Preclinical safety and efficacy of andexanet alfa in animal models.

Essentials There is currently no approved reversal agent for factor Xa (FXa) inhibitors Andexanet alfa has been developed to reverse the anticoagulant effects of FXa inhibitors Andexanet reduced blood loss and anticoagulation markers in rivaroxaban-anticoagulated rabbits Andexanet was well tolerated in monkeys and rats, with no evidence of prothrombotic activity SUMMARY: Background Andexanet alfa is a recombinant modified form of factor Xa (FXa), designed to bind to and reverse the anticoagulant activity of FXa inhibitors. Objectives To evaluate the ability of andexanet to reverse the anticoagulant activity of rivaroxaban, and assess its pharmacokinetics (PK) and toxicity in animal models. Methods The effects of andexanet on blood loss, anti-FXa activity, rivaroxaban unbound plasma concentrations and other coagulation parameters were assessed in a rabbit liver laceration 'treatment' model. Andexanet was administered 10 min after blood loss was initiated. The toxicity of repeated administration of andexanet (up to 60 mg kg-1 day-1 ) was assessed in cynomolgus monkeys. PK parameters were evaluated in rats and monkeys. Results Excess blood loss due to anticoagulation with rivaroxaban was significantly decreased by a single intravenous bolus administration of andexanet at 35 and 75 mg per rabbit, by 75% and 63%, respectively. This correlated with dose-dependent decreases in the unbound fraction of rivaroxaban and anti-FXa activity. Co-administration of rivaroxaban had no significant impact on the PK parameters of andexanet. Andexanet (up to 60 mg kg-1 day-1 ) was well tolerated in monkeys, with no accumulation of andexanet or rivaroxaban. There was a single occurrence of anaphylaxis, which resolved after treatment with diphenhydramine and epinephrine. There was no histological evidence of prothrombotic activity with high-dose andexanet compared with vehicle control, as measured by clot and fibrin deposition in all major organs. Conclusions These data suggest that andexanet is a promising therapy for the reversal of FXa inhibitor-induced anticoagulation, supporting clinical studies in humans.

Antithrombotic and hemostatic capacity of factor Xa versus thrombin inhibitors in models of venous and arteriovenous thrombosis.

Thrombin plays a central role in venous and arterial thrombosis. We utilized two different rabbit models of in vivo thrombosis to investigate the effect of inhibitors of thrombin generation and thrombin activity. The agents tested were specific inhibitors of factor Xa (fXa) [N2-[(phenylmethyl)sulfonyl]-D-arginyl-N-[(1S)-4-[(aminoiminomethyl++ +)a mino]-1-(2-thiazolylcarbonyl)butyl]-glycinamide (C921-78)] and thrombin [D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1-(chloroacetyl)but yl]-L-prolinamide (PPACK)], as well as drugs that affect both thrombin and fXa, unfractionated and low molecular weight (enoxaparin) heparin. The agents administered as constant intravenous infusion were evaluated for antithrombotic efficacy in anesthetized rabbits. All four agents were capable of dose dependent inhibition of thrombosis in venous and arteriovenous thrombosis models. However, due to the more aggressive nature of thrombotic stimulation in the arteriovenous shunt model, complete cessation of thrombus growth was not achieved for any of the agents at the doses tested. Comparison between the agents focused on the differences in extension of coagulation parameters (activated partial thromboplastin time, prothrombin time, thrombin clotting time), changes in hematological parameters, and extension of rabbit cuticle bleeding time at doses required to produce maximum inhibition in the thrombosis models. In the venous thrombosis model at the maximally effective dose, C921-78 had minimal extension of ex vivo clotting parameters, while enoxaparin and unfractionated heparin demonstrated a two to sevenfold increase in activated partial thromboplastin times, and PPACK had a threefold extension of thrombin clotting times. In addition, unlike the other three agents, which exhibited no significant changes in hematological parameters, PPACK demonstrated dose dependent thrombocytopenia. A standardized cuticle bleeding time was used as a measure of perturbation of hemostasis. The agents were evaluated for significant increases in bleeding time at doses up to eight times that needed to completely inhibit venous thrombus formation. Unfractionated heparin displayed a significant bleeding time effect at the dose required to inhibit venous thrombosis (100 u/kg+2 u/kg/min). Enoxaparin and PPACK caused significant bleeding time extensions at four times the fully efficacious venous dose (800 u/kg+8 u/kg/min and 30 microg/kg/min). By contrast, C921-78 did not significantly increase bleeding time even at eight times the maximally effective dose (240 microg/kg+7.2 microg/kg/min). Our results demonstrate that specific inhibition of fXa can be utilized to derive potent antithrombotic activity without disrupting extravascular hemostasis.

Relative efficacy of active site-blocked factors IXa, Xa in models of rabbit venous and arterio-venous thrombosis.

In order to investigate the respective roles of prothrombinase and intrinsic tenase (IXa/VIIIa) in venous thrombosis, we compared the anti-thrombotic efficacy of inhibitors of these two coagulation complexes. The agents tested were dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated bovine factor IXa(IXai) and Glu-Gly-Arg chloromethyl ketone-inactivated human factor Xa (Xai). In vitro formation of active complexes (prothrombinase or tenase) was inhibited by Xai and IXai resulting in IC50 values of 3 nM and 5 nM, respectively. Antithrombotic activity was measured by inhibition of clot accretion on cotton threads placed in the abdominal vena cava of anesthetized rabbits. Intravenous bolus dosing followed by infusion of Xai during the experimental protocol resulted in a dose dependent reduction of clot weight, a dosage of 16.0 micrograms/kg + 0.28 microgram/kg/min being sufficient to produce a 96% inhibition of thrombosis. A much higher dose of IXai (1.0 mg/kg + 17.3 micrograms/kg/min) resulted in a 39% reduction of clot weight. In a rabbit arterio-venous shunt model mimicking arterial thrombosis, the relative efficacy of the two agents was found to be more comparable. The doses required for optimum antithrombotic activity were 128.0 micrograms/kg + 2.2 micrograms/kg/min for Xai and 1.0 mg/kg + 17.3 micrograms/kg/min for IXai. We conclude that, in this study, prothrombinase rather than tenase inhibition was more effective in reducing venous thrombosis and that these effects can be achieved without disruption of extravascular hemostasis.

Hypertonic sodium chloride solutions: effect on hemodynamics and survival after hemorrhage in swine.

We evaluated the hemodynamic response of 12% shed blood replacement with 7.5% sodium chloride after a nonlethal hemorrhage in chronically instrumented swine. Compared to no treatment or Ringer's lactate infusion, the hypertonic salt solution was followed by increased cardiac output. We then evaluated the effect of hypertonic sodium chloride on survival after an otherwise lethal hemorrhage in chronically instrumented and unanesthetized swine. Survival was significantly greater after replacement of 25% of the shed blood with 7.5%, but not with 0.9%, 5.0%, or 10.0% sodium chloride solution. The mechanism appears to be by increased tissue perfusion as indicated by lower lactate values. Resuscitation with low-volume hypertonic sodium chloride may be life saving and life supporting during the initial treatment of trauma patients when conventional therapy is not available.

Regional blood flow during hypothermic arrest.

Little is known about the efficacy of CPR in the setting of hypothermia-induced cardiac arrest. We measured organ blood flow produced by conventional closed-chest CPR in eight swine following normothermic KCl-induced cardiac arrest and in seven swine surface-cooled until cardiac arrest occurred. Radiomicrospheres were injected in the unanesthetized basal state, after five minutes of CPR, and after 20 minutes of CPR. After five minutes of CPR, the cardiac output and cerebral and myocardial blood flows (mean +/- SD) of hypothermic animals were 15.3 +/- 7.5 mL/min/kg, 0.16 +/- 0.11 mL/min/g, and 0.20 +/- 0.15 mL/min/g, respectively. Mean percentage flows were 7%, 15%, and 8%, respectively, of those measured in the unanesthetized prearrest state, and 50%, 55%, and 31%, respectively, of the flow produced during CPR in normothermic animals. Blood flow during hypothermic CPR did not change significantly over time; however, during normothermic CPR, cardiac output and cerebral and myocardial flows decreased so that at 20 minutes there were no significant differences from those values measured in hypothermic animals. The reduction in organ flow produced by external chest compression in hypothermic animals may be a result of the changes in the viscoelastic properties of the thorax that occur during profound hypothermia.

Fluid resuscitation after an otherwise fatal hemorrhage: II. Colloid solutions.

We developed a fixed-volume porcine hemorrhage model that simulates the rapid exsanguination of combat or civilian trauma victims. In this study we compared the ability of colloid resuscitation solutions to prevent death after an otherwise lethal hemorrhage in 100 swine. The shed blood was replaced in a 1:1 ratio with either autologous whole blood (WB), untyped swine fresh frozen plasma (FFP), typed FFP, 5% human serum albumin (ALB), or normal saline (NS). Survival rate analysis indicated that WB was significantly better than FFP (untyped), ALB, or NS but not better than typed FFP. The 24-hour survival rates were: WB = 90%, typed FFP = 79%, untyped FFP = 56%, ALB = 57%, and NS = 25%. All deaths in the untyped FFP group suddenly occurred during or within 15 minutes after treatment in a recovering animal. Deaths in the ALB group steadily occurred for up to 2 1/2 hours after treatment. Analysis of hemodynamic, arterial blood gas, and acid-base data indicated that WB and FFP provided a better acid-buffering capacity in surviving animals than NS or ALB. We conclude that compatible FFP is a better resuscitation agent than ALB after an otherwise fatal hemorrhage because FFP is a better acid buffer.

Early administration of methylprednisolone promotes survival in rats with intra-abdominal sepsis.

We determined how the following drugs affected survival of 350-gm Sprague-Dawley rats subjected to intra-abdominal sepsis according to the method of Wichterman et al (J Surg Res 29:189-201, 1980): gentamicin (4.5 mg/kg/day), clindamycin (30 mg/kg/day), naloxone (2 mg/kg/hr), or methylprednisolone given either as a continuous infusion (2 mg/kg/hr) or as a bolus (30 mg/kg). A control group received only saline in a volume equal to the drug vehicle volume. Treatment was started immediately after cecal ligation and puncture. Drugs not given by bolus were infused by Alzet mini-pump (Model 2001) for 7 days. Percent of original population surviving at 10 days was (size of original population): saline--48% (92), antibiotics--86% (43), naloxone--30% (43), continuous methylprednisolone--14% (43), bolus methylprednisolone--93% (45). Survival of animals receiving either antibiotics or bolus methylprednisolone was significantly increased over the control population.

Naloxone does not prevent death after rapid hemorrhage in swine.

Despite the array of hemorrhage models, animal species and experimental designs, it seems clear that naloxone with or without volume replacement has no place in the treatment of rapidly exsanguinating traumatized patients. In this situation, the adrenergic hemodynamic effects, indirectly due to naloxone, further decrease tissue perfusion leading to a deleterious outcome. We suggest that the membrane stabilizing effects rather than the hemodynamic effects of naloxone should be pursued in the treatment of low perfusion states with longer survival times than rapid hemorrhage, such as septic and cardiogenic shock.