Regular Article

Nonclinical Safety Assessment of PER977: A Small Molecule Reversal Agent for New Oral Anticoagulants and Heparins

International Journal of Toxicology. 2015, Vol. 34(4) 308-317 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1091581815590667 ijt.sagepub.com

Dexter W. Sullivan, Jr1, Shayne C. Gad1, Bryan Laulicht2, Sasha Bakhru2, and Solomon Steiner2

Abstract A new molecular entity, PER977 (di-arginine piperazine), is in clinical development as an anticoagulant reversal agent for new oral anticoagulants and heparins. The good laboratory practices (GLP)-compliant studies were conducted to evaluate the toxicity of PER977 and its primary metabolite, 1,4-bis(3-aminopropyl)piperazine (BAP). PER977 and BAP were negative for systemic toxicity in dogs and rats. PER977 was rapidly eliminated from the blood with little to no accumulation. PER977 was negative for genotoxicity and did not alter neurological, respiratory, or cardiovascular function. Maximum tolerated doses for PER977 were 40 (rat) and 35 mg/kg (dog), and greater than 80 mg/kg (rat) for BAP. The no observable adverse effect level (NOAEL) for 14-day intravenous exposure to both rats and dogs was 20 mg/kg/d. For BAP, the NOAELs for 14-day intravenous exposure to rats and dogs were 5 and 20 mg/kg, respectively. Based on these results, a safe and conservative dose level of 19.4 mg/d was used for the PER977 first in human study. Keywords anticoagulant, NOAC, drug, reversal agent, BAP, PER977

Introduction Annually, millions of patients in the United States require anticoagulation for atrial fibrillation or thromboembolic events,1 prophylactic management following joint replacement procedures,2 or atrial fibrillation.3 The new oral anticoagulants (NOACs) that directly inhibit coagulation factor Xa or IIa have numerous advantages over warfarin: rapid therapeutic effectiveness, ease of dosing, and lack of monitoring requirements. However, each is associated with a risk of major bleeding. In addition, a major drawback to their use is the lack of a reversal agent. This is germane in cases of overdose, trauma, emergency surgery, or elective invasive procedures.4 A safe and efficacious reversal agent for NOACs would allow for rapid emergency response to overdose or trauma, minimize the time patients are off their anticoagulant prior to elective procedures, and provide a level of confidence regarding restarting anticoagulant therapy. To address this significant unmet clinical need, Perosphere, Inc has developed PER977 (Figure 1), a small molecule anticoagulant reversal agent for NOACs and heparins. PER977 (di-arginine piperazine) is a synthetic water soluble new molecular entity that directly combines with heparin and related anticoagulant drugs (ie, Xa and IIa inhibitors), allowing rapid reestablishment of normal blood coagulation. This reversal effect is due to the direct binding of the anticoagulant molecule without binding to blood coagulation factors or to other proteins in the blood.5,6 If PER977 is shown to be safe

and efficacious in humans, it has the potential to not only offer a completely unmet need for a reversal drug with which to reverse the NOACs (specifically the Xa and IIa inhibitors) but also has the potential to provide an improved reversal drug for older anticoagulants such as unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) for which protamine is only partially successful. Prior to its first use in humans, the potential nonclinical toxicity of PER977 was evaluated in a battery of good laboratory practices (GLP) and International Conference on Harmonisation (ICH)-compliant in vivo and in vitro assays. In rat and dog models, maximum tolerated dose (MTD) and 14-day repeat dose toxicity studies as well as the core safety pharmacology studies (respiratory and central nervous system in rats and cardiovascular in dogs) were performed. Additionally, to evaluate the genotoxic potential of PER977, a bacterial reverse mutation assay, an in vitro mammalian chromosome aberration test in CHO cells, and an in vivo mouse micronucleus assay

1 2

Gad Consulting Services, Raleigh, NC, USA Perosphere Inc, Danbury, CT, USA

Corresponding Author: Dexter W. Sullivan, Jr, Gad Consulting Services, 4008 Barrett Drive, Suite 201, Raleigh, NC 27609, USA. Email: [email protected]

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

Sullivan et al

309

Figure 1. Structure for PER977 (2-amino-5-guanidine-pentanoic acid (3-f4-[3-(2-amino-5-guanidino-pentanolyamino)-propyl]-piperazin-1ylgpropyl)amide), C22H48O2N12, molecular weight: 512 Da.

were performed. Finally, 1,4-bis(3-aminopropyl)piperazine (BAP), the primary metabolite of PER977, was evaluated for toxicity in rat and dog models (MTD in rats and 14-day studies in rats and dogs). The US Food and Drug Administration deemed this safety information as supportive of safe usage in humans. Perosphere opened an investigational new drug (IND 117 224) and has advanced the clinical development of PER977 through Phase 2.

Materials and Methods To determine the most appropriate animal species, in vitro assays were conducted to compare the metabolic profiles of PER977 between species and indicated that rat and dog models were consistent with humans (data not shown). The primary clearance pathway for PER977 was identified as peptide cleavage first of a single arginine to monoarginine piperazine and then cleavage of the second arginine to BAP. For PER977, the MTD and 14-day repeat dose toxicity studies in rats and dogs were conducted at Sinclair Research Center (Auxvasse, Missouri), and the safety pharmacology studies were conducted at Calvert Labs (Scott Township, Pennsylvania). The genotoxicity studies were conducted at Nelson Laboratories (Murray, Utah). For BAP, the MTD study in rats and 14-day repeat dose toxicity studies in rats and dogs were conducted at Calvert Labs. Consistent with the intended clinical route, nonclinical safety studies were conducted by the intravenous (IV) route. All studies were conducted in compliance with Good Laboratory Practices and housing, and room conditions were consistent with Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) standards unless stated otherwise.

cellulose acetate membrane) filter and bottled into lypophilization vials. The vials were sterilized by autoclaving at 121 C (250 F) for 30 minutes. The pH values of all solutions were again always confirmed to be approximately 7, and the solutions were stored at refrigerated conditions of 2 C to 8 C (36 F-46 F) until preparation of additional dose solutions if necessary. The ICH-compliant stability studies were conducted to ensure the suitability of PER977 used in the nonclinical studies. The BAP, the primary metabolite for PER977, was supplied by Sigma-Aldrich, and dosing solutions were then prepared weekly for IV administration. Dosing preparations were stored frozen at approximately 21 C to 29 C (6 F to 20 F) following preparation and thawed overnight in the refrigerator at 2 C to 6 C (36 F-43 F) prior to use. As with PER977, ICHcompliant stability studies were conducted to ensure the suitability of BAP used in the nonclinical studies.

Chemicals and Materials For the MTD, 14-day repeat dose and core safety studies in rats and dogs, the control article was a 0.9% saline solution (pH adjusted to approximately 7.4). It was filtered through a 0.2 mm filter and autoclaved at 121 C (250 F) for 30 minutes. The final pH value was confirmed to be 6.77. The Ames test (Salmonella typhimurium reverse mutation assay; Nelson Laboratories) used S-9 specific chemicals controls, 2-aminofluorene and 2-aminoanthracene, and the strain specific nonmetabolic chemical controls, sodium azide, mitomycin-C, and 4-nitro-0-phenylene-diamine. For the chromosomal aberrations assay (Nelson Laboratories), cyclophosphamide and mitomycin C were used as the positive controls. All chemicals and materials were manufactured according to current good manufacturing practices.

Test Articles The new molecular entity PER977 was supplied by Perosphere Biopharmaceuticals, Inc (Mount Kisco, New York). Various stock solutions were prepared by dissolving lyophilized PER977 in water for injection. The pH value was adjusted to 7.4 using 2 mol/L NaOH. For final dose solutions, the stock solutions were diluted accordingly with 0.9% saline. The pH values were always verified to be approximately 7. All dose solutions were filtered through a 0.2 mm (surfactant-free

Animals For the PER977 MTD rat study, 17 male Sprague-Dawley (SD) rats of 10 to 11 weeks old were obtained from Harlan (Indianapolis, Indiana). The mean body weights ranged from 280 to 345 g prior to the first dosing. Drinking water and commercial rodent diet (Harlan Teklad certified rodent diet) were provided ad libitum. For the PER977 MTD dog study, 7 (5 male and 2 female) Beagle dogs of approximately 10 months old were

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

310

International Journal of Toxicology 34(4)

obtained from Ridglan Farms (Mt Horeb, Wisconsin). Mean body weights ranged from 9.48 to 11.32 kg prior to dosing on day 1. A total of 250 g/d commercial diet (Purina dog chow) was provided. For the BAP MTD rat study, 18 male SD rats of 9 to 11 weeks old were obtained from Harlan. Mean body weights ranged from 288 to 390 g prior to the first day of dosing. Drinking water and commercial rodent diet (Harlan Teklad certified rodent diet) were provided ad libitum. For the PER977, 14day repeat dose rat study, 140 (70 male and 70 female) SD rats of approximately 8 weeks old were obtained from Harlan. Mean body weights ranged from 140 to 250 g prior to the first dosing. Drinking water and commercial rodent diet (Harlan Teklad certified rodent diet # 2018C) were provided ad libitum. For the PER977, 14-day repeat dose dog study, 24 (12 male and 12 female) Beagle dogs of 10 to 11 months old obtained from Ridglan Farms. Mean body weights ranged from 7.71 to 11.90 kg prior to the first dosing. Drinking water was provided ad libitum. A total of 250 g/d commercial diet (Purina Dog Chow) were provided throughout the study. For the BAP 14-day repeat dose rat study, 60 (30 male and 30 female) SD rats of approximately 8 weeks old were obtained from Harlan. Mean body weights ranged from 235 to 264 g prior to the first dosing. Drinking water and commercial rodent diet (Harlan Teklad certified rodent diet # 2018C) were provided ad libitum. For the BAP 14-day repeat dose dog study, 18 (9 male and 9 female) Beagle dogs of approximately 6 months old were obtained from Marshall BioResources (North Rose, New York). The mean body weights ranged from 8.2 to 10 kg for males and 6.2 to 8 kg for females prior to the first dosing. Drinking water was provided ad libitum. A total of 400 g/d PMI canine diet were provided throughout the study. For the cardiovascular function test, 4 male Beagle dogs of 52 to 70 months old were obtained from Marshall BioResources and individually housed in compliance with United States Department of Agriculture (USDA) Guidelines. The acclimatization period was 5 days prior to testing. Initial mean body weights ranged from 11.6 to 12.9 kg prior to treatment initiation. Drinking water was provided ad libitum. A total of 200 to 400 g/d commercial diet (Harlan Teklad Canine Diet, Harlan Laboratories, Madison, WI, USA) were provided throughout the study. For the neuropharmacological study, 40 male SD rats of 7 weeks old were obtained from Charles River Laboratories. Mean body weights ranged from 185 to 240 g prior to the first dosing. Drinking water and certified rodent diet (Teklad) were provided ad libitum. For the respiratory safety pharmacology study, 24 male rats of 9 to 10 weeks old were obtained from Charles River Laboratories and housed in compliance with the National Research Council ‘‘Guide for the Care and Use of Laboratory Animals.’’ Mean body weights were 308 to 347 g. Drinking water and certified rodent diet (Teklad) were provided ad libitum, except for during data collection.

Experimental Design Maximum tolerated dose study of PER977 in SD rats. In keeping consistent with the 3Rs principles (replacement of animals whenever feasible, reduction of the number of animals, and

refinement of experimental methods), the judicious use of animals was exercised in all of the MTD studies. Seventeen male SD rats were randomly assigned to 5 groups. PER977 was administered once intravenously to 3 rats at an initial dose level of 10 mg/kg. The rats were observed postdose for clinical signs of toxicity and for mortality/morbidity. Body weights were recorded prior to selection, prior to dosing, and on day 3/4. Gross necropsy was also performed on day 3/4. Following a minimum of 24 hours, the next dose level (20 mg/kg) was administered to an additional 3 rats after which the rats were again observed postdose for clinical signs of toxicity. This procedure was repeated at dose levels of 40 and 60 mg/kg, until an MTD was determined. An additional group of 5 males was intravenously administered PER977 at 40 mg/kg to confirm the findings. Maximum tolerated dose study of PER977 in beagle dogs. Six (4 male/2 female) beagle dogs were randomly assigned to 3 groups. PER977 was administered once intravenously to 1 male dog at an initial dose level of 47 mg/kg (target dose of 50 mg/kg). The dog was observed postdose for clinical signs of toxicity and for mortality/morbidity. Body weights were recorded prior to dosing and daily through day 6 after which the animal was euthanized with Fatal Plus (Vortech, Vortech Pharmaceuticals, Dearborn, MI, USA) at a dose level of at least 1 mL/4.5 kg. Death was assured through exsanguination. A scheduled terminal necropsy was performed, and organ weights were collected for the brain, heart, kidneys, liver with gall bladder, lungs with mainstream bronchi, and spleen. The procedure was repeated at dose levels of 25 and 35 mg/kg until an MTD was determined. An additional group of 4 animals (2 males and 2 females) was intravenously administered PER977 at 35 mg/kg to confirm the findings. Maximum tolerated dose study of BAP in SD rats. Eighteen male SD rats were randomly assigned to 6 groups. The BAP was administered once intravenously to 3 rats at an initial dose level of 5 mg/kg. The rats were observed postdose for clinical signs of toxicity and for mortality/morbidity. Body weights were recorded prior to selection, prior to dosing, and on day 3. Gross necropsy was also performed on day 3/4. Following a minimum of 24 hours, the next dose level (10 mg/kg) was administered to an additional 3 rats after which the rats were again observed postdose for clinical signs of toxicity. This procedure was repeated at higher dose levels (20, 40, 60, and 80 mg/kg) until an MTD was determined. All animals were euthanized by CO2 asphyxiation on day 3 following dose administration. 14-Day repeat dose of PER977 in SD rats. A total of 122 SD rats were used. The study had 2 portions: the toxicity study (main study/80 rats) and the toxicokinetic study (TK study/42 rats). Main (toxicity) study: following about 10 days of acclimation, 80 SD rats (40 males and 40 females) were randomly assigned to 4 groups, which were intravenously dosed for 14 days with either vehicle or 1 of the 3 PER977 dose solutions (0.08, 0.8, and 8.0 mg/mL). The daily dose volume was set at 2.5 mL/kg for all 4 groups. The daily PER977 dose levels were 0.2, 2.0,

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

Sullivan et al

311

and 20.0 mg/kg, respectively. The selected dose levels were based on the results of a dose range finding (DRF) study. These main study rats were euthanized on day 15. For the toxicokinetic portion of the study, 42 SD rats (21 males and 21 females) were randomly assigned to 4 groups with 3 rats per gender assigned to the vehicle control group and 6 rats per gender for each PER977 dose group of 0.2, 2.0, and 20.0 mg/kg. All animals received 14 consecutive daily IV administrations. Blood samples for TK analyses were collected from the TK group 2 to 4 on days 1 and 14 at the following time points: predose (day 14 only), and 5, 15, 30, 60, 120, and 240 minutes following dose administration. For control animals, only 1 sample was collected, 60 minutes following the dose administrations on days 1 and 14. All rats were observed twice daily for signs of morbidity and mortality and once daily for clinical signs until study termination. Body weights were recorded prior to the initial dosing, on days 0 and 7, and on day 14 prior to termination. Food consumption was monitored throughout the study. Ophthalmological examinations were performed during acclimation and on day 14. On day 15, rats were euthanized by carbon dioxide asphyxiation. Following euthanasia, blood samples for evaluation of hematology, coagulation, and serum chemistry parameters were collected, and the organs and tissues normally examined in general nonclinical toxicity studies were removed, weighed, and examined for gross and microscopic pathological anomalies.7,8 14-Day repeat dose of PER977 in beagle dogs. A total of 24 Beagle dogs, 3 dogs/gender/group designated to 4 different treatment groups, were used in the main study. Following an acclimation period of 9 days, study animals were fasted overnight and on day 1 through 14 received IV bolus injections of vehicle or 0.2, 2, and 20 mg/kg of PER977. Two toxicokinetic collections were performed (days 1 and 14; predose, 3, 7, 10, 15, 30, and 45 minutes and 1, 2, 4, 8, and 24 hours postdose). All animals were observed twice daily for signs of morbidity and mortality (days 9 to 15) and once daily for clinical signs beginning on day 1 (predose) until study termination. Animals were given a physical examination by a staff veterinarian during acclimation to determine each animal’s suitability for study. All animals in the main study were also examined prior to termination. Examinations included, but were not limited to, examination of the skin and external ears, eyes, abdomen, neurological, behavior, and general body condition. Body weights were recorded twice during the acclimation period on days 9 and 1 and on days 7 and 15 prior to termination. Animals were fed once daily, and food consumption was monitored throughout the study. Ophthalmological examinations were performed during acclimation and on day 15 prior to termination. During acclimation and on day 14, electrocardiogram (ECG) recordings were performed on all animals. On days 1 through 14, breathing rate and the cutaneous bed were monitored for all animals. Breathing was categorized as normal or labored directly following injection, and the color of the gums was categorized as normal or altered (specifying the duration and color of the observed change if

noted) as a means of monitoring the cutaneous bed. Urine volume samples were collected during acclimation and at termination for urinalysis. On day 15, animals were euthanized. Following sacrifice of the animals, blood samples for evaluation of hematology, coagulation, and serum chemistry parameters were collected, and select organs and tissues were removed, weighed, and examined for gross and microscopic pathological anomalies.7,8 14-Day repeat dose of BAP in SD rats. Following about 10 days of acclimation, 30 SD rats (15 males and 15 females) were randomly assigned to 3 groups, which were intravenously dosed for 14 days with either vehicle or 1 of the 2 BAP dose solutions (5 and 10 mg/ mL). The daily dose volume was set at 2 mL/kg for the control and high-dose groups and 1 mL/kg for the low-dose group. The daily BAP dose levels were 5 and 20 mg/kg, respectively. The selected dose levels were based on the results of a DRF study. These main study rats were euthanized on day 15. All rats were observed twice daily for signs of morbidity and mortality and once daily for clinical signs until study termination. Body weights were recorded prior to the initial dosing, on days 1 and 7, and on day 14 prior to termination. Food consumption was monitored throughout the study. Ophthalmological examinations were performed during acclimation and on day 15. Blood samples for evaluation of hematology, coagulation, and serum chemistry parameters were collected on day 15 prior to animals being euthanized by carbon dioxide asphyxiation. Following euthanasia, select organs and tissues were removed, weighed, and examined for gross and microscopic pathological anomalies.7,8 14-Day repeat dose of BAP in beagle dogs. A total of 18 Beagle dogs, 3 dogs/gender/group designated to 3 different treatment groups, were used in the main study. Following an acclimation period of 12 days, study animals were fasted overnight and on day 1 through 14 received IV bolus injections of 0 (vehicle), 5, or 20 mg/kg of BAP. All animals were observed twice daily for signs of morbidity and mortality throughout the study and once daily for clinical signs beginning on day 1 (predose) until study termination. Animals were given a physical examination by a staff veterinarian during acclimation to determine each animal’s suitability for study. All animals in the main study were also examined prior to termination. Examinations included, but were not limited to, examination of the skin and external ears, eyes, abdomen, neurological, behavior, and general body condition. Body weights were recorded at the time of randomization and prior to dose administration on days 1, 8, and 14. Animals were fed once daily, and food consumption was monitored throughout the study. Ophthalmological examinations were performed during acclimation and on day 13 (males) or day 12 (females). During acclimation and on day 14, ECG recordings were performed on all animals. On day 15, blood samples for evaluation of hematology, coagulation, and serum chemistry parameters were collected after which animals were euthanized, and selected organs and tissues were removed, weighed, and examined for gross and microscopic pathological anomalies.7,8

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

312

International Journal of Toxicology 34(4)

Evaluation of cardiovascular function after PER977 IV administration in conscious telemetered male beagle dogs. For the cardiovascular function test, 4 male beagle dogs received IV bolus injections of PER977 in a Latin square design where each animal received 0, 0.2, 2, and 20 mg/kg of PER977 with 3 days washout between doses, supported by short half-life data. All dosing occurred over an 11-day period. Animals were observed once daily for clinical signs. Body weights were recorded prior to the first dose administration on each dosing day and used for dose volume calculation purposes. Cardiovascular function parameters were collected prior to each dose to determine baseline values for cardiovascular parameter calculations. Following each dose administration (washout period of at least 3 days was allowed between doses), parameters were recorded continuously for at least 22 hours with the exception of day 4 when recording failed beyond 9 hours following dosing. The reason for failed recording could not be determined. The following parameters were analyzed: body temperature, systolic arterial pressure, diastolic arterial pressure, mean arterial pressure, heart rate, P duration, QRS interval, R amplitude, and QT interval. The ECGs were evaluated by a board certified veterinary cardiologist who examined 1 minute tracings of the ECGs obtained at 15 minutes prior to dosing and at 30 minutes, 1, 2, 4, 8, 12, and 22 hours postdose. For the first 4 hours, 15 minute means were calculated for cardiovascular function parameters with 60-minute means calculated thereafter. Mean data were analyzed as the average of percentage change from vehicle value. Neuropharmacological Profile of PER977 in SD Rats. For the neuropharmacological profile test, the Modified Irwin Testing Battery was performed.9 Three groups of 10 male SD rats each received PER977 by IV bolus injection at doses of 0.2, 2, or 20 mg/kg (2.5 mL/kg volumes) and the control group (10 male rats) received 0.9% saline. After dosing, the animals were placed in a fixed environment consisting of a Plexiglas enclosure where animals were free to move. The enclosure was placed on absorbent paper which detects excretions. The rats were observed for apparent neuropharmacological or clinical signs at 5, 15, and 30 minutes and 1, 2, 3, 4, and 24 hours following treatment. Evaluation of Respiratory Function Following Single Dose PER977 in Conscious Rats. For the respiratory function test, 24 male rats (6/ dose group) were trained in the head-out plethysmographic chamber for at least 15 minutes before the day of the experiment. On the day of dosing, each animal was placed in its plethysmographic chamber, and baseline values were obtained for 5 minutes following an approximately 5-minute stabilization period. The rats were then removed from the chamber and dosed with vehicle, or 0.2, 2, or 20 mg/kg PER977 by IV bolus injection. Following dosing, each animal was returned to its designated plethysmograph chamber and the respiratory parameters were recorded 15 minutes (+3 minutes), 1, 2, and 4 hours (+5 minutes) following dose administration of test article/vehicle.

Animals were allowed to stabilize in the plethysmograph for at least 5 minutes before each reading was taken. Tidal volume, minute volume, and respiratory rate were acquired, recorded, and analyzed using DSI Dataquest Open ART (V2.3) & PONEMAH Physiology Platform (V4.2.0). Genotoxicity. The ability of PER977 to induce mutagenicity in S typhimurium tester strains TA97a, TA98, TA100, TA102, and TA1535, was assessed in the presence and absence of rat microsomal fraction S9 mix. PER977 was diluted in 0.9% saline and tested at 0.05, 0.16, 0.5, 1.6, and 5 mg/mL. An aliquot of saline was included and tested as the negative control both with and without metabolic activation. The S-9 specific chemical controls (2-aminofluorene and 2-aminoanthracene) were tested with S-9 metabolic activation only. Strain specific nonmetabolic chemical controls (Sodium Azide, Mitomycin-C, and 4-nitro-0-phenylene-diamine) were only tested in the absence of S-9 metabolic activation. A positive mutagenic response was indicated by demonstration of a clear dose-related response when dilutions were tested and reversion rates greater than 200% of the solvent control in strains TA97a, TA100, and TA102, and reversion rates greater than 300% of the solvent control in strains TA98 and TA1535. The potential of PER977 to induce structural chromosome aberrations in cultured mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells. Based on dose determination studies, PER977 was diluted in saline and final test concentrations of 0.5, 1.6, and 5 mg/mL were evaluated for genotoxicity. The negative control consisted of 1 mL of saline added to the test system. The positive control for the nonactivated system (mitomycin C) and for the activated system (cyclophosphamide) was also evaluated. For cell preparation, CHO cells were seeded into 75 m2 cell culture flasks and incubated with Kaighn modification of Ham’s F-12 medium þ 10% fetal bovine serum (F12K10) until 40% to 60% confluent. For nonactivated systems, the negative control, positive control, and test articles were tested in duplicate. Fetal bovine serum was added at 10% to each flask. The positive control, mitomycin C, was added at 20 mL for a final concentration of 0.166 mg/mL per flask. The final volume in each flask was 10 mL. All of the flasks were incubated for 16 to 18 hours at 37 C (99 F) with 5% + 1% CO2. For the metabolic activation system, the S9 homogenate was prepared from 8- to 10-week-old SD rats injected intraperitoneally with Aroclor 1254. S9 mix was added at 0.65 mL to each flask for a final S9 concentration of 1.5%. The negative control, positive control, and test articles were tested in duplicate. The positive control, cyclophosphamide, was added at 10 mL for a final concentration of 10 mg/mL per flask. The final volume in each flask was 10 mL. All of the flasks were exposed for 3 to 4 hours at 37 C + 1 C with 5% + 1% CO2 for 15 to 21 hours. Prior to cell harvest, cells were examined for cytotoxicity. The cell monolayers were examined microscopically. The flasks were scored at to the degree of discernable morphological cytotoxicity on a relative scale of 0 to 4 with 0 representing

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

Sullivan et al

313

no reactivity (no cell lysis, intracytoplasmic granules) and 4 representing severe reactivity (nearly complete cell destruction). At the end of the exposure and expression periods, the cells were arrested in metaphase with colcemid for 2 hours + 30 minutes. The cells were then removed from the flasks and treated with a 75 mmol/L KCl hypotonic solution and fixative. The cells were dropped onto clean and cold microscope slides. The chromosomes were stained with Giemsa stain and coverslipped with Permount. The cells were then examined microscopically for chromosomal aberrations. The potential genotoxicity of PER977 in vivo was evaluated in CD1 mice. A total of 88 (44 per gender) CD1 mice (Harlan) were administered 0, 0.2, 2.0, or 20 mg/kg PER977 by a single IV dose on study day 1. All animals were observed immediately postdose on day 1 and daily thereafter. The animals were weighed during the acclimation period for dosing calculations and prior to termination. Bone marrow from 5 animals per sex per dose group was collected and evaluated for micronucleus formation within the polychromatic erythrocytes (PCEs) population at 24 and 48 hours postdose.

Statistical Analyses For the 14-day rat and dog studies, the raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each end point by gender and group. Data were analyzed for test article effects by a one-way analysis of variance (ANOVA). If the ANOVA indicated significant between-group differences (P < .05), a group pairwise analysis was performed using Dunnett test, with group 1 designated as the control group. All differences found to have a significance of P < .05 were reported. Commercially available statistical analysis software (JMP 8.0.2.2) was used to perform the statistical analysis. Total and differential leukocyte counts were log 10 transformed and urine pH and specific gravity were ranked prior to statistical analysis. No statistical analysis was performed for cardiovascular function or behavioral tests, as the data generated are not quantitative enough to allow for meaningful comparison. Body temperatures were presented as mean + scanning electron microscope and statistically significant effects were determined by ANOVA with Tukey HSD multiple comparison test. A P value  .05 denotes statistical significance. For the respiratory study in rats, values for the test article-treated group were compared back to the vehicle controls and baseline using a 2-way repeated measures ANOVA followed by a Bonferroni multiple comparison test (SigmaStat, v2.03). Differences with P values .05 were considered statistically significant. For the bacterial reverse mutation assays, significance was indicated by demonstration of a clear dose-related response when dilutions were tested and a doubling in reversion rates compared to control for strains TA97a, TA100, and TA102, and a tripling in reversion rates compared to control for strains TA98 and TA1535. For the in vitro chromosome aberration assay, significance was determined when the positive results

were higher than the critical chi-square value of 3.841 at a confidence interval of 95%. For the micronucleus assay, the raw data were tabulated within each time interval, and the mean was calculated for each end point by gender and group. The differences in response between the sexes were assessed using t test within group. Group mean comparisons for all pairs were performed using Tukey test. All differences found to have a significant P < .05 were reported. Statistical analysis software (SAS Institute Inc., Cary, NC, USA) (JMP 8.0.2.2) was used to perform the statistical analysis.

Results Maximum Tolerated Dose Study of PER977 in SD Rats No mortality was seen at doses up to 40 mg/kg. Administration of 60 mg/kg PER977 resulted in the death of 1/3 rats at approximately 3 minutes postdose. No clinical observations were seen following IV administration of 10 or 20 mg/kg PER977. Administration of 40 mg/kg PER977 resulted in immediate postdose decreased activity in all 3 animals. By 1 hour postdose, red stained snares were seen in 2 of the 3 animals with all animals returning to normal by 24 hours postdose. Prostration, labored breathing, and intermittent flailing were observed in 60 mg/kg animals immediately following dosing. Mortality was observed in 1/3 60 mg/kg animals, and 1 of the remaining 2 animals had red stained nares at 1 hour postdose. The remaining 2 animals were normal by 24 hours postdose. In the confirmatory dose of 40 mg/kg, decreased activity was seen immediately following dosing administration and red stained nares observed at 1 hour postdose. Based on the results of this study, the MTD following a single IV administration of PER977 to SD rats was 40 mg/kg.

Maximum Tolerated Dose Study of PER977 in Beagle Dogs Following exposure to 47 mg/kg PER977, the animal developed neurologic and vascular reactions to the injection. Therefore, the subsequent dose was decreased by approximately 50% (25 mg/kg). No similar findings were seen in the dog treated with 25 mg/kg PER977. Therefore, the dose was escalated to 35 mg/kg for the third animals. Although there were mild indications of reaction to treatment administration at this dose, they were not considered to be physiologically serious, and a target dose of 35 mg/kg PER977 was selected for animals designated for the dose confirmation phase of this study. For the confirmation phase of the study, 4 animals (2 males and 2 females) were dosed intravenously with 35 to 37 mg/kg PER977 for the dose confirmation test. Hypersalivation and/or loss of balance were observed immediately during/following dose administration. However, these findings were resolved within several minutes postdose. All animals throughout the study demonstrated normal appetence and maintained body weights. Besides occasional findings of soft or loose stool, there were no indications of treatment-related effects in any

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

314

International Journal of Toxicology 34(4)

animals after the dosing day. It was concluded that 35 mg/kg was an acceptable target dose level in Beagle dogs.

Table 1. Select Clinical Observations in Rats Administered PER977 During 14-Day Study.a

Maximum Tolerated Dose Study of BAP in SD Rats

Gender Dose, mg/kg/d

No mortality was seen following dosing of 5, 10, 20, 40, 60, or 80 mg/kg BAP. No clinical observations were seen following IV administration of up to 40 mg/kg BAP. Administration of 60 or 80 mg/kg BAP resulted in immediate postdose decreased activity in all animals. All animals returned to normal by 24 hours postdose. Based on the results of this study, the MTD following a single IV administration of BAP to SD rats was determined to be greater than 80 mg/kg.

14-Day Repeat Dose of IV PER977 in SD Rats There was no morbidity/mortality observed during the study. There were clinical observations of apathy and decreased locomotive activities noted in the PER977 2.0 and 20.0 mg/kg treatment groups. Nasal discharge or swelling was observed across groups, but with higher incidence in the treatment group of 20.0 mg/kg (Table 1). These observations were observed typically within 30 minutes postdose and were not observed beyond that. No PER977 treatment-related effects on body weights or food consumption were seen. A full listing of hematology and serum chemistry values can be found in Supplementary Tables 1 and 2 for females and males, respectively. Following the 14-day study period, female rats in the 0.2 mg/kg/d group had statistically significant higher hematocrits and male rats in the 20.0 mg/kg/d group had statistically significant increased absolute and relative monocyte counts when compared to vehicle controls. However, the differences were not considered clinically significant or related to administration of PER977. There were no significant effects for any other hematology parameters, including coagulation parameters (Prothrombin time (PT) and activated partial thromboplastin times [APTTs]). Elevated serum sodium concentrations were seen in female animals in all dose groups and in male animals for the highest dose (20.0 mg/kg) as compared to controls. Other statistically significant serum chemistry results (higher glucose in male rats and lower glucose in female rats in the 2.0-mg/kg dose group, lower creatinine in male rats after 2.0 mg/kg dose, lower triglycerides in female rats of all PER977 dose groups, higher globulin, total protein and cholesterol in male animals of the high-dose group of 20 mg/kg, and higher globulin in females of the 0.2-mg/kg group) were considered to be incidental variations which were without toxicological relevance. No PER977 treatment-related differences in the ophthalmologic examination were observed, and no study drug-related differences in organ weights or relative organ weights were noted between treatment and control groups. There were no treatment related gross necropsy findings or histopathologic observations in tissues at the completion of the 14-day treatment period. In conclusion, repeat dose administration up to 20 mg/kg of PER977 for 14 days in SD rats was well tolerated and

Male 0

0.2

2

Female 20

0

0.2

2

20

Slight/moderate nasal 2/2 6/4 17/8 31/10 2/2 10/6 7/4 21/10 swelling Nasal discharge 2/2 2/2 3/2 9/7 0 3/2 4/3 6/5 Slight apathy 0 0 2/2 7/5 0 0 1/1 1/1 Decreased locomotive 0 0 2/2 2/2 0 0 1/1 2/2 activity a The numbers are expressed as total number of observations/number of animals concerned, N ¼ 10.

Table 2. Toxicokinetic Parameters in Sprague-Dawley Rats Administered PER977. Dose level, mg/kg 0.2 2 20

Day

Cmax, ng/mL

t1/2 min

AUClast, min  ng/mL

Tlast, min

1 14 1 14 1 14

– 73 587 716 14168 20515

– – 9.3 – 13.2 –

– 183 12241 4884 149818 215882

30 5 60 15 60 60

The dashes indicate insufficient data to determine.

did not show systemic toxicity. The no observable adverse effect level (NOAEL) in rats in this study was determined to be 20 mg/kg. The toxicokinetic evaluation results for this study are summarized in Table 2. All animals in this study arm received their target doses over a 14-day period. On day 1, after IV dose administration of PER977 at 2 and 20 mg/kg, the elimination half-life values were 9.3 and 13.2 minutes, respectively. Tlast was no longer than 60 minutes. PER977 exposure increased as the dose level increased from 2 to 20 mg/kg (1:10-fold). A 1:24 fold increase in mean Cmax and a 1:12-fold increase in AUClast were observed. On day 14, the elimination half-life values were not determined due to insufficient data. Tlast ranged from 5 to 60 minutes. As the dose level increased from 0.2 to 2 and further to 20 mg/kg (1:10:100-fold), a 1:10:281-fold increase in mean Cmax and a 1:27:1180-fold increase in AUClast were observed. The study drug concentration in the day 14 predose samples was below the limit of quantitation, indicating little to no drug accumulation with repeat IV administration of PER977.

14-Day Repeat Dose of IV PER977 in Beagle Dogs No morbidity or mortality was observed over the course of the study. During the course of the study, clinical observations were noted at doses 2 mg/kg/d including lip licking, gait disturbance, and inability to stand. These findings were often resolved within 5 to 10 minutes postdose and always within 30 minutes of dosing. No treatment-related differences in

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

Sullivan et al

315

Table 3. Gross Necropsy Findings in Beagle Dogs Administered PER977.a Dose, mg/kg/d 0 0 0

0.2 0.2 0.2 0.2 2 2 20

Sex

Findings

Male Hemo lymph node—perirenal Female Thymus obscured. Cranial mediastinal hemorrhage, extensive Female Pituitary cyst (2)—3 mm and 1 mm doral aspect; left cranial lube of lung—focal pneumonia, 1 cm at margin Male Prostate hyperplasia—dose site collected Male Dose site hemorrhage—dose site collected Female Fibrous adhesion on right caudal lung lobe, with focal pneumonia, purulent Female Dose site hemorrhage—dose site collected Female Spleen—capillary fibrosis 1.5 cm diameter Female Pituitary cyst—dorsorostral aspect—collected on pituitary 1 mm Female Dose site hemorrhage—collected dose site vein

a

Animals not listed had no abnormal findings.

Table 4. Toxicokinetic Parameters in Beagle Dogs Administered PER977. Dose level, mg/kg 0.2 2 20

Day

Cmax, ng/mL

t1/2, min

AUClast, min  ng/mL

Tlast, min

1 14 1 14 1 14

167 163 2967 3291 58549 56528

– – 4.8 5.7 8.9 15.1

865 861 19455 22254 601187 559213

8.5 8.8 23.3 26.7 60 80

The dashes indicate insufficient data to determine.

body weights or body weight changes, food consumption, ocular abnormalities, ECG and blood pressure or respiratory rates, clinical pathology (hematology, serum chemistry, and coagulation parameters), organ weights (relative and absolute), or histopathological changes were observed. Incidental gross necropsy findings (Table 3) were considered not to be test article related. Therefore, the NOAEL for this study was determined to be 20 mg/kg/d. Measurable concentrations of PER977 were present in serum samples up to 10-, 30-, and 60 minutes post-PER977 dosing on day 1 and up to 15-, 30-, and 120 minutes postPER977 dosing on day 14 in all PER977 dosing groups (0.2, 2.0, and 20 mg/kg). The toxicokinetic results are summarized in Table 4. There were no gender differences on day 1 or day 14 PER977 toxicokinetic parameters. At 0.2 mg/kg, there was an insufficient number of quantifiable plasma PER977 concentrations to determine t1/2 and its dependent parameters. After IV administration, PER977 was rapidly eliminated from blood with a mean t1/2 of 5 to 9 minutes on day 1 and 6 to 15 minutes on day 14. It seemed that Tlast was prolonged as dose increased from 2 to 20 mg/kg, with corresponding approximately 60% to 70% decreases in plasma clearance and approximately 40% to

50% decrease in the volume of distribution. There was a dose related and linear increase in PER977 systemic exposure as indicated by mean Cmax and AUClast. The percentage of area under the curve determined by extrapolation of the last quantifiable PER977 plasma concentration was less than 7% in all animals. Day 14 predose samples had PER977 concentrations below the limit of quantitation indicating that there was no accumulation of PER977 with 14 consecutive daily IV doses.

14-Day Repeat Dose of IV BAP in SD Rats All animals survived until the end of the study. No clinical observations were noted at doses of 5 and 20.0 mg/kg BAP. No treatment-related effects were seen on body weight, body weight gain, or food consumption. No opthalmological findings were noted during the study. Following the 14-day study period, there were no treatment-related effects for any hematology parameters. A full listing of coagulation and serum chemistry parameters data are listed in Supplementary Tables 3 and 4 for males and females, respectively. Although not statistically significant due to the large standard deviation, on day 15, APTTs were biologically increased in high-dose males and females when compared to controls (males: control 10.06 seconds, high dose 21.78 seconds; females: control 8.97 seconds, high dose 29.93 seconds). Elevated serum sodium concentrations were seen in female animals in both BAP dose groups as compared to controls. Increased albumin levels were seen in high-dose females when compared to controls. These differences were not considered biologically relevant as the changes occurred only in females, did not show a doserelated effect, and were within normal biological range. No treatment-related changes in absolute or relative organ weights were seen. There were no treatment-related gross necropsy findings. No treatment-related microscopic findings were seen. All findings, including unilateral corneal dystrophy in 2 highdose animals, were considered incidental background findings of no toxicological significance because they were also noted in control animals or are known background findings in animals of this species, strain, and age. Based on the increased APTT in both male and female rats following treatment with 20 mg/kg BAP, the NOAEL for this study was considered to be 5 mg/kg.

14-Day Repeat Dose of IV BAP in Beagle Dogs No morbidity/mortality was observed over the course of the study, and all animals survived to scheduled sacrifice. There were no treatment-related clinical observations during the course of the study following IV administration of up to 20 mg/kg BAP. No treatment-related differences in body weights or body weight changes, food consumption, or ocular abnormalities, ECG, clinical pathology (hematology, serum chemistry, and coagulation parameters), organ weights (relative and absolute), gross necropsy findings, or histopathological changes were observed. Therefore, the NOAEL for this study was determined to be 20 mg/kg/d.

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

316

International Journal of Toxicology 34(4)

Safety Pharmacology

Table 5. Respiratory Function of PER977 in Conscious Rats.a

Evaluation of Cardiovascular Function After PER977 IV Administration in Conscious Telemetered Male Beagle Dogs. No treatment-related clinical signs were seen during the cardiovascular function study in dogs. Intravenous administration of PER977 did not induce any dose-related effects on heart rate, systolic, diastolic, or mean arterial blood pressure in conscious telemetered beagle dogs. The qualitative review of the ECGs showed that PER977 at doses up to 20 mg/kg/d did not have any toxicological effects on cardiac rhythm or ECG morphology. No treatment-related changes were seen for the evaluated cardiovascular function parameters.

Dose, mg/kg

Neuropharmacological Profile of PER977 in SD Rats. No neuropharmacological or clinical signs were observed through 24 hours postdose in any rats receiving the vehicle or IV PER977 at doses of 0.2, 2, or 20 mg/kg throughout the study. Body temperatures were comparable to controls at 30 minutes after dosing. Evaluation of Respiratory Function Following Single Dose PER977 in Conscious Rats. The IV administration of PER977 at 0.2 mg/kg produced statistically significant decreases in respiratory rate at 2 and 4 hours following dosing compared to the baseline values. These changes were not significant when compared to the vehicle control group (Table 5). The respiratory rate was not affected by administration of PER977 at 2 or 20 mg/kg. Since the decreases in respiratory rate (0.2 mg/kg dose at 2 and 4 hours postdose) were not observed in the mid- and high-dose level groups (2.0 and 20 mg/kg), the decreases in respiratory rate observed in the 0.2-mg/kg dose groups were not considered biologically relevant. No changes in tidal volume or minute volume were seen at any dose when compared to predose values or to vehicle control group at any time point. Genotoxicity Testing. For the S typhimurium reverse mutation assays, PER977 concentrations of 0.05, 0.16, 0.5, 1.6, and 5 mg/mL did not produce a 2-fold or 3-fold increase in the number of revertants or produce a clear dose-related response in any of the test strains. The tested concentrations of PER977 did not meet the criteria for a potential mutagen. In the chromosome aberration assay in CHO cells, PER977 at concentrations of 0.5, 1.6, and 5 mg/mL did not show cytotoxic reactivity in CHO cells. The mitotic indices for nonactivated and activated test article solutions did not show differences to the control. In the mammalian erythrocyte micronucleus assay, one 20mg/kg female animal died immediately after dose administration. There were no clinical findings for all study animals. At the highest dose (20 g/kg), 10 of 11 animals had slight to marked hypoactivity and respiratory distress postdose administration. On average, micronucleated PCEs in the bone marrow ranged from 2 to 7 at 24 hours and 3 to 6 at 48 hours postdose administration among all groups. There were no significant differences in the number of micronucleated cells in PER977-treated animals compared to the control group at

0

0.2

2

20

Time, min 0 15 60 120 240 0 15 60 120 240 0 15 60 120 240 0 15 60 120 240

Respiratory rate, breaths/min 187.72 198.37 197.81 199.77 172.19 211.46 167.41 191.23 160.31 161.88 197.59 202.16 193.44 180.73 169.52 188.94 175.27 193.10 212.69 183.91

+ 7.79 + 10.86 + 7.74 + 3.47 + 10.77 + 11.33 + 23.33 + 13.25 + 17.68b + 16.39b + 9.83 + 16.37 + 15.25 + 11.99 + 19.76 + 10.85 + 14.97 + 11.62 + 11.55 + 17.89

Tidal volume, mL

Minute volume, mL/min

1.03 + 1.02 + 0.98 + 1.01 + 1.08 + 0.97 + 0.94 + 0.86 + 0.91 + 0.95 + 0.97 + 0.93 + 0.93 + 1.02 + 1.02 + 0.96 + 0.95 + 1.06 + 0.89 + 1.00 +

188.11 + 195.42 + 186.52 + 196.01 + 180.68 + 195.06 + 153.38 + 159.00 + 136.63 + 148.15 + 187.65 + 179.95 + 173.49 + 174.57 + 164.07 + 177.92 + 158.46 + 195.49 + 179.49 + 176.91 +

0.08 0.07 0.09 0.03 0.05 0.07 0.07 0.08 0.09 0.11 0.06 0.05 0.04 0.08 0.07 0.06 0.04 0.04 0.05 0.05

13.19 8.41 13.57 5.19 10.16 5.86 24.39 21.55 21.23 19.89 13.49 11.70 20.17 14.82 19.83 18.51 12.42 5.59 9.84 18.20

Abbreviation: SEM, scanning electron microscopy. a Values are reported as mean + SEM, N ¼ 6. b Statistically significant (P  .05) change from time 0.

either sampling time (24 or 48 hours postdose), indicating that PER977 does not cause cytogenetic damage in CD1 mice after a single IV administration of up to 20 mg/kg. Based on these test results, PER977 is not considered to be genotoxic.

Discussion Although the NOACs have numerous advantages, they are associated with a risk of major bleeding and thus limited by the lack of a proven reversal agent. PER977 has demonstrated the ability to completely reverse apixaban, dabigatran, edoxaban, rivaroxaban, LMWH, fondaparinux, and UFH in vitro and/or in vivo in rats, and ex vivo in human plasma and whole blood.5,6 Described here are some of the nonclinical studies that were performed prior to first-in-human (FIH) clinical trials to establish the safety of PER977, a new molecular entity currently under development as a reversal agent for NOACs and heparins. The nonclinical safety assessment was designed based on the ICH M3(R2) Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.10 The studies described here are single dose and 14-day repeat dose toxicity studies for PER977 and BAP in rats and dogs, evaluation of respiratory function in rats following a single dose of PER977, neuropharmacological profile of PER977 in rats, evaluation of cardiovascular function after PER977 administration in Beagle dogs, as well as a standard genotoxicity testing (Ames test, and chromosome aberration test). The repeat dose toxicity testing in rats and dogs did not show any treatment-related toxicological findings and confirmed an NOAEL of 20 mg/kg dose in each species for

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015

Sullivan et al

317

PER977 which is over 60 times the starting dose chosen for the FIH study (19.4 mg/d). Similarly, no treatment-related effects were seen in rats or dogs administered BAP daily over 14 days at levels up to 5 mg/kg or 20 mg/kg, respectively (equivalent to or greater than levels of the metabolite achieved by administration of the parent compound). Toxicokinetic testing did not show any accumulation of PER977 with any dose level after repeat dose administration over 14 days. Up to the highest dose tested (20 mg/kg), PER977 did not show any treatment-related effects on the respiratory function or any neurological toxicity in rats and did not indicate any treatment-related effects on the cardiovascular function in dogs. Furthermore, no genotoxic potential was identified in the in vitro standard Ames test, chromosome aberration test, or the in vivo mouse micronucleus assay. No target organ toxicities were identified in any of the studies. The safety profile established in rats, dogs, and in vitro allowed for the safe use of PER977 in human clinical trials which are currently ongoing.11 Said clinical studies have not demonstrated any safety issues associated with PER977 administered alone or following anticoagulants. Authors’ Note Sullivan contributed to conception and design; contributed to acquisition, analysis, and interpretation; drafted the manuscript; and critically revised the manuscript; Gad contributed to conception and design; contributed to acquisition, analysis, and interpretation; and critically revised the manuscript. Laulicht contributed to conception and design; contributed to acquisition, analysis, and interpretation; and critically revised the manuscript. Bakhru contributed to conception and design; contributed to acquisition, analysis, and interpretation; and critically revised the manuscript. Steiner contributed to conception and design; contributed to acquisition, analysis, and interpretation; and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Bryan Laulicht, Sasha Bakhru, and Solomon Steiner are employees of Perosphere Biopharmaceuticals, Inc.

Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Perosphere Biopharmaceuticals, Inc.

Supplemental Material The online [appendices/data supplements/etc] are available at http:// ijt.sagepub.com/supplemental

References 1. Centers for Disease Control and Prevention. Venous Thromboembolism: A Public Health Concern. Web site. http://www.cdc.gov/ NCBDDD/dvt/data.html. Accessed December 22, 2014. 2. Centers for Disease Control and Prevention. Number, Rate and Standard Error of all Listed Surgical and Nonsurgical Procedures for Discharges From Short-Stayhospitals by Selected Procedure Categories: United States, 2010. Web site. http:// www.cdc.gov/nchs/data/nhds/4procedures/2010pro4_numberrate. pdf. Accessed December 22, 2014. 3. Centers for Disease Control and Prevention. Atrial Fibriallation Fact Sheet. Web site. http://www.cdc.gov/dhdsp/data_statistics/fact_ sheets/docs/fs_atrial_fibrillation.pdf. Accessed January 13, 2014. 4. MacLean S, Mulla S, Akl EA, et al., Patient values and preferences in decision making for antithrombotic therapy: a systematic review: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012;141(suppl 2):e1S-e23S. 5. Laulicht BE, Bakhru SH, Jiang X. Antidote for new oral anticoagulants: mechanism of action and binding specificity of PER977. Presented at: 24th Congress of the International Society on Thrombosis and Haematosis, June 29-July 4, 2013; Amsterm. Abstract. Web site. http://www.eventure-online.com/eventure/publicAbstractView.do? id¼226718&congressId¼6839. Accessed December 22, 2014. 6. Laulicht BE, Bakhru SH, Lee C. Small molecule antidote for anticoagulants. Circulation. 2012;126(suppl 21):A11395. Abstract. 7. Bregman CL, Adler RR, Morton DG, Regan KS, Yano BL, Society of Toxicologic Pathology. Recommended tissue list for histopathologic examination in repeat-dose toxicity and carcinogenicity studies: a proposal of the Society of Toxicologic Pathology (STP). Toxicol Pathol. 2003;31(2):252-253. 8. Sellers RS, Morton D, Michael B, et al. Society of Toxicologic Pathology position paper: organ weight recommendations for toxicology studies. Toxicol Pathol. 2007;35(5):751-755. 9. Irwin S. Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse. Psychopharmacologia. 1968;13(3): 222-257. 10. International Conference on Harmonisation of Technical Requirements for Registartion of Pharmaceuticals for Human Use (ICH). ICH M3(R2) Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals; 2009. Web site. http://www.ich.org/fileadmin/ Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/ M3_R2/Step4/M3_R2__Guideline.pdf. Accessed December 22, 2014. 11. Ansell JE, Bakhru SH, Laulicht BE, et al. Use of PER977 to reverse the anticoagulant effect of edoxaban. N Engl J Med. 2014;371(22):2141-2142.

Downloaded from ijt.sagepub.com at UNIV OF WINNIPEG on August 10, 2015