Acute and subacute toxicology

Immediately after sacrifice in the hemorrhage/ resuscitation pig study (Drobin et al., 2004), approximately 2 hours after dosing, tissues were harvested, fixed and submitted to blinded histo-logic examination (n = 8 animals in each group). These studies showed no signs of abnormal exudates or transudates, no edema of the lungs or other organs, or any other significant findings. In samples of heart, ileum, kidneys, lung, pancreas, spleen, liver and skeletal muscle, no pathological lesions were found in any animal or in any organ that could be attributed to MP4.

The effects of transfusion with MP4 following 30 per cent blood volume removal was performed in 30 rats. The endpoints were survival, clinical pathology, clinical observations and histopathology, measured 14 days after dosing with MP4, pentastarch or lactated Ringer's solution. No significant differences were found in regard to body weight gain and food consumption. In all animals except those that received MP4, abnormalities were noted which included thinness, mild depression and rough hair/coat. There were no significant between-group differences with regard to clinical chemistry or hematology measurements. In particular, there were no significant changes in amylase or lipase. Minimal to moderate alveolar histiocytosis and mild to minimal renal tubular epithelial cell vacuolation were seen in the animals that received MP4. Animals that received pentastarch also demonstrated accumulation of vacuolated macrophages in spleen and lymph nodes. The study concluded that infusion of MP4 is without adverse consequences.

Central nervous system toxicity

Concern has been raised in this regard because of unexpected negative results in a clinical trial of DCLHb in stroke patients (Saxena et al., 1998). In one study, the middle cerebral artery (MCA) was ligated in rats and reperfused after 2 hours with MP4 or lactated Ringer's solution. After euthanasia, no differences were found between the treatment groups in regard to infarct size, and neurological scores were significantly improved in the MP4 group at 24 hours compared to the 3-hour time point.

In a second study, MP4, lactated Ringer's solution, stroma-free hemoglobin (SFH) or whole blood was injected into the cisterna magna of anesthetized rats. MP4 had no effect on cerebral blood flow, but flow was reduced in the SFH and blood groups. Intracranial pressure rose transiently in all animals, but remained elevated only in blood-treated animals. SFH elevated mean arterial pressure - an effect not seen with MP4, blood or lactated Ringer's solution.

In a third study, cortical neurons from fetal mice were exposed to MP4 or SFH in culture.The results showed that release of lactate dehydrogenase (LDH) was significantly higher for the cultures exposed to MP4 compared to SFH. However, interpretation of these experiments is obscured because more metHb was present in the MP4 cultures compared to the SFH cultures. Since oxidation of MP4 is minimal in vivo, it is difficult to relate the results of this study to potential clinical toxicity.

Myocardial histology following 30 per cent blood volume exchange in primates

In November 2000, Baxter Healthcare disclosed that they had observed subendocardial necrosis in certain species, including swine (Burhop and Estep, 2001). Although the significance of this lesion was not known (Baxter proposed it was a result of NO scavenging), the FDA requested that Sangart perform the study in primates to establish whether MP4 caused the same lesion. Sixteen monkeys were studied (eight males and eight females). Animals received a single 30 per cent exchange transfusion with either MP4 or lactated Ringer's solution, then were observed for clinical signs, and changes in body weight, food consumption, behavior, electrocardiogram, serum chemistry, hematology, coagulation and urinalysis. Twelve animals were euthanized on Day 3 and the remaining animals were euthanized on Day 13.

No animal died as a result of administration of MP4, and no changes were noted in body weight, food consumption or behavior for animals that received MP4. No electrocardiographic abnormalities were attributable to the product. Significant rises were noted in serum lactate dehydrogenase (LDH) and aspartate aminotransferase (AST), even after correction for spectrophotometric interference by the hemoglobin itself. There were no histopathological findings in skeletal muscle. There were no treatment-related effects on coagulation parameters (PT, aPTT, platelet count), and changes in urinalysis were limited to the presence of very small amounts of detectable hemoglobin on Day 3.

Gross necropsy did not reveal any alterations attributable to MP4 administration, at either Day 3 or 13, including organ weights. Microscopic changes were limited to the presence of foamy macrophages in the spleen and bone marrow, presumed due to phagocytosis of MP4 or a breakdown product.

In a separate study, 21 ml/kg of MP4 was administered to rhesus monkeys via exchangetransfusion at 5m/min with continuous ECG monitoring. With the exception of one incident, all monkeys appeared electrocardiographically normal throughout the 7-day post-dose monitoring period. A single transient episode (approximately 5 seconds) of ventricular bigeminy in one animal was not considered to be related to treatment with MP4, and may have occurred in response to the exchange transfusion and/or stress during chair restraint.There were no alterations in blood pressure, heart rate or body temperature in any animals during or following infusion of MP4.

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