Vehicle ACT. 180 min. Time After Current Initiation (min) 180 min. Vehicle. UFH (75 U/kg) Anti-Factor Xa. Time After Current Initiation (min)
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1 Efficacy of, a Rationally-Engineered Low-Molecular-Weight Heparin, in a Canine Model of Arterial Thrombosis Ian D Fier, Mark A Nedelman, J Luis Guerrero, Ganesh Venkataraman, and Yi Wei Qi Momenta Pharmaceuticals, Inc., Cambridge, MA; Advanced Research Models, Norton, MA ABSTRACT Background: is a novel, rationally-engineered, low-molecular-weight heparin (LMWH) under development for stable angina and acute coronary syndromes with favorable pharmacokinetics for subcutaneous or intravenous administration, potent anti-factor Xa and IIa activity, constant anti-factor Xa:IIa ratio over time, rapid reversibility, and monitorability using standard point-of-care assays. Methods: Right and left femoral arteries of beagle dogs were instrumented with intravascular electrodes and perivascular Doppler flow probes. Fifteen minutes after initiating continuous saline infusion, thrombogenic injury was induced in the right (control) artery by applying an electrical current ( µa) to the intima that was maintained continuously for 8 minutes. Following occlusion of the control artery, an IV bolus plus 9 minutes continuous infusion of (7.5, 75 or 5 anti-xa IU/kg + anti-xa IU/kg/min, n=6/dose) or unfractionated heparin (; 75 U/kg + U/kg/min, n=6) was administered, and the left (active) artery was injured in a similar manner. Results: Incidence of occlusion was higher in arteries treated with saline (/) or (5/6) than with (/6 low; /6 middle; /6 high dose [P<.5, vs. [5 IU/kg]), despite comparable anticoagulation levels (ACT, PT, aptt) between and (5 anti-xa IU/kg). Time to occlusion was significantly prolonged by and at all doses compared to saline (P<.), but only minimal increases in cutaneous bleeding time were observed in all and treatment groups. exhibited consistent anti-factor Xa:IIa ratios over time at all doses. Conclusions:, a novel LMWH with potent anti-factor Xa and IIa activity, monitorability, and reversibility, demonstrated superior efficacy at 5 anti-xa IU/kg to a standard dose of, without increased bleeding risk, in a canine model of deep arterial thrombosis induced by severe electrolytic injury. INTRODUCTION Heparin derivatives, including unfractionated heparin () and low-molecular-weight heparin (LMWH), play a central role in treatment paradigms for acute coronary syndromes (ACS), particularly as adjunctive therapy during PCI., Both classes of agents have potent antithrombotic effects, but also limitations. has unpredictable patient-dependent pharmacokinetics and the potential to induce platelet aggregation and heparin-induced thrombocytopenia. LMWH has reduced inhibition of Factor IIa, limited reversibility with protamine sulfate, and decreased monitorability by point-of-care assays (ie, activated clotting time [ACT] assays). (Figure ) is a novel rationally-engineered LMWH under development for the treatment of stable angina and ACS with favorable pharmacokinetics for subcutaneous or intravenous administration, potent anti-factor Xa and IIa activity, constant anti-factor Xa:IIa ratio over time, rapid reversibility, and monitorability using standard point-of-care assays. OBJECTIVE To examine the antithrombotic and anticoagulant effects of in a canine model of acute arterial thrombosis. METHODS ANIMALS AND REAGENTS This study was carried out at Charles River Laboratories (Worcester, MA, USA) using purpose-bred, male, beagle dogs supplied by Marshall BioResources (North Rose, NY, USA). Husbandry conditions and surgical procedures were managed in accordance with the Guide for the Care and Use of Laboratory Animals. Euthanasia was conducted in compliance with accepted American Veterinary Medical Association guidelines. 5 was supplied by Momenta Pharmaceuticals, Inc. (Cambridge, MA, USA) and reconstituted in.9% sterile saline. Unfractionated heparin (heparin sodium injection, USP; 5 U/mL in.9% sterile saline) was obtained from Baxter Healthcare Corporation (Deerfield, IL, USA). ANIMAL INSTRUMENTATION Figure. interactions with coagulation proteins. -induced conformational changes in antithrombin (AT-III) and the linear heparin chain leads to capture and inactivation of thrombin (Factor IIa) and Factor Xa. Intravascular electrodes were inserted through the left and right femoral artery walls, positioned in direct contact with the intima, and connected to a constant amperage power source. A stenotic device and a perivascular Doppler flow probe were positioned immediately distal and proximal, respectively, to the intravascular electrode. Catheters were inserted into the carotid artery for monitoring continuous arterial blood pressure, blood flow, and heart rate and into the jugular vein for blood sample collection. An intravenous line was inserted into a peripheral vein for study treatment infusions. ELECTROLYTIC INJURY-INDUCED THROMBOSIS The study design, shown in Figure, was based on the model system developed by Lucchesi and colleagues. 6-8 For each artery, the distal stenotic device was adjusted to limit reactive hyperemia to 8% of the baseline response to physical occlusion. In addition, mean arterial pressure and heart rate were maintained at approximately 7 mm Hg and beats per minute, respectively, via isoflurane anesthetic management. Total occlusion was defined as a Doppler flow % of baseline for at least minute without cycling. Figure. Study design. RESULTS Right Femoral Artery (Control) Continuous electrical current ( µa) to intima.9% saline infusion n = 5 min 9 min Left Femoral Artery (Treated) Continuous electrical current ( µa) to intima 5 lu/kg + IU/kg/min 75 lu/kg + IU/kg/min 7.5 lu/kg + IU/kg/min 75 U/kg + U/kg/min Bolus + Continuous Infusion 5 min 9 min HEMATOLOGY AND COAGULATION PARAMETERS Activated clotting time (ACT), prothrombin time (PT), activated partial thromboplastin time (aptt), anti-factor Xa and IIa levels, and cutaneous bleeding time (CBT) were determined by standard methodologies at 5 minutes, 6 minutes, and either 8 minutes or the time of full occlusion, if earlier. STATISTICAL ANALYSIS Values were reported as means ± standard error (SE) unless otherwise noted. Means were compared using the Student s t test assuming equal variance in different treatment groups. Animals were monitored by Doppler flow for up to 8 minutes post current initiation. Full occlusion was defined as a reduction in Doppler flow to % of baseline values for at least minute without cycling., unfractionated heparin. P<.5 vs. control; P<.5 vs.. 8 min 8 min The incidence of occlusion was compared between treatment groups by calculating the odds ratio relative to control and using a z test to derive the P-value. Figure. Percentage of animals with fully occluded femoral arteries. Animals with Occluded Arteries (%) 8 6 (/) (75 U/kg) (5/6) (7.5 lu/kg) (/6) (75 lu/kg) (/6) (5 lu/kg) (/6) Table. Summary of Selected Hematologic Endpoints Treatment ACT (sec) Anti-Factor Xa (IU±SD) Anti-Factor IIa (IU±SD) Control 6 ± 8. ±.. ±. (7.5 IU/kg) ± 7. ±.6.6 ±. (75 IU/kg) 8 ±.66 ±.7.7 ±. (5 IU/kg) ± 8. ±..6 ±.9 (75 U/kg) 6 ± ±..95 ±.9 ACT, activated clotting time; IU, international unit; SD, standard deviation,, unfractionated heparin. Recorded at 6 min after initiation of test article infusion; Bolus dose (see Figure ); P<.5, vs. ; P<., vs. control. Figure. Coagulation activity vs. time as assessed by ACT (left), aptt (center), and PT assays (right) ACT The vehicle control group shown in the graphs subsequently received at 5 IU/kg. Error bars are ± SE., unfractionated heparin. aptt values > seconds not included. Figure 6. Correlation of anti-factor Xa and IIa activities. Anti-Factor IIa Activity (IU/mL) (75 U/kg) Anti-Factor Xa (75 U/kg) 5 (5 lu/kg) aptt Figure 5. Anti-Factor Xa activity (left) and anti-factor IIa activity (right) vs. time. (5 lu/kg) Individual points represent data from a single animal. All animals in all treatment groups are shown. Correlation coefficients (r ) were.89 and.65 in the and unfractionated heparin () groups, respectively. Anti-Factor IIa Activity (lu/ml) (75 lu/kg) (75 lu/kg) Anti-Factor IIa (7.5 lu/kg) The vehicle control group shown in the graphs subsequently received at 5 IU/kg. Error bars are ± SE., unfractionated heparin. 9 8 PT (7.5 lu/kg) Figure 7. Anti-Factor Xa:IIa ratio over time. Anti-Factor Xa:lla Ratio Error bars are + SE (top halves only are shown to maximize clarity)., unfractionated heparin. CONCLUSIONS (75 U/kg) (5 lu/kg) (75 lu/kg) (7.5 lu/kg) Time (min), a novel LMWH with potent anti-factor Xa and IIa activity, monitorability, and reversibility, had significantly greater antithrombotic efficacy at all tested concentrations than vehicle (Figure ). at 5 IU/kg had significantly greater antithrombotic efficacy than at 75 U/kg (Figure ). The enhanced antithrombotic activity of was not associated with increased cutaneous bleeding time relative to the group (data not shown). dose-dependent inhibition of clotting was observed in all coagulation assays (Figure and Table ). Unlike other LMWHs, may therefore have the potential to be assayed by the point-of-care ACT assay, which could simplify its use in patients undergoing PCI to treat stable angina and ACS. exhibited dose-dependent inhibition of both Factor Xa and IIa (Figure 5 and Table ). Anti-Factor IIa activity increased linearly with anti-factor Xa activity for both and, although the correlation coefficient was greater for (r =.89) than (r =.65) (Figure 6). The prominent anti-factor IIa activity observed in these experiments further distinguishes from currently available LMWHs. The ratio of anti-factor Xa activity to IIa activity over time was generally more constant with than, consistent with the known variable metabolism of the large and polydisperse molecules (Figure 7). By combining potent anti-factor Xa and IIa activity, consistent antithrombotic effects over time, easy monitorability, reversibility, and simpler pharmacokinetics, has several key positive features of both LMWH and and, therefore, may be an attractive option in the future for the treatment of stable angina and ACS. REFERENCES. Antman EM, Anbe DT, Armstrong PW, Bates ER, Green LA, Hand M, et al. ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 999 Guidelines for the Management of Patients with Acute Myocardial Infarction). Circulation. ;:e8-9.. Braunwald E, et al. ACC/AHA Guideline Update for the Management of Patients With Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. Available at: Hirsh J, Warkentin TE, Shaughnessy SG, Anand SS, Halperin JL, Raschke R, et al. Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest. ;9:6S-9S.. Institute of Laboratory Animal Resources. Guide for the care and use of laboratory animals. 7th ed. Washington, D.C.: National Academy Press; Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc. ;8: Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in the ambulatory canine: a model for in vivo evaluation of anti-thrombotic agents. Thromb Res. 98;7: Rote WE, Davis JH, Mousa SA, Reilly TM, Lucchesi BR. Antithrombotic effects of DMP 78, a platelet GPIIb/IIIa receptor antagonist, in a canine model of arterial thrombosis. J Cardiovasc Pharmacol. 99;: Huang J, Driscoll EM, Gonzales ML, Park AM, Lucchesi BR. Prevention of arterial thrombosis by intravenously administered platelet PT receptor antagonist AR-C699MX in a canine model. J Pharmacol Exp Ther. ;95:9-9. Poster presented at the 56th Annual Scientific Session of the American College of Cardiology, March 7, 7, New Orleans, Louisiana.
2 Efficacy of, a Rationally-Engineered Low-Molecular-Weight Heparin, in a Canine Model of Arterial Thrombosis Ian D Fier, Mark A Nedelman, J Luis Guerrero, Ganesh Venkataraman, and Yi Wei Qi Momenta Pharmaceuticals, Inc., Cambridge, MA; Advanced Research Models, Norton, MA
3 ABSTRACT Background: is a novel, rationally-engineered, low-molecular-weight heparin (LMWH) under development for stable angina and acute coronary syndromes with favorable pharmacokinetics for subcutaneous or intravenous administration, potent anti-factor Xa and IIa activity, constant anti-factor Xa:IIa ratio over time, rapid reversibility, and monitorability using standard point-of-care assays. Methods: Right and left femoral arteries of beagle dogs were instrumented with intravascular electrodes and perivascular Doppler flow probes. Fifteen minutes after initiating continuous saline infusion, thrombogenic injury was induced in the right (control) artery by applying an electrical current ( µa) to the intima that was maintained continuously for 8 minutes. Following occlusion of the control artery, an IV bolus plus 9 minutes continuous infusion of (7.5, 75 or 5 anti-xa IU/kg + anti-xa IU/kg/min, n=6/dose) or unfractionated heparin (; 75 U/kg + U/kg/min, n=6) was administered, and the left (active) artery was injured in a similar manner. Results: Incidence of occlusion was higher in arteries treated with saline (/) or (5/6) than with (/6 low; /6 middle; /6 high dose [P<.5, vs. [5 IU/kg]), despite comparable anticoagulation levels (ACT, PT, aptt) between and (5 anti-xa IU/kg). Time to occlusion was significantly prolonged by and at all doses compared to saline (P<.), but only minimal increases in cutaneous bleeding time were observed in all and treatment groups. exhibited consistent anti-factor Xa:IIa ratios over time at all doses. Conclusions:, a novel LMWH with potent anti-factor Xa and IIa activity, monitorability, and reversibility, demonstrated superior efficacy at 5 anti-xa IU/kg to a standard dose of, without increased bleeding risk, in a canine model of deep arterial thrombosis induced by severe electrolytic injury. INTRODUCTION Heparin derivatives, including unfractionated heparin () and low-molecular-weight heparin (LMWH), play a central role in treatment paradigms for acute coronary syndromes (ACS), particularly as adjunctive therapy during PCI., Both classes of agents have potent antithrombotic effects, but also limitations. has unpredictable patient-dependent pharmacokinetics and the potential to induce platelet aggregation and heparin-induced thrombocytopenia. LMWH has reduced inhibition of Factor IIa, limited reversibility with protamine sulfate, and decreased monitorability by point-of-care assays (ie, activated clotting time [ACT] assays). (Figure ) is a novel rationally-engineered LMWH under development for the treatment of stable angina and ACS with favorable pharmacokinetics for subcutaneous or intravenous administration, potent anti-factor Xa and IIa activity, constant anti-factor Xa:IIa ratio over time, rapid reversibility, and monitorability using standard point-of-care assays. OBJECTIVE Figure. interactions with coagulation proteins. -induced conformational changes in antithrombin (AT-III) and the linear heparin chain leads to capture and inactivation of thrombin (Factor IIa) and Factor Xa. To examine the antithrombotic and anticoagulant effects of in a canine model of acute arterial thrombosis. METHODS ANIMALS AND REAGENTS This study was carried out at Charles River Laboratories (Worcester, MA, USA) using purpose-bred, male, beagle dogs supplied by Marshall BioResources (North Rose, NY, USA). Husbandry conditions and surgical procedures were managed in accordance with the Guide for the Care and Use of Laboratory Animals. Euthanasia was conducted in compliance with accepted American Veterinary Medical Association guidelines. 5 was supplied by Momenta Pharmaceuticals, Inc. (Cambridge, MA, USA) and reconstituted in.9% sterile saline. Unfractionated heparin (heparin sodium injection, USP; 5 U/mL in.9% sterile saline) was obtained from Baxter Healthcare Corporation (Deerfield, IL, USA). ANIMAL INSTRUMENTATION Intravascular electrodes were inserted through the left and right femoral artery walls, positioned in direct contact with the intima, and connected to a constant amperage power source.
4 A stenotic device and a perivascular Doppler flow probe were positioned immediately distal and proximal, respectively, to the intravascular electrode. Catheters were inserted into the carotid artery for monitoring continuous arterial blood pressure, blood flow, and heart rate and into the jugular vein for blood sample collection. An intravenous line was inserted into a peripheral vein for study treatment infusions. ELECTROLYTIC INJURY-INDUCED THROMBOSIS The study design, shown in Figure, was based on the model system developed by Lucchesi and colleagues. 6-8 For each artery, the distal stenotic device was adjusted to limit reactive hyperemia to 8% of the baseline response to physical occlusion. In addition, mean arterial pressure and heart rate were maintained at approximately 7 mm Hg and beats per minute, respectively, via isoflurane anesthetic management. Total occlusion was defined as a Doppler flow % of baseline for at least minute without cycling. Figure. Study design. Right Femoral Artery (Control) Continuous electrical current ( µa) to intima.9% saline infusion n = 5 min 9 min 8 min Left Femoral Artery (Treated) Continuous electrical current ( µa) to intima 5 lu/kg + IU/kg/min 75 lu/kg + IU/kg/min 7.5 lu/kg + IU/kg/min 75 U/kg + U/kg/min Bolus + Continuous Infusion 5 min 9 min 8 min HEMATOLOGY AND COAGULATION PARAMETERS Activated clotting time (ACT), prothrombin time (PT), activated partial thromboplastin time (aptt), anti-factor Xa and IIa levels, and cutaneous bleeding time (CBT) were determined by standard methodologies at 5 minutes, 6 minutes, and either 8 minutes or the time of full occlusion, if earlier. STATISTICAL ANALYSIS Values were reported as means ± standard error (SE) unless otherwise noted. Means were compared using the Student s t test assuming equal variance in different treatment groups. The incidence of occlusion was compared between treatment groups by calculating the odds ratio relative to control and using a z test to derive the P-value. RESULTS Figure. Percentage of animals with fully occluded femoral arteries. Animals with Occluded Arteries (%) 8 6 (/) (75 U/kg) (5/6) (7.5 lu/kg) (/6) (75 lu/kg) (/6) (5 lu/kg) (/6) Animals were monitored by Doppler flow for up to 8 minutes post current initiation. Full occlusion was defined as a reduction in Doppler flow to % of baseline values for at least minute without cycling., unfractionated heparin. P<.5 vs. control; P<.5 vs..
5 Table. Summary of Selected Hematologic Endpoints Treatment ACT (sec) Anti-Factor Xa (IU±SD) Anti-Factor IIa (IU±SD) Control 6 ± 8. ±.. ±. (7.5 IU/kg) ± 7. ±.6.6 ±. (75 IU/kg) 8 ±.66 ±.7.7 ±. (5 IU/kg) ± 8. ±..6 ±.9 (75 U/kg) 6 ± ±..95 ±.9 ACT, activated clotting time; IU, international unit; SD, standard deviation,, unfractionated heparin. Recorded at 6 min after initiation of test article infusion; Bolus dose (see Figure ); P<.5, vs. ; P<., vs. control. Figure. Coagulation activity vs. time as assessed by ACT (left), aptt (center), and PT assays (right). (75 U/kg) (5 lu/kg) (75 lu/kg) (7.5 lu/kg) ACT 5 aptt 9 PT The vehicle control group shown in the graphs subsequently received at 5 IU/kg. Error bars are ± SE., unfractionated heparin. aptt values > seconds not included. Figure 5. Anti-Factor Xa activity (left) and anti-factor IIa activity (right) vs. time. 8 (75 U/kg) (5 lu/kg) (75 lu/kg) (7.5 lu/kg) Anti-Factor Xa.5 Anti-Factor IIa Anti-Factor IIa Activity (lu/ml)..5.5 The vehicle control group shown in the graphs subsequently received at 5 IU/kg. Error bars are ± SE., unfractionated heparin. Figure 6. Correlation of anti-factor Xa and IIa activities. Anti-Factor IIa Activity (IU/mL) Individual points represent data from a single animal. All animals in all treatment groups are shown. Correlation coefficients (r ) were.89 and.65 in the and unfractionated heparin () groups, respectively.
6 Figure 7. Anti-Factor Xa:IIa ratio over time. (75 U/kg) (5 lu/kg) (75 lu/kg) (7.5 lu/kg) Anti-Factor Xa:lla Ratio Error bars are + SE (top halves only are shown to maximize clarity)., unfractionated heparin. CONCLUSIONS Time (min), a novel LMWH with potent anti-factor Xa and IIa activity, monitorability, and reversibility, had significantly greater antithrombotic efficacy at all tested concentrations than vehicle (Figure ). at 5 IU/kg had significantly greater antithrombotic efficacy than at 75 U/kg (Figure ). The enhanced antithrombotic activity of was not associated with increased cutaneous bleeding time relative to the group (data not shown). dose-dependent inhibition of clotting was observed in all coagulation assays (Figure and Table ). Unlike other LMWHs, may therefore have the potential to be assayed by the point-of-care ACT assay, which could simplify its use in patients undergoing PCI to treat stable angina and ACS. exhibited dose-dependent inhibition of both Factor Xa and IIa (Figure 5 and Table ). Anti-Factor IIa activity increased linearly with anti-factor Xa activity for both and, although the correlation coefficient was greater for (r =.89) than (r =.65) (Figure 6). The prominent anti-factor IIa activity observed in these experiments further distinguishes from currently available LMWHs. The ratio of anti-factor Xa activity to IIa activity over time was generally more constant with than, consistent with the known variable metabolism of the large and polydisperse molecules (Figure 7). By combining potent anti-factor Xa and IIa activity, consistent antithrombotic effects over time, easy monitorability, reversibility, and simpler pharmacokinetics, has several key positive features of both LMWH and and, therefore, may be an attractive option in the future for the treatment of stable angina and ACS. REFERENCES. Antman EM, Anbe DT, Armstrong PW, Bates ER, Green LA, Hand M, et al. ACC/AHA guidelines for the management of patients with ST-elevation myocardial infarction: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Committee to Revise the 999 Guidelines for the Management of Patients with Acute Myocardial Infarction). Circulation. ;:e8-9.. Braunwald E, et al. ACC/AHA Guideline Update for the Management of Patients With Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. Available at: Hirsh J, Warkentin TE, Shaughnessy SG, Anand SS, Halperin JL, Raschke R, et al. Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest. ;9:6S-9S.. Institute of Laboratory Animal Resources. Guide for the care and use of laboratory animals. 7th ed. Washington, D.C.: National Academy Press; Report of the AVMA Panel on Euthanasia. J Am Vet Med Assoc. ;8: Romson JL, Haack DW, Lucchesi BR. Electrical induction of coronary artery thrombosis in the ambulatory canine: a model for in vivo evaluation of anti-thrombotic agents. Thromb Res. 98;7: Rote WE, Davis JH, Mousa SA, Reilly TM, Lucchesi BR. Antithrombotic effects of DMP 78, a platelet GPIIb/IIIa receptor antagonist, in a canine model of arterial thrombosis. J Cardiovasc Pharmacol. 99;: Huang J, Driscoll EM, Gonzales ML, Park AM, Lucchesi BR. Prevention of arterial thrombosis by intravenously administered platelet PT receptor antagonist AR-C699MX in a canine model. J Pharmacol Exp Ther. ;95:9-9. Poster presented at the 56th Annual Scientific Session of the American College of Cardiology, March 7, 7, New Orleans, Louisiana.
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