Life Sciences, Vol. 47, pp. 2089-2095 Printed in the U.S.A.
Pergamon Press
INHIBITION OF SEROTONIN-AMPLIFIED HUMAN PLATELET AGGREGATION BY KETANSERIN, RITANSERIN, AND THE ERGOLINE 5HT2 RECEPTOR ANTAGONISTS - LY53857, SERGOLEXOLE, AND LY237733 P. Anne McBride, J. John Mann, Esther Nimchinsky and Marlene L. Cohen* Laboratory of Psychopharmacology Cornell University Medical College New York, New York 10021 *Lilly Research Laboratories Eli Lilly and Company Lilly Corporate Center Indianapolis, Indiana 46285 (Received in final form September 27, 1990)
Summary_ Human platelets are known to possess 5HT2receptors which, when activated, amplify the aggregation response produced by other aggregating agents. Several 5HT2 receptor antagonists, including ketanserin and ritanserin, are known to antagonize serotonin-mediated aggregation of human platelets. In the present study, we document the ability of three ergoline 5HT2 receptor antagonists, LY53857, sergolexole, and LY237733, to antagonize the serotonergic component of the human platelet aggregation response. Potencies of the ergoline esters (LY53857 and sergolexole) and the ergoline amide (LY237733) to inhibit serotonin-amplified platelet aggregation responses were similar to the potencies of ketanserin and ritanserin under the conditions of our study. Furthermore, all five 5HT2 receptor antagonists were capable of fully inhibiting the serotonergic component of the platelet aggregation response. In contrast to these potent ergoline esters and amides, 1-isopropyl dihydrolysergic acid (up to 10-SM), a putative metabolite of the ergoline esters, was ineffective under these in vitro conditions. These data are consistent with the high potency of these ergolines as antagonists of 5HT2 receptors and further support the involvement of 5HT2 receptors on human platelets in the amplifying response to serotonin. Human platelets possess 5HT2 receptors which, when activated, amplify the aggregation responses produced by other aggregating agents such as adenosine diphosphate (ADP) and epinephrine. Several potent 5HT2 receptor antagonists, including the modified butyrophenones ketanserin and ritanserin, show high affinity for 5HT2 receptors on human platelets (1,2,3),inhibit serotonin-induced amplification of human platelet aggregation (3,4), and inhibit serotonin-induced increases in phosphoinositide turnover (5). Both ketanserin and ritanserin (Figure 1) show relatively high affinity for adrenergic, histaminergic, and dopaminergic receptors, in addition to their potency as 5HT2 receptor antagonists (6,7). Several other more selective 5HT2 receptor antagonists have now been identified. LY53857 was the first of a series of ergoline 5HT2 receptor antagonists that possessed high affinity at 5HT2 receptors with minimal affinity at histamine, ~tl, or dopamine receptors (8,9). LY53857 is a mixture of four isomers that have been well-characterized (10). Using this agent as a prototype for the development of potent and selective 5HT2 receptor antagonists, two additional ergoline 5HT2 receptor antagonists have been identified, sergolexole (LY281067) (11) and LY237733 (12). Both these latter ergolines are potent 5HT2 receptor antagonists 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
5HT 2 Receptor and Ergoline Antagonists
2090
Vol. 47, No. 23, 1990
C--O--CH--CH-'CH3
H~
o
0
F. . ~ ' ~
II
_ ~ N ..CHt_CH _ N ~
H:
H,
H KETANSERIN
CH1--C-CH~
O
C LY53857
~
C--OH _c.,
-,-'C H
RITANSERIN
SERGOLEXOLE
CH,--CIH~-N CH, LY237733
-=CH~
CH3_~,H,_.N.~...~ CH~ 1-1SOPROPYLDIHYDROLYSERGICACID
HG. I Structures of 5HT2 receptor antagonists possessing greater oral potency in rats as 5HT2 receptor antagonists than LY53857. Figure 1 indicates the chemical structures of these three potent 5HT2 receptor antagonists. LY53857 and sergolexole are ergoline esters whereas LY237733 is an ergoline amide. In animals, both LY53857 and sergolexole may be hydrolyzed to 1-isopropyl dihydrolysergic acid (11), a metabolite which possesses lower affinity at 5HT2 receptors in vitro than LY53857 or sergolexole (13). However, 1-isopropyl dihydrolysergic acid was found in high concentration in plasma of rats after the oral administration of both ergoline esters. In vivo studies suggested that 1isopropyl dihydrolysergic acid, although possessing lower 5HT2 receptor affinity, might contribute to the biological effects seen after LY53857 and sergolexole (13). The present study was designed to examine the relative potencies of these ergoline 5HT2 receptor antagonists at the human platelet 5HT2 receptor, and to compare the potency of these three ergoline 5HT2 receptor antagonists to 1-isopropyl dihydrolysergic acid, a putative metabolite, and to two other prototypic non-ergoline 5HT2 receptor antagonists, ritanserin and ketanserin. Methods Subjects The study was performed with blood donated by 26 subjects (12 males, 14 females) who were in excellent physical and emotional health. The subjects ranged in age from 22 to 41 years with a mean age of 27.8 + 1.2 years. Subjects were entirely drug free for at least 2 weeks prior to venipuncture (including aspirin and ibuprofen), and denied histories of substance abuse. All were non-smokers. Subjects were all hospital employees or medical students. In experiments which evaluated the inhibitory potencies of pharmacologic compounds, blood was accepted for use only if 1.0 I.tM serotonin augmented the ADP-induced platelet aggregation response by at least 50%. Blood from 6 potential donors was rejected for use because the serotonin-mediated component of the aggregation response was insufficient. In the case of 6 subjects who participated in the study, platelets were used to evaluate the inhibitory potencies of more than one drug. However, subjects were never included more than once in studies involving a given compound. We have previously established that the magnitude of the serotonin-amplified platelet aggregation response is not associated with gender or age in healthy adults (3; unpublished data).
Vol. 47, No. 23, 1990
5HT2 Receptor and Ergoline Antagonists
2091
Assay Procedures Studies of platelet 5HT2 receptor-mediated aggregation were performed by a modification of our previously published method (3). In brief, 45 ml of blood was drawn between 9:00 a.m. and 10:00 a.m. via a 19-gauge needle into a plastic syringe containing 5.0 ml of 3.8% sodium citrate. Platelet-rich plasma (PRP) was isolated by centrifugation (180 g for 15 min at 20"C). The concentration of platelets was measured and then adjusted to 2.50 x 10S/ml with autologous platelet-poor plasma. The turbidometric measurement of platelet aggregation was performed with a dual-channel aggregometer. One hundred percent light transmission was defined with autologous platelet-poor plasma. Platelet-rich plasma (0.45 ml) was incubated at 37"C in siliconized cuvettes for one minute with continuous stirring. Platelet aggregation was then induced by the addition of one of the following reagents: (1) serotonin alone; (2) a sufficient concentration of ADP to produce a standardized, threshold degree of aggregation (15-20% of maximal aggregation response); or (3) ADP at the concentration determined in Step 2 followed immediately by the addition of serotonin. The ADP concentration was 4.3 -+0.4 ~tM (range 0.6 12.0 IxM). PRP was stored in a closed container until each aliquot was used. The relationship between serotonin concentration and the magnitude of the 5HT2 receptor-mediated aggregation response was assessed with platelets from 3 subjects using concentrations ranging from 10-4M to 10-gM. A serotonin concentration of 1.0 I.tM was used in inhibitory studies since this was the lowest concentration which reliably amplified platelet aggregation induced by ADP. As is typical in humans, serotonin alone induced only negligible platelet aggregation responses in all subjects who participated in this study. Inhibition of 5HT2 receptor-mediated platelet aggregation was assessed by adding each drug to PRP two minutes before the addition of ADP and 1.0 ktM serotonin. Percent inhibition of the serotonin-mediated component of the aggregation response was determined (the response to ADP alone was subtracted from the amplified response to ADP plus serotonin before the percent inhibition was calculated). Drug concentrations spanned the range necessary to achieve zero to maximal inhibition ( a typical study included 8-10 concentrations of drug ranging between 10-SM and 10-SM). The effect of each drug on the aggregation response to ADP alone was also assessed. Since the magnitude of platelet aggregation responses declines with time, responses to ADP alone and to ADP plus 1.0 I.tM serotonin were remeasured in conjunction with the assessment of the inhibitory effect of each drug concentration. Each drug was evaluated with platelets from 6-8 subjects. Platelet aggregation responses were quantified by measurement of the area under each five minute turbidometric tracing using a computerized planimeter. To minimize experimental error, the responses to ADP plus 1.0 ~tM serotonin obtained immediately before, immediately after, and at the time of the assessment of the inhibitory effect of each drug concentration were averaged. Proximate aggregation responses to ADP alone were also averaged. Ritanserin, 1-isopropyl dihydrolysergic acid, sergolexole, and LY237733 were not water soluble at a concentration of 0.5 raM, the most concentrated of the drug solutions added to PRP. Thus, the three ergolines were dissolved in 5% methanol and ritanserin in 60% methanol plus 0.5 N HCI. Ketanserin tartrate and LY53857 were dissolved in normal (0.9%) saline. Normal saline was used in subsequent serial (1:10) dilutions of each of the compounds. Drugs were added to PRP in a volume of .0025 to .01 ml. The final concentration of methanol in PRP did not exceed 0.6%. The addition of .01 ml of 100% methanol had no effect on the magnitude of ADP-induced or serotonin-amplified platelet aggregation responses. Statistical Analyses IC50's were calculated for each platelet donor for each of the drugs by linear regression using only the coordinates which constituted the linear segment of the dose-response curves. These values were then grouped according to drug and the group means were compared by an analysis of variance at the 95% confidence level. Statistical significance was assumed when P
Pergamon Press
INHIBITION OF SEROTONIN-AMPLIFIED HUMAN PLATELET AGGREGATION BY KETANSERIN, RITANSERIN, AND THE ERGOLINE 5HT2 RECEPTOR ANTAGONISTS - LY53857, SERGOLEXOLE, AND LY237733 P. Anne McBride, J. John Mann, Esther Nimchinsky and Marlene L. Cohen* Laboratory of Psychopharmacology Cornell University Medical College New York, New York 10021 *Lilly Research Laboratories Eli Lilly and Company Lilly Corporate Center Indianapolis, Indiana 46285 (Received in final form September 27, 1990)
Summary_ Human platelets are known to possess 5HT2receptors which, when activated, amplify the aggregation response produced by other aggregating agents. Several 5HT2 receptor antagonists, including ketanserin and ritanserin, are known to antagonize serotonin-mediated aggregation of human platelets. In the present study, we document the ability of three ergoline 5HT2 receptor antagonists, LY53857, sergolexole, and LY237733, to antagonize the serotonergic component of the human platelet aggregation response. Potencies of the ergoline esters (LY53857 and sergolexole) and the ergoline amide (LY237733) to inhibit serotonin-amplified platelet aggregation responses were similar to the potencies of ketanserin and ritanserin under the conditions of our study. Furthermore, all five 5HT2 receptor antagonists were capable of fully inhibiting the serotonergic component of the platelet aggregation response. In contrast to these potent ergoline esters and amides, 1-isopropyl dihydrolysergic acid (up to 10-SM), a putative metabolite of the ergoline esters, was ineffective under these in vitro conditions. These data are consistent with the high potency of these ergolines as antagonists of 5HT2 receptors and further support the involvement of 5HT2 receptors on human platelets in the amplifying response to serotonin. Human platelets possess 5HT2 receptors which, when activated, amplify the aggregation responses produced by other aggregating agents such as adenosine diphosphate (ADP) and epinephrine. Several potent 5HT2 receptor antagonists, including the modified butyrophenones ketanserin and ritanserin, show high affinity for 5HT2 receptors on human platelets (1,2,3),inhibit serotonin-induced amplification of human platelet aggregation (3,4), and inhibit serotonin-induced increases in phosphoinositide turnover (5). Both ketanserin and ritanserin (Figure 1) show relatively high affinity for adrenergic, histaminergic, and dopaminergic receptors, in addition to their potency as 5HT2 receptor antagonists (6,7). Several other more selective 5HT2 receptor antagonists have now been identified. LY53857 was the first of a series of ergoline 5HT2 receptor antagonists that possessed high affinity at 5HT2 receptors with minimal affinity at histamine, ~tl, or dopamine receptors (8,9). LY53857 is a mixture of four isomers that have been well-characterized (10). Using this agent as a prototype for the development of potent and selective 5HT2 receptor antagonists, two additional ergoline 5HT2 receptor antagonists have been identified, sergolexole (LY281067) (11) and LY237733 (12). Both these latter ergolines are potent 5HT2 receptor antagonists 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
5HT 2 Receptor and Ergoline Antagonists
2090
Vol. 47, No. 23, 1990
C--O--CH--CH-'CH3
H~
o
0
F. . ~ ' ~
II
_ ~ N ..CHt_CH _ N ~
H:
H,
H KETANSERIN
CH1--C-CH~
O
C LY53857
~
C--OH _c.,
-,-'C H
RITANSERIN
SERGOLEXOLE
CH,--CIH~-N CH, LY237733
-=CH~
CH3_~,H,_.N.~...~ CH~ 1-1SOPROPYLDIHYDROLYSERGICACID
HG. I Structures of 5HT2 receptor antagonists possessing greater oral potency in rats as 5HT2 receptor antagonists than LY53857. Figure 1 indicates the chemical structures of these three potent 5HT2 receptor antagonists. LY53857 and sergolexole are ergoline esters whereas LY237733 is an ergoline amide. In animals, both LY53857 and sergolexole may be hydrolyzed to 1-isopropyl dihydrolysergic acid (11), a metabolite which possesses lower affinity at 5HT2 receptors in vitro than LY53857 or sergolexole (13). However, 1-isopropyl dihydrolysergic acid was found in high concentration in plasma of rats after the oral administration of both ergoline esters. In vivo studies suggested that 1isopropyl dihydrolysergic acid, although possessing lower 5HT2 receptor affinity, might contribute to the biological effects seen after LY53857 and sergolexole (13). The present study was designed to examine the relative potencies of these ergoline 5HT2 receptor antagonists at the human platelet 5HT2 receptor, and to compare the potency of these three ergoline 5HT2 receptor antagonists to 1-isopropyl dihydrolysergic acid, a putative metabolite, and to two other prototypic non-ergoline 5HT2 receptor antagonists, ritanserin and ketanserin. Methods Subjects The study was performed with blood donated by 26 subjects (12 males, 14 females) who were in excellent physical and emotional health. The subjects ranged in age from 22 to 41 years with a mean age of 27.8 + 1.2 years. Subjects were entirely drug free for at least 2 weeks prior to venipuncture (including aspirin and ibuprofen), and denied histories of substance abuse. All were non-smokers. Subjects were all hospital employees or medical students. In experiments which evaluated the inhibitory potencies of pharmacologic compounds, blood was accepted for use only if 1.0 I.tM serotonin augmented the ADP-induced platelet aggregation response by at least 50%. Blood from 6 potential donors was rejected for use because the serotonin-mediated component of the aggregation response was insufficient. In the case of 6 subjects who participated in the study, platelets were used to evaluate the inhibitory potencies of more than one drug. However, subjects were never included more than once in studies involving a given compound. We have previously established that the magnitude of the serotonin-amplified platelet aggregation response is not associated with gender or age in healthy adults (3; unpublished data).
Vol. 47, No. 23, 1990
5HT2 Receptor and Ergoline Antagonists
2091
Assay Procedures Studies of platelet 5HT2 receptor-mediated aggregation were performed by a modification of our previously published method (3). In brief, 45 ml of blood was drawn between 9:00 a.m. and 10:00 a.m. via a 19-gauge needle into a plastic syringe containing 5.0 ml of 3.8% sodium citrate. Platelet-rich plasma (PRP) was isolated by centrifugation (180 g for 15 min at 20"C). The concentration of platelets was measured and then adjusted to 2.50 x 10S/ml with autologous platelet-poor plasma. The turbidometric measurement of platelet aggregation was performed with a dual-channel aggregometer. One hundred percent light transmission was defined with autologous platelet-poor plasma. Platelet-rich plasma (0.45 ml) was incubated at 37"C in siliconized cuvettes for one minute with continuous stirring. Platelet aggregation was then induced by the addition of one of the following reagents: (1) serotonin alone; (2) a sufficient concentration of ADP to produce a standardized, threshold degree of aggregation (15-20% of maximal aggregation response); or (3) ADP at the concentration determined in Step 2 followed immediately by the addition of serotonin. The ADP concentration was 4.3 -+0.4 ~tM (range 0.6 12.0 IxM). PRP was stored in a closed container until each aliquot was used. The relationship between serotonin concentration and the magnitude of the 5HT2 receptor-mediated aggregation response was assessed with platelets from 3 subjects using concentrations ranging from 10-4M to 10-gM. A serotonin concentration of 1.0 I.tM was used in inhibitory studies since this was the lowest concentration which reliably amplified platelet aggregation induced by ADP. As is typical in humans, serotonin alone induced only negligible platelet aggregation responses in all subjects who participated in this study. Inhibition of 5HT2 receptor-mediated platelet aggregation was assessed by adding each drug to PRP two minutes before the addition of ADP and 1.0 ktM serotonin. Percent inhibition of the serotonin-mediated component of the aggregation response was determined (the response to ADP alone was subtracted from the amplified response to ADP plus serotonin before the percent inhibition was calculated). Drug concentrations spanned the range necessary to achieve zero to maximal inhibition ( a typical study included 8-10 concentrations of drug ranging between 10-SM and 10-SM). The effect of each drug on the aggregation response to ADP alone was also assessed. Since the magnitude of platelet aggregation responses declines with time, responses to ADP alone and to ADP plus 1.0 I.tM serotonin were remeasured in conjunction with the assessment of the inhibitory effect of each drug concentration. Each drug was evaluated with platelets from 6-8 subjects. Platelet aggregation responses were quantified by measurement of the area under each five minute turbidometric tracing using a computerized planimeter. To minimize experimental error, the responses to ADP plus 1.0 ~tM serotonin obtained immediately before, immediately after, and at the time of the assessment of the inhibitory effect of each drug concentration were averaged. Proximate aggregation responses to ADP alone were also averaged. Ritanserin, 1-isopropyl dihydrolysergic acid, sergolexole, and LY237733 were not water soluble at a concentration of 0.5 raM, the most concentrated of the drug solutions added to PRP. Thus, the three ergolines were dissolved in 5% methanol and ritanserin in 60% methanol plus 0.5 N HCI. Ketanserin tartrate and LY53857 were dissolved in normal (0.9%) saline. Normal saline was used in subsequent serial (1:10) dilutions of each of the compounds. Drugs were added to PRP in a volume of .0025 to .01 ml. The final concentration of methanol in PRP did not exceed 0.6%. The addition of .01 ml of 100% methanol had no effect on the magnitude of ADP-induced or serotonin-amplified platelet aggregation responses. Statistical Analyses IC50's were calculated for each platelet donor for each of the drugs by linear regression using only the coordinates which constituted the linear segment of the dose-response curves. These values were then grouped according to drug and the group means were compared by an analysis of variance at the 95% confidence level. Statistical significance was assumed when P
