Study Animals SANGUINATE Dose Levels / Regimen Parameters Observations
Determination of Glomerular Filtration Rate and Renal Blood Flow Following Single Intravenous Administration of SANGUINATE in Rats Rat (n=222) 160, 280, 400 mg/kg Single dose IV Glomerular Filtration Rate (GFR) and Renal Blood Flow (RBF) There were no abnormal clinical observations within the study time period to suggest any acute toxic effect of treatment with the test article.  Within 24 hours of SANGUINATE single-dose intravenous administration, no changes were observed in glomerular filtration rate (GFR) or renal blood flow (RBF) measured clearance.
Five−Day Repeat Dose Toxicity Study of SANGUINATE (PEGylated Bovine Hemoglobin) in Ratswith a 14−Day Recovery Period Rat (n=294) 100, 200, 400 mg/kg 5-day repeat dose; 5 min iv infusion Food consumption, body and organ weight measurements, clinical observations, functional observational battery, hematology, blood chemistry, coagulation, urinalysis, immunogenicity, and histopathology including special staining. No test article related effects on standard parameters.  Significant differences for clinical chemistry parameters including increased creatinine, decreased albumin, and decreased alkaline phosphatase activity.  Histopathological results revealed no macroscopic and/or microscopic treatment related findings, and no target organs and /or treatment related findings.  Iron staining was exclusively in the brain (minimal to mild) and in the kidneys (minimal to moderate), but was not considered biologically relevant.  SANGUINATE was well tolerated up to and including 400 mg/kg after single and after 5 days intravenous administration.  No NOAEL.
Maximum Feasible Dose Study of SANGUINATE in Rats Rat (n=20) 2400 mg/kg 5-day repeat dose (MFD); slow iv push Body weights and clinical observations, blood and urine collected for clinical pathology, gross necropsy and select target organs for histopathology. Treatment related findings were noted in clinical observations, and in the evaluation of clinical pathology and histopathology.  No adverse effects on body weights.  Urine discolored and slight increase in the WBC and RBC.  Clinical signs included excretion of red/brown fluid from the urogenital area and red fluid/staining around eyes, and piloerection.  There were changes in the percentage of lymphocytes, and an increase in the percentage of neutrophils in both sexes and monocytes in males. A decrease in hemoglobin was also seen.  The values of total bilirubin and creatinine were increased, and albumin and total protein were significantly decreased.  Microscopic evaluation showed treatment related findings that were limited to the kidneys and heart in both sexes.  The evaluations showed overt signs of systemic toxicity.
Six Months Repeat Dose Toxicity Study of SANGUINATE (PEGylated Bovine Hemoglobin) in Rats With a 30−Day Recovery Period Rat (n=506) 100, 200, 400, 2400 mg/k 6-mo monthly, repeat dose; 5 min iv infusion Food consumption, body and organ weight measurements, clinical and ophthalmic observations, clinical signs of neurotoxicity, hematology, serum chemistry, coagulation, urinalysis, immunogenicity, and histopathology including special staining. Prolonged bleeding was noted in groups receiving SANGUINATE, and the negative controls DPH and Hextend, following intravenous dosing and/or retro-orbital blood sample collection.  This procedure-related bleeding was not seen in the NaCl control groups.  Significant, dose-dependent, albumin, total protein, total bilirubin, AST, ALP, amylase, calcium, creatinine and BUN effects were seen, not on all days.  Recovery groups presented no significant abnormalities, indicating recovery from any treatment-related effects.  Microscopic evaluation of liver and kidneys did not confirm any test article related effects on these or other organs compared to the controls.
Nine Months Repeat Dose Toxicity Study of SANGUINATE (PEGylated Bovine Hemoglobin) in Minipigs with a 30-Day Recovery Period Pig (n=86) 100, 200, 400, 1600 mg/kg 9-mo monthly; 10-15 min iv infusion Food consumption, body and organ weight measurements, clinical and ophthalmic observations, electrocardiographic exams, hematology, serum chemistry, coagulation, urinalysis, immunogenicity, and histopathology including special staining. Majority of assessments showed no specific effects including body weights, ophthalmic exams, the amount of oxy- and deoxy-hemoglobin in whole blood, electrocardiographic exams, and immunogenicity.  Some differences in organ weights (heart, liver, adrenal, and brain) were observed, but were not considered biologically relevant.  Iron staining was observed in the brain and kidney and in one animal in the carotid aortas and jugular vein, but was not considered biologically relevant.  TNF-α and MDA staining had no clear dose trend.  The only clinical sign considered related was diarrhea, observed in 25% of the animals assigned to the 1600 mg/kg SANGUINATE group.  Some differences in hematology and clinical chemistry parameters were observed.  A significant increase in prothrombin time and activated partial thromboplastin time was observed but not clinically relevant.  A NOAEL could not be determined.
Evaluation of Cardiovascular (Hemodynamic) and Pulmonary Function Following Intravenous Administration of SANGUINATE™ (PEGylated Bovine Hemoglobin) in Conscious Telemetered Male Cynomolgus Monkeys Monkey (n=4) 100, 200, 400, 1200 mg/kg 5-day repeat dose; 5-10 min iv injection Clinical observations, clinical pathology, histopathology, toxicokinetics, ECG, hemodynamics, and pulmonary parameters Observations included loose or soft feces, delayed/prolonged bleeding times, facial and inguinal erythema, pink skin color, petichiae on the leg, decreased activity and white foamy/frothy feces, small red areas on the abdomen and extremities.  Clinical chemistry showed no Troponin, increased creatinine, increased BUN, decreased amylase , decreased ALK, and slightly decreased ALT.  There were no microscopic findings that indicated direct test article toxicity.  100 and 200 mg/kg were not associated with changes in heart rate, blood pressure, ECG or pulmonary parameters.  400 mg/kg was associated with minimal increases in arterial pressure after the first dose and slight increases in heart rate and arterial pressure following the fifth dose.  1200 mg/kg/day was associated with decreased heart rate and increased arterial pressure.  Following the fifth day of dosing at 1200 mg/kg/day, increases in heart rate, arterial pressure and QTc were noted as well as decreases in respiratory rate.
Table 2: Summary of SANGUINATE Toxicity and Safety Program.