SC-54684A: An Orally Active Inhibitor of Platelet Aggregation
Background Intravenous therapy has been shown to be beneficial in the prevention of acute platelet-associated thrombotic events. However, orally active agents would be advantageous for chronic therapy. Fibrinogen receptor antagonists block the fibrinogen/platelet interaction and thus inhibit a step required for thrombus formation. To date, no orally active fibrinogen binding inhibitors have been characterized. SC-54684A, now in clinical trial, is the orally active prodrug of a potent and specific fibrinogen binding antagonist.
Methods and Results We measured inhibition of 125I-fibrinogen binding to activated platelets and inhibition of aggregation in platelet-rich plasma to selected agonists and showed IC50s of 1.0×10−8 and 3 to 7×10−8 mol/L, respectively. Specificity of the active moiety was determined by studying its effect on the binding of (1) neutrophils to interleukin (IL)-1β–stimulated endothelial cells, (2) endothelial cells to fibronectin, and (3) vitronectin to isolated vitronectin and fibrinogen receptors. No effect was observed on the binding neutrophils to IL-stimulated endothelial cells or endothelial cell binding to fibronectin. There was a fivefold separation between binding to isolated receptors of vitronectin and fibrinogen. Collagen-induced aggregation was inhibited by 80%, and bleeding time was increased ≈2.5-fold when the active moiety was infused to steady state at 0.2 μg/kg per minute in dogs. When the ester prodrug was given orally and the active moiety was given intravenously, the oral systemic activity was ≈20%. Pharmacokinetic analysis after intravenous infusion of the prodrug or active moiety showed that the prodrug was rapidly converted to the active moiety; the active moiety had a t of 6.5 hours. When the prodrug was administered both orally and intravenously, the systemic availability of the active moiety was 62%.
Conclusions SC-54684A, an orally active antiplatelet drug now in clinical trial, is shown to be a potent, specific fibrinogen binding inhibitor that blocks platelet aggregation to a wide variety of known stimuli and has good bioavailability in animals.
Several important thrombotic diseases such as unstable angina and myocardial infarction have been shown to be related to platelet aggregation leading to thrombus formation. Although intravenous drugs may be useful in acute situations, long-term administration for prevention of secondary events may require the use of orally active agents. Currently available oral antiplatelet agents, such as aspirin and ticlopidine, have been shown to be beneficial in reducing myocardial reinfarction and mortality after coronary occlusions.1 The protection afforded by these agents is remarkable because aspirin and ticlopidine have only limited platelet inhibitory activity. Aspirin inhibits only the cyclooxygenase pathway leading to thromboxane production,2 and ticlopidine interferes with the ability of adenosine diphosphate (ADP) to stimulate platelets.3 4 In each case, these agents do not completely inhibit such important platelet agonists as thrombin, collagen (which is exposed in injured vessels), or shear forces (which occur in injured or atherosclerotic vessels5 ). Nevertheless, these agents exert protective effects, suggesting that substances that are effective against a wider variety of agonists could yield even greater protection.
Inhibitors of fibrinogen (fgn) binding to its receptor glycoprotein (GP) IIb/IIIa on activated platelets represent a class of compounds that inhibit platelet aggregation to all known stimuli. Several reports in the literature have described molecules that are active in inhibiting platelet aggregation when administered intravenously (7E3, Integrelin, DPM 728, SC-49992).6 7 8 9 10 11 12 In this report, we describe the pharmacological properties of the ester prodrug SC-54684A (SCp, Fig 1⇓, a hydrochloride salt in which the carboxylic acid of the aspartate is converted to an ethyl ester) and the active moiety, SC-54701A (SCa), in which the carboxylate is not esterfied. SC-54684A is currently in clinical trial as an antithrombotic agent for chronic use.
We show that SCa is a potent inhibitor of fgn binding to platelets, resulting in inhibition of platelet aggregation. The prodrug ethyl ester (SCp) is readily absorbed after oral administration and is rapidly metabolized to SCa. Furthermore, we show that SCa is specific for GP IIb/IIIa compared with other integrins sharing the same β3-subunit. We believe that SCp has greater bioavailability than other orally active agents thus far reported and that SCp has a sufficiently long half-life to make it a viable candidate for clinical development as an oral agent for treatment of thrombotic diseases resulting from platelet aggregation.
SCa and SCp were prepared at Searle. SCp, ethyl 3-(S)-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate, was synthesized by reacting commercially available 4-aminobenzamidine hydrochloride with succinic anhydride followed by a mixed anhydride coupling with ethyl 3(S)-amino-4-pentynoate.13 The (S) ethyl 3-amino-4-pentynoate was prepared through the addition of 1-lithio-2-trimethylsilylacetylene to 4-benzoyloxyazetidinone followed by isolation and treatment of the product with anhydrous HCl in ethanol, which afforded the racemic material that was separated using classic techniques and subsequently deprotected. SCa was prepared by cleavage of SCp, using pig liver esterase (Sigma) in phosphate buffer (pH 7.4, 0.1 mol/L, Sigma).
Semipurified unlabeled fgn (Kabi, Division of Helena Laboratories) was prepared as previously described.14 Platelet agonists included ADP (BioData) and collagen (equine tendon, Chronolog).
Dogs (beagles; weight, 7 to 10 kg) were obtained from White Eagle Laboratories, Doylestown, Pa. Platelet-rich plasma (PRP) was prepared from blood obtained from dogs and from normal human volunteers who reported that they had not taken nonsteroidal anti-inflammatory agents for at least 14 days.
Human vitronectin receptors (αvβ3) were purified from placenta and human fibrinogen receptors (αIIbβ3) were purified from outdated platelets as previously described (References 15 and 16, respectively). Human vitronectin was purified from fresh frozen plasma.17 Biotinylated human vitronectin was prepared by coupling NHS-biotin from Pierce Chemical Co to purified vitronectin.18 Assay buffer, OPD substrate tablets, and radioimmunoassay grade bovine serum albumin (BSA) were obtained from Sigma. Antibiotin antibody was obtained from Calbiochem. Linbro microtiter plates were obtained from Flow Labs. Soybean trypsin inhibitor and all cell culture and buffer reagents were purchased from Sigma. Cell culture plasticware was purchased from Costar, except the 96-well nontissue culture grade microtiter plates were purchased from Dynatech. The CellTiter 96 Assay was purchased from Promega. Human umbilical vein endothelial cells (HUVEC, for the endothelial cell adhesion assay) at passage 1 were purchased from Cell Systems.
Fgn binding was assessed in a manner similar to that of Marguerie et al19 and Plow et al,20 as modified by Nicholson et al.14 Responses at each concentration were averaged, and the standard error was calculated. The IC50 was estimated from each individual dose-response curve. The IC50s were averaged, and standard error was calculated.
In Vitro Platelet Aggregation: Inhibition of Aggregation of Human and Dog PRP
PRP was prepared as previously described,14 and platelet aggregation was measured as an increase in light transmission. Agonists included ADP and collagen: ADP, 20 μmol/L; collagen in human, 4 μg/mL; and collagen in dogs, 33 μg/mL. All agonists were titrated to concentrations greater than that required for maximal aggregation. Responses at each concentration were averaged, and the standard error was calculated. The IC50s were estimated from the dose-response curves, and averaged and standard error was calculated.
Neutrophil–Endothelial Cell Adhesion Assay
Isolation and fluorescent labeling of human neutrophils. Isolation and labeling of neutrophils with Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyamine perchlorate, Molecular Probes) were as described by Look et al.21
Cell adhesion assay. Monolayers of HUVECs (Clonetics Corp) were grown on fibronectin-coated, 96-well tissue culture plates and activated with interleukin (IL)-1β for either 4 or 20 hours in complete endothelial growth medium (Clonetics Corp) containing 5% fetal bovine serum. Subsequently, the monolayers were washed three times with 300 μL per well Hanks’ balanced salt solution (HBSS) containing Ca2+/Mg2+ and 0.1% BSA to ensure complete removal of the cytokine. One hundred microliters of SCa (in HBSS/BSA) then was added to the appropriate wells and the plates incubated at 37°C for 30 minutes, after which a microscopic analysis was carried out to confirm that there was no obvious effect on endothelial cell morphology. Dil-labeled human neutrophils were added to each well (3×105/50 μL) and the plate returned to the 37°C incubator for 20 minutes to allow the neutrophils to settle and bind to the HUVEC. Unbound neutrophils were removed by three buffer washes (300 μL per well). During the third wash, a clear adhesive plate sealer was placed over the buffer-filled wells, and the plate was inverted and centrifuged at 200g for 3 minutes. After removing the sealer (maintaining the plate in an inverted position) and decanting the buffer by shaking, the wells were washed an additional time, decanted, and the plate centrifuged in an inverted position to remove all residual liquid from the wells.
The number of neutrophils bound was determined by solubilizing each well in 100 μL of 0.5% Triton-X100 for 45 minutes (37°C) and transferring 80-μL samples to a fluoricon assay plate (Baxter Healthcare Corp) for quantitation of fluorescence in a Pandex fluorescence concentration analyzer plate reader (λex=545 nm, λem=575 nm). Cell number was established by comparison to a standard curve of the Dil-labeled neutrophils prepared by a 12-step serial dilution on a separate 96-well plate.
Endothelial Cell Adhesion Assay
HUVECs were grown to passage 4 in MCDB131, 4% fetal bovine serum, 1% human serum, 1 μg/mL gentamycin sulfate. Tissue culture plates (Costar, 24-well) were fibronectin coated for 2 hours at room temperature with 20 μg/mL human fibronectin in Dulbecco’s Ca2+/Mg2+-free phosphate-buffered saline. The solution was aspirated and cells plated into the wells at a 1:4 split and cultured for 3 days in 5% CO2 at 37°C. Subconfluent cell cultures (3 days after seeding at a 1:4 split) were removed from the incubator, medium aspirated, and replaced with 0.6 mL of complete medium containing 250 μmol/L GRGDSP or 50 μmol/L SCa. (Note: Previous experiments with bovine pulmonary artery cells [data not shown] indicated that cells sensitive to 100 μmol/L GRGDSP detached more rapidly when sparse rather than confluent cultures were used. Human cells were unaffected by GRGDSP.) The plate was returned to the incubator for 1 hour and 50 minutes. The plate then was removed and medium aspirated from each well just before taking a photomicrograph using a ×20 phase objective on a Nikon diaphot phase microscope equipped with a Nikon 8008 camera set for automatic exposure with TMAX 100 film (Kodak). Photography was completed within 10 minutes.
The concentration of the active agent in canine plasma was estimated using plasma from treated dogs to inhibit PRP from donor dogs. Blood was collected in citrate (9:1) from nontreated dogs and centrifuged (500g, 3 minutes, ×2) to yield PRP. The remaining blood sample was centrifuged at 2000g for 10 minutes to obtain platelet-poor plasma (PPP), used to set baseline in an aggregometer and for dilution of samples. Plasma (225 μL) from treated dogs was mixed with 225 μL of PRP from donor dogs, and aggregation was determined.14 Percent inhibition was calculated, and the concentration of active compound in plasma from treated dogs was estimated by comparing the inhibition observed with a standard curve prepared using plasma that had been spiked with known amounts of SCa. If the plasma from the treated animals produced 80% to 100% inhibition, the plasma was diluted with PPP to produce results that were within the 20% to 80% range of the standard curve.
Ex Vivo Platelet Aggregation and Determination of Oral Systemic Activity
Dogs received either SCp administered orally (single dose) by gelatin capsule or SCa (1 mg/kg in 4 mL of saline/water [50:50]) injected intravenously into the cephalic vein over 1 minute. Before dosing, two control blood samples (2×2 mL) were drawn by venipuncture from the cephalic vein. Blood was centrifuged at 266g for 6 minutes to prepare PRP. Percent aggregation before administration of compound (baseline) was established. Blood samples were drawn at selected time intervals after administration of compound for the first 14 hours and again at 24 hours after dosing for determination of platelet aggregation. The remaining blood was centrifuged for 2 minutes at 12 000g to prepare PPP for determination of the concentration of compound in the plasma samples by bioassay (see above). Inhibition of collagen-induced platelet aggregation was determined by comparing aggregation responses in the samples containing compound with the responses of samples before administration of compound (baseline).
Oral Systemic Activity
Concentration of active compound in plasma samples was determined by bioassay from both the oral study and the intravenous study. These concentrations were plotted versus the time the sample was taken and the area under the curve (AUC) was calculated. AUCs for intravenous (IV) and oral (PO) treatments were corrected for the respective doses, and oral systemic activity (OSA) was expressed in percentage as (PO AUC÷IV AUC)×100.
Bleeding time was assessed in the upper lip of anesthetized beagles. An 18-gauge×2-in. catheter (Becton Dickinson) was placed in the antecubital vein on the forelimb for the infusion of drug or 0.9% NaCl. The infusion was continued for 5 hours (0.15 mL/min, Harvard programmable infusion pump model 22). A catheter was placed in the jugular vein to obtain blood samples for the determination of platelet aggregation. Pharmacodynamic steady state (inhibition of platelet aggregation) was achieved at approximately 2 hours. When steady state was achieved, the bleeding time measurement was determined according to the methods described by Jergens et al24 using a spring-loaded device (Simplate II, Organon Teknika Corp) to make the incisions (maximum time of observation, 30 minutes). The bleeding times from the two cuts were averaged and used as the bleeding time value for that particular test. Statistical analysis of bleeding times was carried out by the nonparametric (ranks) maximum t test method.25 Significance was assumed when P<.05 was found.
In one experiment, 14C-labeled ester ([14C]-SCp) and acid ([14C]-SCa) were each administered intravenously to one female beagle dog at a dose of 2.5 mg/kg. The same compounds were also administered intragastrically as a solution to two female beagle dogs at the same dose. Blood was collected at selected times between 0 and 24 hours after administration. Urine and feces were collected each day for 4 days after administration. The total 14C radioactivity was counted in blood, plasma, urine, and some fecal samples. The plasma from each of the dogs (intravenous or oral, acid or ester) was prepared, and corresponding samples were pooled and profiled by high-pressure liquid radiochromatography (HPLRC). The ester and acid concentrations were estimated using the HPLRC profiles. Samples of 24- and 48-hour urine and feces were also analyzed by HPLRC.
In Vitro Platelet Effects
SCa inhibited fgn binding with an IC50 of 1.0±0.2×10−8 mol/L, as shown in the dose-response curve (Fig 2⇓). With respect to platelet aggregation, the dose-response curves in Fig 3⇓ illustrate that the IC50 for SCa in human PRP was 7.0±0.4×10−8 mol/L when collagen was used as the agonist and 3.5±0.3×10−8 mol/L with ADP as the agonist. This figure also illustrates that inhibition in dog PRP was similar to that observed in human PRP, with IC50s of 6.1±0.8×10−8 mol/L and 6.7±0.5×10−8 mol/L for ADP and collagen, respectively. In addition, Fig 3⇓ presents data on the comparison of the activity of SCa with SCp. SCp had an IC50 of 4.6×10−6 mol/L in collagen-stimulated dog PRP. (Note: This amount of activity can be accounted for by the known 1% contamination of SCp with the free acid SCa.) The relative lack of activity in response to SCp confirms that the ester acts as a prodrug, which requires in vivo metabolism for conversion to the active moiety.
We have examined the specificity of SCa for GP IIb/IIIa in a variety of models. The effect of SCa was determined on the binding of neutrophils to HUVEC stimulated with IL-1. In response to IL-1, the HUVECs express endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) in a time-dependent fashion.26 ELAM-1 binds to a carbohydrate containing moiety (sialyl Lewisx structure) on neutrophils, while ICAM-1 binds to the heterodimeric B2 integrins (CD11a/CD18 and CD11b/CD18). SCa at concentrations up to 10 μmol/L had no effect on the binding of neutrophils to endothelial cells expressing either ELAM or ICAM (data not shown).
SCa competed with vitronectin for binding to isolated vitronectin receptors (αvβ3) with an IC50 of 1.6±0.1×10−5 mol/L compared with its inhibition of binding to isolated fibrinogen receptors (αIIbβ3) with an IC50 of 4.7±0.7×10−10 mol/L (Fig 4⇓). Thus, SCa is about 34 000 times more specific for αIIbβ3 than for αvβ3.
SCa did not affect spreading or attachment of subconfluent HUVECs when compared with cells treated with vehicle (phosphate-buffered saline). After 2 hours of incubation with SCa, no differences were observed between the experimental and control chambers (comparisons not shown). The endothelial cells remained well spread, with distinct ruffling edges. The peptide GRGDSP also did not disrupt HUVEC attachment or spreading (data not shown), which suggests that non-RGD integrins may be involved in human endothelial cell–substratum interactions.
Control bleeding time was 2.66±0.52 minutes (Fig 5⇑). Bleeding time was significantly increased (approximately 2 times control) at 0.10 μg/kg per minute, which resulted in approximately 35% inhibition of platelet aggregation induced by collagen. At a dose of 0.2 μg/kg per minute when collagen-induced aggregation was inhibited by 80%, bleeding time was increased about 2.5-fold. At 0.3 μg/kg per minute, when aggregation was inhibited by >85%, bleeding times were 5 times control (15 minutes). No animals had bleeding times over the 20 minutes.
In Vivo Oral Systemic Activity
When administered orally, SCp completely inhibited platelet aggregation for more than 24 hours after a single dose of 7.5 mg/kg (data not shown). Likewise, when administered intravenously at 1 mg/kg, platelet activity was completely inhibited for more than 8 hours, and inhibition was still detectable 24 hours later (data not shown). AUCs were determined from plasma concentrations determined by bioassay plotted against time (Fig 6⇓). A comparison of the AUC for SCp administered orally and the AUC for SCa administered intravenously indicated an OSA of 18.7%.
We administered 14C-labeled compound (both SCa and SCp) (Table⇓) to dogs both by the intragastric and intravenous routes and measured the amount of radiolabeled compound in the plasma as a function of time after each route of administration. (Note: The study was repeated using unlabeled compound. Data from this experiment [mean±SD] are in parentheses after the 14C-labeled data.) Half-life data are presented in Fig 7⇑. Shown in Fig 8⇓ are the amounts of SCp or SCa detected in the plasma after intragastric or intravenous administration of either SCp or SCa.
After intravenous administration of SCa, the half-life of the β-phase for elimination of SCa (two-compartment open model) was 6.5 hours (4.7±1.0 hours) (Fig 7⇑ and Table⇑), the total plasma clearance was 0.3 L/h per kilogram (0.42±0.02 L/h per kilogram) and the volume of distribution (Vd/area) was 2.8 L/kg (2.9±0.7 L/kg). The unchanged compound was the only radioactive constituent in every plasma sample and in the 0- to 48-hour urine sample. Seventy-five percent of the administered radioactive dose was recovered in the urine after 4 days. In contrast, after intravenous administration of SCp, the apparent elimination half-life (two-compartment open model) was 1.6 hours (1.4±0.8 hours) (Fig 7⇑ and Table⇑), with a plasma clearance of 2.05 L/h per kilogram (2.02±0.32 L/h per kilogram) and a volume of distribution (Vd/area) of 4.6 L/kg (4.3±3.5 L/kg). Most of the dose was rapidly transformed into the free acid. Only a small amount of the ester was eliminated in the urine, accounting for 11.6% of the radioactivity recovered in 0- to 24-hour urine. The acid was the major radioactive compound in urine, accounting for 73.8% of the radioactivity excreted in 0- to 24-hour urine.
In contrast to intravenously administered SCa, the radioactivity eliminated in the urine when SCp was administered intravenously was only 40.5% of the dose, while 52.2% of the dose was excreted in the feces, suggesting that a significant amount of SCp was eliminated by biliary excretion. In 0- to 24-hour feces, the radioactivity was almost exclusively recovered as the radiolabeled free acid (92.9%). Only 4.3% was detected as the ester. These data may suggest that after excretion of SCp in the bile, the compound may be metabolized into SCa in the lumen of the intestine.
After oral administration of SCp, the mean percentage of the dose excreted in the urine was 28.6±14.8%. In the 0- to 48-hour urine, the acid and the ester accounted for 23.8±15.5% and 0.9±0.3% of the dose, respectively, showing that most of the radioactivity was excreted in the urine as radiolabeled free acid.
When the acid [14C]-SCa was given orally, the mean [14C]-free acid maximum plasma concentration (Cmax) was 0.205 μg/mL. This value was close to the Cmax observed after administration of the ester (0.172 μg/mL); however, the time to reach the maximum plasma concentration was shorter (1.5 hours).
In each animal, when the total radioactivity plasma concentration was ≥≈100 ng/mL, the concentration of total radioactivity in blood was approximately equal to or greater than the plasma concentration. This indicates a considerable distribution of the total radioactivity in red blood cells and/or platelets at concentrations ≥100 ng/mL.
After oral administration, the absolute bioavailability of ester prodrug [14C]-SCp expressed in terms of the ester in the plasma was low (11.8%, Fig 8a⇑). Compound was, however, present in greater quantity as the free acid form (Figs 8b⇑ and 8c⇑). The systemic availability of the active metabolite [14C]-SCa after oral administration of the prodrug [14C]-SCp was 21.3% (systemic availability=AUC1/AUC2 where AUC1=AUC0-24h of SCa when SCp was administered IG and AUC2=AUC0-24h of SCa when the active molecule SCa was administered intravenously) (Fig 8b⇑). In contrast, when we administered SCp intragastrically and intravenously and measured SCa after both routes of administration, the AUC ratio was 61.5% (Fig 8c⇑). The time to reach the maximum plasma concentration of the acid (tmax) was 3 hours, and the mean maximum plasma concentration of the SCa (Cmax) was 0.172 μg/mL. A plateau was observed from 3 to 5 hours.
The absolute bioavailability of [14C]-SCa given intragastrically was only 8.5% (Fig 8d⇑), considerably lower than when SCp was given intragastrically, indicating that the ester prodrug is needed to increase absorption after oral administration.
Intravenous agents for prevention of platelet aggregation are useful for the treatment of acute thrombotic events such as reocclusion after thrombolytic therapy or emergency angioplasty in an acute-care setting. However, intravenous therapy is not appropriate for chronic treatment, ie, prevention of thrombotic disorders including recurrence of myocardial infarction or unstable angina. For chronic therapy, an orally active agent is desirable because of ease of administration and greater patient compliance. SC-54684A is currently in clinical trial for chronic therapy in the prevention of platelet-associated thrombus formation.
SCp is a prodrug with excellent oral availability that has been shown to inhibit platelet aggregation in human and animal platelets in vitro (as the active moiety) as well as in intact dogs. The compound is potent and effectively inhibits ex vivo platelet aggregation in dogs at reasonable doses. (In a separate study,27 we have determined that 2 mg/kg BID at steady state leads to a minimum of 50% platelet inhibition during a 24-hour period.) In this study, we have reported that a single oral dose of 7.5 mg/kg resulted in complete inhibition of platelet aggregation for more than 24 hours. Likewise, platelet aggregation was completely inhibited for >8 hours after a single dose of 2.5 mg/kg. SCp leads to an active metabolite that inhibits platelet aggregation by inhibiting fibrinogen binding and should, therefore, be effective in response to all known platelet agonists. Agents that act through this mechanism should provide greater protection compared with other compounds, such as aspirin, which only inhibit platelet aggregation in response to specific stimuli.28
Recently, reports have appeared in which peptide mimetics29 30 were described as being orally available. However, this is the first report in which a detailed analysis of bioavailability is reported, and the level of absorption is higher than reported for other agents.29 Our data suggest that >50% of the administered prodrug SCp is absorbed after oral administration and that about half that amount is converted to the active agent SCa.
Low bioavailability is an undesirable characteristic because it can lead to variation in absorption between individuals or even in a single individual as a function of external factors, such as diet. The oral systemic activity of the active compound after administration of the prodrug exceeded 20%. On the other hand, for purposes of safety, it is desirable that a large fraction of the administered compound be absorbed. After oral administration of the ester, the systemic availability was measured to be >60% ([SCp IG/SCp IV] measuring plasma concentrations of SCa), suggesting that even though not all of the prodrug was converted to biologically active material, >60% of the dose was absorbed, limiting the potential for increased levels of absorption due to dietary effects or individual variations.
Since an agent such as SCa has the potential of being used chronically for an extended period of time, it was expected that a high degree of specificity would lead to fewer side effects. We have shown that SCa is highly specific for the platelet αIIbβ3 integrin relative to αvβ3, which may be an indication of increased safety. In contrast, the antibody 7E3 in development is not specific to αIIbβ3 relative for αvβ3.31 32 Furthermore, after conversion of SCp to its active form, there is little further metabolism, suggesting that there is little opportunity for the formation of toxic metabolites.
Bleeding time has been advanced as an important measurement, even though there is considerable controversy as to the correlation of bleeding time measurements with clinical blood loss.33 Some studies have reported inhibition of aggregation with no increase in bleeding time,34 while still others report greater levels of bleeding associated with platelet inhibition.11 In an animal model, the correlation between the level of inhibition determined in ex vivo platelet preparations and the bleeding time found at the time of blood sampling may be influenced by the type of agonist and concentrations used in the aggregation studies. For example, the lack of effects on bleeding time at 100% inhibition of ex vivo platelet aggregation reported for TP9201 was measured using ADP at concentrations titrated to give 80% of maximal aggregation (≈5 μmol/L).34 When results were reported for DMP728, increased bleeding time was reported at levels that inhibited ex vivo aggregation in response to 10 μmol/L ADP.11 In the present studies, we used a supramaximal dose of collagen (33 μg/mL) to produce ex vivo platelet aggregation. We found a small increase in bleeding time at doses that inhibited this aggregation response 80%. However, it is difficult to relate these results to each other because of the differences in methodology. Nevertheless, it is apparent that some compounds appear to produce beneficial effects in a variety of animal models without substantially increasing template bleeding times.
In addition, the efficacious dose at which the bleeding time measurements are made could be influenced by the choice of animal model. TP9201 was reported to be effective in preventing clot formation in an inverted artery model at doses that did not increase bleeding time.34 This model has been described as being a very platelet-rich model.35 In contrast, the canine electrical damage model is associated with a less homogenous thrombus that is formed by a fibrin/platelet clot.36 The effectiveness of a compound in preventing thrombus formation may be influenced by the nature of the injury, which leads to different forms of thrombus. For example, compounds directed solely against platelet aggregation, such as inhibitors of fibrinogen binding, may appear more potent in models that are highly platelet dependent. While, for example, DMP728 led to increased bleeding time at efficacious doses, the increase may be a reflection of a higher concentration of compound required to prevent thrombus formation in a model that is not exclusively platelet dependent. We believe that the present data suggest that SC-54684A represents a highly bioavailable agent that only minimally increases bleeding times at doses that lead to significant inhibition of platelet aggregation. Whether these results will be translated into less bleeding in a clinical situation is not known, however, particularly because the clinical relevance between increases in template bleeding time and clinical events remains controversial.
SCp is a prodrug of a potent, specific, and relatively stable metabolite, SCa. After oral administration, a significant amount of the compound is absorbed, leading to inhibition of platelet aggregation. Clinical trials with SCp have shown it to be potent and safe in humans.
- Received May 27, 1994.
- Accepted August 19, 1994.
- Copyright © 1995 by American Heart Association
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