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The use of ex vivo platelet aggregation to confirm the in vivo α2-adrenoreceptor antagonist effect of idazoxan in man
N. 6. MUIR, f. C. DOXEY, M. E. HAVLER,AND ]- M, CLIFFORD
The a~gr~gat~~~ of human platelets induced by adrenaline has been used as a test system tu investigate the in viva effect of the ~~-adrenoreceptor an~aganist idazoxan during initial intravenous studies with increasing doses. The ~nh~b~~~~ effect of idazoxan in vitro was confirmed; add&n of idazoxan to platelet suspensicrns prior ta adrenaline caused a competitive ~~hjb~~~on of the ag~r~~ato~ response by specific antagonism of the plateSet ~~-adr~nor~c~~tor. Fo~~~w~n~ intravenous infusions of increasing doses of idazoxan to volunteers, a dose-dependent ~nhjbjtion af the ex viva a~gregato~ response to adrenaline was observed in isolated piatefet suspensions compared to predose values, The ~nbibito~ effects of idazoxan in vivc~declined in a biphasic manner with a more rapid faff over the first hour. This reflects the kinefics of the drug in plasma and the s~rnilo~arjth~~c nature of the co~c~~trat~on-response line observed in vitro. l~t~~v~~o~s doses of 100 and 300 f*g/kg were demonstrated to be effective antagoonist doses of the platelet ol,-adrenoreceptor in healthy votunteers. Key Words:
Idazoxan; aggregation; Ptatetets; Man
ldaroxan ~RX78~~94~2Q(1,4-benzodioxanyl))-2-imidazotine hydrochl~ride~ is a research compound postulated to act as a presynaptic ~~~)-adrenoreceptor antagonist, and it has been shown to have potent, selective, and specific antagonistic effects at a2-adrenoreceptors in animals (Chapleo et al., 1981; Mouilte et al., 1981; Uoxey et al., 1983; Hannah et ;al., 1983). The aggregatary response of isolated human platelets to adrenaline is mediated primarily by an interaction with a2-adrenoreceptors (Grant and Scrutton, 1979,198O; Hsu et al., 19791, and idazoxan has been shown to be a potent competjt~ve ~~h~b~to~ of this response (Kerry and Scrutton, 2983). The present studies investigated the use of this isolated cell system as a method for d~rn~~strat~ng in vivu GYQ-adrenoreceptor a~~ag~~jsrn and far jd~nt~~~ng an effective ~*-a~tagonjstic dose level during initiaf intravenous studies with increasing doses of idazoxan.
96
N. C. Muir et al.
METHODS Reagents The acid citrate-dextrose anticoagulant (IO mM citrate) was prepared by combination of 9.3 ml 1 M trisodium citrate, 7 ml O.? M citric acid, and 4.2 g of dextrose and dilution to 100 ml with distilled water. Solutions of 25 and 250 PM adrenaline in 0.1% citric acid were prepared daily from a stock solution of 1 mM adrenaline ~Sigma), which was stored frozen. Idazoxan was obtained from the Chemical Synthesis Laboratory of Reckitt and Colman. Preparation
and Aggregation
of Platelet Suspensions
Preparation of platelet suspensions and measurement of platelet aggregation followed a previously published method (Kerry and Scrutton, 1983) with minor modifications. Blood samples were withdrawn by an indwelfing venous cannula or by slow venipuncture using a 19-ga. needle from a forearm vein. The blood was carefully run down the inside wall of a 10- or SO-ml plastic centrifuge tube containing one-tenth the blood volume of acid citrate-dextrose and was mixed by slow inversion of the capped tube. The titrated blood was centrifuged (15Og, 20 min), and the upper platelet-rich plasma (PRP) layer was carefully removed using a plastic disposable pipette (Elkay Lab. Products, Hampshire). The PRP could be stored at room temperature in a tightly capped plastic tube for up to 3 hr before deterioration in the aggregation response was observed. An aliquot (1 ml) of the remaining blood was centrifuged for 2 min in an Eppendorf benchtop microcentrifuge (10,OOOg) to obtain platelet-free plasma (PFP). Aggregation studies were carried out using a Payton Associates dual-channel aggregation module (Centronics, Croydon, Surrey) with silanized glass cuvettes and stir bars. The response was recorded on a dual~channel recorder, each channel of which was cafibrated with 0.25ml portions of PRP and PFP, setting transmittance to 15 and 95%‘ respectively (level 2, range 4). The calibration and all subsequent measurements were carried out at 37°C using a stirrer speed of 900 rpm. Samples of PRP were placed in a glass cuvette containing a magnetic stir bar and preincubated for 2 to 3 min to achieve thermal equilibrium before addition of the adrenaline stimulus (0.5-10 ~1) using a Hamilton-type syringe. The slope of the initial phase of the aggregation response was taken as the measure of platelet aggregation. The inhibitory effect of idazoxan on adrenaline-induced aggregation was examined in vitro; idazoxan was included in the PRP 1 min before the addition of adrenaline. The inhibition of the adrenaline aggregatory response produced by idazoxan was measured by comparison of the slope of the initial aggregation phase in the presence and absence of the antagonist. Clinical Study Design Five healthy adult male volunteers ranging in age from 28 to 35 years and in weight from 69 to 96 kg received single intravenous infusions of idazoxan (I or IO mglml in sterile water) at several different dose levels, ranging from 0.03 to 390 L&kg, administered on separate occasions at least 1 week apart in a dose-rising manner. The entire dose was infused over a 15 to 20-min period. Two of the five volunteers
ldazoxan and Platelet Aggregation
received each dose up to 30 pglkg. The IOO- and 300-@kg doses were administered to all five volunteers. Blood samples were removed prior to dosing (20 ml) and at 1 and 20 min after the end of infusion {IO ml) at doses up to 100 pglkg for preparation of platelet suspensions and measurement of the aggregatory response. After 300pglkg doses, samples (15 ml) were taken at 1, IO, and 20 min, and 3, 5, and 6 hr after dosing. A portion of each blood sample (5 ml) was placed in a heparinized tube, the plasma separated by centrifugation and stored deep frozen until required for determination of plasma levels of idazoxan using a specific high performance liquid chromatography (HPLC) assay method (Muir et al., 1986). The remainder was used for preparation of platelets. RESULTS In Vitro Studies The stimulation of aggregation by adrenaline in each volunteer showed an approximately linear loglo concentration-response relationship over a 0.1-2 PM adrenaline range and a reduced response at adrenaline concentrations above a certain limiting value (Figure 1). The concentration-response curves were similar in any NO IOAZOXAN
*T
1
il.01
0. IpN IOAZOXAN
e c
:;
l 1.00
0. 10 ~NALX~
C~TRATION
10.00
ccl
1cJo.00
FIGURE 1. The relationship between the primary aggregatory response of human platelets to adrenaline alone and in the presence of 0.1 PM idazoxan. Data points are single estimations using PRP from one volunteer, but are typical of those produced.
97
$8
N. C. Muir et al. one individual measured on separate occasions, but marked differences were observed between individuals in the maximum response and, to a lesser extent, in the adrenaline concentration producing maximal response. Addition of idazoxan in vitro caused a competitive antagonism in the response to adrenaline resulting in a parallel shift in the concentration-response curve (Figure I). The extent of inhibition at one adrenaline concentration was dependent upon the added idazoxan concentration, and a linear relationship between log,, idazoxan concentration and percentage inhibition could be obtained at that particular adrenaline concentration (Figure 2). Clinical Studies The minimum adrenaline concentration producing a maximal response (usually 2 or 4 PM) in control samples of PRP taken from each volunteer prior to each occasion of idazoxan dosing was used as the aggregatory stimulus to prepare an in vitro idazoxan concentration-response curve (also in control PRP) for the same individual. These idazoxan concentration-response lines again showed only slight differences for any one individual volunteer tested on separate occasions, but larger
FIGURE 2. The inhibitor effect of idazoxan on the primary a~regato~ response to adrenaline (2 pm) of human platelets. Each point represents single estimates (I = 0.999 by linear regression analysis).
ldazoxan and Platelet A~regation
99
X I~IBITION OF AGGREGATION
t
Oi 0
1
2
3 TIRE
4
AFTER EN0 OF INFUSION
5
6
I 7
WURS)
FIGURE 3. The percentage inhibition of the platelet aggregation response to an adrenaline stimulus after a 300~pg/kg idazoxan infusion. Each point represesents the mean ( 2 SEM) of the values in five volunteers. Adrenaline concentrations are as shown in Table 1.
differences occurred between individuals. The PRP prepared from blood samples taken from each volunteer after intravenous infusions of idazoxan was challenged, using the same adrenaline concentration and the percentage inhibition of the resultant ex vivo aggregatory response calculated relative to a control response to adrenaline in a sample of PRP obtained priorto dosing. No inhibition of the response to adrenaline was seen after doses of 0.03, 0.1, 0.3, or 1 pg/kg of idazoxan. One of the two volunteers receiving the 3qglkg dose showed a decreased adrenaline response 1 min after completion of the drug infusion, as did both volunteers receiving the IO- and 30qg/kg doses. By 20 min, however, the response had returned to that of the control. Following both the IOO- and 3~-~~kg idazoxan infusions, the aggregatory response to adrenaline was greatly inhibited 1 min after dosing compared to control predose values (Table I), this inhibitor effect had decreased 20 min after the end of the drug infusion. The results confirm that intravenous doses of 100 and 300 P&kg of idazoxan are effective antagonist doses at the a2-adrenoreceptor of platelets of healthy volunteers. Furthermore, the extent of the inhibition of adrenaline-induced aggregation following the high dose of idazoxan (300 pg/kg i.v.) was greater than that observed after 100 pgikg i.v. of the drug, indicating that the effects
100
N. C. Muir et al.
of the antagonist were dose related. Following the 300-pg/kg dose, the inhibitory effects of idazoxan declined in a biphasic manner with a more rapid fall in effect occurring over the first hour of dosing. Thereafter, the effects decreased more slowly, approximately 50% inhibition of aggregation being apparent 3-6 hr after dosing (Figure 3). The idazoxan concentration-response lines prepared in vitro using PRP obtained from each volunteer prior to dosing allowed quantitation of the inhibition in terms of equivalent plasma concentrations of idazoxan following an intravenous infusion of 300 pg/kg. Values obtained by this method were in relatively good agreement with plasma levels of idazoxan determined by a specific HPLC method following the administration (Figure 4) of the same intravenous dose of the antagonist. The biphasic nature of the inhibition of the platelet aggregation response to adrenaline probably reflects the distribution and elimination kinetics of the drug in plasma and the semilogarithmic nature of the concentration-response line observed in vitro leading to significant inhibition remaining at later time points when the idazoxan plasma levels have declined below 50 ng/ml.
500
IOAZOXAN CNWWLI EQUIVALENTS BY PLATELET AGGREG.
IDAZ~~N~~/ML) --e-r*-
400
p0 ?5 f x I3 ;: 200
100
0
j
.
i
. TM
4
.
i
AFTER EN0 OF INFlJSJON
.
i
.
B-y--i
G+OURS)
FIGURE 4. The calculated idazoxan equivalents by platelet aggregation and idazoxan plasma levels by HPLC after a 300-p&kg fusion. Each point represents the mean ( rf:SEM) of the values in five volunteers. Adrenaline concentrations are as shown in Table 1.
ldazoxan and Platelet Aggregation TABLE 1 The Percentage Inhibition of the Primary Aggregatory Response to Adrenaline Human Platelets at 1 and 20 Minutes After Intravenous Infusions of ldazoxan ADRENALINE CONC.
DOSAGE (?m )LG*KG-I)
VOL
@MY
1 MIN
20 MIN
1 2 3 4 5 Mean SEM
2 1 2 2 4 -
63 91 85 51 70 72 k 7.3
16 53 39 49 41 40 26.4
B Adrenaline concentrations prior to dosing (see text).
ADRENALINE CONC.
in
DOSAGE (300 @‘KC-‘)
(ILMY
1 MIN
20 MIN
2 2 2 4 4
88 94 91 79 96 89 23.0
70 77 72 49 76 69 25.1
-
employed were the minimum concentration
producing maximal response
DISCUSSION In vitro idazoxan caused a competitive inhibition of the primary aggregation response to adrenaline in platelet suspensions from human volunteers. The results were in agreement with previous observations (Kerry and Scrutton, 1983) and have been attributed to specific antagonism by idazoxan of platelet az-adrenoreceptors. Following the intravenous infusion of idazoxan to volunteers, a dose-dependent inhibition of the ex vivo aggregatory response to adrenaline was observed in isolated platelet suspensions compared to control predose samples. The extent of inhibition reflected the concentration of drug in plasma as measured by a specific HPLC assay, but considerable inhibition of the aggregatory response was still seen at later times after the highest dose (300 Pg/kg), when plasma levels of drug had declined significantly, due to the nature of the concentration-response curve. The results demonstrate that intravenous doses of 100 and 300 pgfkg of idazoxan effectively antagonize the aggregatory effects of adrenaline on the platelet o12-adrenoreceptors of healthy volunteers. Subsequent studies in healthy volunteers have demonstrated that there is a good correlation between the effective c+-antagonist doses of idazoxan determined using inhibition of adrenaline-induced platelet aggregation and other systems. Clifford et al. (1982) demonstrated that intravenous doses of 100 and 300 pg/kg of idazoxan antagonized the sedation, hypotension, and dry mouth produced by clonidine in healthy volunteers. Similarly, Brown et al. (1985) demonstrated that idazoxan (200 pg/kg i.v.) antagonized the elevations in growth hormone produced by guanfacine. Finally, Elliott et al. (1984) found that idazoxan (200 kg/kg i.v.) antagonized the pressor effects of ~-methyl-noradrenaline &-agonist) without markedly affecting phenylephrine (~~-agonist) responses. inhibition of platelet aggregation induced by a fixed dose of adrenaline has been successfully used to demonstrate both in vitro and in vivo antagonistic effects of idazoxan at c+-adrenoreceptors. This approach has limitations in that it may mask
101
102
N. C. Muir et al. low levels of inhibition if supramaximal doses of adrenaline are employed or produce variable results if the dose of adrenaline occupies different positions on the dose-response curve. In the present studies, the lowest concentration of adrenaline producing a maximal response was used, since it ensured reproducible control responses and allowed measurement of the antagonistic effect over a wide range. This ex vivo technique should prove valuable for the identification of effective antagonist doses for this class of compound particularly in dose-ranging studies in healthy volunteers in which it is not desirable to inject a challenge drug (e.g., clonidine). Subsequent studies in healthy volunteers in which a variety of systemic challenges have been used confirmed that the effective c+antagonist dose range for idazoxan is the same as that established using adrenaline-induced aggregation.
REFERENCES Brown MJ, Struthers AD, Burrin ]M, DiSilvio L, Brown DC (1985) The physiological and pharmacological role of presynaptic a- and B-adrenoreceptors in man. Br J C/in Pharmacol2O:M9658. Chapleo CB, Doxey JC, Myers PL, Roach AG (1981) RX781094, a new potent, selective antagonist of cy,-adrenoreceptors. Br ] Pharmacol74:842P. Clifford JM, Day MD, Orwin fM (1982) Reversal of clonidine induced miosis by the az-adrenoreceptor antagonist RX781094. Br ] Clin Pharmacol 14:99-101. Doxey JC, Roach AG, Smith CFC (1983) Studies on RX781094: a selective, potent and specific antag onist of a,-adrenoreceptors. Br J Pharmacol 78:489-505. Elliott HL, iones CR, Vincent J, Lawrie C, Reid JL (1984) The alpha adrenoreceptor antagonist properties of idazoxan in normal subjects. Clin Pharmacoi Ther 36:190-196. Grant JA, Scrutton MC (1979) Novel a2-adrenoreceptors primarily responsible for inducing platelet aggregation. Nature 277:659-661. Grant IA, Scrutton MC (1980) Interaction
of selec-
tive a-adrenoreceptor agonists and antagonists with human and rabbit platelets. BrJ Pharmacol 71:121-134. Hannah JAM, Hamilton CA, Reid JL (1983) RX781094: a new potent a,-adrenoreceptor antagonist: in vivo and in vitro studies in the rabbit. Naunyn Schmiedebergs Arch Pharmacol 322~221-227. Hsu CH, Knapp DR, Halushka PV (1979) The effects of alpha-adrenergic agents on human platelet aggregation. 1 Pharmacof Exp Ther 208:366-370. Kerry R, Scrutton MC (1983) Effects of 781094, a new selective cu2-adrenoreceptor antagonist, on the aggregatory responses of human blood platelets and on binding of [‘Hldihydroergocryptine to these cells. Br J Pharmacol79:401-407. Mouille P, Dabire H, Fournier B, Schmitt H (1981) A further attempt to characterize the a*-adrenoreceptor blocking properties of (imidazolyl-2-j2-benzodioxane I:4 (170 150) in pithed rats. Eur I Pharmacol73:367-370. Muir NC, Lloyd-Jones JG, Nichols ID, Clifford JM (1986) The pharmacokinetics after intravenous and oral administration in man of the cu2-adrenareceptor antagonist idazoxan (RX781094) Eur / Clin Pharmacol29: 743-745.
The a~gr~gat~~~ of human platelets induced by adrenaline has been used as a test system tu investigate the in viva effect of the ~~-adrenoreceptor an~aganist idazoxan during initial intravenous studies with increasing doses. The ~nh~b~~~~ effect of idazoxan in vitro was confirmed; add&n of idazoxan to platelet suspensicrns prior ta adrenaline caused a competitive ~~hjb~~~on of the ag~r~~ato~ response by specific antagonism of the plateSet ~~-adr~nor~c~~tor. Fo~~~w~n~ intravenous infusions of increasing doses of idazoxan to volunteers, a dose-dependent ~nhjbjtion af the ex viva a~gregato~ response to adrenaline was observed in isolated piatefet suspensions compared to predose values, The ~nbibito~ effects of idazoxan in vivc~declined in a biphasic manner with a more rapid faff over the first hour. This reflects the kinefics of the drug in plasma and the s~rnilo~arjth~~c nature of the co~c~~trat~on-response line observed in vitro. l~t~~v~~o~s doses of 100 and 300 f*g/kg were demonstrated to be effective antagoonist doses of the platelet ol,-adrenoreceptor in healthy votunteers. Key Words:
Idazoxan; aggregation; Ptatetets; Man
ldaroxan ~RX78~~94~2Q(1,4-benzodioxanyl))-2-imidazotine hydrochl~ride~ is a research compound postulated to act as a presynaptic ~~~)-adrenoreceptor antagonist, and it has been shown to have potent, selective, and specific antagonistic effects at a2-adrenoreceptors in animals (Chapleo et al., 1981; Mouilte et al., 1981; Uoxey et al., 1983; Hannah et ;al., 1983). The aggregatary response of isolated human platelets to adrenaline is mediated primarily by an interaction with a2-adrenoreceptors (Grant and Scrutton, 1979,198O; Hsu et al., 19791, and idazoxan has been shown to be a potent competjt~ve ~~h~b~to~ of this response (Kerry and Scrutton, 2983). The present studies investigated the use of this isolated cell system as a method for d~rn~~strat~ng in vivu GYQ-adrenoreceptor a~~ag~~jsrn and far jd~nt~~~ng an effective ~*-a~tagonjstic dose level during initiaf intravenous studies with increasing doses of idazoxan.
96
N. C. Muir et al.
METHODS Reagents The acid citrate-dextrose anticoagulant (IO mM citrate) was prepared by combination of 9.3 ml 1 M trisodium citrate, 7 ml O.? M citric acid, and 4.2 g of dextrose and dilution to 100 ml with distilled water. Solutions of 25 and 250 PM adrenaline in 0.1% citric acid were prepared daily from a stock solution of 1 mM adrenaline ~Sigma), which was stored frozen. Idazoxan was obtained from the Chemical Synthesis Laboratory of Reckitt and Colman. Preparation
and Aggregation
of Platelet Suspensions
Preparation of platelet suspensions and measurement of platelet aggregation followed a previously published method (Kerry and Scrutton, 1983) with minor modifications. Blood samples were withdrawn by an indwelfing venous cannula or by slow venipuncture using a 19-ga. needle from a forearm vein. The blood was carefully run down the inside wall of a 10- or SO-ml plastic centrifuge tube containing one-tenth the blood volume of acid citrate-dextrose and was mixed by slow inversion of the capped tube. The titrated blood was centrifuged (15Og, 20 min), and the upper platelet-rich plasma (PRP) layer was carefully removed using a plastic disposable pipette (Elkay Lab. Products, Hampshire). The PRP could be stored at room temperature in a tightly capped plastic tube for up to 3 hr before deterioration in the aggregation response was observed. An aliquot (1 ml) of the remaining blood was centrifuged for 2 min in an Eppendorf benchtop microcentrifuge (10,OOOg) to obtain platelet-free plasma (PFP). Aggregation studies were carried out using a Payton Associates dual-channel aggregation module (Centronics, Croydon, Surrey) with silanized glass cuvettes and stir bars. The response was recorded on a dual~channel recorder, each channel of which was cafibrated with 0.25ml portions of PRP and PFP, setting transmittance to 15 and 95%‘ respectively (level 2, range 4). The calibration and all subsequent measurements were carried out at 37°C using a stirrer speed of 900 rpm. Samples of PRP were placed in a glass cuvette containing a magnetic stir bar and preincubated for 2 to 3 min to achieve thermal equilibrium before addition of the adrenaline stimulus (0.5-10 ~1) using a Hamilton-type syringe. The slope of the initial phase of the aggregation response was taken as the measure of platelet aggregation. The inhibitory effect of idazoxan on adrenaline-induced aggregation was examined in vitro; idazoxan was included in the PRP 1 min before the addition of adrenaline. The inhibition of the adrenaline aggregatory response produced by idazoxan was measured by comparison of the slope of the initial aggregation phase in the presence and absence of the antagonist. Clinical Study Design Five healthy adult male volunteers ranging in age from 28 to 35 years and in weight from 69 to 96 kg received single intravenous infusions of idazoxan (I or IO mglml in sterile water) at several different dose levels, ranging from 0.03 to 390 L&kg, administered on separate occasions at least 1 week apart in a dose-rising manner. The entire dose was infused over a 15 to 20-min period. Two of the five volunteers
ldazoxan and Platelet Aggregation
received each dose up to 30 pglkg. The IOO- and 300-@kg doses were administered to all five volunteers. Blood samples were removed prior to dosing (20 ml) and at 1 and 20 min after the end of infusion {IO ml) at doses up to 100 pglkg for preparation of platelet suspensions and measurement of the aggregatory response. After 300pglkg doses, samples (15 ml) were taken at 1, IO, and 20 min, and 3, 5, and 6 hr after dosing. A portion of each blood sample (5 ml) was placed in a heparinized tube, the plasma separated by centrifugation and stored deep frozen until required for determination of plasma levels of idazoxan using a specific high performance liquid chromatography (HPLC) assay method (Muir et al., 1986). The remainder was used for preparation of platelets. RESULTS In Vitro Studies The stimulation of aggregation by adrenaline in each volunteer showed an approximately linear loglo concentration-response relationship over a 0.1-2 PM adrenaline range and a reduced response at adrenaline concentrations above a certain limiting value (Figure 1). The concentration-response curves were similar in any NO IOAZOXAN
*T
1
il.01
0. IpN IOAZOXAN
e c
:;
l 1.00
0. 10 ~NALX~
C~TRATION
10.00
ccl
1cJo.00
FIGURE 1. The relationship between the primary aggregatory response of human platelets to adrenaline alone and in the presence of 0.1 PM idazoxan. Data points are single estimations using PRP from one volunteer, but are typical of those produced.
97
$8
N. C. Muir et al. one individual measured on separate occasions, but marked differences were observed between individuals in the maximum response and, to a lesser extent, in the adrenaline concentration producing maximal response. Addition of idazoxan in vitro caused a competitive antagonism in the response to adrenaline resulting in a parallel shift in the concentration-response curve (Figure I). The extent of inhibition at one adrenaline concentration was dependent upon the added idazoxan concentration, and a linear relationship between log,, idazoxan concentration and percentage inhibition could be obtained at that particular adrenaline concentration (Figure 2). Clinical Studies The minimum adrenaline concentration producing a maximal response (usually 2 or 4 PM) in control samples of PRP taken from each volunteer prior to each occasion of idazoxan dosing was used as the aggregatory stimulus to prepare an in vitro idazoxan concentration-response curve (also in control PRP) for the same individual. These idazoxan concentration-response lines again showed only slight differences for any one individual volunteer tested on separate occasions, but larger
FIGURE 2. The inhibitor effect of idazoxan on the primary a~regato~ response to adrenaline (2 pm) of human platelets. Each point represents single estimates (I = 0.999 by linear regression analysis).
ldazoxan and Platelet A~regation
99
X I~IBITION OF AGGREGATION
t
Oi 0
1
2
3 TIRE
4
AFTER EN0 OF INFUSION
5
6
I 7
WURS)
FIGURE 3. The percentage inhibition of the platelet aggregation response to an adrenaline stimulus after a 300~pg/kg idazoxan infusion. Each point represesents the mean ( 2 SEM) of the values in five volunteers. Adrenaline concentrations are as shown in Table 1.
differences occurred between individuals. The PRP prepared from blood samples taken from each volunteer after intravenous infusions of idazoxan was challenged, using the same adrenaline concentration and the percentage inhibition of the resultant ex vivo aggregatory response calculated relative to a control response to adrenaline in a sample of PRP obtained priorto dosing. No inhibition of the response to adrenaline was seen after doses of 0.03, 0.1, 0.3, or 1 pg/kg of idazoxan. One of the two volunteers receiving the 3qglkg dose showed a decreased adrenaline response 1 min after completion of the drug infusion, as did both volunteers receiving the IO- and 30qg/kg doses. By 20 min, however, the response had returned to that of the control. Following both the IOO- and 3~-~~kg idazoxan infusions, the aggregatory response to adrenaline was greatly inhibited 1 min after dosing compared to control predose values (Table I), this inhibitor effect had decreased 20 min after the end of the drug infusion. The results confirm that intravenous doses of 100 and 300 P&kg of idazoxan are effective antagonist doses at the a2-adrenoreceptor of platelets of healthy volunteers. Furthermore, the extent of the inhibition of adrenaline-induced aggregation following the high dose of idazoxan (300 pg/kg i.v.) was greater than that observed after 100 pgikg i.v. of the drug, indicating that the effects
100
N. C. Muir et al.
of the antagonist were dose related. Following the 300-pg/kg dose, the inhibitory effects of idazoxan declined in a biphasic manner with a more rapid fall in effect occurring over the first hour of dosing. Thereafter, the effects decreased more slowly, approximately 50% inhibition of aggregation being apparent 3-6 hr after dosing (Figure 3). The idazoxan concentration-response lines prepared in vitro using PRP obtained from each volunteer prior to dosing allowed quantitation of the inhibition in terms of equivalent plasma concentrations of idazoxan following an intravenous infusion of 300 pg/kg. Values obtained by this method were in relatively good agreement with plasma levels of idazoxan determined by a specific HPLC method following the administration (Figure 4) of the same intravenous dose of the antagonist. The biphasic nature of the inhibition of the platelet aggregation response to adrenaline probably reflects the distribution and elimination kinetics of the drug in plasma and the semilogarithmic nature of the concentration-response line observed in vitro leading to significant inhibition remaining at later time points when the idazoxan plasma levels have declined below 50 ng/ml.
500
IOAZOXAN CNWWLI EQUIVALENTS BY PLATELET AGGREG.
IDAZ~~N~~/ML) --e-r*-
400
p0 ?5 f x I3 ;: 200
100
0
j
.
i
. TM
4
.
i
AFTER EN0 OF INFlJSJON
.
i
.
B-y--i
G+OURS)
FIGURE 4. The calculated idazoxan equivalents by platelet aggregation and idazoxan plasma levels by HPLC after a 300-p&kg fusion. Each point represents the mean ( rf:SEM) of the values in five volunteers. Adrenaline concentrations are as shown in Table 1.
ldazoxan and Platelet Aggregation TABLE 1 The Percentage Inhibition of the Primary Aggregatory Response to Adrenaline Human Platelets at 1 and 20 Minutes After Intravenous Infusions of ldazoxan ADRENALINE CONC.
DOSAGE (?m )LG*KG-I)
VOL
@MY
1 MIN
20 MIN
1 2 3 4 5 Mean SEM
2 1 2 2 4 -
63 91 85 51 70 72 k 7.3
16 53 39 49 41 40 26.4
B Adrenaline concentrations prior to dosing (see text).
ADRENALINE CONC.
in
DOSAGE (300 @‘KC-‘)
(ILMY
1 MIN
20 MIN
2 2 2 4 4
88 94 91 79 96 89 23.0
70 77 72 49 76 69 25.1
-
employed were the minimum concentration
producing maximal response
DISCUSSION In vitro idazoxan caused a competitive inhibition of the primary aggregation response to adrenaline in platelet suspensions from human volunteers. The results were in agreement with previous observations (Kerry and Scrutton, 1983) and have been attributed to specific antagonism by idazoxan of platelet az-adrenoreceptors. Following the intravenous infusion of idazoxan to volunteers, a dose-dependent inhibition of the ex vivo aggregatory response to adrenaline was observed in isolated platelet suspensions compared to control predose samples. The extent of inhibition reflected the concentration of drug in plasma as measured by a specific HPLC assay, but considerable inhibition of the aggregatory response was still seen at later times after the highest dose (300 Pg/kg), when plasma levels of drug had declined significantly, due to the nature of the concentration-response curve. The results demonstrate that intravenous doses of 100 and 300 pgfkg of idazoxan effectively antagonize the aggregatory effects of adrenaline on the platelet o12-adrenoreceptors of healthy volunteers. Subsequent studies in healthy volunteers have demonstrated that there is a good correlation between the effective c+-antagonist doses of idazoxan determined using inhibition of adrenaline-induced platelet aggregation and other systems. Clifford et al. (1982) demonstrated that intravenous doses of 100 and 300 pg/kg of idazoxan antagonized the sedation, hypotension, and dry mouth produced by clonidine in healthy volunteers. Similarly, Brown et al. (1985) demonstrated that idazoxan (200 pg/kg i.v.) antagonized the elevations in growth hormone produced by guanfacine. Finally, Elliott et al. (1984) found that idazoxan (200 kg/kg i.v.) antagonized the pressor effects of ~-methyl-noradrenaline &-agonist) without markedly affecting phenylephrine (~~-agonist) responses. inhibition of platelet aggregation induced by a fixed dose of adrenaline has been successfully used to demonstrate both in vitro and in vivo antagonistic effects of idazoxan at c+-adrenoreceptors. This approach has limitations in that it may mask
101
102
N. C. Muir et al. low levels of inhibition if supramaximal doses of adrenaline are employed or produce variable results if the dose of adrenaline occupies different positions on the dose-response curve. In the present studies, the lowest concentration of adrenaline producing a maximal response was used, since it ensured reproducible control responses and allowed measurement of the antagonistic effect over a wide range. This ex vivo technique should prove valuable for the identification of effective antagonist doses for this class of compound particularly in dose-ranging studies in healthy volunteers in which it is not desirable to inject a challenge drug (e.g., clonidine). Subsequent studies in healthy volunteers in which a variety of systemic challenges have been used confirmed that the effective c+antagonist dose range for idazoxan is the same as that established using adrenaline-induced aggregation.
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