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The majority of women experience vasomotor symptoms (VMS), such as hot flashes and night sweats, during the menopausal transition. Recent evidence strongly suggests a connection between neurokinin 3 (NK3) receptor signaling and VMS associated with menopause. The NK3 receptor antagonist fezolinetant is currently in phase 3 development for treatment of moderate to severe VMS associated with menopause. We investigated the pharmacological effects of repeated administration of fezolinetant on levels of sex hormones and gonadotropins, neuronal activity in the hypothalamus, and skin temperature as an index of hot flash-like symptoms in ovariectomized rats as a model of menopause. Ovariectomized rats exhibited several typical menopausal symptoms: hyperphagia, increased body weight, significantly decreased plasma estradiol levels, increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, and significantly increased skin temperature. Increased c-Fos expression (an indirect marker of neuronal activity) in median preoptic nucleus (MnPO) hypothalamic neurons was also observed in ovariectomized rats. Repeated oral administration of fezolinetant (1–10 mg/kg, twice daily) for 1 week dose-dependently reduced plasma LH levels without affecting estradiol or FSH levels, inhibited the activation of MnPO neurons, and attenuated hot flash-like symptoms. In addition, fezolinetant dose-dependently reduced hyperphagia and weight gain in ovariectomized rats. These preclinical findings suggest that fezolinetant attenuates hot flash-like symptoms via inhibition of neuronal activity in the MnPO of ovariectomized rats and provides further support for the ongoing clinical development of fezolinetant for the treatment of VMS associated with menopause.

The oral direct factor Xa inhibitor darexaban administered intraduodenally prevented venous thrombus formation in both rats and rabbits with no effect on bleeding. The indirect parenteral Factor Xa inhibitor fondaparinux exerted similar properties, only prolonging bleeding time at extremely high doses. In contrast, the thrombin inhibitor ximelagatran and low-molecular-weight heparin enoxaparin prolonged bleeding time at antithrombotic doses. Studies using human platelets showed darexaban glucuronide, a darexaban metabolite that predominantly determines darexaban antithrombotic effects in vivo, had no effect on platelet activation and aggregation, while heparin and enoxaparin activated platelets. Melagatran, heparin, and enoxaparin all inhibited thrombin-induced platelet aggregation at clinically relevant concentrations. Taken together, these results suggest that thrombin-inhibiting drugs may increase the risk of bleeding, while darexaban may have potential as an orally available antithrombotic agent with a wide therapeutic window.

Mirabegron, a selective β(3)-adrenoceptor agonist, facilitates urine storage function by exerting a relaxing effect on bladder smooth muscle. Here, we investigated the effect of mirabegron on bladder function during the storage phase. We assessed the effect of mirabegron on the resting intravesical pressure in anesthetized rats and also tested antimuscarinics (oxybutynin and tolterodine) under the same experimental conditions. Mirabegron dose-dependently decreased the resting intravesical pressure, while oxybutynin and tolterodine showed no statistically significant effects on resting intravesical pressure. We also investigated the effect of mirabegron on bladder function using cystometry technique in conscious rats with bladder outlet obstruction. While mirabegron dose-dependently decreased the frequency of nonvoiding contractions, considered an index of abnormal response in bladder storage, no significant effects were noted on the amplitude of nonvoiding contractions, micturition pressure, threshold pressure, voided volume, residual volume, or bladder capacity. Neither oxybutynin nor tolterodine affected the frequency of nonvoiding contractions; however, oxybutynin increased residual volume and tended to decrease voided volume in a dose-dependent manner, and tolterodine dose-dependently decreased voided volume. Taken together, these results shed light on the suggestion of mirabegron as a therapeutic agent, compared with antimuscarinics, with its most prominent effect being the facilitation of bladder storage.

SummaryWe evaluated the relationship between antithrombotic effects and pharmacodynamic (PD) marker changes produced by the novel factor (F)Xa inhibitors darexaban (YM150) and rivaroxaban in a rabbit model of plaque disruption-induced arterial thrombosis. Animals were subjected to catheter-induced endothelial denudation via the femoral artery followed by a two-week high-cholesterol diet. Plaque disruption was induced by balloon angioplasty, and then stasis was achieved by ligation at the distal side of the injured segment. Darexaban and rivaroxaban were administered orally 1 hour (h) before and 9 h after plaque disruption, and their antithrombotic effects were evaluated 24 h after the initiation of ligation. Prothrombin time (PT), activated partial thromboplastin time (APTT), and plasma FXa activity were measured using blood samples collected before and 1 h after administration. Darexaban and rivaroxaban significantly reduced thrombus formation. The thrombus weight obtained in the 30 mg/kg darexaban group was comparable to that in the 1 mg/kg rivaroxaban group (2.17 ± 0.63 and 3.23 ± 1.64 mg, respectively, vs. 8.01 ± 1.08 mg in the control group). Plasma FXa activity correlated with the antithrombotic effects of darexaban and rivaroxaban, while PT only correlated with those of darexaban. Our findings suggest that the degree of plasma FXa inhibition may be useful for predicting antithrombotic effects of darexaban and rivaroxaban in arterial thrombosis. PT may also be useful in evaluating antithrombotic effects of darexaban in particular.

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fXa inhibitory activity and its oral anticoagulant activity, the linker between benzamidine and the central benzene ring was modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a (YM-202054), and 41c (YM-203558) exhibited potent fXa inhibitory activity and oral anticoagulant activity. In particular, YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0 h. Additionally, these compounds showed a high degree of selectivity for other serine proteases.

Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anticoagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effective oral anticoagulant activity in mice.

We previously demonstrated that recombinant human interleukin-11 (rHuIL-11) induced osteoblast differentiation of C3H10T1/2 progenitor cells and also acted synergistically with recombinant human bone morphogenetic protein-2 (rHuBMP-2) in performing the same function. In this study, we investigated the effect of rHuIL-11 and rHuBMP-2 on bone formation in a rat ectopic model. When placed in rats, implants consisting of polymer-coated gelatin sponges containing various concentrations of rHuBMP-2 showed a dose-dependent increase in calcium content. This was confirmed by radiographic analysis of the implants. Although implants containing rHuIL-11 alone did not accumulate calcium, implants containing a combination of rHuBMP-2 and rHuIL-11 had significantly higher calcium levels than those containing rHuBMP-2 alone. This increase was rHuIL-11 dose dependent. The synergistic effect of 20 micrograms rHuIL-11 and 6 micrograms rHuBMP-2 on bone formation was estimated to be 1 week in advance of that of 6 micrograms rHuBMP-2 alone. Histologic examination revealed that the combination of rHuIL-11 and rHuBMP-2 caused spindle cells to accumulate around implants and induced cell infiltration into implants. Bone formation occurred faster in implants with the combination of rHuIL-11 and rHuBMP-2 compared with rHuBMP-2 alone. These results suggest that rHuIL-11 acts synergistically with rHuBMP-2 to more rapidly stimulate bone formation compared with rHuBMP-2 alone. This novel combined therapy may be of great clinical benefit in bone healing.

The antithrombotic effects of YM466, a synthetic direct inhibitor of human factor Xa (FXa), were evaluated in an arterio-venous shunt thrombosis model in squirrel monkeys. YM466 significantly inhibited thrombus formation after continuous IV zx infusion at doses of 3–30 µg/kg/h. Although prothrombin time (PT) was significantly prolonged at doses of 10 and 30 µg/kg/h, this effect was small (1.2- and 1.4-fold of the control, respectively). Furthermore, YM466 had no effect on template bleeding time and activated partial thromboplastin time at any dose. Plasma anti-FXa activity increased in a dose-dependent manner correlating with the antithrombotic effect of YM466. Thrombin-antithrombin III complex levels decreased to less than normal levels at 30 µg/kg/h of YM466. Mean plasma concentrations of YM466 measured by LC/MS/MS were 2.5, 10.5, 27.4, and 75.5 ng/ml at doses of 1, 3, 10, and 30 µg/kg/h, respectively. These results suggest that YM466 is a safe antithrombotic agent with low bleeding risk, and that plasma anti-FXa activity may be a suitable parameter for monitoring its antithrombotic effect. Drug Dev. Res. 58:190–195, 2003. © 2003 Wiley-Liss, Inc.

Factor Xa (FXa) is a serine protease which plays a pivotal role in the coagulation cascade. The inhibition of FXa has received great interest as a potential target for the development of new antithrombotic drug. Herein we describe a series of novel 7-amidino-2-naphthoanilide and 7-amidino-2-naphthalensulfonanilide derivatives which are potent FXa inhibitors. These scaffolds are rigid and are allowed to adopt an L-shape conformation which was estimated as the active conformation based on a docking study of YM-60828 with FXa. Optimization of the side chain at the central aniline nitrogen of 7-amidino-2-naphthoanilide has led to several potent and orally active FXa inhibitors. 5h (YM-169964), the best compound of these series, showed potent FXa inhibitory activity (IC 50 =3.9 nM) and effectively prolonged prothrombin time by 9.6-fold ex vivo at an oral dose of 3 mg/kg in squirrel monkeys.

The pharmacological properties of YM-57029 ({4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine–glycine–aspartic acid–serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 μg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

We examined the biochemical properties of YM-57029 ({4-[4-(4-Carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate) and the pharmacodynamics and pharmacokinetics of its prodrug, YM128 (Ethyl (Z)-(4-{4-[4-(N 2 -hydroxycarbamimidoyl)phenyl]-3-oxopiperazin-1-yl}piperidino)acetate), an orally-active glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist. YM-57029 strongly inhibited aggregation of human platelets induced by various agonists, with IC50 values ranging from 3.6 to 51 nM. YM-57029 specifically inhibited fibrinogen binding to purified GPIIb/IIIa about 1,000-fold more potently than Arg-Gly-Asp-Ser (RGDS). Moreover, YM-57029 effectively inhibited an Arg-Gly-Asp (RGD) peptide binding to platelets, suggesting that YM-57029 competed with the RGD sequence of ligand. YM-57029 or YM128 dose-dependently inhibited ex vivo platelet aggregation after iv bolus injection or oral administration to beagle dogs and cynomolgus monkeys. However, YM128 exerted more potent and prolonged inhibitory effects on platelet aggregation than YM-57029 after oral administration to cynomolgus monkeys. Furthermore, YM-57029 prolonged template bleeding time at a dose that inhibited ex vivo platelet aggregation during cumulative iv infusion to cynomolgus monkeys. Metabolic and pharmacokinetic studies showed that YM128 effectively converted into YM-57029 in liver microsomes from humans as well as dogs and monkeys, and that bioavailabilities of YM128 in dogs and monkeys were 32.3 and 22.2%, respectively. These results suggest that YM128, a prodrug of YM-57029, may be a valuable GPIIb/IIIa antagonist with good bioavailability in humans. Drug Dev. Res. 55:149-161, 2002. © 2002 Wiley-Liss, Inc.

The antithrombotic effects of YM-75466 ([N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-naphthyl)methyl]sulfamoyl]acetic acid monomethane sulfonate), a novel orally-active factor Xa inhibitor, and its effects on bleeding time and coagulation time were studied in rats and compared with those of warfarin. Both agents were orally administered. In the venous thrombosis model, YM-75466 and warfarin inhibited thrombus formation dose-dependently, with ID50 values of 3.3 and 0.56 mg/kg, respectively. Ex vivo study showed that both YM-75466 and warfarin prolonged prothrombin time dose-dependently, with doses, causing a two-fold prolongation of prothrombin time in the control group, of 89 and 0.38 mg/kg, respectively. In bleeding time studies, YM-75466 and warfarin prolonged bleeding time dose-dependently, with doses, causing a two-fold prolongation of bleeding time in the control group, of >100 and 0.43 mg/kg, respectively. These results show that the antithrombotic effects of YM-75466 are markedly separate from its effects on bleeding time and coagulation time compared with warfarin.

We compared the antithrombotic effect of anti-GPIIb/IIIa antibody Fab fragment YM337 with that of a thromboxane A2 synthetase inhibitor, sodium ozagrel. With the monkeys under halothane anesthesia, the right middle cerebral artery was observed via a transorbital approach without cutting the dura mater. Photoillumination (wavelength 540 nm) was applied to the middle cerebral artery, and then rose bengal (20 mg kg(-1)) was administered intravenously. The experimental drugs were intravenously injected 15 min before rose bengal injection and followed by continuous infusion for 3 h after dye injection. The thrombotic occlusion induced by this photochemical reaction in monkey middle cerebral artery was reproducible. YM337 significantly prolonged the time to first occlusion and the total time of arterial patency during the 3-h observation period after dye injection. In contrast, sodium ozagrel had no significant effect. YM337 but not sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation. However, while sodium ozagrel significantly inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Neurological deficit in the YM337-treated animals was significantly milder than that in the control group. The area of infarct in the YM337 treatment animals was smaller than that in the control group. The novel selective GPIIb/IIIa antagonist YM337 was effective in ameliorating the decrease in patency of the middle cerebral artery and reducing the area of cerebral infarction in monkeys.

Platelet aggregation plays a important role in the thrombotic cerebral infarction. The final common mechanism in the formation of a platelet aggregate is the linking of adjacent platelets by fibrinogen binding to the platelet integrin alpha 11b beta 3. In this study, we evaluated the effect of the disintegrin, triflavin, in a rat middle cerebral artery thrombosis model. Thrombus at the left middle cerebral artery in rat was induced by photochemical reaction between rose bengal and green light, which caused endothelial injury at the site of irradiation. We measured the time to occlusive thrombus formation and the size of ischaemic cerebral damage. Triflavin dose dependently prolonged the time to occlusive thrombus formation in this model. Triflavin also reduced the size of ischaemic cerebral damage on examination at 24 h after photochemical reaction. Triflavin dose dependently inhibited ADP- and collagen-induced platelet aggregation and platelet retention in the collagen-coated beads method ex vivo. These effects were thought to result from the blockade of platelet integrin alpha IIb beta 3. Blockade of platelet integrin alpha IIb beta 3 may be useful in the prevention of cerebral arterial thrombosis.

1. 1. The potential antiplatelet agent use of the whole molecule, F(ab) 2 and Fab fragments of humanized antiglycoprotein (GP) IIb/IIIa monoclonal antibody, hC4G1, were investigated in rhesus monkeys. 2. 2. Fab completely inhibited platelet aggregation 1 hr after an IV bolus administration of 1 mg/kg without a decrease in platelet count or prolongation of bleeding time, and the duration of inhibition was much shorter than that of F(ab) 2 3. 3. These results suggest that the Fab fragment of hC4G1 may be a more useful antiplatelet agent in patients with acute thromboembolic diseases than the whole molecule or F(ab) 2 fragments.

Here, we investigated the anticoagulant effects of darexaban in mice and human plasma in vitro, effects of darexaban in thrombosis and bleeding models in mice, and reversal effects of anti-inhibitor coagulant complex (ACC) and recombinant factor VIIa (rFVIIa) on anticoagulant effects of darexaban. In mice, darexaban inhibited FXa activity in plasma with an ED50 value of 24.8 mg/kg. Both darexaban and warfarin prolonged prothrombin time (PT) at 3 mg/kg and 0.3 mg/kg/day, respectively. PT and activated partial thromboplastin time (aPTT) prolonged by darexaban were dose-dependently reversed by intravenously-administered rFVIIa, significantly so at 1 mg/kg. In a pulmonary thromboembolism (PE) mouse model, both darexaban and warfarin dose-dependently reduced the mortality rate, significantly so at 10 mg/kg and 3 mg/kg/day, respectively. In a FeCl3-induced venous thrombosis (VT) mouse model, darexaban (0.3-10 mg/kg) dose-dependently decreased the thrombus protein content, significantly so at doses of 3 mg/kg or higher. In a tail-transection mouse model, darexaban had no significant effect on the amount of blood loss at doses up to 10 mg/kg, while warfarin showed a dose-dependent increase in blood loss, significantly so from 1 mg/kg/day. Darexaban and its metabolite darexaban glucuronide significantly prolonged PT and aPTT in human plasma in vitro, and while rFVIIa concentration-dependently reversed the prolonged PT in this plasma, ACC dose-dependently reversed both PT and aPTT changes prolonged by darexaban. Taken together, these results suggest that darexaban has a potential to be an oral anticoagulant with a better safety profile than warfarin, and that rFVIIa and ACC may be useful as antidotes to darexaban in cases of overdose.

We have developed two monoclonal antibodies (MAbs), B6A3 and C4G1. The whole molecules of the two MAbs inhibited in vitro human platelet aggregation induced by either ADP, collagen or thrombin, and their F(ab')2 fragments inhibited ex vivo platelet aggregation induced by ADP in monkey. The concentrations necessary for complete inhibition were 5 and 1 microgram/ml for B6A3 and C4G1, respectively. The Fab fragment of C4G1 but not B6A3 inhibited platelet aggregation. B6A3 and C4G1 bound to activated platelets with dissociation constants of 0.25 and 0.82 nM, respectively. B6A3 recognized an epitope on beta 3, which was sensitive to reduction and alkylation of cystine residues, and C4G1 recognized a conformational epitope on the alpha IIb beta 3 complex, which was sensitive to EDTA. The binding of fibrinogen to activated platelets was inhibited by both MAbs. However, the binding of fibrinogen to isolated alpha IIb beta 3 was inhibited by the whole molecule of C4G1 but not B6A3, although both MAbs bound to the isolated alpha IIb beta 3. The binding of these MAbs to the isolated alpha IIb beta 3 was not inhibited by either Arg Gly Asp Ser (RGDS) or fibrinogen gamma-peptide. In addition, B6A3 but not C4G1 bound to human endothelial cells. These MAbs should contribute to the elucidation of the mechanism of platelet aggregation.

The thrombolytic activity of a novel modified t-PA analog, YM866, was compared with that of recombinant t-PA (t-PA) in rabbits with experimental jugular vein thrombosis. Thrombus was induced in an isolated segment of the jugular vein from a mixture of whole autologous blood and thrombin. Thirty minutes after the induction of thrombus, YM866 was administered by iv bolus injection, while t-PA was given by the same method or by a 60-min iv infusion. Thrombi were removed 70 min after iv bolus injection or 10 min after the termination of iv infusion, and thrombus size was measured by total protein content in the isolated thrombus. Both YM866 and t-PA exhibited dose-dependent thrombolysis; the thrombolytic activity of YM866, however, was 4 times greater than that of t-PA. The improved thrombolytic activity of YM866 correlated with its relatively higher antigen levels in plasma, which result from its prolonged biological half-life. Depletion of plasma fibrinogen to less than 20% of baseline levels was observed in all groups. Template bleeding time was not significantly altered in any group. These results suggest that YM866 offers the potential for clinical use in thrombolytic therapy by iv bolus injection for patients with venous thromboembolic diseases. © 1994 Wiley-Liss, Inc.

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 μM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 μM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID₅₀ values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

The thrombolytic activity of a novel modified t-PA, YM866, was compared with that of a recombinant t-PA in a canine model of copper coil-induced coronary thrombosis. The coronary thrombus was allowed to age for 1, 3 or 6 hr before either drug was administered. YM866 was administered by i.v. bolus injection, while t-PA was given by the same method, as well as by 60-min i.v. infusion. YM866 showed thrombolytic activity 2 to 4 times as potent as that of t-PA when administered by bolus injection, the difference in thrombolytic effect being obvious in the 3- and 6-hr-old thrombi. Coronary reperfusion was achieved more rapidly with YM866 than with i.v. infusion of t-PA. In animals injected with doses of more than 0.1 mg/kg of YM866, no acute reocclusion occurred. Depletion of plasma fibrinogen to 70% of baseline levels was observed in animals given 0.2 mg/kg YM866, 0.4 mg/kg t-PA by bolus, and 0.6 mg/kg t-PA via infusion. The residual plasma YM866 and t-PA antigen 30 min after bolus injection was 25% and 3% of the peak levels, respectively. YM866, administered by i.v. bolus injection, was thus confirmed to exert a thrombolytic effect superior to that of bolus injection and infusion of t-PA, without systemic fibrinolytic activation. These results suggest the potential clinical applicability of YM866 as a thrombolytic agent that can be administered by i.v.bolus injection for acute myocardial infarction.

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.

Factor Xa (fXa) is a serine protease involved in the coagulation cascade, which has received great interest as a potential target for the development of new antithrombotic drugs. Herein we report a novel series of fXa inhibitors in which the 1,4-diazepane moiety was designed to interact with the S4 aryl-binding domain of the fXa active site. Compound 13 (YM-96765) showed potent fXa inhibitory activity (IC50 = 6.8 nM) and effective antithrombotic activity without prolonging bleeding time.

The effects of YM337, the Fab fragment of a humanized anti-glycoprotein IIb/IIIa (GPIIb/IIIa) monoclonal antibody C4G1, on in vitro platelet function and binding properties were compared with those of abciximab, the Fab fragment of the human/murine chimeric anti-GPIIb/IIIa monoclonal antibody 7E3. Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin. Further, both inhibited human platelet adhesion to von Willebrand factor, fibrinogen, fibronectin and subendothelial matrix with similar potency. Fibrinogen binding to washed platelets was dose-dependently inhibited by both agents. In binding assay using 125I-YM337 and 125I-abciximab, Kd values determined with platelet-rich plasma were 6.74 +/- 0.56 nM for YM337 and 6.65 +/- 1.45 nM for abciximab, and the number of binding sites were 42,700 +/- 3,000 for YM337 and 76,000 +/- 5,400 for abciximab. GPIIb/IIIa was precipitated from the solubilized fraction of platelets by both agents. In contrast, integrin alphavbeta3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337. Fibrinogen binding to purified GPIIb/IIIa was dose-dependently inhibited by both agents. In contrast, vitronectin binding to purified integrin alphavbeta3 was dose-dependently inhibited by abciximab but not by YM337, supporting the idea that abciximab reacts to integrin alphavbeta3. Therefore, YM337 was suggested to bind to a different epitope of GPIIb/IIIa from abciximab. These results suggest that YM337 specifically acts on platelet GPIIb/IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab.

SummaryRecent clinical studies have shown that the interruption of platelet function appears to be effective for treatment of coronary occlusive diseases. For this purpose, a Fab fragment of humanized anti-platelet glycoprotein (GP) IIb/IIIa monoclonal antibody (YM 337) that exhibited an in vivo antithrombotic effect without prolongation of bleeding time in monkeys was previously characterized. In this study, the effect of YM 337 under physiological flow conditions with high or low shear rate was evaluated. The antiplatelet effects of YM 337 under varying wall shear rates were examined with the whole blood flow system. This technique allows real time visualization of the formation of fluorescence-labeled platelet thrombi on a collagen surface in a parallel plate flow chamber mounted on an epifluorescence microscope. The process of thrombus growth was also recorded in video tape and subjected to computer-assisted image analysis. We found that YM 337 displayed a high shear-preferential antiplatelet effect, while ReoPro, a control anti-GP IIb/IIIa antibody, did not show such shear dependency. The present findings therefore suggest that YM 337 might be a useful antiplatelet agent which can block pathological thrombotic events occurring under high shear, such as in coronary occlusive diseases.

SummaryWe examined the effect of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, on thrombolysis with tissue-type plasminogen activator in a copper coil-induced coronary thrombosis model in rhesus monkeys. Fifty minutes after the formation of an occlusive thrombus, a test drug was administered by either i.v. bolus injection followed by continuous infusion (YM337, 0.25 mg/kg + 1.5 μg/kg/min) or i.v. bolus injection (aspirin, 17 mg/kg). Sixty minutes after induction of the occlusive thrombus, thrombolysis was initiated with tPA at a total dose of 0.5 mg/kg intravenously administered over 60 min, with 10% given as an initial bolus. The median time to reperfusion was significantly shortened by YM337 [saline, 60 min (n = 5); aspirin, 45 min (n = 5); YM337, 30 min (n = 5)]. The incidence of reocclusion was significantly decreased by YM337 (saline, 4/4; aspirin, 5/5; YM337, 1/5), and the median time to reocclusion was significantly prolonged by YM337 [saline, 30 min (n = 4); aspirin, 30 min (n = 5); YM337, 180 min (n = 5)]. YM337 significantly reduced the thrombus protein content at the end of experiment. ADP-induced platelet aggregation was completely inhibited by YM337. These results suggest that YM337 may be of clinical value as an adjunctive agent in thrombolytic therapy for patients with acute myocardial infarction.

We directly compared the effects of YM337, the Fab fragment of the humanized monoclonal antibody C4G1, on platelet aggregation and template bleeding time with those of abciximab, the Fab fragment of the human/murine chimeric monoclonal antibody 7E3, in rhesus monkeys. The duration of inhibition of platelet aggregation by abciximab after i.v. bolus injection was much longer than that by YM337. Although YM337 significantly prolonged template bleeding time at 5 min after i.v. bolus injection, this action recovered within 1 h after injection. In contrast, although abciximab also prolonged template bleeding time, the duration of this effect was sustained. In a dose-escalating continuous infusion study, we evaluated the relationship between inhibition of platelet aggregation and prolongation of template bleeding time. Platelet aggregation was inhibited by over 80% by both agents at 3 microg/kg per min, and template bleeding time was prolonged to about 30 min at 30 microg/kg per min for YM337 and 10 microg/kg per min for abciximab. Interestingly, plasma concentrations between inhibition of platelet aggregation and prolongation of template bleeding time did not overlap with YM337, but did overlap with abciximab. These results suggest that YM337 allows easier control of antiplatelet activity with less effect on bleeding time than abciximab, and has a wider therapeutic window than abciximab.

We report here the synergistic antithrombotic effect of aurintricarboxylic acid in combination with a snake venom-derived disintegrin, triflavin, in a photochemically induced thrombosis model in rats. The time to initiation of thrombus was prolonged by i.v. bolus injection of aurintricarboxylic acid at 10 mg/kg. In contrast, time to occlusion was dose-dependently prolonged by both agents, this prolongation being significant with aurintricarboxylic acid at 10 mg/kg i.v. and with triflavin at more than 3 mg/kg i.v. Interestingly, the combination of aurintricarboxylic acid at 3 mg/kg i.v. and triflavin at 1 mg/kg i.v. prolonged not only the initiation of thrombus, but also the time to occlusion.

SummaryThe antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 μg/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 μg/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation.Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 μg/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 ± 5.2 min) or sodium ozagrel (9.4 ± 3.0 min) compared with saline (13.3 ± 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model.These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.

A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab')2 fragment of hC4G1 (F(ab')2 hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab')2 hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab')2 hC4G1 were directed against the C-terminal region of F(ab')2 fragment common to all human and humanized IgG antibodies. Natural homo-reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity-purified human homoreactive antibodies enhanced phagocytosis of platelets treated with the F(ab')2 hC4G1. Monkey plasma with high homo-reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab')2 hC4G1 to a monkey with low antibody activity. These results suggest that F(ab')2 of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo-reactive antibodies.

We investigated the antithrombotic effects of a F(ab')2 fragment of a humanized anti-platelet glycoprotein (GP) IIb/IIIa monoclonal antibody, YM207, in a platelet-rich thrombosis model. Thrombus was induced in a mesenteric venule of squirrel monkeys by irradiation with filtered light in combination with i.v. administration of a fluorescent dye (sodium fluorescein). Time to occlusive thrombus formation was significantly prolonged by i.v. bolus injection of 0.3 mg/kg of YM207. At doses of 1 mg/kg and 3 mg/kg, complete occlusion did not occur during 60 min of the observation. Platelet aggregation in platelet-rich plasma was inhibited by YM207 at doses 0.3 mg/kg or more. A good correlation was obtained between inhibition of platelet aggregation and the percentage of GPIIb/IIIa receptors blocked by YM207. Bleeding time was significantly prolonged at a dose of 3 mg/kg. Platelet counts showed no significant change. These results suggest that YM207 is effective in preventing platelet-rich thrombus formation without inducing the prolongation of bleeding time or a significant decrease in platelet count. YM207 may be a useful therapeutic agent for the treatment of thrombotic disorders.

Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.

We compared the thrombolytic activity of a novel modified tissue-type plasminogen activator (t-PA; del 92-173, 275Arg-->Glu), YM866, with that of t-PA in a platelet-rich thrombosis model. Thrombus was induced in guinea pig mesenteric artery by irradiation with filtered light in combination with intravenous (i.v.) administration of fluorescent dye. When occlusion by the thrombus extended to 99% of the luminal area of the vessel, test drug (YM866, t-PA, or saline) was administered by i.v. bolus injection under heparinization. Both YM866 and t-PA exhibited dose-dependent thrombolytic activity; however, the improvement in occlusion rate and the incidence of successful thrombolysis induced by YM866 were three times higher than those induced by t-PA. With YM866 1 mg/kg, alpha 2-plasmin inhibitor levels decreased significantly to 58% of saline group values, but no change was noted in fibrinogen levels. YM866 antigen levels at this dose were seven times higher than those of t-PA. These results suggest that YM866 in single bolus injection is a thrombolytic agent superior to t-PA in platelet-rich thrombi without systemic fibrinolytic activation and that this efficacy is due to the prolonged half-life (t1/2) of the drug.

We compared the thrombolytic activity of YM866, a novel modified tissue-type plasminogen activator, with that of t-PA by intracoronary administration in a canine thrombosis model of copper coil-induced 6-hr-old thrombi. Either drug was administered by a single injection (10 min) or multiple injection (4 x 10 min) under heparinization (300 IU/kg, i.v.). The reperfusion rate of YM866 was 4 times higher than that of t-PA when administered by single injection. Time to reperfusion of YM866 by single injection was shorter than that of either agent by multiple injection. No group showed any decrease in plasma fibrinogen levels. No acute reocclusion was seen in animals with YM866 by single injection. The improved thrombolytic activity of YM866 by single injection correlated with the relatively higher antigen levels of this agent due to its prolonged biological half-life. These results suggest that single intracoronary administration of YM866 is a safe and effective thrombolytic method for rapid recanalization and lowered acute reocclusion without activation of systemic fibrinolysis.

YM150, an oral, direct factor Xa inhibitor, is currently being evaluated in Phase II studies as prophylaxis for venous thromboembolism in patients undergoing orthopedic surgery. In the present study, we compared the antithrombotic effect of YM150 with the effects of antithrombin-dependent indirect factor Xa inhibitors, enoxaparin and fondaparinux, and a direct thrombin inhibitor, ximelagatran, in ferric chloride (FeCl3)-induced venous and arterial thrombosis models in rats. We also evaluated the bleeding time in a rat tail transection model. Prior to any experimentation, male Sprague-Dawley rats, which had been fasting for at least 12 h, were anesthetized with urethane (1 g/kg, i.p.) or sodium pentobarbital (50 mg/kg, i.p.). YM150 and ximelagatran were administered intra-duodenally, and both enoxaparin and fondaparinux were given subcutaneously, 30 min prior to induction of thrombus or tail transection. All animals were kept warm with a heating pad during the experiments. Venous and arterial thromboses were produced, respectively, by the 5 min application of 8% FeCl3 soaked filter paper to the external surface of the inferior vena cava and 35% FeCl3 soaked filter paper to the abdominal aorta. The venous thrombosis model was supplemented by using a silk thread venous stenosis. To measure bleeding time, the tail was transected 5 mm from its tip. Blood was carefully blotted each 30 sec with a filter paper. Once a blood stain was observed, we defined bleeding as blood flow sustained over 30 sec. Bleeding time was defined as the sum of the bleeding periods during the 60 min observation in each animal. Administration of intra-duodenal YM150 significantly inhibited both venous and arterial thrombus formation at doses of 10 mg/kg or greater, and 3 mg/kg or greater, respectively. This indicated that YM150 promoted an antithrombotic effect at similar dose ranges for venous and arterial thromboses. In contrast, YM150 did not prolong the bleeding time at doses up to 30 mg/kg. Venous thrombus formation was inhibited by subcutaneous enoxaparin at doses of 100 IU/kg or greater and fondaparinux at doses of 0.03 mg/kg or greater. Arterial thrombus formation was inhibited by subcutaneous administration of 1000 IU/kg enoxaparin and 3 mg/kg fondaparinux. The results indicated that 10–100 times higher doses of these antithrombotics were needed to inhibit arterial thrombosis. Furthermore, enoxaparin at doses of 300 IU/kg or greater and fondaparinux at doses of 1 mg/kg or greater, significantly prolonged the bleeding time, suggesting that these two medications may be associated with increased risk of hemorrhage at concentrations used to prevent arterial thrombosis. At doses of 1 mg/kg or greater, intra-duodenal ximelagatran inhibited both venous and arterial thrombus formation. The dose-response curve for ximelagatran tended to be steeper than that for other anticoagulants tested. Antithrombotic doses of ximelagatran (1 mg/kg or greater), produced similar prolongations of bleeding time as those seen with administration of enoxaparin and fondaparinux. In conclusion, YM150, an oral direct factor Xa inhibitor, shows promise as an antithrombotic drug with potentially wider safety margins than current antithrombin-dependent factor Xa inhibitors and a thrombin inhibitor.

The objective of this study was to evaluate the effects of direct factor Xa inhibitor, YM150 and its major in vivo metabolite, YM-222714, on clot formation and clot lysis compared with other anticoagulants, such as a direct thrombin inhibitor (melagatran), a pentasaccharide (fondaparinux), low molecular weight heparin (enoxaparin) and unfractionated heparin. To assess clot lysis, the tissue plasminogen activator (tPA)-induced clot lysis assay was used with human plasma triggered by low and high levels of tissue factor (TF). Under low TF conditions, clot formation was completely prevented by melagatran at 1 μmol/L, by fondaparinux at all concentrations examined (0.1 to 1 μg/mL), by enoxaparin at 0.3 and 1 IU/mL and by heparin at 0.1 and 0.3 U/mL. Even under high TF conditions, 0.3 U/mL heparin prevented any clot formation. Although melagatran, fondaparinux, enoxaparin, and heparin potently prevented plasma clot formation under low TF conditions, under high TF conditions they were less effective at prolonging the clotting time. Under both low and high TF conditions, YM150 and YM-222714 prolonged the clotting time in a concentration dependent manner at concentrations between 0.3 and 3 μmol/L. YM150 and YM-222714 significantly accelerated clot lysis under both low and high TF conditions, but their effects were most evident under high TF conditions. Lower concentrations of melagatran (0.1 and 0.3 μmol/L) enhanced clot lysis under low TF conditions, but under high TF conditions, enhancement of clot lysis required higher melagatran concentrations (0.3 μmol/L or more). Under high TF conditions, fondaparinux enhanced clot lysis only at the highest concentration tested (1 μg/mL). Enoxaparin and heparin enhanced clot lysis under low TF conditions at the lowest test concentrations (0.1 IU/mL and 0.03 U/mL, respectively). Both also enhanced clot lysis under high TF conditions, but their effect reached statistical significance only at higher concentrations (1 IU/mL and 0.1 U/mL, respectively). These results suggested that direct factor Xa inhibitors, YM150 and YM-222714, exert stable anticoagulant effects independently of TF concentration. Both inhibitors enhanced tPA-induced fibrinolysis in human plasma clotted via the extrinsic coagulation pathway. Useful characteristics of YM150 and YM-222714, such as a linear dose response and reliable anticoagulation independent of TF concentration, may lead to the creation of an anticoagulant that is easier to use in the clinical setting than existing products. Potentially beneficial antithrombotic effects, which can be promoted by accelerating endogenous fibrinolytic pathways, may further aid in the prevention or treatment of thrombosis.