Quantitation of drug levels and platelet receptor ... - Wiley Online Library

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thromboxane A, with a platelet receptor dissociation constant (estimated by Schild ... dose of a thromboxane receptor antagonist that would be expected to beĀ ...
Quantitation of drug levels and platelet receptor blockade caused by a thromboxane antagonist SQ 28,668 is a structural analog of thromboxane A,. It inhibits the effects of thromboxane in vitro. Fiftysix healthy male subjects were given either placebo or three equal daily doses of SQ 28,668 ranging from 25 to 1200 mg. Plasma drug concentrations increased in a dose-dependent manner. The shape of the plasma drug concentration-time curve was consistent with enterohepatic recirculation. The effects of SQ 28,668 on ex vivo platelet aggregation suggested that SQ 28,668 is a specific competitive antagonist of thromboxane A, with a platelet receptor dissociation constant (estimated by Schild analysis) of about 19 nmol/L. Approximately 94%occupation of thromboxane receptors by SQ 28,668 was required to produce a small but measurable increase of the template bleeding time. Dose-ranging studies of antithrombotic drugs are difficult and expensive. For this reason, a method was developed that allows estimation of the dose of a thromboxane receptor antagonist that would be expected to be therapeutically equivalent to a given dose of aspirin. (CLINPHARMACOLTHER1986;40:634-42.)

Lawrence T. Friedhoff, M.D., Ph.D., J. Manning, M.S., P. T. Funke, Ph.D., E. Ivashkiv, M.S., J. Tu, Ph.D., W. Cooper, M.S., and D. A. Willard, M.D. Princeton, N. J Thromboxane A, (TXA,) is a potent inducer of platelet aggregation. ' Clinical studies suggest that drugs proCOOH ducing a decrease in the effects of TXA, may have therapeutic benefit for patients with coronary bypass grafts, unstable angina, membranoproliferative glomerulonephritis, or cerebral i~chemia.~"Conclusive demonstration of the benefit of TXA, blockade has been hampered because the blocking agents available in the past were nonspecific, altering production of other autacoids in addition to TXA,.6 SQ 28,668 is [~S-[~CY,~P(~Z),~P(~E,~R,~S),~CY]]7-[3-(3-hydroxy-4-phenyl- 1-pentenyl)-7-oxabicyclor2.2.11-hept-2-yl]-5-heptenoic acid (see structure). Structure of SQ 28,668 In vitro, SQ 28,668 is a specific TXA, antagonist; it is a potent inhibitor of platelet aggregation induced by guinea pigs, SQ 28,668 inhibits thromboxane-mediated arachidonic acid, collagen, and the TXA analog SQ vasoconstri~tion.~ 26,538.This inhibition in vitro is not associated with The purpose of this study was to quantitate the physsignificant changes in platelet cyclic adenosine monoiologic effects of increasing levels of TXA, blockade phosphate, cyclooxygenase, or thromboxane synthetase induced by SQ 28,668 and to relate them to plasma a ~ t i v i t y SQ . ~ 28,668 prevents coronary thrombosis in anesthetized pigs with critical coronary stenoses.' In drug concentrations in a way that might allow quantitative comparisons with the effects of other drugs that inhibit the production or effects of TXA,. To accomFrom the Squibb Institute for Medical Research, Princeton. plish this required evaluation of the drug's pharmacoReceived for publication Jan. 24, 1986; accepted June 3 , 1986. kinetics and specificity as an inhibitor of the human Reprint requests: Lawrence T. Friedhoff, M.D., Ph.D., Squibb Institute for Medical Research, P.O. Box 4000, Princeton, NJ 08540. platelet TXA, receptor, as well as correlation of the

VOLUME 40 NUMBER 6

drug's pharmacokinetics with its pharmacodynamics and assessment of drug binding to plasma proteins. The method used to compare the potency of various inhibitors can also be used to quantitate the role of TXA, in maintaining normal hemostasis.

METHODS Fifty-six healthy male volunteers were enrolled in this study. They ranged in age from 18 to 35 years (mean age 25 years) and in weight from 49.5 to 93.0 kg (mean weight 73.8 kg). Subjects were enrolled in groups of eight, one group at a time. Six subjects in each group received three equal daily doses of SQ 28,668 administered orally; two subjects in each group were randomly assigned to receive matching placebo medication in a single-blind study design. Daily doses of 25, 50, 100, 200, 400, 800, and 1200 mg were studied. The safety of SQ 28,668 was assessed with frequent physical examinations, vital sign measurements, and a battery of laboratory tests. Bleeding time was assessed with a Simplate I1 device (General Diagnostics, Morris Plains, N.J.). For each subject, bleeding times were measured by the same nurse who was unaware of the treatment randomization. Blood for ex vivo platelet aggregation studies was drawn with a 19-gauge butterfly needle and plastic syringes containing 1/10 volume of 3.8% sodium citrate. Immediately after the citrated blood was obtained, platelet-rich plasma was prepared by centrifugation at 200 x g for 10 minutes at 25' C. Aliquots of plateletrich plasma were placed in aggregometer cuvettes. A single 0.7 ml aliquot of platelet-rich plasma was placed in a plastic test tube and centrifuged at 8800 x g for 2 minutes at room temperature. The supernatant platelet-poor plasma was then removed and placed in an aggregometer cuvette. Platelet-rich and platelet-poor plasma was stored at room temperature until used. Platelet aggregometry was performed with a ChronoLog Model 550 platelet aggregometer (set at 37' C) with a Model 703 chart recorder (Chrono-Log Corp., Havertown, Pa.). This instrument measures changes in the optical density of platelet-rich plasma. The value measured is "normalized" using the optical density of platelet-poor plasma and the result is plotted as a function of time. Aggregation of the platelets in plateletrich plasma causes a decrease in optical density that approaches the optical density of platelet-poor plasma as platelet aggregation becomes complete. The maximal slope of the optical density-time curve trace during the 3-minute period of observation was recorded. All ag-

Thromboxane antagonist 635 gregation studies were conducted with siliconized cuvettes and were completed within 3. hours of venipuncture. Platelet aggregation responses to collagen (10 kg/ ml; Collagenreagent Horm, Hormonchemie, Munich, W. Germany), arachidonic acid (800 pmoliL; Sigma Chemical Co., St. Louis, Mo.), adenosine diphosphate (10 ~ r n o l i L Sigma ; Chemical Co.), and the thromboxane agonist U46,619 (0.1, 0.5, 1.5, 5, 15, 50, and 150 pmol/L; Cayman Chemical Co., Denver Colo.) were assessed. When adenosine diphosphate was used as to aggregate platelets, indomethacin (100 ~ m o l i L )was added to prevent synthesis of TXA,. Platelet counting was performed with a Baker Series 810 Whole Blood Platelet Analyzer (Baker Instruments Corp., Bethlehem, Pa.) and was completed within 12 hours of venipuncture. For platelet-rich plasma challenged with arachidonic acid, adenosine diphosphate plus indomethacin, or collagen, only one concentration of each agonist was used. The baseline means for the treated groups were compared with the placebo group mean, using a modified t test (Dunnett's test). The treated vs. control group mean changes from baseline at each timepoint after dosing were similarly compared. Because these data did not appear to be normally distributed, all statistical tests were carried out on the rank-transformed data. A subject's platelet aggregation data was excluded from these analyses if his aggregation response at baseline appeared to be too small to allow accurate estimation of inhibitory drug effects. For platelet-rich plasma challenged with adenosine diphosphate, one subject was excluded because of inadequate baseline aggregation. For plasma challenged with arachidonic acid, two subjects likewise were excluded and three others were excluded because of a total absence of platelet aggregation at baseline. For platelet-rich plasma challenged with U-46,619, seven concentrations of agonist were used, yielding responses that nearly always ranged from negligible to maximal. These dose-response data permitted the estimation of the ED,, concentration (i.e., the concentration of U46,619 causing 50% maximal platelet aggregation). The quantity R, defined as the ratio of the ED,, at each posttreatment time point to that at baseline - 1, was then calculated. R is a measure of the shift of the U46619 dose-response curve. To calculate geometric 1 was computed. An addimeans, the quantity R tional quantity, F, defined as F = R/(R I), also was computed. According to the theory of Schild, F is the fraction of thromboxane receptors occupied by a drug

+

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CLIN PHARMACOL THER DECEMBER 1986

[Plasma SQ 28,668 Concentration) =

.

-

[0.965810 y - 0.0221921 [Concentration of SQ 29,9691 [Urine SQ 28,668 Concentration] =

.

[0.944162 .y - 0.0145871 [Concentration of SQ 29,9691

(Area Under The Peak at 455.4)

= (Area Under The Peak at 458.4)

Fig. 1. Ions monitored in SQ 28,668 assay.

such as SQ 28,668" and can also be thought of as the fractional inhibition of thromboxane activity (see Discussion). Areas under the R and F time curves were calculated from 0 to 8 hours after dosing, using the trapezoidal rule. The ED,, concentration was estimated by fitting the responses to each of the seven concentrations of U46,619 to the cumulative normal distribution function by nonlinear least-squares regression. Data obtained at concentrations higher than the concentration at which the maximum response occurred were excluded from the curve fitting. The estimated ED,, was then obtained directly from the fitted curve. In one subject given 8 0 0 mg SQ 28,668 and in two subjects given 1200 mg, platelet aggregation at 1 hour was inhibited even when challenged with the highest concentration of U-46,619 (150 pmollL). In these instances the ED,, was estimated conservatively as 150 p.mol/L. ED,, values estimated in this way were not used for the Schild plots. Schild plots for each subject were performed according to standard methods.I0 Samples with SQ 28,668 concentrations below 50 nglml were excluded from the analysis because for such samples the shift of the platelet aggregation dose-response curve was too small to be quantitated accurately. Data points for samples with an R value greater than 100 were excluded because they fell far from the line obtained from the rest of the data. Plots were performed only for subjects who had at least three data points. The pA, and K, values were corrected for the fact that blood samples used for platelet aggregation studies were diluted with citrate anticoagulant solution. For measurement of drug levels, SQ 28,668 in dilute neutral aqueous solutions of plasma and urine was absorbed on C,, solid-phase columns (Analytichem

International, Inc., Harbor City, Calif.) and eluted with n-butyl acetate. The samples were esterified with 4 ~1 of pentafluorobenzyl bromide in 50 p1 of acetonitrile and in the presence of 10 p1 of N,N-diisopropylethyl amine at 40' C for 20 minutes. After removal of the excess of the reagent, samples were reacted with 250 p1 of N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) at 60" C for 1 hour to form the trimethylsilyl ether of the pentafluorobenzyl ester of SQ 28,668. These samples were then analyzed by GCInegative ion chemical ionization MS" with a HewlettPackard 5985B G U M S (Hewlett-Packard Co., Palo Alto, Calif.). The GC was equipped with a 3.5 m by 0.23 mm inside diameter Chrompack bonded phase CP SIL 8 CB fused silica capillary column (Chrompack, Inc., Bridgewater, N.J.) inserted to within '/s inch of the MS source by means of a heated (290" C) transfer line. At 290" C the injector was operated in the spitless mode with a total flow of 100 mllmin of helium carrier gas. The diverter valve was closed for 0.8 minute and an aliquot of 0.5 p1 of the BSTFs-15A solution was injected. The starting oven temperature of 200" C was held for 0.8 minute and raised at a rate of 30" Clmin to 300' C. Methane was delivered to the source at a temperature of 250" C and a flow rate of 17 mllmin giving a pressure of about 0.75 torr as measured with a thermocouple gauge. The ionizing current was 300 pamps at an ionizing voltage of 230 eV. The ions depicted in Fig. 1 at 455- and 458- were monitored to give the ratio of SQ 28,668 to SQ 29,969 (trideuterated SQ 28,668) internal standard added at the beginning of the extraction procedure. The calibration curves used to determine SQ 28,668 in plasma and urine were obtained by least-square fit of ratios measured at 2, 1.5, 1.O, 0.5, 0.25, 0.125, 0.0625, 0.0313, and 0.0 for SQ 2 8 , 6 6 8 l S Q 29,969. The ratios were measured three times and the average was used. The retention time was 2.4 minutes and the sensitivity was about 10 pg injected on the column. Protein-free filtrate was prepared with Amicon Centrifree devices (Amicon Corp., Danvers, Mass.). The SQ 28,668 content of these samples was measured by an RIAi2 that allowed use of smaller sample volumes than the G U M S procedure. Bleeding times were analyzed by a two-tailed paired t test on the logarithms of the ratios of postdose to predose values. Comparisons between dose groups were made by Dunnett's test. Pharmacokinetic parameters, including the AUC, were computed according to standard procedures. l3

VOLUME 40 NUMBER 6

Table I. Pharmacokinetic parameters

Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Dose (mg)

c,,,

t,,,,,,

fngW

(hr)

AUC (hr . ng/ml)*

tl,, (hr)*

25 0 50 0 100 0 200 0 400 0 800 0 1200 0

58.8 6.6 243.8 39.5 297.5 53.9 749.2 67.2 1721.3 326.5 3479.7 885.2 5296.7 1373.4

0.97 0.13 0.69 0.17 1.22 0.22 1.36 0.19 0.91 0.14 1.86 0.48 1.92 0.45

159t 16 380$ 76 766$ 105 2025t 214 3536t 605 9580 2157 13765 1967

5.5t 1.4 2.43: 0.4 1.83: 0.2 4.3t 1.9 3.v 0.7 2.47 0.5 3.2 0.4

*Because of the second rise in the plasma concentration-time curve. AUC and t ~ , , could not be accurately estimated for all subjects

tN

= 5.

Table 11. Mean (tSE) urinary SQ 28,668 concentrations (ngtml) Time after dose (hr)

Dose fwd

0-4

4-8

As part of the safety portion of this study, serum creatinine levels and 24-hour creatinine clearances were determined before, during, and after drug treatment. The means for each dose level of SQ 28,668 were compared by an analysis of covariance technique, which adjusted for differences on the predose day. Dunnett's multiple comparison test was then used to compare the means for each of the doses with that for placebo. Student's paired t tests were performed (using the pooled error terms from the above analyses) to compare the mean within each dose level on each study day with its corresponding value before dosing. The procedures of this study were approved by the Institutional Review Committee of the Medical Center at Princeton, Princeton, N. J.

RESULTS The plasma levels of SQ 28,668 measured after the first dose are shown in Fig. 2 and the derived phar-

8-12

12-24

macokinetic parameters are shown in Table I. The mean maximum SQ 28,668 plasma concentration (C,,,) varied from 59 to 5297 nglml, depending on the dose administered. The mean time of the maximum concentration (t,,,) varied from 0.7 to 1 .9 hours and tended to be longer after larger doses. A second rise in the plasma concentration-time curve at about 6 to 8 hours suggests possible enterohepatic recirculation of SQ 28,668. The ratio of the AUC to the dose tended to increase for the larger doses. Values for the terminal half-life of the plasma concentration-time curves (tl,,) were difficult to estimate accurately (because of the second rise in the plasma level-time curve) but means ranged from 1.8 to 5.5 hours. Protein binding of SQ 28,668 was about 87%. Table I1 summarizes the urinary excretion data. Maximal urinary concentrations were generally achieved 0 to 4 hours after dosing. An average of less than 0,5% of the administered dose was recovered in the urine during the 24 hours after drug dosing.

CLlN PHARMACOL THER DECEMBER 1986

638 Friedhoff et al.

TIME (HOURS)

Fig. 3. Mean inhibition of arachidonate (800 ~mol1L)-induced platelet aggregation measured in platelet-rich plasma obtained after the initial dose of SQ 28,668.

0.1

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6

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Fig. 2. Mean plasma SQ 28,668 concentrations as a function of time after the initial dose of SQ 28,668.

SQ 28,668 had no effect on the platelet count of platelet-rich plasma but caused a dose- and time-dependent inhibition of arachidonic acid-induced platelet aggregation (Fig. 3). Collagen-induced platelet aggregation was inhibited slightly (20% to 30%) 1 to 4 hours after dosing with the highest doses; adenosine diphosphate-induced aggregation was not affected by SQ 28,668 (data not shown). Fig. 4 shows the mean effect of a 1200 mg dose of SQ 28,668 on U46,619-induced platelet aggregation, measured ex vivo, as a function of time after dosing. SQ 28,668 caused a time-dependent shift to the right in the response curve. The same maximal level was achieved at all times, except 1 hour after dosing, when thromboxane inhibition was very profound. Inhibition of U46,619-induced platelet aggregation was quantitated by calculating R = [ED,, postdosel ED,, predosel-1, where ED,, postdose and ED,, predose are the concentrations of U46,619 required to produce a 50% maximal aggregation response at postdose and predose times, respectively. An additional param-

U46sl9 CONCENTRATION (pM)

Fig. 4. Maximum slope of the platelet aggregation trace as a function of the added U46,619 concentration and the time after a single 1200 mg dose of SQ 28,668 was administered.

eter, F = R/(R + I), that measures the fractional inhibition of thromboxane activity and the fraction of thromboxane receptors occupied by SQ 28,668" was also calculated. F and R + 1 increased in a dose-dependent manner (Figs. 5 and 6) as did the areas under the R and F time curves (Fig. 7). The areas under the R time curves were approximately proportional to the AUCs. Because SQ 28,668 caused a shift of the aggregation response curve usually without affecting the maximal response, the data of Figs. 2 and 6 were analyzed according to the method of Schild." The mean slope of the Schild plots was - 1.024 and the mean of the intercepts gave a pA, of 6.83 and a K, of

VOLUME 40 NUMBER 6

Dose 0 1200 mg

x 800 mg 0 400 mg

200 mg A 100 mg A 50 mg 0 25 mg 0 Placebo

TIME (HOURS)

Fig. 5. Mean F value as a function of time after SQ 28,668 dosing. F is a measure of inhibition of the TXA, receptor (see text). Negative F values indicate potentiation.

147 nmol/L. When corrected for protein binding of SQ 28,668 (87%), the PA, and KB were 7.72 and 19 nmol/L, respectively. Bleeding time measured 1 hour after SQ 28,668 dosing (Fig. 8) tended to be prolonged by doses of 400 mg or more. The data of Figs. 5 , 6 , and 8 suggested that bleeding time prolongation was associated with doses that caused geometric mean R + 1 values of about 21 or greater (or F values of 0.94 or greater). Doses that caused geometric mean R 1 values of about 8 or less ( F values of 0.86 or less) did not prolong bleeding time. Bleeding times measured 48 hours after the last dose were not affected by SQ 28,668. SQ 28,668 had no consistent effect on serum creatinine or creatinine clearance. No adverse reactions attributed to SQ 28,668 were noted during this study.

+

DISCUSSION Pharmacokinetic analysis of SQ 28,668 evaluated after single doses of 25 to 1200 mg revealed that the AUC increased disproportionately to the administered dose, suggesting that the pharmacokinetics of this drug are nonlinear. The t,,, ranged from 0.7 to 1.9 hours, tending to be longer after the higher doses. This is consistent with the idea that larger doses are more slowly absorbed or eliminated than the smaller doses. Dosing with SQ 28,668 in healthy male volunteers caused a dose-dependent inhibition of ex vivo platelet aggregation induced by U46,619 and arachidonic acid. Collagen-induced aggregation was slightly inhibited at short times after the highest doses of SQ 28,668, whereas adenosine diphosphate-induced aggregation

TIME (HOURS) 48 HOURS AFTER THE LAST (THIRD) DOSE

Fig. 6. Shift of the U46,619-induced platelet aggregation dose-response curve. R = concentration of U46,619 required to produce 50% maximal aggregation before dosing divided by the concentration required after dosing, minus 1 .

was not affected. U46,619 and arachidonic acid-induced platelet aggregation are mediated entirely via the platelet TXA, receptor,14.15whereas collagen-induced aggregation is mediated partially via this receptor.16 Adenosine diphosphate-induced aggregation (in the presence of indomethacin, which blocks second-phase aggregation) should be completely independent of t h r ~ m b o x a n e .Thus ' ~ the results of this study are consistent with the hypothesis that SQ 28,668 acts as a specific TXA, antagonist when administered to normal subjects. Schild analysis of the platelet aggregation data was consistent with the hypothesis that this antagonism is competitive. The apparent K, for SQ 28,668 was 147 nmol/L. Since SQ 28,668 is 87% protein bound, the true KB for the free compound would be about 19 nmol/L, a figure that is in excellent agreement with the dissociation constant of 20 nmolL reported for in vitro competitive binding studies of platelet membranes." This agreement suggests that SQ 28,668 is not metabolized to substances with significant thromboxane inhibitory activity. Assessment of the efficacy of antiplatelet drugs requires the study of large numbers of patients for long periods of time.'" For this reason, dose-ranging studies

640 Friedhof

et

CI.IN PHARMACOL THEK DECEMBER 1986

al.

0.801

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Dose of SQ 28,868 (mg) 'Significantly different than 1 (p