Intravascular thrombosis is one of the most frequent pathological events and a major cause of morbidity and mortality in Western civilization. Factors that stimulate thrombosis include vascular damage, stimulation of platelets, and activation of the coagulation cascade. Platelet activation and the resulting aggregation has been shown to be associated with various pathologic conditions including cardiovascular and cerebrovascular thromboembolic disorders such as unstable angina, myocardial infarction, transient ischemic attack, stroke and atherosclerosis (1-8). The contribution of platelets to these disease processes stems from their ability to form aggregates or platelet thrombi as a consequence of arterial wall injury (9). Injury of blood vessel walls could occur in either the short or long term by various pathophysiologic processes. Platelets are then activated by a number of activators or agonists that are released from within platelets or from the injured arterial walls, with the subsequent adherence, aggregation to the disrupted vessel surface, and resultant formation of an occlusive thrombus in the lumen of the vessel (9,10).
Recently a common pathway for all platelet agonists was identified: this is the platelet glycoprotein IIb/IIIa complex (GPIIb/IIIa), which is the membrane protein mediating platelet aggregation (11-13). GPIIb/IIIa in activated platelets is known to bind four soluble adhesive proteins; fibrinogen, von Willebrand factor (VWF), fibronectin, and vitronectin. The binding of fibrinogen and VWF to GPIIb/IIIa causes platelets to aggregate. The binding of fibrinogen is mediated in part by the Arg-Gly-Asp (RGD) recognition sequence, which is common to the adhesive proteins that bind to GPIIb/IIIa. Several RGD-containing peptides have been shown to block fibrinogen binding and prevent the formation of platelet thrombi (14,15). However, their therapeutic utilities are limited by the low affinity or the lack of oral bioavailability or both. Recent studies in humans with a monoclonal antibody for GPIIb/IIIa (7E3) suggested the antithrombotic benefit of GPIIb/IIIa antagonism (16,17). Several other selective GPIIIb/IIIa antagonists, including integrelin, tirofiban (MK383), and lamifiban are in advanced stages of clinical development, aimed primarily for intravenous use in the treatment and prevention of acute ischemic heart diseases (18-20). Recent clinical studies with orally active GPIIb/IIIa antagonists, including xemilofiban (SC54684) and fradafiban (BIBU104) demonstrated an oral antiplatelet activity in humans on their administration 2-3 times orally per day (21,22). These factors prompted us to develop a more potent compound with a slower dissociation rate from human GPIIb/IIIa receptors for the treatment of thromboembolic disorders.
We describe the antiplatelet efficacy, platelet GPIIb/IIIa binding kinetics, specificity, and oral antiplatelet efficacy of the α-sulfonamide isoxazoline analog, DMP 802.
MATERIALS AND METHODS
Adenosine 5′-diphosphate (ADP), collagen, epinephrine, and other reagents used but not specifically mentioned were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.). Arachidonic acid was purchased from Nu Check Prep (Elusian, MN, U.S.A.). 125I-Fibrinogen and radiolabeled DMP 802 were custom synthesized at DuPont NEN (Boston, MA, U.S.A.). DMP 802 (Fig. 1) was synthesized at DuPont Merck Pharmaceutical Co. (Wilmington, DE, U.S.A.).
Light-transmittance aggregometry assay. Venous blood was obtained from healthy human donors who were drug and aspirin free for ≥2 weeks before blood collection or from mongrel dogs, swine, guinea pigs, hamsters, rabbits, or rats, as previously described (23,24). In brief, blood was collected into citrated Vacutainer tubes. The blood was centrifuged for 10 min at 1,000 rev/min in a Sorvall RT6000 Tabletop Centrifuge with H-1000 B rotor (Dupont, Wilmington, DE, U.S.A.) at room temperature, and platelet-rich plasma (PRP) was removed. The remaining blood was centrifuged for 10 min at 2,500 rev/min at room temperature, and platelet-poor plasma (PPP) was removed. Samples were assayed on a PAP-4 Platelet Profiler (Biodata Corp., Horsham, PA, U.S.A.) by using PPP as the blank (100% transmittance). Two hundred microliters of PRP (2-3 × 108 platelets/ml) was added to each micro test tube, and transmittance was set to 0. Twenty microliters platelet agonist ADP (10 μM final concentration) was added to each tube, and the aggregation profiles were plotted (percentage transmittance vs. time). Twenty microliters of DMP 802 was added at different concentrations ranging from 0.001 to 100 μM for 8 min before the addition of ADP (10-100 μM), with final concentration depending on the species. Results were expressed as percentage inhibition of agonist-induced platelet aggregation or IC50 (μM).
Platelet125I-fibrinogen binding assay. Human PRP (h-PRP) was applied to a size-exclusion sepharose column to prepare human gel-purified platelets (h-GPPs) as previously described (24,25). Aliquots of h-GPP (1-2 × 108 platelets/ml) along with 1 mM calcium chloride were added to removable 96-well plates, 125I-fibrinogen (26.5 μCi/mg) was added, and the h-GPPs were activated by addition of ADP, epinephrine, and sodium arachidonate at 100 μM each. The 125I-fibrinogen bound to the activated platelets was separated from the free form by centrifugation, and then counted on a gamma counter. Nonspecific binding (due to entrapment of 125I-fibrinogen), in either the presence or the absence of the inhibitors, was shown (in the absence of agonists) to be in the range of 4-6% of total 125I-fibrinogen binding to agonist-activated platelets. Percentage inhibition of 125I-fibrinogen binding to activated platelets was calculated by dividing the specific binding in the presence by that of the absence of the inhibitors multiplied by 100 as shown in the equation.
For IC50 determination, DMP 802 was added at various concentrations (0.0001-1.0 μM) before platelet activation.
GPIIb/IIIa Receptor-biotinylated fibrinogen binding assay. GPIIb/IIIa receptors were purified from human platelets according to D'Souza et al. (13). The purified GPIIb/IIIa protein was coated on enzyme-linked immunosorbent assay (ELISA) plates, and the competition between DMP 802 and biotinylated fibrinogen on the binding to the coated GPIIb/IIIa receptors was determined. Bound fibrinogen was detected by using an antibiotin alkaline phosphatase conjugate and paranitrophenol detection at 405 nM. For IC50 determination, DMP 802 was added at various concentrations to plates coated with purified GPIIb/IIIa protein, which was followed by the addition of biotinylated fibrinogen. Percentage inhibition of biotinylated fibrinogen binding in relation to DMP802 concentration was calculated as shown in the equation, and then the IC50 for DMP802 was determined.
Degree and extent of binding to unactivated platelets. Venous blood was obtained from healthy human donors into citrated Vacutainer tubes. The blood was centrifuged for 15 min at 150 g (850 r/min in a Sorvall RT6000 Tabletop Centrifuge with H-1000 B rotor) at room temperature, and platelet-rich plasma (PRP) was removed. Unstimulated platelets (6 ml of PRP) were pretreated for 10 min at 22°C with different GPIIb/IIIa antagonists at 10 μM or saline and then applied to 80 ml sepharose CL-4B (2.5-cm diameter siliconized column). Platelets were eluted and collected in Tyrode's buffer. Binding of 125I-fibrinogen to platelets was performed as described by Mousa et al. (23). In brief, aliquots of h-GPPs at 2-3 × 108 platelets/ml along with 1 mM calcium chloride were added to removable 96-well plates, 125 I-fibrinogen (26.5 μCi/mg) was added, and the h-GPPs were either unactivated or activated by addition of the previously described platelet agonists including ADP, epinephrine, and sodium arachidonate at 100 μM each. The 125I-fibrinogen bound to the activated platelets was separated from the free form by centrifugation, and then counted on a gamma counter. Nonspecific binding (due to entrapment of 125I-fibrinogen), in either the presence or the absence of DMP 802, was shown (in the absence of agonists) to be in the range of 4-6% of total 125I-fibrinogen binding to agonist-activated platelets. The effects of pretreatment of platelets with DMP 80 on size-exclusion column separation of unbound DMP 802 from platelets on the degree and extent of its association to platelet was determined. The degree and extent of association of DMP 802 to platelets reflect the degree of shift to the right in the 125I-fibrinogen-binding curve to activated platelets.
Binding affinity to activated versus unactivated platelets. This assay was used to determine a compound's saturable binding to platelets by using PRP. Citrated whole blood (5-ml draw, Vacutainer tubes) was collected from healthy, aspirin-free, human subjects, canine, baboon, or swine and centrifuged for 10 min at 150 g (22°C, Sorvall RT6000 Table Top Centrifuge). PRP was removed, pooled, and platelets were counted by using a Coulter T540 Hematology Analyzer (Coulter Corporation, Miami, FL, U.S.A.). Saline (810 μl, 0.9% USP, Baxter) and 40 μl of radiolabeled [3H]DMP 802 of different concentrations were added to assay tubes, followed by 50 μl of PRP. Samples were incubated for 10 min at 22°C. ADP (100 μl, 10 μM) was added to all samples, followed by incubation for 10 min at 22°C. Platelets were harvested through Whatman 934AH GFB filters (Whatman Int. Ltd., Maidstone, England) that had been presoaked (30 min) in 0.2% polyethylenimine (PEI). Filters were washed quickly 3 times with 5 ml of ice-cold saline, removed, and placed into scintillation vials. Six milliliters of DuPont NEN formula 989 per vial was added; vials were allowed to stand for 60 min, shaken, and counted by using a liquid scintillation counter. The binding affinity (Kd) to either activated or unactivated platelets for the various GPIIb/IIIa antagonists was calculated by using a Scatchard plot.
Dissociation rates: Citrated whole blood (5-ml draw, Vacutainer tubes) was collected from healthy, aspirin-free, human subjects. A portion of this blood was treated with [3H]DMP 802, and the rest was processed for PRP and PPP. One tube from each subject was centrifuged for 10 min (150 g), the resulting PRP removed, and platelet counts determined. This count was used to normalize the radiolabeled platelets. Designated individual tubes of whole blood were treated for 60 min, ± activation (ADP, 10 μM), with 40 nM of [3H]DMP 802. To help ensure sample viability during this period, the blood was maintained on a rocker. After this 60-min incubation period, the tubes were centrifuged for 10 min (150 g). The resulting 3H-radioligand/PRP was carefully removed and centrifuged an additional 10 min (∼250 g). The resulting PPP was removed and the platelet pellet resuspended (∼1.6 × 8/ml) in fresh PPP. Of this suspension, 500 μl was transferred to wells of a 24-well plate [5% bovine serum albumin (BSA) block]. Cold ligand (100 μM) was added to the appropriate wells and, at a designated time point (0, 2, 15, 30, 60, 90, or 120 min), bound [3H]DMP 802 was removed from the wells by centrifugation for 2 min (10,000 g). Supernatant fluid was discarded and platelet pellet containing bound [3H]DMP 802 was counted by using a beta counter. Counts recovered were compared with the control (t = 0) and presented as percentage [3H]DMP 802 platelet bound over time, after addition of excess cold DMP 802.
Integrin specificity studies
Purified αvβ3 receptors biotinylated vitronectin binding assay. Purified receptor obtained from human placenta was diluted with coating buffer and coated (100 μl/well) on Costar (3590) high-capacity binding plates overnight at 4°C. Coating solution was discarded, and plates were washed once with blocking/binding (B/B) buffer. One hundred ten microliters of 1.0% BSA in B/B buffer was applied for 60 min at room temperature. Thirty microliters of biotinylated vitronectin plus 50 μl of either inhibitor or B/B buffer with 1.0% BSA was added to each well and incubated for 25 min at room temperature. Plates were washed twice with B/B buffer and incubated 1 h at room temperature with antibiotin alkaline phosphatase (100 μl/well) in B/B buffer containing 1.0% BSA. Finally, plates were washed twice with B/B buffer (50 mM TRIS-HCl, 100 mM NaCl, 2 mM CaCl2, 1 mM MgCl2·6 H2O, 1 mM MnCl2·4 H2O, and stored at 4°C + 1% BSA, pH 7.4) followed by the addition of 100 μl of phosphatase substrate (1.5 mg/ml). Reaction was stopped by adding 2N NaOH (25 μl/well), and developed color was read at 405 nm.
SK-BR-3 Cell-vitronectin (αvβ5-mediated) adhesion assay. A Costar 3590 plate was coated with 100 μl of vitronectin (0.25 μg per well) overnight at 4°C. After overnight coating, each well was washed twice with 200 μl phosphate-buffered saline (PBS), and nonspecific binding was blocked by adding 200 μl of PBS + 50% BSA per well for 1 h at room temperature. SK-Breast cancer cell line (SK-BR-3) cells (ATCC, Rockville, MD, U.S.A.) were detached with 0.005% trypsin/0.1% EDTA, washed, and resuspended in serum-free McCoy's 5A standard medium (Gibco BRL) at 1 × 106 cells/ml. Cells were labeled with 2 μM calcein-AM (Molecular Probes 3100, 50 μg per vial) for 30 min at 37°C in a humidified incubator. After calcein labeling, cells were washed twice with 40 ml of McCoy's 5A medium and centrifuged for 15 min at 300 r/min × 2. Cells (1 × 106 cells/ml) were preincubated with either 150 μl of test compounds or medium, gently mixed, and then incubated for 15 min at room temperature. Drug-treated SK-BR-3 cells were added to the assay plate in duplicate and incubated for 60 min on a shaker at room temperature. Plates were covered with foil to prevent photobleaching of the dye from labeled cells. After the drug/cell and matrix interaction, medium was gently removed from the wells and they were washed twice with 200 μl McCoy's 5A medium. After washing, 100 μl of McCoy's 5A was added to each well, and the fluorescence was read on a Cytofluor 2300 at sensitivity 2, Ex = 485 nM and Em = 530 nm.
Jurkat-CSI α4β1-mediated) adhesion assay. Fibronectin, containing CS1 or CS1 fragment (100 μl), was plated onto 96-well plates (Costar 3590, high binding, flat bottom) at 50 μg/ml overnight at 4°C. The protein solution was removed from the wells and washed twice with PBS containing protease inhibitor. The nonspecific binding was blocked by adding 150 μl of PBS plus 1% BSA for 2 h at room temperature or overnight. On the day of experiment, Jurkat cells were obtained and centrifuged for 10 min at 1,200 rev/min. After centrifugation (at 300 rev/min × 2 for 10 min), Jurkat cells in ∼2 ml of standard RPMI medium plus ∼20-30 ml medium were then resuspended in ∼2 ml medium and counted in a Coulter counter. Cells were labeled by adding 2 μM calcein-AM per 3-5 × 106 cells/ml at 37°C for 30 min with occasional shaking. DMP 802 (100 μM) was incubated for 30 min at 37°C in a dark incubator. Labeled Jurkat ∼2 × 105 cells were added to each well and incubated at room temperature for 1 h on a rotator. After incubation, wells were washed twice with RPMI, and then 150 μl RPMI was added back into the wells and fluorescence read in Cytofluor 2300 within 1 h; Ex, 485 nm (filter B); Em, 530 nm (filter B).
Purified α5β1 receptor-biotinylated fibronectin binding assay. Purified receptor obtained from human placenta was diluted (1:2,000) with coating buffer and coated (100 μl/well) onto Costar (3590) high-capacity binding plates overnight at 4°C. Coating solution was discarded, and plates were washed once with buffer (B/B buffer). Wells were then blocked with 200 μl (B/B buffer containing 1% BSA). After washing once with B/B buffer, 100 μl of biotinylated fibronectin (2 nM; 1:5, 125 in the binding buffer) was added plus 11 μl of either inhibitor or B/B buffer containing 1.0% BSA to each well and incubated for 1 h at room temperature. Plate was washed twice with B/B buffer and incubated for 1 h at room temperature with 100 μl antibiotin alkaline phosphatase (1:12,000 dilution) in B/B buffer. Plates were washed twice with B/B buffer and then incubated for 1 h at room temperature with 100 μl alkaline phosphatase substrate. Color was developed at room temperature for ∼45 min. The reaction was stopped by adding 2N NaOH (25 μl/well), and absorbance was read at 405 nm.
Oral antiplatelet effects in dogs
Experimental procedures. Fasted, conscious mongrel dogs of either sex weighing between 8 and 12 kg were used in the study. For basal platelet-aggregatory response (ex vivo), two venous blood samples (3 ml each) were drawn by venipuncture from a catheterized cephalic vein of each animal. Percentage platelet aggregation in response to 100 μM ADP was assessed as previously described. DMP 802 was administered orally (single dose) in soft gelatin capsules at 0.05-0.2 mg/kg, p.o. (n = 4-5). Blood samples obtained at 0, 1, 2, 4, 6, 12, and 24 h after single oral doses of DMP 802 were placed on a platform rocker until assayed for percentage platelet aggregation, as previously described. Additionally, blood samples were withdrawn at different intervals (0, 1, 3, 6, 12, 24, 27, 30, 48, 51, 54, 72, 78, 96, 97, 99, 102, 108, and 168 h) after an oral dose of 0.05 mg/kg (n = 5) once a day for 5 days. The oral antiplatelet efficacy of DMP 802 was calculated by comparing percentage aggregation response of samples after DMP 802 administration to that of basal samples obtained from the same animal. Platelet count was carried out with 100 μl of citrated whole blood by using Coulter Counter T-540.
Pharmacokinetics in dogs
The pharmacokinetic characteristics of DMP 802 were investigated in dogs after single intravenous (i.v.) and oral (p.o.) doses (four dogs per group). After administration of an i.v. bolus dose of 0.025-0.4 mg/kg and orally at 0.1 mg/kg, blood samples were withdrawn in EDTA tubes for plasma measurements of DMP 802. Plasma concentrations of DMP 802 were determined by solid-phase extraction followed by LC/MS/MS assays for plasma samples obtained from dogs treated either intravenously or orally with DMP 802. By using 0.2 ml of dog plasma for extraction, the minimal quantifiable limit of the assays was 2 ng/ml in dog PPP (harvested from blood collected in anticoagulant EDTA). Mean concentrations were reported as zero if all values were below quantifiable limit (2 ng/ml).
Data are expressed as mean ± SEM. The design of the experimental protocol in certain sections of the study allowed each animal to serve as its own control with regard to the basal values (percentage aggregation). Data were analyzed by either paired or group analysis by using Student's t test or analysis of variance (ANOVA) when applicable; differences were considered significant at p < 0.05.
In vitro antiplatelet (GPIIB/IIIA) efficacy
Platelet-aggregation studies. In the human PRP light-transmittance assay, DMP 802 inhibited platelet aggregation induced by 10-100 μM ADP with an IC50 of 0.029 ± 0.0042 μM (Fig. 2). Additionally, in PRP from different species including canine, guinea pig, swine, hamster, rabbit, and rat, DMP 802 inhibited platelet aggregation induced by ADP with IC50 values of 0.025-1.0 μM (Fig. 2). DMP 802 was shown to be less potent in inhibiting aggregation of platelets obtained from New Zealand rabbits and rats (IC50, 0.88 ± 0.22 and 1.0 ± 0.08 μM, respectively) as compared with platelets obtained from other species (Fig. 2). A steep dose-response relation is shown for DMP 802 in inhibiting platelet aggregation in PRP obtained from the various species (Fig. 2).
Platelet125I-fibrinogen-binding assay. In the h-GPP 125I-fibrinogen-binding assay, DMP 802 inhibited 125I-fibrinogen binding to activated GPP with an IC50 of 0.012 ± 0.003 μM (Fig. 3).
GPIIb/IIIa receptor-biotinylated fibrinogen binding. In a purified GPIIb/IIIa receptor enzyme-linked immunosorbent assay (ELISA), DMP 802 demonstrated direct inhibition of fibrinogen binding, with an IC50 of 0.00021 ± 0.00005 μM.
Degree and extent of binding to unactivated platelets. DMP 802 demonstrated a tight association to unactivated human and canine platelets after purification of GPP by using size-exclusion chromatography, as evident from the shift in the 125I-fibrinogen-binding inhibition curves (Fig. 4A and B). DMP 802 did not affect the nonspecific binding of 125I-fibrinogen to unactivated platelets (Fig. 4A and B).
Binding affinity to activated versus unactivated platelets. DMP 802 binds with high affinity to both activated and unactivated human platelets with a Kd = 0.61 ± 0.17 and 0.57 ± 0.21 nM, respectively (Fig. 5).
Dissociation rates: DMP 802 demonstrated tight association with unactivated human, baboon, or canine platelets (t1/2 of dissociation, 32 ± 2, 32 ± 13, and 11 ± 1 min, respectively). In contrast, DMP 754 demonstrated relatively lesser binding affinity to unactivated human, baboon, or canine platelets (t1/2 of dissociation, 7 ± 0, 8 ± 1, and 1.4 ± 0.1 min, respectively) as compared with DMP 802 (Table 1).
In vitro integrin specificity
αvβ3 Receptor-binding assay. In purified αvβ3 receptors ELISA, DMP 802 demonstrated no significant inhibitory efficacy of vitronectin binding at concentrations ≤10 μM, with an IC50 >10 μM (Table 2).
SK-BR-3 Cell-vitronectin (αvβ5-mediated) adhesion assay. DMP 802 was ineffective in inhibiting αvβ5-mediated adhesion to vitronectin at concentrations ≤10 μM with <15% inhibition (Table 2).
α4β1/CS1-Mediated adhesion. DMP 802 was ineffective in inhibiting α4β1-mediated adhesion to the CS1 domain in fibronectin at concentrations ≤100 μM (Table 2).
α5β1-Fibronectin ELISA. DMP 802 was ineffective in inhibiting α4β1-mediated adhesion to fibronectin at concentrations up to 10 μM with <5% inhibition (Table 2).
Oral antiplatelet efficacy in dogs
Single oral dosing and antiplatelet efficacy. Oral antiplatelet efficacy of DMP 802 was examined after a single oral doses ranging from 0.05 to 0.2 mg/kg in mongrel dogs. Antiplatelet efficacy was demonstrated after oral dosing as evident from the ex vivo inhibition of platelet aggregation induced by 100 μM ADP (Fig. 6). A submaximal antiplatelet efficacy was shown at 0.05 mg/kg, p.o., with maximal antiplatelet efficacy at 0.1-0.2 mg/kg, p.o. (Fig. 6).
Repeated oral daily dosing and antiplatelet efficacy. The antiplatelet efficacy of DMP 802 was determined after repeated oral dosing at 0.05 mg/kg, daily, for 5 days in conscious dogs (Fig. 7). DMP 802 resulted in repeatable antiplatelet effects on once daily oral administration at extremely low unit dose (0.05 mg/kg) for the 5 days of the study in dogs. DMP 802 resulted in a sustained 70-90% inhibition of ex vivo platelet aggregation for the first 10 h after dosing, which was followed by a decline to 20-30% inhibition by 24 h without significant residual buildup from previous dosing.
Pharmacokinetics in dogs
The pharmacokinetic characteristics of DMP 802 were investigated in dogs after single i.v. and p.o. doses (Table 3). After administration of an i.v. bolus dose of 0.025-0.4 mg/kg in dogs, plasma concentrations of DMP 802 declined polyexponentially. The mean terminal elimination phase half-life (t1/2) was 13-17 h. The apparent systemic plasma clearance (CL) increased from 0.6 to 4.5 ml/min/kg at doses ranging from 0.025 to 0.4 mg/kg, respectively (data not shown). The apparent volume of distribution at steady-state (Vss) was dose dependent, increasing from 0.8 to 4.2 L/kg as the dose increased from 0.025 to 0.4 mg/kg, respectively. The pharmacokinetics of DMP 802 in dogs appeared to be dose dependent. This nonlinear characteristic in dogs may be related to saturable binding of DMP 802 to platelets (26).
Peak plasma concentrations of DMP 802 were attained within 1.2 h after single p.o. doses of DMP 802 at 0.1 mg/kg. Based on the low-dose i.v. data, the apparent bioavailability (F) of DMP 802 was 14.9% in dogs (Table 3). The postabsorptive plasma concentrations of DMP 802 declined monoexponentially as a function of time. The apparent t1/2 values of DMP 802 in dogs after p.o. administration was 11.9 h (Table 3).
Platelet activation and the resulting aggregation has been shown to be associated with various pathologic conditions including cardiovascular and cerebrovascular thromboembolic disorders, such as unstable angina, myocardial infarction, transient ischemic attack, stroke, and atherosclerosis (1,4,5,8). The contribution of platelets to these disease processes stems from their ability to form aggregates or platelet thrombi as a consequence of arterial injury (1,4,5,8). It has been well recognized that the platelet GPIIb/IIIa, by its binding to circulating fibrinogen, is the final common pathway for all agonist-induced platelet-aggregate formation (11,12). The binding of fibrinogen is mediated in part by the RGD recognition sequence, which is common to other adhesive proteins that bind to GPIIb/IIIa receptors or other integrins (11-13). Several RGD-containing peptides, as well as the monoclonal antibody (7E3) against the platelet GPIIb/IIIa, have been shown to block fibrinogen binding and prevent the formation of platelet thrombi (14,15,17,18,23,24). Selective GPIIb/IIIa antagonists, including integrelin, tirofiban (MK383), and lamifiban, are in advanced stages of clinical development, aimed primarily for i.v. use in the treatment and prevention of acute ischemic heart diseases (18,20,27). Recent clinical studies with orally active GPIIb/IIIa antagonists including xemilofiban (SC54684) and fradafiban (BIBU104) demonstrated oral antiplatelet activity in humans on their administration 2-3 times per day (21,22). These factors prompted us to develop a potent and selective GPIIb/IIIa antagonist for the treatment of the different thromboembolic disorders. Under the same aggregation-assay conditions, aspirin, ticlopidine, and hirudin were shown to be effective mainly against single agonists including arachidonic acid, ADP, or thrombin, respectively, in inhibiting platelet aggregation in human PRP with an IC50 of 10, 50, and 0.1 μM, respectively (23). Unlike those currently available antiplatelet drugs such as aspirin and ticlopidine, DMP 802 demonstrated high and similar potency in inhibiting platelet-aggregation regardless of the agonist used. DMP 802 is shown to be a competitive inhibitor with high affinity in inhibiting fibrinogen binding to platelet GPIIb/IIIa receptors. Additionally, DMP 802 demonstrated a high degree of selectivity toward the platelet GPIIb/IIIa receptors as compared with the closely related vitronectin receptors on endothelial cells or other adhesion receptors. This is unlike the linear RGDS peptide, which recognizes multiple integrin receptors. The high affinity and specificity of DMP 802 to the platelet GPIIb/IIIa might be very important for achieving an optimal efficacy/safety ratio. Additionally, DMP 802 demonstrated high affinity for both activated and unactivated platelets, along with relatively slow dissociation rates, suggesting a possible prolonged duration of in vivo antiplatelet effects. This is in contrast to current intravenous GPIIb/IIIa antagonists such as integrelin, tirofiban, lamifiban, and DMP 728, which have a short duration of antiplatelet effects associated with their relative fast dissociation rates from human platelets (18,20,27,28). DMP 802 demonstrated a dose-dependent oral antiplatelet efficacy with an extended duration of antiplatelet efficacy and absolute oral bioavailability of 14.9% in dogs. Despite a 14.9% oral bioavailability of DMP 802, it exhibited a potent and reproducible antiplatelet profile on repeated daily oral administration at low unit dose of 0.05 mg/kg for 5 days. This might be due to its tight binding to resting platelets as well as its slow rate of dissociation from platelets.
In conclusion, DMP 802 inhibited platelet aggregation, inhibited fibrinogen binding to activated human platelets, associated with high and similar affinity to unactivated/activated human platelets, and demonstrated a high degree of specificity for the human platelet GPIIb/IIIa without any apparent interferences with other known and tested adhesion molecules. DMP 802 demonstrated a potent oral antiplatelet efficacy at 14.9% oral bioavailability in dogs.
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