SummaryThe inhibitory effects of argatroban on clot- or fibrin-bound human thrombin were studied using the thrombin-specific chromogenic substrate S2238 (200 μM). These effects were compared to those of recombinant hirudin (rHV2 Lys 47) and the heparin/antithrombin III complex. Argatroban concentration-dependently inhibited the cleavage of S2238 by a thrombin solution, which had been titrated to give the same change in OD405 nm as fibrin-bound thrombin, with an IC50 of 1.1 μM with 90% inhibition at 8 μM. rHV2 Lys 47 and heparin had IC50 values of 1.2 nM and 0.003 U/ml respectively under these conditions. However, when the compounds were tested against fibrin-bound thrombin, argatroban had an IC50 of 2.8 μM with 65% inhibiton at 8 μM, whereas rHV2 Lys 47 had an IC50 of 23 nM (with only 56% inhibition at 200 nM), and heparin had an IC50 of 0.5 α 0.38 U/ml (with only 58% inhibition at 5 U/ml); i. e. the two compounds were 19 and 168 times less active against fibrin-bound thrombin than against thrombin in solution. The differences between the inhibitory effects of the compounds against thrombin bound to a plasma clot were even more striking in that the IC50 of argatroban was increased from 1.1 (vs. thrombin in solution) to 2.7 μM, while, although rHV2 Lys 47 and heparin had IC50 values of 2.8 nM and 0.004 U/ml against thrombin in solution, they had little (32% inhibition by 4 pM rHV2 Lys 47) or no effect (even at 5.0 U/ml heparin) against the amidolytic activity of a plasma clot. We conclude that argatroban could present advantages over hirudin and heparin in the treatment of pathologies where the enzymatic activity of clot-bound thrombin may play a significant role.

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AimsWe investigated the relative pharmacokinetics, pharmacodynamics, and safety of the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor alirocumab following injection at three different sites.MethodsSixty healthy subjects (39 male, 21 female; age 20–45 years) were randomized to receive a single subcutaneous injection of alirocumab 75 mg via 1-mL prefilled pen into the abdomen, upper arm, or thigh (NCT01785329). Subjects were followed for 85 days ± 2 days following study drug administration. Pharmacokinetic (PK) parameters for the systemic exposure of alirocumab were calculated, and levels of free PCSK9 were assessed. Percentage changes from baseline in LDL-C were compared between injection site groups using linear mixed-effects models.ResultsAlirocumab concentration–time profiles were similar, and free PCSK9 levels were reduced to approximately zero between Day 3 and Day 4 postinjection in all groups. LDL-C levels reached nadir on Day 15 postinjection in all groups with mean percentage reductions of 48.4% (abdomen), 39.5% (upper arm), and 45.6% (thigh) at this time point. A similar effect on LDL-C levels was seen across the entire time course of the study at all three injection sites. Treatment-emergent adverse events were experienced by 8/20 (abdomen), 11/20 (upper arm), and 13/20 (thigh) subjects. There were 2 mild/transient injection site reactions. There were no serious adverse events.DiscussionA single subcutaneous administration of alirocumab 75 mg via prefilled pen was well tolerated with similar pharmacokinetics and pharmacodynamics when injected into the abdomen, upper arm, or thigh.ConclusionThese results suggest that alirocumab can be interchangeably injected in the abdomen, upper arm, or thigh.

1 Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and prothrombolytic activity in animals. We describe the antithrombotic eects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate. 2 The activity of MA33H1 against rat PAI-1 was con®rmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in rat venous (thromboplastin+stasis in the abdominal vena cava) and arterial (electric current applied to a carotid artery) thrombosis models. The eects on tailtransection bleeding time were studied. 3 MA33H1 at 100 ng ml 71 inhibited both human (44.1%) and rat PAI-1 (49.7%). This eect was concentration-dependent. Its eect on human PAI-1 was not signi®cantly inhibited by 1 mg ml 71 ®brin or a &7 fold molar excess of vitronectin (1 nM). Inhibition of rat PAI-1 was unchanged by ®brin, but vitronectin reduced inhibition from 0.5 nM. 4 In the venous thrombosis model, MA33H1 signi®cantly reduced thrombus weights by 38 and 58.6% at 50 and 100 mg kg 71 min 71 i.v. respectively. This eect was inhibited by tranexamic acid. In the arterial model, MA33H1 signi®cantly increased the delay to occlusive thrombus formation by 58 and 142% at 50 and 100 mg kg 71 min 71 i.v., and did not aect bleeding time at 300 mg kg 71 min 71 i.v. 5 Thus, a monoclonal antibody which transforms PAI-1 to a t-PA substrate prevents thrombus formation in the rat with no eect on bleeding time at a higher dose.

SummaryClot-associated thrombin retains amidolytic activity, and is resistant to inhibition by heparin, but not to low molecular weight thrombin inhibitors. We show that clot-associated thrombin induces platelet aggregation, is resistant to heparin:antithrombin III, less so to recombinant hirudin (rHV2Lys47) but not to argatroban, an active-site directed thrombin inhibitor. Fibrin clots prepared with human fibrinogen and thrombin were used to aggregate rabbit washed platelets assessed by single platelet counting, thromboxane B2 (TXB2) immunoassay and scanning electron microscopy. Fibrin clots decreased platelet counts, and released TXB2. Electron microscopy showed platelet aggregates on the clot surface. Argatroban concentration-dependently inhibited such aggregation with IC50s of 21 nM and 13 nM versus aggregation and TXB2 release respectively. The IC50s of Argatroban against fluid-phase thrombin producing similar aggregation were 12 nM (aggregation) and 33 nM (TXB2). rHV2Lys47 was less active against clot-induced aggregation (IC50 = 1.8 nM) than against fluid-phase thrombin (IC50 = 0.06 nM). Heparin had an IC50 of 0.02 mU/ml against aggregation induced by fluid-phase thrombin, but much greater concentrations are required to inhibit clot-induced aggregation (IC50 = 48 mU/ml). These data provide a basis for the superiority of direct-acting thrombin inhibitors over heparin in platelet rich thrombi.

SSR182289A competitively inhibits human thrombin (K i ϭ 0.031 Ϯ 0.002 M) and shows good selectivity with respect to other human proteases, e.g., trypsin (K i ϭ 54 Ϯ 2 M), factor Xa (K i ϭ 167 Ϯ 9 M), and factor VIIa, factor IXa, plasmin, urokinase, tPA, kallikrein, and activated protein C (all K i values Ͼ250 M). In human plasma, SSR182289A demonstrated anticoagulant activity in vitro as measured by standard clotting parameters (EC 100 thrombin time 96 Ϯ 7 nM) and inhibited tissue factor-induced thrombin generation (IC 50 of 0.15 Ϯ 0.02 M). SSR182289A inhibited thrombin-induced aggregation of human platelets with an IC 50 value of 32 Ϯ 9 nM, but had no effect on aggregation induced by other platelet agonists. The anticoagulant effects of SSR182289A were studied by measuring changes in coagulation markers ex vivo after i.v. or oral administration in several species. In dogs, SSR182289A (0.1-1 mg/kg i.v. and 1-5 mg/kg p.o.) produced dose-related increases in clotting times. After oral dosing, maximum anticoagulant effects were observed 2 h after administration with increases in thrombin time, 2496 Ϯ 356%; ecarin clotting time (ECT), 1134 Ϯ 204%; and activated partial thromboplastin time (aPTT), 91 Ϯ 20% for the dose of 3 mg/kg p.o., and thrombin time, 3194 Ϯ 425%; ECT, 2017 Ϯ 341%; and aPTT, 113 Ϯ 9% after 5 mg/kg p.o. Eight hours after administration of 3 or 5 mg/kg SSR182289A, clotting times were still elevated. SSR182289A also showed oral anticoagulant activity in rat, rabbit, and macaque. Hence, SSR182289A is a potent, selective, and orally active thrombin inhibitor.

ABSTRACT

Argatroban was evaluated in three models of thrombosis in the rabbit: the Wessler model (thromboplastin plus stasis of the left jugular vein), an arteriovenous shunt model, and a model of arterial thrombosis induced by endothelial and intimal damage of the left femoral artery. Calcium heparin was used as a comparator. Both substances inhibited thrombus formation in the Wessler model with ID50 values of 0.32 and 0.16 mg/kg intravenous bolus for argatroban and heparin respectively, with similar changes in thrombin time (4 to 5 times control) and activated partial thromboplastin time (APTT) (1.6 to 2.1 times control) for both substances at antithrombotic doses. The ID50 values of both substances were 2.4 micrograms/kg/min (argatroban) and 0.5 microgram/kg/min (heparin). When they were administered by continuous infusion, no significant effects on the APTT were noted. In the arteriovenous shunt, the ID50 values for argatroban and heparin (respectively) were 0.16 and 0.07 mg/kg intravenous bolus, and 4.5 and 2.8 micrograms/kg/min intravenous infusion. Vessel clamping followed immediately by electrical stimulation (5 mA direct current, 5 minutes) of the left femoral artery leads to the formation of an occlusive thrombus approximately 30 minutes after clamping. Argatroban infused for 60 minutes before the vascular lesion and throughout the 90 minute observation period led to a dose-dependent delay in arterial occlusion with significant effects seen at 5 micrograms/kg/min with five of eight animals showing normal femoral blood flow at 90 minutes postlesion at 20 micrograms/kg/min; no significant increases (Dunnett's test) in APTT were noted with argatroban. Heparin was without effect even at 40 micrograms/kg/min, despite an eight-fold increase in APTT at 20 micrograms/kg/min and values of more than 300 seconds at 40 micrograms/kg/min. Thus, in models of arterial but not venous thrombosis, argatroban is a more potent antithrombotic agent than heparin on a weight basis, with antithrombotic effects at a much lower degree of systemic anticoagulation.