Journal Logo

Articles

Pharmacokinetics and Pharmacodynamics of SM-20302, a GPIIb/IIIa Receptor Antagonist, in Anesthetized Dogs

Rebello, Sam S.; Huang, Jinbao; Shiu, Wayne J.; Saito, Kumi; Kaneko, Munekiyo; Saitoh, Yoshikazu; Lucchesi, Benedict R.

Author Information
Journal of Cardiovascular Pharmacology: September 1998 - Volume 32 - Issue 3 - p 485-494
  • Free

Abstract

Activation of platelets and aggregation is an important phenomenon in the pathogenesis of cardiovascular diseases (1). Plaque rupture with subsequent platelet activation and thrombosis is a hallmark of acute ischemic syndromes of unstable angina and acute myocardial infarction (2). Platelet aggregation can be induced by activation of distinct receptors. For example, aspirin inhibits the cyclooxygenase-dependent formation of proaggregatory prostaglandin endoperoxides and thromboxane A2(3). Ticlopidine and clopidogrel block the adenosine diphosphate (ADP)-mediated activation of platelets (4-6). However, various pathways of platelet activation converge in a final common event, which is the surface expression of glycoprotein (GP) IIb/IIIa receptors and their cross-linking with soluble fibrinogen. During aggregation, platelets form a complex with soluble fibrinogen by their integrin receptors. The platelet GPIIb/IIIa complex, also known as the αIIbβ3, is the recognized receptor that constitutes the final pathway in aggregation (7,8). Pharmacologic blockade of the GPIIb/IIIa receptors, therefore, offers a effective means of achieving anticoagulation, irrespective of the mode of platelet activation. The clinical efficacy of 7E3, an antibody to the GPIIb/IIIa receptor, in angioplasty and restenosis (9,10) offers substantive evidence that the GPIIb/IIIa-receptor antagonists may serve as useful therapeutic agents in the prevention of arterial thrombosis.

In an attempt to develop a new class of GPIIb/IIIa-receptor antagonists, we performed a preclinical study examining the in vitro and in vivo actions of SM-20302, a synthetic GPIIb/IIIa-receptor antagonist reported to produce inhibition of ex vivo platelet aggregation in rhesus monkeys, guinea pigs, and mice (11). SM-20302 specifically inhibits fibrinogen binding to the GPIIb/IIIa receptor without influencing binding to fibronectin and vitronectin. Administration of SM-20302 was found to be less hemorrhagic than aspirin and ticlopidine in a mouse model of hydrochloric acid/ethanol-induced gastrointestinal bleeding (11). The primary aim of this investigation was to study the pharmacokinetic and pharmacodynamic characteristics of SM-20302 in anesthetized beagle dogs. Our results indicate that SM-20302 is a effective and specific antiplatelet agent targeted to the platelet GPIIb/IIIa receptor and possesses a favorable pharmacokinetic and pharmacodynamic profile in vivo.

METHODS

Chemical agents

SM-20302 was supplied by Sumitomo Pharmaceuticals, Inc. (Osaka, Japan) as a white powder, which was dissolved in 0.02N HCl/0.9% sodium chloride solution and administered intravenously in 1- to 3-ml volumes. The chemical structure of SM-20302 is shown in Fig. 1. Sodium citrate, ADP, arachidonic acid (AA), epinephrine, and standard reagents were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Heparin sodium injection, USP (1,000 U/ml) was purchased from Elkins-Sinn, Inc. (Cherry Hill, NJ, U.S.A.).

FIG. 1
FIG. 1:
Chemical structure of SM-20302 (MW = 525.5).

Experimental design

The study conformed to the position of the American Heart Association on research animal use adopted on November 11, 1984, by the American Heart Association. The procedures followed in this study were in accordance with the guidelines of the University of Michigan (Ann Arbor) Committee on the Use and Care of Animals, and with the Guide for Care and Use of Laboratory Animals, U.S. Department of Health, Education, and Welfare Publication No. NIH 78-23.

Healthy, male, purpose-bred beagle dogs (9-15 kg; HRP Inc., Kalamazoo, MI, U.S.A.) were used for the study. Dogs that satisfied the following preestablished criteria were included in the final protocol: (a) a circulating platelet count of ≥100,000/μl; (b) demonstrated ability for epinephrine-primed platelets to aggregate in response to both AA and ADP before administration of SM-20302; and (c) absence of heart worms on final postmortem examination. The latter criterion was used because heartworm-positive dogs possess anticoagulant substances that prolong the time to occlusion and therefore interfere in the experimental evaluation of new antithrombotic drugs. Dogs were anesthetized with sodium pentobarbital (30 mg/kg, i.v.), intubated, and ventilated with room air at a tidal volume of 30 mg/kg at a rate of 12 breaths/min (ventilator; Harvard Apparatus, South Natick, MA, U.S.A.). Catheters were inserted into the right femoral vein and right jugular vein for blood sampling and drug administration, respectively.

After a 60-min stabilization period, SM-20302 was administered intravenously in varying doses of 30 (n = 2), 100 (n = 4), 300 (n = 4), and 1,000 (n = 4) μg/kg. Blood samples (5-10 ml) were collected in sodium citrate (3.7% solution; one part of citrate to nine parts of blood) and heparin (100 U/ml solution; one part of heparin to nine parts of blood) in plastic syringes. For pharmacokinetic assessment, citrated blood samples (5 or 10 ml) were obtained at 0 (predrug), 2, 15, 30, 45, 60, 90, 120, 180, 240, 300, 360, 420, and 480 min after SM-20302 administration. Blood samples were centrifuged at 2,000 g for 10 min to obtain plasma. Plasma samples were stored at −70°C until the day of assay. For pharmacodynamic evaluation, citrated and heparinized blood samples (10 ml) were obtained at 0 (predrug), 60, 120, 240, 360, and 480 min after SM-20302 administration.

Ex vivo platelet-aggregation studies

Citrated and heparinized blood samples were processed as follows. The whole blood cell count was determined with an H-10 cell counter (Texas International Laboratories, Inc., Houston, TX, U.S.A.). Platelet-rich plasma (PRP), the supernatant present after centrifugation of anticoagulated whole blood at 1,000 r/min for 5 min (140 g), was diluted with platelet-poor plasma (PPP) to achieve a platelet count of 200,000/mm3. PPP was prepared after the PRP was removed by centrifuging the remaining blood at 2,000 g for 10 min and discarding the bottom cellular layer. Citrated PPP samples were frozen at −70°C for later drug assays. Ex vivo platelet aggregation was assessed by established spectrophotometric methods with a four-channel aggregometer (BioData-PAP-4; Bio Data Corp., Hatboro, PA, U.S.A.) by recording the increase in light transmission through a stirred suspension of PRP maintained at 37°C. Platelet-aggregation profiles were obtained with ADP (20 μM) and AA (0.65 mM). A subaggregatory concentration of epinephrine (550 nM) was used to prime the platelets before the addition of the platelet agonists. Values for platelet aggregation were expressed as percentage of light transmission standardized to PRP and PPP samples, yielding 0% and 100% light transmission, respectively.

Measurement of ionized calcium in PRP

The inhibitory effect of SM-20302 (10 ng/ml) on ADP-induced platelet aggregation was examined in the presence of varying calcium in heparinized PRP. The ionized calcium in the PRP medium was altered by using sodium citrate. The final concentration of ionized calcium in the PRP medium used for platelet aggregation studies was determined by an ion analyzer (Nova-6; Nova Biomedical Instruments, Waltham, MA, U.S.A.).

Estimation of plasma concentration of SM-20302

SM-20302 was analyzed by high-performance liquid chromatography (HPLC) after extraction from citrated plasma samples. Plasma (1 ml) was mixed with 50 μl of SL-10579 (10 μg/ml; internal standard for SM-20302) solution and 3 ml of sodium acetate buffer (10 mM; pH 5). The mixture was applied to a Bond Elut certify-II column (Varian, Sugarland, TX, U.S.A), which was preconditioned with methanol followed by 10 mM sodium acetate buffer. The column was rinsed with 3 ml of 10 mM sodium acetate buffer and 3 ml of 10% aqueous methanol solution. The eluate was evaporated at room temperature and dissolved in 0.2 ml of the mobile phase (0.1% trifluoroacetic acid in 22% of aqueous acetonitrile solution). Chromatography was performed by using a Puresil C18 column (Waters Corp., Milford, MA, U.S.A.) (4.6 mm i.d. × 25 cm, 5 μm), flow rate of 1 ml/min, sample injection volume of 50 μl, and column temperature of 35°C. The eluate was monitored at 230 nm by a UV detector (Shimadzu SPD-10AD, Kyoto, Japan). Plasma concentration of SM-20302 was calculated from a standard curve prepared by spiking known quantities of the drug in blank plasma samples.

Validation of dosing regimen in a canine model of coronary artery thrombosis

To validate the pharmacokinetics and pharmacodynamics, we designed a bolus-plus-infusion regimen for four anesthetized dogs with the aim of maintaining a concentration that would approximate the median inhibitory concentration (IC50) value of SM-20302 for inhibiting platelet aggregation in heparinized PRP (hPRP). Dogs were anesthetized and instrumented as described earlier. The thrombosis model (12) used for this part of the study was used successfully for related studies by other investigators (13-16). In brief, the heart was exposed and suspended in a pericardial cradle. The left circumflex coronary artery was isolated, and a flow probe (model 1.5RB24; Transonic Systems Inc., Ithaca, NY, U.S.A.) was placed around the artery. An external stenosis was produced by securing a suture-ligature around the artery and an adjacent 18-gauge hypodermic needle and then removing the needle. An intracoronary electrode was fashioned from the tip of a 25-gauge hypodermic needle (4 mm in length) attached to a 30-gauge Teflon-insulated, silver-coated copper wire. The needle-tip electrode was inserted into the arterial wall at a site distal to the flow probe and in close proximity to the stenosis. Electrolytic injury was induced by connecting the electrode to the positive pole (anode) of a dual-channel square-wave generator (Grass S88 stimulator and a Grass Constant Current Unit, model CCU1A; Grass Instrument Co., Quincy, MA, U.S.A.). The current delivered to the vessel was monitored continuously on an ammeter and maintained at 150 μA for 3 h during the infusion of SM-20302. Blood flow was monitored continuously to determine the time to occlusion. Template bleeding time was determined with the use of a device (Surgicutt; International Technidyne Corporation, Edison, U.S.A.), which made a uniform incision 5 mm long and 1 mm deep on the upper surface of the tongue. The tongue lesion was blotted with filter paper every 30 s until the transfer of blood to the filter paper ceased. In all the animals, bleeding-time determinations were carried out for a maximal period of 30 min.

Data analysis

A two- or three-compartment pharmacokinetic model was fitted to the SM-20302 concentration-versus-time profiles by using the software program PCNONLIN (version 4.0; Statistical Consultants, Lexington, KY, U.S.A.). Parameter estimations were made for all animals by using nonlinear regression and weighting the residuals by 1/c(t), where c(t) represents the predicted plasma concentration of SM-20302. The kinetic parameters such as clearance (CL), initial volume of distribution (V1), volume of distribution at steady state (Vss), mean residence time (MRT), distribution half-life (T1/2α), and terminal half-life (T1/2last) were calculated by using standard equations (17). The PCNONLIN output was confirmed by noncompartmental analysis wherein the linear regression of the terminal concentration-time data was used to calculate the CL, MRT, and Vss.

To investigate the pharmacodynamics of the antiplatelet effects of SM-20302, the observed platelet-inhibition and measured plasma-concentration data from all the dogs were pooled and fitted to a sigmoid Emax model by using the software program PCNONLIN (version 4.0; Statistical Consultants). The following equation was used to calculate the parameters. Equation (1)

where E is the predicted effect (percentage platelet inhibition), Emax is the maximal effect (percentage platelet inhibition), IC50 is the plasma concentration of SM-20302 that produces 50% of the maximal inhibition, and γ is the Hill coefficient, which measures the steepness of curve. Reciprocal weighting (1/E) was used to obtain weighted least-squares estimates of Emax, IC50 and γ.

Statistics

The data are expressed as mean ± SEM. A one-way analysis of variance (ANOVA) was used to analyze the pharmacokinetic data. Similarly, effect of SM-20302 on ADP/AA-induced platelet aggregation and coronary artery blood flow was investigated by ANOVA. Fisher's protected least-significant difference (PLSD) and Bonferroni/Dunn post hoc analysis were used to determine significance at p < 0.05. In all analyses, values were determined to be statistically different at a level of p < 0.05.

RESULTS

Pharmacokinetic profile of SM-20302

All 14 dogs completed the study successfully. The plasma-concentration profile of intravenous SM-20302 is shown in Fig. 2. The administration of increasing doses of SM-20302 was associated with a dose-dependent increase in the plasma concentration. For the 30- and 100-μg/kg doses, the kinetics of SM-20302 could be described by using a two-compartment model. For the 300- and 1,000-μg/kg doses, a three-compartment model was adequate. Table 1 summarizes the estimated pharmacokinetic parameters obtained in the experimental animals. Model-dependent parameters such as the C0 and total area under the curve (AUC) were increased dose dependently, whereas model-independent parameters such as the CL, V1, T1/2α, and T1/2last were constant and dose independent, indicating linear kinetics. Across the wide range of doses used, the initial phase of distribution had an half-life of 4 min and initial volume of distribution of 124-208 ml/kg. The steady-state volume of distribution, Vss, was large and variable, ranging from 965 to 1,700 ml/kg. One-way ANOVA comparing the model-independent parameters indicated that the plasma clearance (p = 0.42) and terminal half-life (p = 0.88) were statistically similar. The mean plasma clearance and terminal half-life for SM-20302 in dogs were found to be 7.54 ml/min/kg and 188 min, respectively.

FIG. 2
FIG. 2:
Mean plasma concentration of SM-20302 versus time data in dogs. A dose-dependent increase in C0 was observed. The disposition kinetics of SM-20302 could be described by a two- or three-compartment model. Values are expressed as mean ± SEM.
TABLE 1
TABLE 1:
Pharmacokinetic parameters for SM-20302 in anesthetized dogs

Pharmacodynamics of platelet inhibition

All platelet-aggregation determinations were performed in citrated PRP (cPRP) and hPRP samples. The percentage platelet aggregation induced by ADP in cPRP before the drug administration was 81, 65, 79, and 75 in the 30-, 100-, 300-, and 1,000-μg/kg dose groups, respectively (Figs. 3-5). Similarly, percentage platelet aggregation induced by ADP in hPRP before the drug administration were 84, 81, 78, and 69. There was no statistically significant difference in the aggregation profiles of ADP in cPRP and hPRP at baseline. Similar results were obtained with AA.

FIG. 3
FIG. 3:
Effect of intravenous SM-20302 (100 μg/kg; n = 4) on ex vivo platelet aggregation induced by adenosine diphosphate (ADP; 20 μM) and arachidonic acid (AA; 0.65 mM). Platelet aggregations were performed in citrated platelet-rich plasma (cPRP) and heparinized platelet-rich plasma (hPRP) (see Methods). Values are expressed as mean ± SEM of the percentage platelet-aggregation values. Preselected time intervals represent time elapsed after drug administration.
FIG. 4
FIG. 4:
Effect of intravenous SM-20302 (300 μg/kg; n = 4) on ex vivo platelet aggregation induced by adenosine diphosphate (ADP; 20 μM) and arachidonic acid (AA; 0.65 mM). Platelet aggregation were performed in citrated platelet-rich plasma (cPRP) and heparinized platelet-rich plasma (hPRP) (see Methods). Values are expressed as mean ± SEM of the percentage platelet-aggregation values. Preselected time intervals represent time elapsed after drug administration.
FIG. 5
FIG. 5:
Effect of intravenous SM-20302 (1,000 μg/kg; n = 4) on ex vivo platelet aggregation induced by adenosine diphosphate (ADP; 20 μM) and arachidonic acid (AA; 0.65 mM). Platelet aggregations were performed in citrated platelet-rich plasma (cPRP) and heparinized platelet-rich plasma (hPRP) (see Methods). Values are expressed as mean ± SEM of the percentage platelet-aggregation values. Preselected time intervals represent time elapsed after drug administration.

Because the 30-μg/kg dose had an insignificant effect on the ex vivo platelet aggregation, only two animals were included in this group, and they were excluded from the statistical analysis. Single intravenous administration of increasing doses of SM-20302 had marked but reversible effects on the ex vivo platelet aggregation (Figs. 3-5). Maximal inhibition of aggregation was observed at 30 min after SM-20302 administration. The maximal percentage inhibition produced by the 30- (data not shown because of insignificant effect), 100-, 300-, and 1,000-μg/kg doses in response to ADP-induced aggregation in cPRP were 88, 93, 95, and 91, respectively. Similarly, the maximal percentage inhibition produced by the 30-, 100-, 300-, and 1,000-μg/kg doses in response to AA-induced aggregation in cPRP was 90, 94, 91, and 95, respectively. A dose-response relation was not observed in the extent of platelet inhibition in cPRP. In contrast, when platelet aggregations were conducted in hPRP, a dose-related effect on ex vivo platelet aggregation was observed. By using ADP as an agonist, the maximal percentage inhibition produced in hPRP was 44, 56, 59, and 89 for 30-, 100-, 300-, and 1,000-μg/kg doses, respectively. Similarly, with AA as an agonist, the maximal percentage inhibition produced was 45, 72, 70, and 84. The inhibitory effect produced by 30 μg/kg returned to baseline values within 60 min (data not shown). On the other hand, the inhibition produced by the 1,000-μg/kg dose persisted for 6 h after the administration of SM-20302.

To estimate the in vivo IC50, the plasma-concentration data from each group were pooled and fitted to a sigmoid Emax model, shown in Fig. 6. The curved lines represent the simulated profile of platelet inhibition. It was noted that the curve for hPRP (r2 = 0.72) was displaced to the right of that for cPRP (r2 = 0.61). The estimated IC50 values for inhibiting ADP-induced aggregation in cPRP and hPRP were 19.38 and 79.16 ng/ml, respectively. Similarly, the estimated IC50 values for inhibiting AA-induced aggregation in cPRP and hPRP were 14.05 and 89.95 ng/ml, respectively. The Hill coefficient for the concentration-effect relations ranged from 2 to 3, indicating a steep concentration-response relation. Administration of a wide dose range of SM-20302 did not affect the cell counts (platelets, WBC, RBC) of the dogs under study (Table 2).

FIG. 6
FIG. 6:
Pharmacodynamics of SM-20302-induced platelet inhibition in citrated platelet-rich plasma (cPRP) and heparinized platelet-rich plasma (hPRP). Pooled plasma concentration values and the observed percentage inhibition of platelet aggregation were related by using a sigmoid Emax model. A fourfold to sixfold difference in median inhibitory concentration (IC50) was observed for SM-20302 in cPRP and hPRP.
TABLE 2
TABLE 2

Relation between calcium and extent of platelet inhibition

A subthreshold concentration of SM-20302 (10 ng/ml) was selected to study the potentiation of platelet inhibition in the presence of varying calcium concentrations. When the ionized calcium was diminished in the hPRP samples, a progressive increase in the inhibition of ADP-induced platelet aggregation was observed (Fig. 7). The maximal and minimal values of inhibition were observed when the ionized calcium concentration was 0.035 and 0.9 mM, respectively.

FIG. 7
FIG. 7:
Effect of calcium on the inhibitory action of SM-20302 (10 ng/ml). Adenosine diphosphate (ADP)-induced platelet aggregation was examined by using heparinized platelet-rich plasma (hPRP) containing varying concentrations of ionized calcium. Serial decrease in calcium was associated with a progressive increase in platelet inhibition.

Design and validation of dosing regimen

Based on the estimated pharmacokinetic parameters, an infusion regimen was designed. A bolus dose of 100 μg/kg was calculated to achieve rapidly a concentration that would approximate the IC50 value of SM-20302 for inhibiting platelet aggregation in hPRP. The bolus dose was immediately followed by a continuous infusion of 1 μg/kg/min for 3 h. Figure 8 indicates that the bolus-plus-infusion regimen gave rise to a mean plasma concentration of 64.5 ng/ml during the infusion period. As predicted earlier, the maximal inhibition of platelet aggregation in cPRP by using ADP and AA was 99 and 93%, whereas that in hPRP was 67 and 64%, respectively. It was observed that the extent of platelet inhibition produced by SM-20302 in hPRP closely paralleled its plasma concentration during the duration of infusion.

FIG. 8
FIG. 8:
Validation of a dosing regimen for SM-20302. Plasma concentration of SM-20302 during a 3-h infusion (100 μg/kg + 1 μg/kg/min) in dogs is shown on the right Y-axis. Effect of intravenous SM-20302 on ex vivo platelet aggregation induced by adenosine diphosphate (ADP; 20 μM) and arachidonic acid (AA; 0.65 mM) is shown on the left Y-axis. Platelet aggregation were performed in citrated platelet-rich plasma (cPRP) and heparinized platelet-rich plasma (hPRP; see Methods). Values are expressed as mean ± SEM. Preselected time intervals represent time during drug infusion.

Electrolytic injury to the coronary artery in saline-treated dogs (n = 6) produced a progressive decline in the blood flow. All dogs had coronary artery occlusion in 62 ± 8 min (Fig. 9). Infusion of SM-20302 (n = 4) for 3 h maintained the vessel patency despite vessel-wall injury (p < 0.05). The template bleeding time was increased from a baseline (predrug) value of 2.5 to 30 min after 1 h of infusion. At 3 h, the bleeding time declined to 20 ± 5 min.

FIG. 9
FIG. 9:
Antithrombotic efficacy of the 100 μg/kg + 1 μg/kg/min infusion regimen of SM-20302. Coronary artery blood flow was maintained during the infusion of SM-20302 despite persistent vessel-wall injury. Values are expressed as mean ± SEM. Preselected time intervals represent time during drug infusion.

DISCUSSION

The platelet GPIIb/IIIa-receptor complex interacts with soluble adhesive ligands such as fibrinogen, von Wille-brand factor (vWF), fibronectin, and vitronectin, an interaction that serves a central role in platelet aggregation and arterial thrombus formation. The adhesive ligands contain an Arg-Gly-Asp (RGD) recognition sequence necessary for binding to the GPIIb/IIIa receptor. Antagonists of the GPIIb/IIIa receptors include monoclonal antibodies (7E3), polypeptides containing RGD (bitistatin) or KGD sequence (integrilin), cyclic RGD mimetic peptides (MK-383 and TP-9201), and synthetic compounds (SC-54684, Ro 43-8857, and L-738,167). The pharmacodynamic profile of platelet inhibition induced by these antagonists, except 7E3, parallels their pharmacokinetic half-life in vivo. The reported biological half-life for 7E3 in humans is 7 h (18). Gold et al. (19) observed that intravenous administration of 7E3 (0.2 mg/kg) produced complete inhibition of the platelet aggregation at 1 h and a bleeding-time prolongation of >30 min. The inhibition of platelets was protracted over 48-72 h, and some patients had high levels of immunoglobulin G (IgG), suggestive of an immunologic response. Despite these effects, 7E3 is reported to reduce the incidence of ischemic complications (9) and restenosis (10) in patients undergoing angioplasty. Administration of MK-383 at the rate of 0.4 μg/kg/min for 1 h and 0.2 μg/kg/min for 4 h produced a 4.6- and 5.5-fold extension in bleeding time and complete inhibition of platelets during the infusion. However, the half-life of MK-383 was found to be 1.6 h, and the inhibition of platelets was reversible (20). In the RESTORE trial, intravenous administration (bolus-plus-36 h infusion) of MK-383 showed a significant 38% and an insignificant 24% relative reduction in the clinical composite outcome (death) on days 2 and 30, respectively (21).

Because current agents pose potential problems of immunogenicity and short survival time in the circulation, there is a continued effort to develop synthetic GPIIb/IIIa antagonists possessing a relatively long half-life and favorable platelet-inhibition and bleeding-time profiles. In our study, we examined the pharmacokinetic and pharmacodynamic properties of SM-20302 in anesthetized beagle dogs. Increasing doses of SM-20302 were well tolerated by the dogs and had no effect on the circulating blood cell counts. It appears that SM-20302 exhibits linear kinetics in beagle dogs. This observation is supported by the fact that the model-dependent parameters such as the C0 and total AUC were increased dose dependently, whereas model-independent parameters such as the CL, V1, T1/2α, and T1/2last were constant and dose independent. Although the plasma clearance values were statistically insignificant between the four dose groups, the 30-μg/kg dose group dogs showed a slightly higher value. This may be due to underestimation of the AUC or variability in the plasma concentration during the terminal phase of elimination or both. The volume of distribution at steady state was large, ranging between 965 and 1,700 ml/kg. Similar high volumes of distribution were reported in dogs for other RGD-mimetics such as L-703,014 (610 ml/kg; 22) and TP-9201 (574-746 ml/kg; 23). The higher volume of distribution perhaps necessitated the use of a three-compartment model to describe the kinetics at larger doses. This may be a result of excessive binding to the blood components or the vasculature. The rapid clearance of SM-20302 resulted in a mean residence time of 145-234 min, which paralleled the terminal half-life (∼3.1 h). The half-life of SM-20302 is relatively longer compared with L-703,014 (1.9 h; 22), MK-383 (1.6 h; 20), and TP-9201 (2.5-2.6 h; 23) but much shorter than that reported for L-738,167 (4 days; 24).

Earlier we examined the effect of SM-20302 on bleeding-time prolongation in anesthetized dogs. It was shown that the maximal prolongations of the template bleeding time induced by the 100-, 300-, 600-, and 1,000-μg/kg doses were 2.5-, 9.5-, 10-, and >10-fold, respectively, and the effect was reversible (25). The pharmacodynamics of SM-20302-induced ex vivo platelet inhibition in our study was examined in hPRP in addition to the conventionally used cPRP. It was reported earlier (26) that the in vivo efficacy of TP-9201, a GPIIb/IIIa-receptor antagonist, correlates well with the ex vivo platelet inhibition in hPRP but not in cPRP. The main reason for this discrepancy is the concentration of ionized calcium in cPRP, which is 18- to 21-fold less than that in hPRP (25). A physiologic concentration of extracellular ionized calcium is needed to maintain the integrity of the GPIIb/IIIa heterodimeric complex on the platelet surface (8,27,) and fibrinogen binding to the activated integrin receptor (28,29,30). Human platelets incubated with extracellular calcium chelators, EDTA and EGTA, undergo morphologic changes (31), fail to aggregate (27,32,33), or bind to fibrinogen after activation by ADP (28,30,34). In some of these experiments, the chelation of calcium was accompanied by dissociation of the integrin receptor into free GPIIb and GPIIIa subunits (27,32). Under hypocalcemic conditions, the response to the platelet agonists can vary depending on the extent of calcium depletion (35).

In our study, the aggregation values in cPRP and hPRP were similar before SM-20302 administration. However, a marked difference in the aggregation profile was observed in cPRP and hPRP after SM-20302 administration. The estimated IC50 value in cPRP was fourfold to sixfold lower than that in hPRP, which may indicate that SM-20302 is a potent antagonist in cPRP. SM-20302 in the cPRP may have acted synergistically with the low extracellular [Ca2+] to reduce the platelet-aggregation response, thereby resulting in a false indication of its ex vivo inhibitory potency. On the other hand, the ex vivo inhibition of platelets in hPRP by SM-20302 may represent its true mode of interaction with the GPIIb/IIIa as that occurring in vivo under normocalcemic conditions. In this study, we also found that serial depletion of ionized calcium in the hPRP medium enhanced the inhibitory actions of a subthreshold concentration of SM-20302. Earlier, we found that the in vitro IC50 of c7E3, MK-383, DMP-728, and SM-20302 for inhibiting ADP- or tartrate-resistant acid phosphatase (TRAP)-induced human platelet aggregation in cPRP was approximately twofold lower than that in D-Phe-Pro-Arg-chloromethyl ketone (PPACK)-anticoagulated blood (36). A similar phenomenon was demonstrated for integrilin (37) by observing that the IC50 values for ADP- and TRAP-induced platelet aggregation in cPRP were fourfold and 7.5-fold lower than those in pPRP.

It is apparent that the IC50 value estimated in cPRP has limited significance, whereas that obtained by using hPRP may reflect truly the potency of SM-20302 in inhibiting platelets in vivo. Therefore dosing regimens for a GPIIb/IIIa antagonist based on its inhibition profile in cPRP can be misleading. This hypothesis is supported by the clinical trials with integrilin (37). By using four different dosing strategies of integrilin in patients for elective percutaneous interventions, it was observed that administration of bolus (90-180 μg/kg) plus infusion (0.5-1 μg/kg/min for 20 h) produced no dose-dependent inhibition of ADP-induced platelet aggregation in cPRP. The maximal inhibition produced by all four dosing strategies was ∼90% (38). In the IMPACT-II study (39), it was noted that despite 70-78% inhibition of ADP-induced platelet aggregation in cPRP, the integrilin therapy did not influence 6-month clinical outcomes after percutaneous transluminal coronary angioplasty (PTCA). From previous efficacy studies with TP-9201, we showed that a partial inhibition (45%) in hPRP was associated with antithrombotic efficacy in vivo, whereas lack of inhibition in hPRP was not (26). In this study, based on the estimated pharmacokinetic parameters for SM-20302, we designed a dosing regimen to achieve and maintain a concentration that would approximate the IC50 value of SM-20302 in hPRP. The extent of platelet inhibition obtained in hPRP during the infusion was 64-67%. Moreover, the fact that SM-20302 maintained the vessel patency despite vessel-wall injury in a canine model of coronary artery thrombosis indicates that indeed the infusion regimen was efficacious in vivo.

In conclusion, the results demonstrate that SM-20302 exhibits linear pharmacokinetics and that its ability to inhibit platelet aggregation in normocalcemic PRP correlates with its in vivo antithrombotic efficacy.

Acknowledgment: This study was supported in part by the Cardiovascular Pharmacology Research Fund. During the tenure of this study, S.S.R was the recipient of an Advanced Postdoctoral Fellowship from the American Heart Association, Michigan Affiliate, and a Merck Pharmacology Postdoctoral Fellowship.

We thank Dr. Gabriel Robbie (University of Illinois at Chicago) for the critical review of the manuscript.

REFERENCES

1. Hennekens CH, Buring JE, Sandercock P, Collins R, Peto R. Aspirin and other antiplatelet agents in the secondary and primary prevention of cardiovascular disease. Circulation 1989;80:749-56.
2. MacIsaac AI, Thomas JD, Topol EJ. Toward the quiescent coronary plaque. J Am Coll Cardiol 1993;22:1228-41.
3. Smith JB, Willis L. Aspirin selectively inhibits prostaglandin production in human platelets. Nature 1971;231:235-7.
4. Herbert JM, Frehel D, Vallee E, et al. Clopidogrel: a novel antiplatelet and antithrombotic agent. Cardiovasc Drug Rev 1993;11:180-98.
5. Dunn FW, Soria J, Soria C, Thomaidis A, Lee H, Caen JP. In vivo effect of ticlopidine on fibrinogen-platelet cofactor activity and binding of fibrinogen to platelets. Agents Actions 1984;15(suppl):97-103.
6. Yao SK, Ober JC, McNatt J, et al. ADP plays an important role in mediating platelet aggregation and cyclic flow variations in vivo in stenosed and endothelium-injured canine coronary arteries. Circ Res 1992;70:39-48.
7. Pytela R, Pierscbacher MS, Ginsberg MH, Plow EF, Rouslahti E. Platelet membrane glycoprotein IIb/IIIa: a member of a family of RGD specific adhesion receptors. Science 1986;231:1559-62.
8. Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb/IIIa complex. Blood 1988;71:831-43.
9. The EPIC Investigators. Evaluation of a chimeric monoclonal antibody c7E3 Fab fragment directed against the platelet glycoprotein IIb/IIIa receptor for preventing ischemic complications of high-risk angioplasty. N Engl J Med 1994;330:956-61.
10. Topol EJ, Califf RM, Weisman HF, et al. Randomized trial of coronary intervention with antibody against platelet IIb/IIIa integrin for reduction of clinical restenosis. Lancet 1994;343:881-6.
11. Sakurama T, Horisawa S, Kurata H, et al. SM-20302, a novel GPIIb/IIIa antagonist, has shown a selective antithrombotic activity without increasing the risk of hemorrhage in vivo [Abstract]. Thromb Haemost 1997;(suppl):7.
12. Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in the ambulatory canine: a model for in vivo evaluation of antithrombotic agents. Thromb Res 1980;17:841-53.
13. Nicolini, FA, Lee P, Malycky JL, et al. Selective inhibition of factor Xa during thrombolytic therapy markedly improves coronary artery patency in a canine model of coronary thrombosis. Blood Coagul Fibrinolysis 1996;7:39-48.
14. Shebuski RJ, Stabilito IJ, Sitko GR, Polokoff MH. Acceleration of recombinant tissue-type plasminogen activator-induced thrombolysis and prevention of reocclusion by the combination of heparin and the Arg-Gly-Asp-containing peptide bitistatin in a canine model of coronary thrombosis. Circulation 1990;82:169-77.
15. Lynch JL, Sitko GR, Lehman DE, Vlasuk GP. Primary prevention of coronary arterial thrombosis with the factor Xa inhibitor rTAP in a canine electrolytic injury model. Thromb Haemost 1995;74:640-5.
16. Chen LY, Nichols WW, Hendricks J, Mehta JL. Myocardial neutrophil infiltration, lipid peroxidation, and antioxidant activity after coronary artery thrombosis and thrombolysis. Am Heart J 1995;129:211-8.
17. Rowland M, Tozer TM, eds. Clinical pharmacokinetics: principles and theory. Malvern, PA: Lea & Febiger, 1986.
18. Kleiman NS, Raizner AE, Jordan R, et al. Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors. J Am Coll Cardiol 1995;26:1665-71.
19. Gold HK, Gimple LW, Yasuda T, et al. Pharmacodynamic study of F(ab′)2 fragments of murine monoclonal antibody 7E3 directed against human platelet glycoprotein IIb/IIIa in patients with unstable angina pectoris. J Clin Invest 1990;86:651-9.
20. Barrett JS, Murphy G, Peerlinck K, et al. Pharmacokinetics and pharmacodynamics of MK-383, a selective non-peptide platelet glycoprotein IIb/IIIa receptor antagonist, in healthy men. Clin Pharmacol Ther 1994;56:377-88.
21. King SB III. Administration of tirofiban (MK-0383) will reduce the incidence of adverse cardiac outcome following PTCA/DCA (RESTORE) [Abstract]. J Am Coll Cardiol 1996;27(suppl A):xxi.
22. Barrett JS, Gould RJ, Ellis JD, et al. Pharmacokinetics and pharmacodynamics of L-703,014, a potent fibrinogen receptor antagonist, after intravenous and oral administration in the dog. Pharm Res 1994;11:426-31.
23. Modi NB, Baughman SA, Paasch BD, Celniker A, Smith SY. Pharmacokinetics and pharmacodynamics of TP-9201, a GPIIb/IIIa antagonist, in rats and dogs. J Cardiovasc Pharmacol 1995;25:888-97.
24. Prueksaritanont T, Gorham LM, Naue JA, Hamill TG, Askew BC, Vyas KP. Disposition of L-738,167, a potent and long-acting fibrinogen receptor antagonist, in dogs: dose-dependent pharmacokinetics. Drug Metab Dis 1997;25:355-61.
25. Rebello SS, Huang J, Saito K, Lucchesi BR. The in vivo efficacy of SM-20302, a GPIIb/IIIa receptor antagonist, correlates with ex vivo platelet inhibition in heparinized blood but not in citrated blood. Arterioscler Thromb Vasc Biol 1998;18:954-60.
26. Rebello SS, Driscoll EM, Lucchesi BR. TP-9201, a glycoprotein IIb/IIIa platelet receptor antagonist, prevents rethrombosis after successful arterial thrombolysis in the dog. Stroke 1997;28:1789-96.
27. Pidard D, Didry D, Kunicki TJ, Nurden AT. Temperature-dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. Blood 1986;67:604-11.
28. Marguerie GA, Plow EF, Edgington TS. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem 1979;254:5357-63.
29. Mustard JF, Packham MA, Kinlough-Rathbone RL, Perry DW, Regoeczi E. Fibrinogen and ADP-induced platelet aggregation. Blood 1978;52:453-66.
30. Peerschke EI, Zucker MB, Grant RA, Egan JJ, Johnson MM. Correlation between fibrinogen binding to human platelets and platelet aggregability. Blood 1980;55:841-7.
31. White JG. Effects of ethylenediamine tetracetic acid (EDTA) on platelet structure. Scand J Haematol 1968;5:241-54.
32. Shattill SJ, Brass LF, Benett JS, Pandhi P. Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex. Blood 1985;66:92-8.
33. Zucker MB, Grant RA. Nonreversible loss of platelet aggregability induced by calcium deprivation. Blood 1978;52:505-14.
34. Lanza F, Stierle A, Gachet C, Cazenave JP. Differential effects of extra- and intracellular calcium chelation on human platelet function and glycoprotein IIb/IIIa complex stability. Nouv Rev Fr Hematol 1992;34:123-31.
35. Cimminiello C, Milani M, Uberti T, Arpaia G, Bonfardeci G. Effects of ticlopidine and indobufen on platelet aggregation induced by A23187 and adrenaline in the presence of different anticoagulants. J Int Med Res 1989;17:514-20.
36. Rebello SS, Huang JB, Faul JD, Lucchesi BR. In vitro assessment of GPIIb/IIIa receptor antagonists and the role of ionized calcium. Circulation 1998 (submitted).
37. Phillips DR, Teng W, Arfsten A, et al. Effect of Ca2+ on GPIIb-IIIa interactions with integrilin. Circulation 1997;96:1488-94.
38. Ohman EM, Harrington RA, Lincoff AM, Kitt MM, Kleiman NS, Tcheng JE. Early clinical experience with integrilin, an inhibitor of the platelet glycoprotein IIb/IIIa integrin receptor. Eur Heart J 1995;16(suppl L):50-5.
39. Resar JR, Brinker JA, Gerstenblith G, et al. Disparity of integrilin inhibition of platelet aggregation and GP IIb/IIIa fibrinogen binding in angioplasty patients [Abstract]. Circulation 1996;94(suppl I):I-98.
Keywords:

Antiplatelet agent; Platelet aggregation; Pharmacokinetics; Pharmacodynamics; GPIIb/IIIa-receptor antagonist

Copyright © 1998 Wolters Kluwer Health, Inc. All rights reserved.