Br. J. clin. Pharmac. (1991), 32, 181-186
A D 0 N I S 030652519100165V
The pharmacodynamics and pharmacokinetics of a novel thromboxane receptor blocking drug vapiprost (GR32191) after single intravenous doses in healthy subjects M. THOMAS', R. J. KEERY2, M. K. CHARTER', N. L. SCULLY3, J. E. CHILTON' & P. LUMLEY2 Departments of 'Clinical Pharmacology, 'Peripheral Pharmacology and 3Biochemical Pharmacology, Glaxo Group Research, Ware, Herts SG12 ODP
1 The effect of single, serially increasing, intravenous doses of a specific thromboxane receptor blocking drug, vapiprost, upon platelet aggregation induced ex vivo by the thromboxane A2 mimetic, U-46619, was examined in 12 healthy males. 2 Subjects received either 1 (n = 1 subject), 2 (n = 6), 3 (n = 2), or 4 (n = 3) administrations of vapiprost within the dose range 0.125 to 16 mg and, in random order, placebo on separate study days at intervals of at least 48 h. 3 All doses of vapiprost produced an immediate antagonism of U-46619-induced platelet aggregation in whole blood. Both the magnitude and duration of the rightward displacement of the concentration-effect curves increased with dose. Although lower doses produced parallel displacements of these curves, with the higher doses the maximum response to U-46619 was reduced such that 50% platelet aggregation was not achieved. After the 16 mg dose of vapiprost, virtually complete suppression of platelet aggregation (up to a concentration of 30 pRM) was seen. This degree of inhibition was maintained for 2 h after dosing, following which there was a gradual return to pre-dose U-46619 sensitivity over the next 12 to 24 h. U-46619-induced platelet aggregation was unaffected by placebo. 4 Across the dose range, vapiprost was rapidly cleared from plasma, with an elimination half-life of 69-84 min and a plasma clearance of 514-721 ml min-'. 5 Vapiprost had no effect upon pulse rate, blood pressure, the ECG, peak expiratory flow rate, nor were drug-related changes in routine laboratory screens seen. No local or systemic side-effects were reported.
Keywords vapiprost thromboxane receptor blockade platelet aggregation pharmacokinetics
U-46619
Introduction
Vapiprost
( [1R-[1ot (Z) ,2pf,3p,5a]]-(+)-7-[5-([1,1 '-bi-
& Lumley 1990). Aggregation produced through nonTxA2-dependent mechanisms, such as that induced by ADP is, however, unaffected. In addition to blocking the platelet TxA2 receptor, vapiprost has also been shown to block the receptor on vascular smooth muscle in man (Maconochie et al., 1988). Upon repeat oral dosing, the drug produces a progressive, cumulative blockade of TxA2 receptor-mediated platelet aggregation which occurs in the absence of any parallel increase in its plasma concentration (Thomas & Lumley, 1990). This phenomenon may be due to a persistent occupation of the TxA2 receptor by vapiprost or to a reduction, by an unknown mechanism, in receptor number (Armstrong
phenyl] - 4 - ylmethoxy) - 3 - hydroxy - 2 - (1 - piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride salt) has been shown to be a potent and specific thromboxane (Tx)A2 receptor blocking drug in vitro using platelets and both vascular and airways smooth muscle preparations from different species (Lumley et al., 1989b). The drug is active in various experimental models of thrombosis (Lumley et al., 1990). When administered orally to healthy subjects, vapiprost produces a rapid, profound and sustained blockade of platelet aggregation ex vivo to the TxA2 mimetic U-46619 (lla, 9a epoxymethanoPGH2) and collagen (Lumley et al., 1989a; Thomas
Correspondence: Dr M. Thomas, Department of Clinical Pharmacology, Glaxo Group Research, Park Road, Ware, Herts SG12 ODP
181
182
M. Thomas et al.
et al., 1990). In all studies performed with vapiprost in man, the drug has been well tolerated and free of serious adverse effects (Thomas & Lumley, 1990). The potential clinical applications for a thromboxane receptor blocking drug include the treatment of thrombotic events and occlusive vascular disease. As vapiprost will be used to prevent or treat chronic disease, it will be given long term by mouth and all of our previous human pharmacological studies have been conducted using this route. However, there may well be circumstances in clinical practice where intravenous administration is indicated. For example, the intravenous route might be preferred in patients in shock or coma in whom absorption from the gastrointestinal tract is likely to be erratic or unreliable. Likewise, the intravenous route offers advantages when a rapid anti-thrombotic action is thought to be desirable, e.g. in the prevention of re-occlusion and re-stenosis of coronary arteries following angioplasty or thrombolysis in acute myocardial infarction. The aims of the present study were to examine for the first time in man the effects of ascending doses of vapiprost given by intravenous infusion upon platelet aggregation induced by U-46619, to make an initial assessment of safety and tolerability and to assess the pharmacokinetic properties of the drug by this route.
Methods
Subjects Twelve healthy male subjects, mean age 33 years (range 24-42 years), took part in a placebo-controlled, crossover study which had been approved by the Glaxo Group Research Ethics Review Committee. Based on the results of a medical history, physical examination, routine laboratory safety screens, and an ECG examination, subjects were shown to be fit and well before admission to the study. Subjects with a history of spontaneous bleeding or any other symptoms suggestive of a bleeding disorder or peptic ulceration, were specifically excluded from the study. Also excluded from the study were smokers and subjects who had taken other drugs, including aspirin or aspirin-like drugs within 14 days of the start of the study. No alcohol or exercise was allowed for 24 h before and after dosing and no tea, coffee or foods containing caffeine were consumed on a study day. After the nature and purpose of the study had been explained to the subjects, they gave consent in writing.
Clinical procedure
Subjects fasted overnight and reported to the laboratory the following morning bringing with them an early morning urine specimen for routine testing. A cannula (Wallace-Y-Can) was inserted into a vein in each arm. One cannula was used to administer the treatment and was removed at the end of the infusion. The other cannula was used to obtain blood samples and was kept patent between sampling times by flushing through with normal saline. Duplicate blood samples were drawn for platelet aggregation studies and samples were obtained
for drug assay, haematological and biochemical safety screens. With subjects resting supine, pulse rate and blood pressure were measured until steady using an automated sphygmomanometer (Narco Scientific) and a baseline of three readings at 5 min intervals was then obtained. At the times pulse rate and blood pressure were measured, 12-lead ECGs were also recorded (Marquette Cardiograph) and immediately afterwards measurements of peak expiratory flow rate (PEFR) were made using a Wrights Peak Flow Meter. When control measurements and blood samples had been taken, subjects received either drug or placebo administered intravenously over 10 min as a 1 ml min-1 infusion (2 ml min-1 for 16 mg dose) under ECG control. At the end of the infusion (time 0), further measurements of pulse rate, blood pressure and PEFR were made, an ECG was recorded and blood samples for aggregation studies and plasma drug assay were obtained. Following discontinuation of the infusion, monitoring continued as follows: pulse rate and blood pressure at, 5, 10, 15, 20, 25, 30, 40, 50 and 60 min, 1.5, 2, 3 and 4 h; ECG at 30 and 60 min, 2, 3 and 4 h; PEFR at 10, 20, 30 and 60 min, 2, 3 and 4 h. Further blood samples for platelet studies were drawn after 30 min, 1, 2, 3, 4, 8, 12 h and in addition at 24 h after the 8 and 16 mg doses, and for drug assay at 5, 10, 20, 30, 40, 50 and 60 min, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 12 h. Subjects were allowed up after 2 h, but lay down again 10 min before the 3 and 4 h cardiovascular measurements. The second cannula was removed after the 12 h blood samples had been taken and the 24 h sample was obtained by direct venepuncture. Further laboratory safety tests were performed 48 h after a dose of vapiprost or placebo and 7 days after the last study day. Vapiprost was formulated as a 1 mg (base) ml-1 solution using ,B-cyclodextrin to improve solubility in the molar ratio of 1:1.4 and isotonicity was adjusted with mannitol (5 % w/v). The pH of the solution was 6.0. The stock solution of vapiprost was diluted with normalsaline to adjust for dose. The following doses of vapiprost were administered in ascending order of magnitude at intervals of at least 48 h (0.125 mg (n = 2 subjects)), 0.25 mg (n = 2), 0.5 mg (n = 3), 1 mg (n = 4), 2 mg (n = 4), 4 mg (n = 3), 8 mg (n = 6) and 16 mg (n = 6), while each subject also received, in random order, placebo (P-cyclodextrin) alone on another occasion. Subjects received between 1 and 4 administrations of vapiprost. Assessment ofplatelet aggregation
Blood samples for platelet studies were anticoagulated with trisodium citrate (final concentration 12.9 mM) and transferred immediately to the laboratory for analysis. U-46619 concentration-effect curves were constructed in whole blood by counting single platelets using a Becton Dickinson Ultra-Flo 100 platelet counter (Lumley & Humphrey, 1981; Thomas & Lumley, 1990). Measurements of aggregation were made in the presence of aspirin (2 mM) to inhibit endogenous production of prostaglandins. The concentration of U-46619 required to produce a 50% reduction in platelet count (EC50) was determined before and then at each time point after dosing. When less than 50% aggregation was obtained,
Antagonism ofplatelet aggregation by i. v. vapiprost the EC50 post-treatment value was taken as 30 FLM, the highest concentration of U-46619 used.
183
6. The plasma clearance of vapiprost (CL) was calculated from CL = D/AUC.
Analysis of vapiprost in plasma Results
Vapiprost was measured in plasma by high performance liquid chromatography (h.p.l.c.) after solid-phase extraction on advanced automated sample processor (AASP) C8 extraction cartridges. The extract was introduced onto a 5 p,M PLRP-S column (Polymer Laboratories) by an AASP, and vapiprost was detected by fluorescence emission at 310 nm after excitation at 254 nm. The working range of the assay was 5-150 ng ml-1 vapiprost when assaying 0.5 ml of the plasma sample. The precision of the assay at its lower limit (5 ng ml-') was 3.7% (n = 6). Samples containing vapiprost at concentrations in excess of 150 ng ml- 1 were assayed after dilution with pooled control plasma. Pharmacokinetic calculations
Platelet aggregation U-46619 induced a concentration-related platelet aggregation in control blood samples from all subjects. Geometric mean EC50 values are shown in Table 1. Vapiprost (0.125 to 16 mg) produced a dose-related antagonism of U-46619-induced platelet aggregation in terms of both the magnitude and duration of the blockade (Table 1). Doses of 0.125 to 1 mg, produced parallel rightward displacements of the U-46619 concentrationeffect curves for platelet aggregation. The mean response in four subjects who received 1 mg is shown in Figure la.
100
The maximum measured concentration of vapiprost in plasma (Cmax) was obtained by inspection of the data. If there were sufficient data to determine adequately an elimination phase, then the following parameters were also derived: 1. The elimination rate constant for vapiprost in plasma (Xj). The number of points to be included in the elimination phase was determined by inspection of a loglinear plot of plasma concentration against time. The rate constant was determined by linear least-squares regression using the logarithmically transformed points. 2. The area under the curve of plasma vapiprost concentration vs time, extrapolated to infinite time (AUC). The portion of the area between times t, and tj+1 was calculated using the logarithmic trapezoidal rule if Ci > Cj,+1 > 0, and the linear trapezoidal rule otherwise, where C, is the concentration measured at time ti. The area was extrapolated to infinite time using C,/XJ, where CQ is the last measured plasma concentration. The extrapolated portion of the profile was checked for consistency with any concentrations below the limit of
40
A-'!e ;9, t.i s {E.. fi +~.°* k.~j /4 !# X
.A .A Aht. w I
Bllh;ft;;
?°
Figure 1 Platelet aggregation U-46619 concentration-effect curves ex vivo before and following administration of intravenous doses of vapiprost; a) 1 mg, b) 16 mg or c) placebo (for subjects receiving 16 mg dose). U-46619-induced aggregation was determined prior to dosing (0), immediately after dosing (@), and at 1 (E), 2 (-), 4 (A) and 6 (v) h (both doses), and at 8 (*), 12 (V) and 24 (A) h, 16 mg dose only. Curves are the mean ± s.e. mean from four subjects in (a) and from the same six subjects in (b) and (c).
184
M. Thomas et al.
Table 1 Geometric mean EC50 values (range) for U-46619-induced platelet aggregation ex vivo before and after intravenous infusion of increasing doses of vapiprost or placebo U-46619 EC50 values (>LM) Time post-infusion (h) Dose (mg)
ControP
0.125 (n = 2)
0.25 (n = 2)
0.4
(0.3-0.5)
0.50 (n = 3)
0.3
4.8
(0.2-0.4)
(2.5-8.2)
1.0 (n = 4)
0.4
6.5
(0.3-0.6)
(5.6-9.4)
(1.9-3.9)
2.0 (n = 4)
0.3 (0.1-1.4)
8.5
5.0 (1.9-19.2)
4.0
3.7
(5.0-30)
(2.0-6.2)
(2.0-5.5)
4.0 (n = 3)
0.2
25.1
8.4
5.7
5.6
3.4
(0.2-0.3)
(17.5-30)
(4.8-14.6)
(3.2-9.5)
(3.8-8.0)
(1.9-5.4)
8.0 (n = 6)
0.2
(0.2-0.5)
30.0c (_)d
16.0 (n = 6)
0.2 (0.1-0.3)
30.0 (-)
Placebob (n = 11)
0.3
0.3
(0.2-0.5)
(0.2-0.5)
0
0.5
1
2
3
4
6
8
12
24
0.2
1.1
0.4 (0.3-0.6)
0.4 (0.2-0.8)
NT
NT
NT
(0.2-0.6)
0.4 (0.3-0.6)
NT
(0.8-1.4)
0.4 (0.3-0.6)
0.4
(0.2-0.2)
2.5 (2.4-2.6)
1.2 (1.1-1.3)
1.0
1.2 (0.7-1.9)
0.6
NT
NT
NT
(0.5-0.8)
0.4 (0.3-0.6)
NT
(0.6-1.5) 2.4
1.9
0.6
0.5
NT
NT
(1.0-3.9)
1.5 (0.7-2.9)
1.2
(1.3-4.5)
(0.7-1.9)
(0.5-0.8)
(0.5-0.5)
4.2
2.8
0.6
NT
(0.8-1.8)
(0.4-0.8)
0.5 (0.4-0.6)
NT
(2.0-5.0)
2.0 (1.2-2.9)
1.4
(2.4-5.9)
2.7
1.7
0.5 (0.4-1.7)
NT
(0.8-5.3)
0.9 (0.4-3.5)
NT
(1.1-6.6)
2.1 (0.9-3.6)
1.2
0.6 (0.5-0.8)
0.4 (0.3-0.4)
NT
(0.6-1.9)
3.7
(2.8-4.8) 3.0
24.0
(7.9-30) 30.0 (-)
0.3
(0.2-0.5)
20.6
19.0
13.9
5.2
1.8
1.3
(5.0-30)
(4.0-30)
(1.9-30)
(1.6-30)
(2.0-16.3)
(0.7-3.4)
(0.4-2.0)
0.5 (0.2-0.7)
30.0 (-)
30.0
21.9 (14.7-30)
15.0 (2.7-30)
7.0 (3.6-30)
3.5 (1.4-5.5)
1.6 (1.3-4.2)
0.6 (0.4-2.3)
NT
NT
NT
NT
(-)
9.7
0.3
0.3
0.3
0.3
(0.2-0.6)
(0.2-0.5)
(0.2-0.6)
(0.1-0.5)
Mean of duplicate control readings. Mean results from the 11 subjects given placebo; one subject was withdrawn from the study for reasons unrelated to the study and did not receive placebo. c Highest concentration of U-46619 tested = 30 p.M. If aggregation not achieved, EC50 assigned this value. d All EC50 values = 30 hence no range given. n = number of subjects. NT = Not tested. a
b
p.M,
The maximum blockade was seen at the end of the infusion following which there was a progressive return towards control values over the next 6 h. The same pattern of activity was reproduced in subjects after 2 and 4 mg, but in one subject given 2 mg and in two subjects after 4 mg, the maximum of the U-46619 concentrationeffect curve was depressed such that 50% aggregation was not achieved at the time the infusion was stopped (Table 1). Depression of the maximum response to U-46619 became more evident with the 8 and 16 mg doses. Thus, with the latter dose (Figure lb), the platelets were un-responsive to the effects of U-46619 for 2 h postinfusion after which time the degree of the blockade gradually reduced, although there was still some antagonism present 12 h after dosing. In contrast to vapiprost, placebo had no effect upon U-46619-induced platelet aggregation (Table 1, Figure lc). The mean effect (U-46619 EC5o) vs time plots for the whole dose range of vapiprost tested is shown in Figure 2.
Pharmacokinetics Plasma concentrations of vapiprost after doses of 0.125 and 0.5 mg were below the assay limit (5 ng ml-'). The
L)
1.0\
0.4 0
2
4
6
8
10
12
24
Time after infusion (h) Figure 2 U-46619 mean EC5o values vs time following intravenous infusion of vapiprost, 0.125 (A), 0.25 (V), 0.5 (e), 1.0 (O), 2 (A), 4 (O), 8 (0) and 16 (V) mg. For numbers of subjects at each dose, see Table 1.
Antagonism ofplatelet aggregation by i. v. vapiprost _
The limitations of the drug assay, coupled with the fact that 50% platelet aggregation to U-46619 was not achieved for up to 2-4 h after the higher doses of vapiprost in the majority of subjects, made it difficult to test (numerically) for a relationship between EC50 values and plasma concentrations of drug. However, there appears to be a good correlation between plasma concentrations of vapiprost and pharmacological effect in that the decline in plasma drug concentrations more or less mirror the U-46619 EC50 time plots (compare Figures 2 and 3). The time at which the limit of assay of plasma vapiprost concentrations (5 ng ml-') is reached seems to correspond to a U-46619 EC50 value of approximately 4 pM (Figures 2 and 3). This point represents a 10 to 20-fold rightward displacement of the aggregation concentration-effect curve to the TxA2 mimetic.
500
CD 0
C
0
L
50
c
(D 0
E co
185
10
Safety and tolerability Time after infusion (h)
Vapiprost had no effects on pulse rate, blood pressure, the ECG or PEFR. No local or systemic adverse events were reported by the subjects nor were there any significant drug-related changes in any of the routine laboratory safety screens.
Figure 3 Plasma vapiprost concentration (median values) vs time following intravenous infusion of 1 (U), 2 (A), 4 (E), 8 (0) and 16 (V) mg vapiprost. For numbers of subjects at each dose, see Table 2. mean plasma concentrations of vapiprost obtained following intravenous infusions of 1, 2, 4, 8 and 16 mg are shown in Figure 3 and the derived pharmacokinetic variables are given in Table 2. The median maximum plasma drug concentration at the end of the infusion increased from 38 ng ml-1 with the 1 mg dose to 431 ng ml-1 after the 16 mg dose. After the infusion was switched off, plasma drug concentrations declined in a biphasic manner at all dose levels, and could be measured after the 16 mg dose for 6 h before falling below the lower limit of assay. Vapiprost was rapidly cleared from plasma with clearance and elimination half-life values of 514 to 721 ml min-' and 68 to 86 min, respectively, across the dose range (Table 2). As with plasma clearance and plasma half-life, other measures of drug disposition such as volume of distribution and mean residence time showed no evidence of dose-related variation.
Discussion The results of this study show that vapiprost administered by intravenous infusion results in a dose-related, profound and sustained antagonism of platelet aggregation induced by the TxA2 mimetic U-46619, ex vivo in healthy male subjects. Furthermore, intravenously administered vapiprost produced no local or systemic side-effects, but in particular no unwanted cardiovascular effects were seen. The pronounced blockade of platelet aggregation and good tolerability seen in the present study is in keeping with previously reported studies with orally administered vapiprost in healthy subjects (Thomas & Lumley, 1990). For this study, vapiprost was formulated as an isotonic
Table 2 Pharmacokinetic variables after intravenous infusions of increasing doses of vapiprost. Results are expressed as median values (range)
Dose (mg)
n
1
4
2 4
8 16
4 3 6 6
(ng ml-' min)
xz (min-1)
t2 (min)
38
1944
514
60
113
(1279-2189)
0.008 (0.007-0.010
84
(31-41)
(67-106)
(457-782)
(48-63)
(90-144)
69
2781
69 (64-86)
59
97
(2593-4035)
0.010 (0.008-0.011)
719
(41-93)
(496-771)
(52-67)
(86-115)
70 (65-95)
617
55
(520-668)
(50-60)
93 (91-125)
Cmax (ng ml-)
AUC
160
6478
0.010
(133-182)
(5991-7688)
(0.007-0.011)
CL (ml min-')
Vss (1)
MRT
(min)
237
12295
0.010
68
651
55
92
(172-333)
(8808-17352)
(0.007-0.011)
(64-94)
(461-908)
(49-67)
(79-130)
431
22198
0.008
86
721
64
105
(211-716)
(19682-31287)
(0.006-0.009)
(74-111)
(511-813)
(55-80)
(88-122)
n = number of subjects.
186
M. Thomas et al.
solution with P-cyclodextrin to increase its solubility at physiological pH. Whilst vapiprost as the hydrochloride salt is relatively soluble in aqueous media at natural pH 2.8, it has a low solubility at pH 7.4 due to formation of the zwitterion. Intravenous administration of aqueous solutions of the drug to animals resulted in local irritancy, tissue necrosis and thrombosis at the site of injection. In contrast, when complexed with ,-cyclodextrin no such effects were observed. In addition, complexing vapiprost in this way did not affect its TxA2-receptor blocking potency determined either in vitro or in vivo in experimental animals (Lumley, unpublished data). In the present study, doses of 8 and 16 mg produced a profound blockade of the platelet TxA2 receptor which was of rapid onset, and persisted for at least 8 h after dosing. The rapid onset of activity with intravenous vapiprost administration offers advantages in conditions such as acute myocardial infarction or in angioplasty where early prevention of platelet aggregation or further deposition of platelets on damaged endothelium is desirable. Once blockade of the TxA2 platelet receptor has been established using the intravenous route it could either be maintained by further intravenous or oral doses, in the latter case provided the patient is able to swallow oral medication. Although the present data suggest that there appears to be a correlation between plasma concentration of drug and antagonism of U-46619-induced platelet aggregation, we know that such a correlation no longer applies upon repeat oral dosing with vapiprost. Thus, a cumulative effect upon U-46619-induced platelet aggregation is produced such that after two or three days a continuous blockade is achieved in the absence of any parallel increase in plasma drug concentration (Thomas & Lumley, 1990). It is therefore likely that repeat intravenous dosing would have a similar cumulative action on platelet aggregation and a sustained blockade of TxA2-receptor-mediated platelet aggregation would be achieved. Although lower doses of vapiprost produced parallel rightward displacements of the U-46619 concentration-
effect curve, consistent with competitive, reversible antagonism, administration of high doses depressed the maximum response to the agonist. This phenomenon is seen with human platelets in vitro (Lumley et al., 1989; Takahara et al., 1990) and has also been reported during ex vivo studies using oral administration of vapiprost (Thomas & Lumley, 1990). Possible explanations for the effect of vapiprost on the maximum aggregatory response to U-46619 have been advanced (Lumley et al., 1989). One feature of the binding of vapiprost to the human platelet TxA2 receptor, is its extremely slow dissociation (Armstrong et al., 1989). Thus, it is possible that in the presence of a high receptor occupancy by vapiprost, U-46619 cannot occupy sufficient TxA2 receptors to elicit a maximum response and so a depression of the concentration-effect curve is seen. The slow dissociation of vapiprost from the TxA2 receptor leading to a 'pseudoirreversible' profile of action may also in part account for its prolonged duration of action in man (Armstrong et al., 1990). The present study was also the first opportunity to investigate some of the pharmacokinetic properties of vapiprost which cannot be determined after oral dosing, in particular plasma clearance which is of the order of 500 to 700 ml min-'. It is known from other studies in man that less than 1% of an oral dose appears as unchanged drug in urine (unpublished data) so it is likely that a large proportion of the plasma clearance is metabolic. Although not designed as a formal intravenous dose-linearity study, the results suggest that there is no significant change in clearance with dose. Inspection of other measures of disposition kinetics such as volume of distribution and the elimination rate constant and the mean residence time also shows no evidence of doserelated variation. In conclusion, the present study has demonstrated that intravenous administration of vapiprost leads to a profound and sustained blockade of the platelet TxA2 receptor in man, and provides a means of achieving virtually instantaneous TxA2 receptor blockade.
References Armstrong, R. A., Lumley, P. & Humphrey, P. P. A. (1989). Characteristics of [3H]-GR32191 binding to the thromboxane (TP) receptor of human platelets. Br. J. Pharmac., Proc. Suppl., 98, 843P. Armstrong, R. A., Lumley P. & Humphrey, P. P. A. (1990). Reduction in the number of tromboxane receptors (Bmax) on human platelets after exposure to GR32191. Br. J. Pharmac., Proc. Suppl., 99, 113P. Charter, M. K. (1989). The estimation of moments: a technical note. J. Pharmacokin. Biopharm., 17, 203-208. Lumley, P., Finch, H., Collington, E. W. C. & Humphrey, P. P. A. (1990). Vapiprost (GR32191). Drugs ofthe Future, 15, 1087-1092. Lumley, P. & Humphrey, P. P. A. (1981). A method for quantifying platelet aggregation and analysing drug-receptor interactions on platelets in whole blood in vitro. J. pharmac. Methods, 6, 153-166. Lumley P., Keery, R. J., Kensington, J. E. & Thomas, M. (1989a). Antagonism of collagen-induced platelet aggregation ex vivo in volunteers by GR32191, a thromboxane A2
receptor blocking drug. Eur. J. clin. Pharmac., 36, Suppl., A298. Lumley, P., White, B. P. & Humphrey, P. P. A. (1989b). GR32191, a highly potent and specific thromboxane A2 receptor-blocking drug on platelets and vascular and airways smooth muscle in vitro. Br. J. Pharmac., 97, 783-794. Maconochie, J. G., Kensington, J. & Lumley, P. (1988). Evaluation of the vascular thromboxane A2 receptorblocking activity of GR32191 in man. Br. J. clin. Pharmac., 26, 662P. Takahara, K., Murray, R., FitzGerald, G. A. & Fitzgerald, D. J. (1990). The response to thromboxane A2 analogues in human platelets. J. biol. Chem., 265, 6836-6844. Thomas, M. & Lumley, P. (1990). Preliminary assessment of a novel thromboxane A2 receptor-blocking drug GR32191 in healthy subjects. Circulation, 81 (Suppl. I). I-53-I-58.
(Received 13 November 1990, accepted 12 February 1991)
A D 0 N I S 030652519100165V
The pharmacodynamics and pharmacokinetics of a novel thromboxane receptor blocking drug vapiprost (GR32191) after single intravenous doses in healthy subjects M. THOMAS', R. J. KEERY2, M. K. CHARTER', N. L. SCULLY3, J. E. CHILTON' & P. LUMLEY2 Departments of 'Clinical Pharmacology, 'Peripheral Pharmacology and 3Biochemical Pharmacology, Glaxo Group Research, Ware, Herts SG12 ODP
1 The effect of single, serially increasing, intravenous doses of a specific thromboxane receptor blocking drug, vapiprost, upon platelet aggregation induced ex vivo by the thromboxane A2 mimetic, U-46619, was examined in 12 healthy males. 2 Subjects received either 1 (n = 1 subject), 2 (n = 6), 3 (n = 2), or 4 (n = 3) administrations of vapiprost within the dose range 0.125 to 16 mg and, in random order, placebo on separate study days at intervals of at least 48 h. 3 All doses of vapiprost produced an immediate antagonism of U-46619-induced platelet aggregation in whole blood. Both the magnitude and duration of the rightward displacement of the concentration-effect curves increased with dose. Although lower doses produced parallel displacements of these curves, with the higher doses the maximum response to U-46619 was reduced such that 50% platelet aggregation was not achieved. After the 16 mg dose of vapiprost, virtually complete suppression of platelet aggregation (up to a concentration of 30 pRM) was seen. This degree of inhibition was maintained for 2 h after dosing, following which there was a gradual return to pre-dose U-46619 sensitivity over the next 12 to 24 h. U-46619-induced platelet aggregation was unaffected by placebo. 4 Across the dose range, vapiprost was rapidly cleared from plasma, with an elimination half-life of 69-84 min and a plasma clearance of 514-721 ml min-'. 5 Vapiprost had no effect upon pulse rate, blood pressure, the ECG, peak expiratory flow rate, nor were drug-related changes in routine laboratory screens seen. No local or systemic side-effects were reported.
Keywords vapiprost thromboxane receptor blockade platelet aggregation pharmacokinetics
U-46619
Introduction
Vapiprost
( [1R-[1ot (Z) ,2pf,3p,5a]]-(+)-7-[5-([1,1 '-bi-
& Lumley 1990). Aggregation produced through nonTxA2-dependent mechanisms, such as that induced by ADP is, however, unaffected. In addition to blocking the platelet TxA2 receptor, vapiprost has also been shown to block the receptor on vascular smooth muscle in man (Maconochie et al., 1988). Upon repeat oral dosing, the drug produces a progressive, cumulative blockade of TxA2 receptor-mediated platelet aggregation which occurs in the absence of any parallel increase in its plasma concentration (Thomas & Lumley, 1990). This phenomenon may be due to a persistent occupation of the TxA2 receptor by vapiprost or to a reduction, by an unknown mechanism, in receptor number (Armstrong
phenyl] - 4 - ylmethoxy) - 3 - hydroxy - 2 - (1 - piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride salt) has been shown to be a potent and specific thromboxane (Tx)A2 receptor blocking drug in vitro using platelets and both vascular and airways smooth muscle preparations from different species (Lumley et al., 1989b). The drug is active in various experimental models of thrombosis (Lumley et al., 1990). When administered orally to healthy subjects, vapiprost produces a rapid, profound and sustained blockade of platelet aggregation ex vivo to the TxA2 mimetic U-46619 (lla, 9a epoxymethanoPGH2) and collagen (Lumley et al., 1989a; Thomas
Correspondence: Dr M. Thomas, Department of Clinical Pharmacology, Glaxo Group Research, Park Road, Ware, Herts SG12 ODP
181
182
M. Thomas et al.
et al., 1990). In all studies performed with vapiprost in man, the drug has been well tolerated and free of serious adverse effects (Thomas & Lumley, 1990). The potential clinical applications for a thromboxane receptor blocking drug include the treatment of thrombotic events and occlusive vascular disease. As vapiprost will be used to prevent or treat chronic disease, it will be given long term by mouth and all of our previous human pharmacological studies have been conducted using this route. However, there may well be circumstances in clinical practice where intravenous administration is indicated. For example, the intravenous route might be preferred in patients in shock or coma in whom absorption from the gastrointestinal tract is likely to be erratic or unreliable. Likewise, the intravenous route offers advantages when a rapid anti-thrombotic action is thought to be desirable, e.g. in the prevention of re-occlusion and re-stenosis of coronary arteries following angioplasty or thrombolysis in acute myocardial infarction. The aims of the present study were to examine for the first time in man the effects of ascending doses of vapiprost given by intravenous infusion upon platelet aggregation induced by U-46619, to make an initial assessment of safety and tolerability and to assess the pharmacokinetic properties of the drug by this route.
Methods
Subjects Twelve healthy male subjects, mean age 33 years (range 24-42 years), took part in a placebo-controlled, crossover study which had been approved by the Glaxo Group Research Ethics Review Committee. Based on the results of a medical history, physical examination, routine laboratory safety screens, and an ECG examination, subjects were shown to be fit and well before admission to the study. Subjects with a history of spontaneous bleeding or any other symptoms suggestive of a bleeding disorder or peptic ulceration, were specifically excluded from the study. Also excluded from the study were smokers and subjects who had taken other drugs, including aspirin or aspirin-like drugs within 14 days of the start of the study. No alcohol or exercise was allowed for 24 h before and after dosing and no tea, coffee or foods containing caffeine were consumed on a study day. After the nature and purpose of the study had been explained to the subjects, they gave consent in writing.
Clinical procedure
Subjects fasted overnight and reported to the laboratory the following morning bringing with them an early morning urine specimen for routine testing. A cannula (Wallace-Y-Can) was inserted into a vein in each arm. One cannula was used to administer the treatment and was removed at the end of the infusion. The other cannula was used to obtain blood samples and was kept patent between sampling times by flushing through with normal saline. Duplicate blood samples were drawn for platelet aggregation studies and samples were obtained
for drug assay, haematological and biochemical safety screens. With subjects resting supine, pulse rate and blood pressure were measured until steady using an automated sphygmomanometer (Narco Scientific) and a baseline of three readings at 5 min intervals was then obtained. At the times pulse rate and blood pressure were measured, 12-lead ECGs were also recorded (Marquette Cardiograph) and immediately afterwards measurements of peak expiratory flow rate (PEFR) were made using a Wrights Peak Flow Meter. When control measurements and blood samples had been taken, subjects received either drug or placebo administered intravenously over 10 min as a 1 ml min-1 infusion (2 ml min-1 for 16 mg dose) under ECG control. At the end of the infusion (time 0), further measurements of pulse rate, blood pressure and PEFR were made, an ECG was recorded and blood samples for aggregation studies and plasma drug assay were obtained. Following discontinuation of the infusion, monitoring continued as follows: pulse rate and blood pressure at, 5, 10, 15, 20, 25, 30, 40, 50 and 60 min, 1.5, 2, 3 and 4 h; ECG at 30 and 60 min, 2, 3 and 4 h; PEFR at 10, 20, 30 and 60 min, 2, 3 and 4 h. Further blood samples for platelet studies were drawn after 30 min, 1, 2, 3, 4, 8, 12 h and in addition at 24 h after the 8 and 16 mg doses, and for drug assay at 5, 10, 20, 30, 40, 50 and 60 min, 1.5, 2, 2.5, 3, 4, 6, 8, 10 and 12 h. Subjects were allowed up after 2 h, but lay down again 10 min before the 3 and 4 h cardiovascular measurements. The second cannula was removed after the 12 h blood samples had been taken and the 24 h sample was obtained by direct venepuncture. Further laboratory safety tests were performed 48 h after a dose of vapiprost or placebo and 7 days after the last study day. Vapiprost was formulated as a 1 mg (base) ml-1 solution using ,B-cyclodextrin to improve solubility in the molar ratio of 1:1.4 and isotonicity was adjusted with mannitol (5 % w/v). The pH of the solution was 6.0. The stock solution of vapiprost was diluted with normalsaline to adjust for dose. The following doses of vapiprost were administered in ascending order of magnitude at intervals of at least 48 h (0.125 mg (n = 2 subjects)), 0.25 mg (n = 2), 0.5 mg (n = 3), 1 mg (n = 4), 2 mg (n = 4), 4 mg (n = 3), 8 mg (n = 6) and 16 mg (n = 6), while each subject also received, in random order, placebo (P-cyclodextrin) alone on another occasion. Subjects received between 1 and 4 administrations of vapiprost. Assessment ofplatelet aggregation
Blood samples for platelet studies were anticoagulated with trisodium citrate (final concentration 12.9 mM) and transferred immediately to the laboratory for analysis. U-46619 concentration-effect curves were constructed in whole blood by counting single platelets using a Becton Dickinson Ultra-Flo 100 platelet counter (Lumley & Humphrey, 1981; Thomas & Lumley, 1990). Measurements of aggregation were made in the presence of aspirin (2 mM) to inhibit endogenous production of prostaglandins. The concentration of U-46619 required to produce a 50% reduction in platelet count (EC50) was determined before and then at each time point after dosing. When less than 50% aggregation was obtained,
Antagonism ofplatelet aggregation by i. v. vapiprost the EC50 post-treatment value was taken as 30 FLM, the highest concentration of U-46619 used.
183
6. The plasma clearance of vapiprost (CL) was calculated from CL = D/AUC.
Analysis of vapiprost in plasma Results
Vapiprost was measured in plasma by high performance liquid chromatography (h.p.l.c.) after solid-phase extraction on advanced automated sample processor (AASP) C8 extraction cartridges. The extract was introduced onto a 5 p,M PLRP-S column (Polymer Laboratories) by an AASP, and vapiprost was detected by fluorescence emission at 310 nm after excitation at 254 nm. The working range of the assay was 5-150 ng ml-1 vapiprost when assaying 0.5 ml of the plasma sample. The precision of the assay at its lower limit (5 ng ml-') was 3.7% (n = 6). Samples containing vapiprost at concentrations in excess of 150 ng ml- 1 were assayed after dilution with pooled control plasma. Pharmacokinetic calculations
Platelet aggregation U-46619 induced a concentration-related platelet aggregation in control blood samples from all subjects. Geometric mean EC50 values are shown in Table 1. Vapiprost (0.125 to 16 mg) produced a dose-related antagonism of U-46619-induced platelet aggregation in terms of both the magnitude and duration of the blockade (Table 1). Doses of 0.125 to 1 mg, produced parallel rightward displacements of the U-46619 concentrationeffect curves for platelet aggregation. The mean response in four subjects who received 1 mg is shown in Figure la.
100
The maximum measured concentration of vapiprost in plasma (Cmax) was obtained by inspection of the data. If there were sufficient data to determine adequately an elimination phase, then the following parameters were also derived: 1. The elimination rate constant for vapiprost in plasma (Xj). The number of points to be included in the elimination phase was determined by inspection of a loglinear plot of plasma concentration against time. The rate constant was determined by linear least-squares regression using the logarithmically transformed points. 2. The area under the curve of plasma vapiprost concentration vs time, extrapolated to infinite time (AUC). The portion of the area between times t, and tj+1 was calculated using the logarithmic trapezoidal rule if Ci > Cj,+1 > 0, and the linear trapezoidal rule otherwise, where C, is the concentration measured at time ti. The area was extrapolated to infinite time using C,/XJ, where CQ is the last measured plasma concentration. The extrapolated portion of the profile was checked for consistency with any concentrations below the limit of
40
A-'!e ;9, t.i s {E.. fi +~.°* k.~j /4 !# X
.A .A Aht. w I
Bllh;ft;;
?°
Figure 1 Platelet aggregation U-46619 concentration-effect curves ex vivo before and following administration of intravenous doses of vapiprost; a) 1 mg, b) 16 mg or c) placebo (for subjects receiving 16 mg dose). U-46619-induced aggregation was determined prior to dosing (0), immediately after dosing (@), and at 1 (E), 2 (-), 4 (A) and 6 (v) h (both doses), and at 8 (*), 12 (V) and 24 (A) h, 16 mg dose only. Curves are the mean ± s.e. mean from four subjects in (a) and from the same six subjects in (b) and (c).
184
M. Thomas et al.
Table 1 Geometric mean EC50 values (range) for U-46619-induced platelet aggregation ex vivo before and after intravenous infusion of increasing doses of vapiprost or placebo U-46619 EC50 values (>LM) Time post-infusion (h) Dose (mg)
ControP
0.125 (n = 2)
0.25 (n = 2)
0.4
(0.3-0.5)
0.50 (n = 3)
0.3
4.8
(0.2-0.4)
(2.5-8.2)
1.0 (n = 4)
0.4
6.5
(0.3-0.6)
(5.6-9.4)
(1.9-3.9)
2.0 (n = 4)
0.3 (0.1-1.4)
8.5
5.0 (1.9-19.2)
4.0
3.7
(5.0-30)
(2.0-6.2)
(2.0-5.5)
4.0 (n = 3)
0.2
25.1
8.4
5.7
5.6
3.4
(0.2-0.3)
(17.5-30)
(4.8-14.6)
(3.2-9.5)
(3.8-8.0)
(1.9-5.4)
8.0 (n = 6)
0.2
(0.2-0.5)
30.0c (_)d
16.0 (n = 6)
0.2 (0.1-0.3)
30.0 (-)
Placebob (n = 11)
0.3
0.3
(0.2-0.5)
(0.2-0.5)
0
0.5
1
2
3
4
6
8
12
24
0.2
1.1
0.4 (0.3-0.6)
0.4 (0.2-0.8)
NT
NT
NT
(0.2-0.6)
0.4 (0.3-0.6)
NT
(0.8-1.4)
0.4 (0.3-0.6)
0.4
(0.2-0.2)
2.5 (2.4-2.6)
1.2 (1.1-1.3)
1.0
1.2 (0.7-1.9)
0.6
NT
NT
NT
(0.5-0.8)
0.4 (0.3-0.6)
NT
(0.6-1.5) 2.4
1.9
0.6
0.5
NT
NT
(1.0-3.9)
1.5 (0.7-2.9)
1.2
(1.3-4.5)
(0.7-1.9)
(0.5-0.8)
(0.5-0.5)
4.2
2.8
0.6
NT
(0.8-1.8)
(0.4-0.8)
0.5 (0.4-0.6)
NT
(2.0-5.0)
2.0 (1.2-2.9)
1.4
(2.4-5.9)
2.7
1.7
0.5 (0.4-1.7)
NT
(0.8-5.3)
0.9 (0.4-3.5)
NT
(1.1-6.6)
2.1 (0.9-3.6)
1.2
0.6 (0.5-0.8)
0.4 (0.3-0.4)
NT
(0.6-1.9)
3.7
(2.8-4.8) 3.0
24.0
(7.9-30) 30.0 (-)
0.3
(0.2-0.5)
20.6
19.0
13.9
5.2
1.8
1.3
(5.0-30)
(4.0-30)
(1.9-30)
(1.6-30)
(2.0-16.3)
(0.7-3.4)
(0.4-2.0)
0.5 (0.2-0.7)
30.0 (-)
30.0
21.9 (14.7-30)
15.0 (2.7-30)
7.0 (3.6-30)
3.5 (1.4-5.5)
1.6 (1.3-4.2)
0.6 (0.4-2.3)
NT
NT
NT
NT
(-)
9.7
0.3
0.3
0.3
0.3
(0.2-0.6)
(0.2-0.5)
(0.2-0.6)
(0.1-0.5)
Mean of duplicate control readings. Mean results from the 11 subjects given placebo; one subject was withdrawn from the study for reasons unrelated to the study and did not receive placebo. c Highest concentration of U-46619 tested = 30 p.M. If aggregation not achieved, EC50 assigned this value. d All EC50 values = 30 hence no range given. n = number of subjects. NT = Not tested. a
b
p.M,
The maximum blockade was seen at the end of the infusion following which there was a progressive return towards control values over the next 6 h. The same pattern of activity was reproduced in subjects after 2 and 4 mg, but in one subject given 2 mg and in two subjects after 4 mg, the maximum of the U-46619 concentrationeffect curve was depressed such that 50% aggregation was not achieved at the time the infusion was stopped (Table 1). Depression of the maximum response to U-46619 became more evident with the 8 and 16 mg doses. Thus, with the latter dose (Figure lb), the platelets were un-responsive to the effects of U-46619 for 2 h postinfusion after which time the degree of the blockade gradually reduced, although there was still some antagonism present 12 h after dosing. In contrast to vapiprost, placebo had no effect upon U-46619-induced platelet aggregation (Table 1, Figure lc). The mean effect (U-46619 EC5o) vs time plots for the whole dose range of vapiprost tested is shown in Figure 2.
Pharmacokinetics Plasma concentrations of vapiprost after doses of 0.125 and 0.5 mg were below the assay limit (5 ng ml-'). The
L)
1.0\
0.4 0
2
4
6
8
10
12
24
Time after infusion (h) Figure 2 U-46619 mean EC5o values vs time following intravenous infusion of vapiprost, 0.125 (A), 0.25 (V), 0.5 (e), 1.0 (O), 2 (A), 4 (O), 8 (0) and 16 (V) mg. For numbers of subjects at each dose, see Table 1.
Antagonism ofplatelet aggregation by i. v. vapiprost _
The limitations of the drug assay, coupled with the fact that 50% platelet aggregation to U-46619 was not achieved for up to 2-4 h after the higher doses of vapiprost in the majority of subjects, made it difficult to test (numerically) for a relationship between EC50 values and plasma concentrations of drug. However, there appears to be a good correlation between plasma concentrations of vapiprost and pharmacological effect in that the decline in plasma drug concentrations more or less mirror the U-46619 EC50 time plots (compare Figures 2 and 3). The time at which the limit of assay of plasma vapiprost concentrations (5 ng ml-') is reached seems to correspond to a U-46619 EC50 value of approximately 4 pM (Figures 2 and 3). This point represents a 10 to 20-fold rightward displacement of the aggregation concentration-effect curve to the TxA2 mimetic.
500
CD 0
C
0
L
50
c
(D 0
E co
185
10
Safety and tolerability Time after infusion (h)
Vapiprost had no effects on pulse rate, blood pressure, the ECG or PEFR. No local or systemic adverse events were reported by the subjects nor were there any significant drug-related changes in any of the routine laboratory safety screens.
Figure 3 Plasma vapiprost concentration (median values) vs time following intravenous infusion of 1 (U), 2 (A), 4 (E), 8 (0) and 16 (V) mg vapiprost. For numbers of subjects at each dose, see Table 2. mean plasma concentrations of vapiprost obtained following intravenous infusions of 1, 2, 4, 8 and 16 mg are shown in Figure 3 and the derived pharmacokinetic variables are given in Table 2. The median maximum plasma drug concentration at the end of the infusion increased from 38 ng ml-1 with the 1 mg dose to 431 ng ml-1 after the 16 mg dose. After the infusion was switched off, plasma drug concentrations declined in a biphasic manner at all dose levels, and could be measured after the 16 mg dose for 6 h before falling below the lower limit of assay. Vapiprost was rapidly cleared from plasma with clearance and elimination half-life values of 514 to 721 ml min-' and 68 to 86 min, respectively, across the dose range (Table 2). As with plasma clearance and plasma half-life, other measures of drug disposition such as volume of distribution and mean residence time showed no evidence of dose-related variation.
Discussion The results of this study show that vapiprost administered by intravenous infusion results in a dose-related, profound and sustained antagonism of platelet aggregation induced by the TxA2 mimetic U-46619, ex vivo in healthy male subjects. Furthermore, intravenously administered vapiprost produced no local or systemic side-effects, but in particular no unwanted cardiovascular effects were seen. The pronounced blockade of platelet aggregation and good tolerability seen in the present study is in keeping with previously reported studies with orally administered vapiprost in healthy subjects (Thomas & Lumley, 1990). For this study, vapiprost was formulated as an isotonic
Table 2 Pharmacokinetic variables after intravenous infusions of increasing doses of vapiprost. Results are expressed as median values (range)
Dose (mg)
n
1
4
2 4
8 16
4 3 6 6
(ng ml-' min)
xz (min-1)
t2 (min)
38
1944
514
60
113
(1279-2189)
0.008 (0.007-0.010
84
(31-41)
(67-106)
(457-782)
(48-63)
(90-144)
69
2781
69 (64-86)
59
97
(2593-4035)
0.010 (0.008-0.011)
719
(41-93)
(496-771)
(52-67)
(86-115)
70 (65-95)
617
55
(520-668)
(50-60)
93 (91-125)
Cmax (ng ml-)
AUC
160
6478
0.010
(133-182)
(5991-7688)
(0.007-0.011)
CL (ml min-')
Vss (1)
MRT
(min)
237
12295
0.010
68
651
55
92
(172-333)
(8808-17352)
(0.007-0.011)
(64-94)
(461-908)
(49-67)
(79-130)
431
22198
0.008
86
721
64
105
(211-716)
(19682-31287)
(0.006-0.009)
(74-111)
(511-813)
(55-80)
(88-122)
n = number of subjects.
186
M. Thomas et al.
solution with P-cyclodextrin to increase its solubility at physiological pH. Whilst vapiprost as the hydrochloride salt is relatively soluble in aqueous media at natural pH 2.8, it has a low solubility at pH 7.4 due to formation of the zwitterion. Intravenous administration of aqueous solutions of the drug to animals resulted in local irritancy, tissue necrosis and thrombosis at the site of injection. In contrast, when complexed with ,-cyclodextrin no such effects were observed. In addition, complexing vapiprost in this way did not affect its TxA2-receptor blocking potency determined either in vitro or in vivo in experimental animals (Lumley, unpublished data). In the present study, doses of 8 and 16 mg produced a profound blockade of the platelet TxA2 receptor which was of rapid onset, and persisted for at least 8 h after dosing. The rapid onset of activity with intravenous vapiprost administration offers advantages in conditions such as acute myocardial infarction or in angioplasty where early prevention of platelet aggregation or further deposition of platelets on damaged endothelium is desirable. Once blockade of the TxA2 platelet receptor has been established using the intravenous route it could either be maintained by further intravenous or oral doses, in the latter case provided the patient is able to swallow oral medication. Although the present data suggest that there appears to be a correlation between plasma concentration of drug and antagonism of U-46619-induced platelet aggregation, we know that such a correlation no longer applies upon repeat oral dosing with vapiprost. Thus, a cumulative effect upon U-46619-induced platelet aggregation is produced such that after two or three days a continuous blockade is achieved in the absence of any parallel increase in plasma drug concentration (Thomas & Lumley, 1990). It is therefore likely that repeat intravenous dosing would have a similar cumulative action on platelet aggregation and a sustained blockade of TxA2-receptor-mediated platelet aggregation would be achieved. Although lower doses of vapiprost produced parallel rightward displacements of the U-46619 concentration-
effect curve, consistent with competitive, reversible antagonism, administration of high doses depressed the maximum response to the agonist. This phenomenon is seen with human platelets in vitro (Lumley et al., 1989; Takahara et al., 1990) and has also been reported during ex vivo studies using oral administration of vapiprost (Thomas & Lumley, 1990). Possible explanations for the effect of vapiprost on the maximum aggregatory response to U-46619 have been advanced (Lumley et al., 1989). One feature of the binding of vapiprost to the human platelet TxA2 receptor, is its extremely slow dissociation (Armstrong et al., 1989). Thus, it is possible that in the presence of a high receptor occupancy by vapiprost, U-46619 cannot occupy sufficient TxA2 receptors to elicit a maximum response and so a depression of the concentration-effect curve is seen. The slow dissociation of vapiprost from the TxA2 receptor leading to a 'pseudoirreversible' profile of action may also in part account for its prolonged duration of action in man (Armstrong et al., 1990). The present study was also the first opportunity to investigate some of the pharmacokinetic properties of vapiprost which cannot be determined after oral dosing, in particular plasma clearance which is of the order of 500 to 700 ml min-'. It is known from other studies in man that less than 1% of an oral dose appears as unchanged drug in urine (unpublished data) so it is likely that a large proportion of the plasma clearance is metabolic. Although not designed as a formal intravenous dose-linearity study, the results suggest that there is no significant change in clearance with dose. Inspection of other measures of disposition kinetics such as volume of distribution and the elimination rate constant and the mean residence time also shows no evidence of doserelated variation. In conclusion, the present study has demonstrated that intravenous administration of vapiprost leads to a profound and sustained blockade of the platelet TxA2 receptor in man, and provides a means of achieving virtually instantaneous TxA2 receptor blockade.
References Armstrong, R. A., Lumley, P. & Humphrey, P. P. A. (1989). Characteristics of [3H]-GR32191 binding to the thromboxane (TP) receptor of human platelets. Br. J. Pharmac., Proc. Suppl., 98, 843P. Armstrong, R. A., Lumley P. & Humphrey, P. P. A. (1990). Reduction in the number of tromboxane receptors (Bmax) on human platelets after exposure to GR32191. Br. J. Pharmac., Proc. Suppl., 99, 113P. Charter, M. K. (1989). The estimation of moments: a technical note. J. Pharmacokin. Biopharm., 17, 203-208. Lumley, P., Finch, H., Collington, E. W. C. & Humphrey, P. P. A. (1990). Vapiprost (GR32191). Drugs ofthe Future, 15, 1087-1092. Lumley, P. & Humphrey, P. P. A. (1981). A method for quantifying platelet aggregation and analysing drug-receptor interactions on platelets in whole blood in vitro. J. pharmac. Methods, 6, 153-166. Lumley P., Keery, R. J., Kensington, J. E. & Thomas, M. (1989a). Antagonism of collagen-induced platelet aggregation ex vivo in volunteers by GR32191, a thromboxane A2
receptor blocking drug. Eur. J. clin. Pharmac., 36, Suppl., A298. Lumley, P., White, B. P. & Humphrey, P. P. A. (1989b). GR32191, a highly potent and specific thromboxane A2 receptor-blocking drug on platelets and vascular and airways smooth muscle in vitro. Br. J. Pharmac., 97, 783-794. Maconochie, J. G., Kensington, J. & Lumley, P. (1988). Evaluation of the vascular thromboxane A2 receptorblocking activity of GR32191 in man. Br. J. clin. Pharmac., 26, 662P. Takahara, K., Murray, R., FitzGerald, G. A. & Fitzgerald, D. J. (1990). The response to thromboxane A2 analogues in human platelets. J. biol. Chem., 265, 6836-6844. Thomas, M. & Lumley, P. (1990). Preliminary assessment of a novel thromboxane A2 receptor-blocking drug GR32191 in healthy subjects. Circulation, 81 (Suppl. I). I-53-I-58.
(Received 13 November 1990, accepted 12 February 1991)
