is important, because an unexpectedly marked bleeding tendency may develop when uremic patients receive large doses of these antibiotics. Whether our results fully explain the discrepancy between in vivo and in vitro effects of SB-PC awaits further investigation. LITERATURE CITED 1. Born, G. V. R. 1962. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature (London) 194:927-929. 2. Brown, C. H., E. A. Natelson, M. W. Bradshaw, C. P. Alfrey, and T. W. Williams. 1975. Study of the effects of ticarcillin on blood coagulation and platelet function. Antimicrob. Agents Chemother. 7:652-657. 3. Brown, C. H., E. A. Natelson, M. W. Bradshaw, T. W. Williams, and C. P. Alfrey. 1974. The hemostatic defect produced by carbenicillin. N. Engl. J. Med. 291:265-270. 4. Cazenave, J. P., M. A. Guccione, M. A. Packham, and J. F. Mustard. 1977. Effects of cephalothin and penicillin G on platelet function in vitro. Br. J. Hematol.
36:135-152.
5. Gordon, D. H. 1970. Carbenicillin in renal failure. Lancet ii:422. 6. McClure, P. D., J. G. Casserly, C. Monsier, and D. Crozier. 1970. Carbenicillin-induced bleeding disorder. Lancet ii: 1307-1308. 7. Mills, D. C. B., I. A. Roff, and G. C. K. Roberts. 1968. The release of nucleotides, 5-hydroxytryptamine and enzyme from human blood platelets during aggregation. J. Physiol. 195:715-729. 8. Nakai, Y., Y. Shirakawa, T. Fujita, Z. Suzuoki, and T. Fugono. 1972. The metabolic fate of a-sulphobenzylpenicillin in rats. Xenobiotica 2:147-157.
Vol. 13, No. 5
Printed in U.S.A.
NOTES Inhibition of Platelet Function by Sulbenicillin and Its Metabolite YASUO
IKEDA,`*
MASAO KIKUCHI,' SHIGEYOSHI MATSUDA,' KEISUKE TOYAMA,' MITSUTO HASEGAWA,' KIYOAKI WATANABE,2 AND YASUHIKO ANDO2
Department of Hematology' and Department of Clinical Pathology,2 School of Medicine, Keio University,
Tokyo, Japan
Received for publication 14 October 1977
The effect of sulbenicillin and its major metabolite, a-sulfobenzylpenicilloic acid, on platelet function was investigated. Sulbenicillin caused inhibition of platelet aggregation and release reaction in the same manner as carbenicillin. aSulfobenzylpenicilloic acid was found to cause much stronger inhibition of platelet function. The results indicate that the strong inhibitory action of a-sulfobenzylpenicilloic acid may also take part in impaired platelet functions following administration of sulbenicillin to humans. There have been a number of reports of inpaired platelet response to aggregating agents when penicillin G (Pc-G) and related antibiotics (carbenicillin, ticarcillin) have been added to platelet-rich plasma (PRP) in vitro or when patients or volunteers received large doses of these antibiotics (2, 3, 6). Reviewing these studies, we realized that the minimal concentration of the drugs that would show their inhibitory effects in vitro far exceeded the blood level of the antibiotics reached during treatment of severe gram-negative infection, but the reason for this discrepancy has not been clarified yet. Sulbenicillin (SB-PC), a newly developed semisynthetic penicillin, was found to cause impaired platelet functions in vitro, and a similar discrepancy of in vivo and in vitro inhibitory effect was also observed. This prompted us to investigate the effect of a-sulfobenzylpenicilloic acid (SB-PA), the major metabolite of SB-PC, on platelet function in comparison to SB-PC. Platelet aggregation was studied by a turbidimetric method according to Born et al. (1). Blood was obtained from normal healthy subjects who had not taken any drugs for at least 10 days. The anticoagulant was 3.8% trisodium citrate dihydrate, 1 part to 9 parts of blood. PRP was prepared by centrifugation at 77 x g for 15 min at room temperature. PRP was incubated with 0.1 volume of SB-PC or SB-PA (Takeda Pharmaceutical Co., Japan) for 1 min before the addition of aggregating agents. Adenosine 5'-diphosphate (ADP) was obtained from Sigma Chemical Co., St. Louis, Mo., epinephrine from
Daiichi Pharmaceutical Co., Japan, and collagen from Hormon-Chemic, Germany. Maximal change in light transmission in control PRP was assigned 100% and in PRP treated with antibiotics was expressed as percentages of the control value. In collagen-induced platelet aggregation, aggregation time was also used as an indicator of platelet response to collagen. Serotonin release was monitored by measuring the release of ['4C]serotonin from platelets prelabeled with this amine according to the method of Mills et al. (7). PRP was incubated with 0.4 ,uM [14C]serotonin (specific activity, 52 mCi/mmol; Radiochemical Centre, Amersham, England) for 60 min at 37°C. Collagen (x32 dilution) or epinephrine (5 ,g/ml) was used to induce release of serotonin. Release was terminated after constant stirring for 5 min at 37°C by immediate centrifugation at 7,000 x g for 1 min. The radioactivity in the supernatant was counted in a liquid scintillation counter. The release was expressed as a percentage of the total amount of radioactivity originally contained in the platelets. The effect of SB-PC and SB-PA on ADPinduced platelet aggregation is shown in Fig. 1. High concentrations of SB-PC or SB-PA (25 mM) grossly inhibited the primary aggregation induced by ADP; the latter showed more pronounced inhibitory effect. In the presence of 10 mM SB-PC or 2 mM SB-PA, nearly normal primary aggregation was observed with subsequent rapid disaggregation. The minimum concentration of SB-PC that inhibited the second-
881
882
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
ary aggregation was found to be 5 mM, whereas that of SB-PA was much lower (less than 1 mM). Similar results were obtained with epinephrine-induced platelet aggregation (Fig. 2). The effect of several concentrations of SB-PC and SB-PA on collagen-induced platelet aggregation is shown in Table 1. Maximal aggregability as well as aggregation time of collagen-induced aggregation was markedly affected both by SB-PC and SB-PA. Again SB-PA showed more pronounced inhibitory effect on collageninduced aggregation as compared with SB-PC. The effect of SB-PC or SB-PA on ['4C]serotonin release induced by epinephrine and collagen is summarized in Table 2. SB-PC at a concentration of 25 mM grossly inhibited epinephrine- or collagen-induced serotonin release from human platelets in vitro at 5 min, whereas SBPA exhibited similar inhibitory effects at 5 mM. Although there are no reports regarding the SB
-
effect of SB-PC on platelet function, it is ekpected that SB-PC would show significant inhibitory effect on platelet function because of their TABLE 1. Effect of SB-PC and SB-PA on collageninduced aggregation of human platelets Concn Concn Agent (M AgeM)
Control SB-PC
0 0.2 1.2 2.5 5.0 10.0 0.2 1.0 2.0 5.0 10.0
SB-PA
aggregability of control) (%
Maximum
ti Agtime (mi) Aggregation)
100 100 100 95 54.5 25 100 75 60.5 12.5
2.0 2.0 2.0 2.2 2.8 3.2 2.0 3.0 3.0 3.2
0
SB - PA
PC
N
"I
C I._
a
._
-E01 ;i
imM 0
1
2
3
4
min .
FIG. 1. The effect of SB-PC and SB-PA on ADP-induced platelet aggregation. Platelet aggregation studies were carried out in the presence of various concentration of SB-PC and SB-PA as described in the text. SB- PA
SB- PC
l.oOr
.E C 50 Epin.phrine 2pg/m!, -
-J
0
1
2
3
4
0
1
2
3
4 min.
FIG. 2. The effect of SB-PC and SB-PA on epinephrine-induced platelet aggregation. PRP was preincubated with various concentrations of SB-PC and SB-PA for 5 min. Platelet aggregation was determined as described in the text.
VOL. 13, 1978
TABLE 2. Effect of SB-PC or SB-PA on serotonin release from human platelets Agent
Control SB-PC SB-PA
x32
Concn
(mM)
Epinephrine, 5 ytg/ml (%)
Coagen
5 25
32.6 30.4 13.0 14.2
50.4 48.6 22.8
5 25
4.5
27.6 9.5
close structural similarities with carbenicillin. The mechanism of Pc-G-induced platelet dysfunction was studied in detail by Cazenave et al. (4), who showed that Pc-G may cause impairment of platelet function by coating the platelet surface, since it inhibited various aspects of platelet function. SB-PC and SB-PA also inhibited primary aggregation as well as secondary aggregation of platelet in the same manner as carbenicillin or Pc-G. SB-PC was also found to inhibit the ability of platelets to adhere to everted rabbit aorta (data not shown). Therefore, the mechanism of SB-PC- and SB-PA-induced inhibition of platelet aggregation appears to be identical to that of carbenicillin or Pc-G. Our interesting finding was that SB-PA, the major metabolite of SB-PC, inhibited platelet aggregation and release of serotonin from platelets in vitro. The inhibitory effect of SB-PC and SB-PA was compared on a molar basis, and SBPA was found to be much stronger than SB-PC. Following administration of high doses to human volunteers, carbenicillin was found to inhibit platelet function (3). Maximal concentration of carbenicillin reached during treatment was estimated to be 100 to 150 ,ug/ml (5). On the other hand, carbenicillin at a concentration of 1,000 ,g/ml showed no inhibitory effect on platelet aggregation in vitro (1). A similar discrepancy of in vivo and in vitro results was observed in SB-PC. The minimal concentration of SB-PC that inhibited platelet aggregation in vitro was 5 mM (2,070 ,g/ml), whereas patients receiving
883 NOTES 32 g of SB-PC per day showed markedly decreased platelet aggregation. (Maximal concentration of SB-PC in blood was approximately 100 to 150 ,ug/ml.) SB-PA, the major metabolite of SB-PC, showed marked inhibitory effect. Pc-G and carbenicillin were also known to have their penicilloic acid derivatives, although the effect of these compounds on platelet function was not examined. Data are currently not available to show the concentration of SB-PA in circulating blood in humans. In rats, however, approximately 10 to 20% of SB-PC was found to be excreted into urine as SB-PA. The fact that SB-PA showed stronger inhibition of platelet function is important, because an unexpectedly marked bleeding tendency may develop when uremic patients receive large doses of these antibiotics. Whether our results fully explain the discrepancy between in vivo and in vitro effects of SB-PC awaits further investigation. LITERATURE CITED 1. Born, G. V. R. 1962. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature (London) 194:927-929. 2. Brown, C. H., E. A. Natelson, M. W. Bradshaw, C. P. Alfrey, and T. W. Williams. 1975. Study of the effects of ticarcillin on blood coagulation and platelet function. Antimicrob. Agents Chemother. 7:652-657. 3. Brown, C. H., E. A. Natelson, M. W. Bradshaw, T. W. Williams, and C. P. Alfrey. 1974. The hemostatic defect produced by carbenicillin. N. Engl. J. Med. 291:265-270. 4. Cazenave, J. P., M. A. Guccione, M. A. Packham, and J. F. Mustard. 1977. Effects of cephalothin and penicillin G on platelet function in vitro. Br. J. Hematol.
36:135-152.
5. Gordon, D. H. 1970. Carbenicillin in renal failure. Lancet ii:422. 6. McClure, P. D., J. G. Casserly, C. Monsier, and D. Crozier. 1970. Carbenicillin-induced bleeding disorder. Lancet ii: 1307-1308. 7. Mills, D. C. B., I. A. Roff, and G. C. K. Roberts. 1968. The release of nucleotides, 5-hydroxytryptamine and enzyme from human blood platelets during aggregation. J. Physiol. 195:715-729. 8. Nakai, Y., Y. Shirakawa, T. Fujita, Z. Suzuoki, and T. Fugono. 1972. The metabolic fate of a-sulphobenzylpenicillin in rats. Xenobiotica 2:147-157.
